Antibody–Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available GlucometersClick to copy article linkArticle link copied!
- Elissa K. LeonardElissa K. LeonardDepartment of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United StatesTranslational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United StatesMore by Elissa K. Leonard
- Miguel Aller PelliteroMiguel Aller PelliteroDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesMore by Miguel Aller Pellitero
- Boris JuelgBoris JuelgRagon Institute of MGH, MIT, and Harvard, Cambridge, Massachusetts 02139, United StatesMore by Boris Juelg
- Jamie B. Spangler*Jamie B. Spangler*Email: [email protected]Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United StatesDepartment of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United StatesTranslational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United StatesDepartment of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesBloomberg−Kimmel Institute for Cancer Immunotherapy, Sidney Kimmel Comprehensive Cancer Center Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United StatesDepartment of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United StatesMore by Jamie B. Spangler
- Netzahualcóyotl Arroyo-Currás*Netzahualcóyotl Arroyo-Currás*Email: [email protected]Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United StatesDepartment of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United StatesMore by Netzahualcóyotl Arroyo-Currás
Abstract
Rapid diagnostics that can accurately inform patients of disease risk and protection are critical to mitigating the spread of the current COVID-19 pandemic and future infectious disease outbreaks. To be effective, such diagnostics must rely on simple, cost-effective, and widely available equipment and should be compatible with existing telehealth infrastructure to facilitate data access and remote care. Commercial glucometers are an established detection technology that can overcome the cost, time, and trained personnel requirements of current benchtop-based antibody serology assays when paired with reporter molecules that catalyze glucose conversion. To this end, we developed an enzymatic reporter that, when bound to disease-specific patient antibodies, produces glucose in proportion to the level of antibodies present in the patient sample. Although a straightforward concept, the coupling of enzymatic reporters to secondary antibodies or antigens often results in low yields, indeterminant stoichiometry, reduced target binding, and poor catalytic efficiency. Our enzymatic reporter is a novel fusion protein that comprises an antihuman immunoglobulin G (IgG) antibody genetically fused to two invertase molecules. The resulting fusion protein retains the binding affinity and catalytic activity of the constituent proteins and serves as an accurate reporter for immunoassays. Using this fusion, we demonstrate quantitative glucometer-based measurement of anti-SARS-CoV-2 spike protein antibodies in blinded clinical sample training sets. Our results demonstrate the ability to detect SARS-CoV-2-specific IgGs in patient serum with precise agreement to benchmark commercial immunoassays. Because our fusion protein binds all human IgG isotypes, it represents a versatile tool for detection of disease-specific antibodies in a broad range of biomedical applications.
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Introduction
Figure 1
Figure 1. Schematic overview of the detection assay to quantify COVID-19-specific antibodies using a commercially available glucometer. A strip coated with SARS-CoV-2 spike protein RBD is incubated with patient serum, and RBD-specific antibodies bind to the strip. The strip is rinsed to remove any nonspecific antibodies and transferred to a solution containing Ab+Inv fusion protein, which binds to the patient antibody captured on the strip. The strip is again rinsed, transferred to a solution of sucrose, and incubated. The amount of sucrose converted to glucose is measured using a commercial glucometer and is proportional to the amount of the patient’s antibodies in the serum.
Results
Design and Purification of Antihuman Antibody–Invertase Fusion Proteins (Ab+Inv)
Figure 2
Figure 2. Design and purification of antibody–invertase (Ab+Inv) fusion proteins. (A) Schematic of Ab+Inv fusion proteins containing an antihuman IgG antibody. The C-terminus of the antibody light chain (LC) was tethered to the N-terminus of invertase via a flexible linker 15 or 25 amino acids in length (LC15 and LC25, respectively) or the C-terminus of the heavy chain (HC) is tethered to the N-terminus of invertase via a flexible linker 17 or 27 amino acids in length (HC17 and HC27, respectively). HC and LC variable and constant domains are labeled. (B) Ab+Inv fusion proteins migrate at slightly larger molecular weights (MW) than expected by SDS-PAGE. The unfused antibody (Ab) and invertase (Inv) alone run at the expected sizes under nonreducing (146 kDa for Ab and 62 kDa for Inv) and reducing (49 kDa for Ab HC, 24 kDa for Ab LC, and 62 kDa for Inv) conditions. Note that the samples for both SDS-PAGE gels were boiled to minimize the divergent migration of invertase and the fusion proteins relative to their expected molecular weights (62 kDa for invertase and ∼266 kDa for the Ab+Inv fusion proteins). The Ab+Inv fusion proteins run somewhat larger than their expected MW (∼266 kDa) under nonreducing conditions, as do the Inv-fused LC (∼84 kDa) and HC (∼110 kDa) under reducing conditions. (C) Yield per liter of transfected cells for each Ab+Inv fusion protein is shown compared to the yield of the unfused antibody (Ab). For LC15, HC17, and HC27, the yield from the pooled peak 1 is shown in a lighter shade and the yield from the pooled peak 2 is shown in the darker shade. Bar height reflects the total yield for both peaks. (D) Representative size-exclusion chromatography (SEC) traces for the four Ab+Inv fusion proteins are shown. The separately pooled fractions that were tested for LC15, HC17, and HC27 (peak 1 and peak 2) are indicated.
Validation of Ab+Inv Binding to Human IgG
Figure 3
Figure 3. Ab+Inv fusion proteins bind hIgG with the same affinity as the unfused antibody. (A) Equilibrium BLI titrations of the soluble HP6017 antibody, Ab+Inv LC15, LC25, HC17, and HC27 against immobilized hIgG. Invertase (Inv) is included as a negative control. (B) KD derived from the three-parameter curve fit of the equilibrium binding data in panel (A) is shown. (C) Association rate (kon) generated from the kinetic BLI curve fit of the data in Figure S2, assuming a 1:1 binding model, is shown.
Validation of Ab+Inv Catalytic Activity
Figure 4
Figure 4. Ab+Inv fusion proteins exhibit equivalent catalytic activity to unfused invertase. (A) Ab+Inv fusion proteins or invertase (Inv) were incubated with 250 mM sucrose for 6 to 24 min. Concentrations of 166 nM Ab+Inv fusion proteins and 330 nM for Inv were used to achieve a molar equivalent amount of the enzyme, n = 1. (B) Various concentrations of unfused Ab, Ab+Inv fusion proteins, or Inv were incubated with 250 mM sucrose for 8 min. The concentration of the Ab+Inv fusion proteins is one-half of the invertase concentration shown on the x-axis, as each fusion protein contains two copies of invertase, n = 2. (C) Unfused Ab, Ab+Inv fusion proteins, or Inv were incubated with various concentrations of sucrose for 15 min. Concentrations of 166 nM Ab+Inv fusion proteins and 330 nM for Inv were used to achieve a molar equivalent amount of the enzyme, n = 2. (D) Unfused Ab, Ab+Inv fusion proteins, or Inv were incubated with 250 mM sucrose for 15 min. Concentrations of 166 nM Ab+Inv fusion proteins and 330 nM for Inv were used to achieve a molar equivalent amount of the enzyme, n = 3. The dotted lines in (A)–(D) indicate the limit of detection for the glucometer (10 mg/dL). Error bars indicate standard deviation in all panels.
Development and Deployment of an Anti-SARS-CoV-2 Antibody Detection Assay
Figure 5
Figure 5. Glucometer-based immunoassay using Ab+Inv fusion protein detects the presence of SARS-CoV-2-targeted antibodies in patient samples. (A) Schematic of the detection workflow for the glucometer-based strip immunoassay, which detects sucrose conversion by Ab+Inv fusion protein LC15 in proportion to SARS-CoV-2 spike protein RBD-targeted antibodies in a test sample. (B) Platform shown in (A) was used to build calibration curves in 10% serum, detecting glucose readouts for various concentrations of the spiked anti-SARS-CoV-2 spike protein RBD antibody. The fitted single curve yields EC50 = 35 nM, with n = 3. The limit of detection (LOD) is 1.2 ± 0.2 nM. (C) This platform was used to measure anti-SARS-CoV-2 antibody titers in patient samples with known seroconversion status (TS1), with n = 3. (D) Schematic of the detection workflow for a benchmark spectrophotometric strip immunoassay, which detects tetramethylbenzidine (TMB) oxidation by horseradish peroxidase (HRP)-labeled anti-hIgG antibodies in proportion to SARS-CoV-2 spike protein RBD-targeted antibodies in a test sample. (E) The strategy shown in (D) was used to build calibration curves in 10% serum, detecting glucose readouts for various concentrations of spiked anti-SARS-CoV-2 spike protein RBD antibodies. The fitted single curve yields EC50 = 2 nM, with n = 3. The LOD is 0.8 ± 0.1 nM. (F) This strategy was used to measure anti-SARS-CoV-2 antibody titers in patient samples with known seroconversion status (TS1), with n = 3. Discrimination between positive and negative samples matched that of the glucometer-based assay. Error bars indicate standard deviation in all panels.
Figure 6
Figure 6. Glucometer-based immunoassay using Ab+Inv fusion protein enables monitoring of anti-SARS-CoV-2 antibody titers in hospitalized patients. A glucometer-based strip immunoassay was used to measure SARS-CoV-2 spike protein RBD-targeted antibody levels in samples from hospitalized patients with COVID-19 (TS2), n = 1. The glucometer assay consistently determined the emergence of anti-SARS-CoV-2 antibody responses at ∼10 days post symptom onset, and responses plateaued at ∼20 days post symptom onset in patients A, B, F, and G. Solid lines are included for visual clarity to highlight IgG titer trends.
Figure 7
Figure 7. Robust clinical agreement observed between glucometer-based and commercial spectrophotometric immunoassays. (A) Longitudinal time course of SARS-CoV-2 spike protein RBD-targeted antibody levels in the hospitalized patient with COVID-19 (patient B from the TS2 cohort), as detected by the glucometer-based immunoassay using Ab+Inv fusion protein, with n = 1. (B) Longitudinal time-course of SARS-CoV-2 spike protein RBD-targeted antibody levels in the hospitalized patient with COVID-19 (patient B from the TS2 cohort), as detected by a commercial spectrophotometric assay (Epitope Diagnostics), with n = 1. (C) To establish a seroconversion threshold for the glucometer-based assay, we computed the average and standard deviation (gray shaded areas) of the glucose output generated by five confirmed SARS-CoV-2 negative samples, obtaining a seroconversion cutoff of 39 ± 32 ng/mL. Extrapolating this threshold to all the data from the TS2 cohort, we were able to accurately stratify by seroconversion status. The short horizontal lines represent the mean and ± standard errors for the two groups. (D) Agreement chart generated using the Bangdiwala method, (38) comparing glucometer-based measurements with a commercial spectrophotometric assay (Coronacheck). A 95% positive and a 96% negative percent agreement were observed.
Discussion
Supporting Information
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- 7Wei, J.; Stoesser, N.; Matthews, P. C.; Ayoubkhani, D.; Studley, R.; Bell, I.; Bell, J. I.; Newton, J. N.; Farrar, J.; Diamond, I.; Rourke, E.; Howarth, A.; Marsden, B. D.; Hoosdally, S.; Jones, E. Y.; Stuart, D. I.; Crook, D. W.; Peto, T. E. A.; Pouwels, K. B.; Eyre, D. W.; Walker, A. S.; the COVID-19 Infection Survey team Antibody Responses to SARS-CoV-2 Vaccines in 45,965 Adults from the General Population of the United Kingdom. Nat Microbiol 2021, 6, 1140– 1149, DOI: 10.1038/s41564-021-00947-3Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1Skt7%252FO&md5=11cc3fc7307c8f79193b7c917f62bd64Antibody responses to SARS-CoV-2 vaccines in 45,965 adults from the general population of the United KingdomWei, Jia; Stoesser, Nicole; Matthews, Philippa C.; Ayoubkhani, Daniel; Studley, Ruth; Bell, Iain; Bell, John I.; Newton, John N.; Farrar, Jeremy; Diamond, Ian; Rourke, Emma; Howarth, Alison; Marsden, Brian D.; Hoosdally, Sarah; Jones, E. Yvonne; Stuart, David I.; Crook, Derrick W.; Peto, Tim E. A.; Pouwels, Koen B.; Eyre, David W.; Walker, A. SarahNature Microbiology (2021), 6 (9), 1140-1149CODEN: NMAICH; ISSN:2058-5276. (Nature Portfolio)Abstr.: We report that in a cohort of 45,965 adults, who were receiving either the ChAdOx1 or the BNT162b2 SARS-CoV-2 vaccines, in those who had no prior infection with SARS-CoV-2, seroconversion rates and quant. antibody levels after a single dose were lower in older individuals, esp. in those aged >60 years. Two vaccine doses achieved high responses across all ages. Antibody levels increased more slowly and to lower levels with a single dose of ChAdOx1 compared with a single dose of BNT162b2, but waned following a single dose of BNT162b2 in older individuals. In descriptive latent class models, we identified four responder subgroups, including a 'low responder' group that more commonly consisted of people aged >75 years, males and individuals with long-term health conditions. Given our findings, we propose that available vaccines should be prioritized for those not previously infected and that second doses should be prioritized for individuals aged >60 years. Further data are needed to better understand the extent to which quant. antibody responses are assocd. with vaccine-mediated protection.
- 8Amanat, F.; Stadlbauer, D.; Strohmeier, S.; Nguyen, T. H. O.; Chromikova, V.; McMahon, M.; Jiang, K.; Arunkumar, G. A.; Jurczyszak, D.; Polanco, J.; Bermudez-Gonzalez, M.; Kleiner, G.; Aydillo, T.; Miorin, L.; Fierer, D. S.; Lugo, L. A.; Kojic, E. M.; Stoever, J.; Liu, S. T. H.; Cunningham-Rundles, C.; Felgner, P. L.; Moran, T.; García-Sastre, A.; Caplivski, D.; Cheng, A. C.; Kedzierska, K.; Vapalahti, O.; Hepojoki, J. M.; Simon, V.; Krammer, F. A Serological Assay to Detect SARS-CoV-2 Seroconversion in Humans. Nat. Med. 2020, 26, 1033– 1036, DOI: 10.1038/s41591-020-0913-5Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptFyqsbY%253D&md5=6f5df9742dec5a6bdb94ac5ccaad043dA serological assay to detect SARS-CoV-2 seroconversion in humansAmanat, Fatima; Stadlbauer, Daniel; Strohmeier, Shirin; Nguyen, Thi H. O.; Chromikova, Veronika; McMahon, Meagan; Jiang, Kaijun; Arunkumar, Guha Asthagiri; Jurczyszak, Denise; Polanco, Jose; Bermudez-Gonzalez, Maria; Kleiner, Giulio; Aydillo, Teresa; Miorin, Lisa; Fierer, Daniel S.; Lugo, Luz Amarilis; Kojic, Erna Milunka; Stoever, Jonathan; Liu, Sean T. H.; Cunningham-Rundles, Charlotte; Felgner, Philip L.; Moran, Thomas; Garcia-Sastre, Adolfo; Caplivski, Daniel; Cheng, Allen C.; Kedzierska, Katherine; Vapalahti, Olli; Hepojoki, Jussi M.; Simon, Viviana; Krammer, FlorianNature Medicine (New York, NY, United States) (2020), 26 (7), 1033-1036CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)We describe a serol. ELISA for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma, and is amenable to scaling. Serol. assays are of crit. importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy, and investigate correlates of protection.
- 9Mendrone-Junior, A.; Dinardo, C. L.; Ferreira, S. C.; Nishya, A.; Salles, N. A.; Almeida Neto, C.; Hamasaki, D. T.; Facincani, T.; Oliveira Alves, L. B.; Machado, R. R. G.; Araujo, D. B.; Durigon, E. L.; Rocha, V.; Sabino, E. C. Correlation between SARS-COV-2 Antibody Screening by Immunoassay and Neutralizing Antibody Testing. Transfusion 2021, 61, 1181– 1190, DOI: 10.1111/trf.16268Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovVKltro%253D&md5=6e7b8dedfc4e77bc7513f9587e7ad7caCorrelation between SARS-COV-2 antibody screening by immunoassay and neutralizing antibody testingMendrone-Junior, Alfredo; Dinardo, Carla Luana; Ferreira, Suzete Cleuza; Nishya, Anna; Salles, Nanci Alves; de Almeida Neto, Cesar; Hamasaki, Debora Toshei; Facincani, Tila; de Oliveira Alves, Lucas Bassolli; Machado, Rafael Rahal Guaragna; Araujo, Danielle Bastos; Durigon, Edison Luiz; Rocha, Vanderson; Sabino, Ester CerdeiraTransfusion (Malden, MA, United States) (2021), 61 (4), 1181-1190CODEN: TRANAT; ISSN:0041-1132. (Wiley-Blackwell)The efficacy of convalescent plasma (CP), an alternative for the treatment of COVID-19, depends on high titers of neutralizing antibodies (nAbs), but assays for quantifying nAbs are not widely available. Our goal was to develop a strategy to predict high titers of nAbs based on the results of anti-SARS-CoV-2 immunoassays and the clin. characteristics of CP donors. A total of 214 CP donors were enrolled and tested for the presence of anti-SARS-CoV-2 antibodies (IgG) using two com. immunoassays: EUROIMMUN (ELISA) and Abbott (Chemiluminescence). Quantification of nAbs was performed using the Cytopathic Effect-based Virus Neutralization test. Three criteria for identifying donors with nAbs ≥ 1:160 were tested: - C1: Curve ROC; - C2: Conditional decision tree considering only the IA results and - C3: Conditional decision tree including both the IA results and the clin. variables. The performance of the immunoassays was similar referring to both S/CO and predictive value for identifying nAbs titers ≥1:160. Regarding the studied criteria for identifying CP donors with high nAbs titers: (a) C1 showed 76.1% accuracy if S/CO = 4.65, (b) C2 presented 76.1% accuracy if S/CO ≥4.57 and (c) C3 had 71.6% accuracy if S/CO was ≥4.57 or if S/CO was between 2.68-4.57 and the last COVID-19-related symptoms were recent (within 19 days). SARS-CoV-2 IgG immunoassays (S/CO) can be used to predict high anti-SARS-CoV-2 nAbs titers. This study has proposed different criteria for identifying donors with ≥1:160 nAbs titers, all with high efficacy.
- 10Oh, H.; Ahn, H.; Tripathi, A. A Closer Look into FDA-EUA Approved Diagnostic Techniques of Covid-19. ACS Infect. Dis. 2021, 7, 2787– 2800, DOI: 10.1021/acsinfecdis.1c00268Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitV2rsbnO&md5=193523ad54754b65632f0b9d5c03e2c1A Closer Look into FDA-EUA Approved Diagnostic Techniques of Covid-19Oh, Hyunju; Ahn, Hyunjeong; Tripathi, AnubhavACS Infectious Diseases (2021), 7 (10), 2787-2800CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)A review. The 2019 coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 virus, caused a worldwide pandemic in 2020 and is the most urgent health issue worldwide. In this review, we highlight the details of Food and Drug Administration-Emergency Use Authorizations approved diagnostics kits, focusing on the similarities and differences. It is essential to understand the currently available options and the advantages and disadvantages each provides to select the appropriate products that maximize the testing efficiency. We believe this work will provide a holistic evaluation of the current COVID-19 diagnostic resources, including variations across the countries, and guide developing novel diagnostic techniques to improve and optimize the current testing options.
- 11Lee, C. Y.-P.; Lin, R. T. P.; Renia, L.; Ng, L. F. P. Serological Approaches for COVID-19: Epidemiologic Perspective on Surveillance and Control. Front. Immunol. 2020, 11, 879, DOI: 10.3389/fimmu.2020.00879Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVWntrvN&md5=eafcca8c0e6b2cb576101ccc604d8384Serological approaches for COVID-19: epidemiologic perspective on surveillance and controlLee, Cheryl Yi-Pin; Lin, Raymond T. P.; Renia, Laurent; Ng, Lisa F. P.Frontiers in Immunology (2020), 11 (), 879CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)A review. Since Dec. 2019, the novel coronavirus, SARS-CoV-2, has garnered global attention due to its rapid transmission, which has infected more than two million people worldwide. Early detection of SARS-CoV-2 is one of the crucial interventions to control virus spread and dissemination. Mol. assays have been the gold std. to directly detect for the presence of viral genetic material in infected individuals. However, insufficient viral RNA at the point of detection may lead to false neg. results. As such, it is important to also employ immune-based assays to det. one's exposure to SARS-CoV-2, as well as to assist in the surveillance of individuals with prior exposure to SARS-CoV-2. Within a span of 4 mo, extensive studies have been done to develop serol. systems to characterize the antibody profiles, as well as to identify and generate potentially neutralizing antibodies during SARS-CoV-2 infection. The vast diversity of novel findings has added value to coronavirus research, and a strategic consolidation is crucial to encompass the latest advances and developments. This review aims to provide a concise yet extensive collation of current immunoassays for SARS-CoV-2, while discussing the strengths, limitations and applications of antibody detection in SARS-CoV-2 research and control.
- 12Porstmann, B.; Porstmann, T.; Nugel, E.; Evers, U. Which of the Commonly Used Marker Enzymes Gives the Best Results in Colorimetric and Fluorimetric Enzyme Immunoassays: Horseradish Peroxidase, Alkaline Phosphatase or β-Galactosidase?. J. Immunol. Methods 1985, 79, 27– 37, DOI: 10.1016/0022-1759(85)90388-6Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXksFagsbk%253D&md5=29951f3bc50194081427a6febbfde135Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or β-galactosidase?Porstmann, Baerbel; Porstmann, T.; Nugel, E.; Evers, U.Journal of Immunological Methods (1985), 79 (1), 27-37CODEN: JIMMBG; ISSN:0022-1759.Comparing the marker enzymes horseradish peroxidase (HRP), alk. phosphatase (AP) and β-galactosidase (βGal) in IgG-coupled form with respect to their temp.-dependent kinetics over a period of 22 h, the temp. of 37° warrants highest substrate turnover of all enzymes at all reaction times using fluorogens. Also, applying chromogens, the optimum temp. for βGal is 37°; for HRP and AP, the optimum temp.depends on the reaction time. The substrate turnover of HRP using ABTS as chromogen is much higher compared to the other enzymes, with respect to both mol enzyme (molar activity) and to gram enzyme (specific activity). The turnover decreases for all enzymes in different degrees after coupling to IgG. The turnover of fluorogenic substrates is lower for all enzymes than the turnover of chromogenic substrates but due to the more sensitive detection of fluorogenic products the detection limits for all conjugates were lowered too; in particular, detection limits for βGal-IgG were lower by a factor of 333 compared to the colorimetric procedure. In a 2-site binding EIA for α1-fetoprotein (AFP), the detection limit for AFP was reduced only by a factor of 2 with all 3 marker enzymes when the fluorimetry assay was used rather than the colorimetry assay. The HRP-IgG conjugates allowed for lowest detection limits for AFP (0.5-1 μg/1), highest anal. sensitivity (slope of std. curves) at shortest periods of substrate reaction compared to the other enzymes.
- 13Gundinger, T.; Spadiut, O. A Comparative Approach to Recombinantly Produce the Plant Enzyme Horseradish Peroxidase in Escherichia coli. J. Biotechnol. 2017, 248, 15– 24, DOI: 10.1016/j.jbiotec.2017.03.003Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXksFChu7s%253D&md5=da0e0766b0c03e31cfdfff6a549d45d9A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coliGundinger, Thomas; Spadiut, OliverJournal of Biotechnology (2017), 248 (), 15-24CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)Horseradish peroxidase (HRP) is used in various biotechnol. and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither prodn. from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titers of 10 mg per L cultivation broth. Thus, the traditional way of prodn. of HRP from plant still prevails. In this study, we revisited the recombinant prodn. of HRP in E. coli and investigated and compared both strategies, (a) the prodn. of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the prodn. of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titer of 100 mg L-1 cultivation broth, and were able to produce 48 mg active HRP per L cultivation broth in the periplasm. In terms of biochem. properties, sol. HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and prodn. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amts., which are then refolded.
- 14Lewis, T. L.; Roth, K. A. Immunohistochemical Detection Methods. In Pathobiology of Human Disease, Elsevier, 2014; pp https://doi.org/10.1016/B978-0-12-386456-7.07405-0 pp 3829– 3840.Google ScholarThere is no corresponding record for this reference.
- 15Vashist, S. K.; Luong, J. H. T. Enzyme-Linked Immunoassays. In Handbook of Immunoassay Technologies; Elsevier, 2018; pp 97– 127.Google ScholarThere is no corresponding record for this reference.
- 16Moshe, M.; Daunt, A.; Flower, B.; Simmons, B.; Brown, J. C.; Frise, R.; Penn, R.; Kugathasan, R.; Petersen, C.; Stockmann, H.; Ashby, D.; Riley, S.; Atchison, C.; Taylor, G. P.; Satkunarajah, S.; Naar, L.; Klaber, R.; Badhan, A.; Rosadas, C.; Marchesin, F.; Fernandez, N.; Sureda-Vives, M.; Cheeseman, H.; O’Hara, J.; Shattock, R.; Fontana, G.; Pallett, S. J. C.; Rayment, M.; Jones, R.; Moore, L. S. P.; Ashrafian, H.; Cherapanov, P.; Tedder, R.; McClure, M.; Ward, H.; Darzi, A.; Elliott, P.; Cooke, G. S.; Barclay, W. S. SARS-CoV-2 Lateral Flow Assays for Possible Use in National Covid-19 Seroprevalence Surveys (React 2): Diagnostic Accuracy Study. BMJ 2021, n423, DOI: 10.1136/bmj.n423Google ScholarThere is no corresponding record for this reference.
- 17Nguyen, V.-T.; Song, S.; Park, S.; Joo, C. Recent Advances in High-Sensitivity Detection Methods for Paper-Based Lateral-Flow Assay. Biosens. Bioelectron. 2020, 152, 112015 DOI: 10.1016/j.bios.2020.112015Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFOjt7w%253D&md5=636c5c3be6ce03fc803cd92598430209Recent advances in high-sensitivity detection methods for paper-based lateral-flow assayNguyen, Van-Thuan; Song, Seungri; Park, Seungkyung; Joo, ChulminBiosensors & Bioelectronics (2020), 152 (), 112015CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Paper-based lateral-flow assays (LFAs) have achieved considerable com. success and continue to have a significant impact on medical diagnostics and environmental monitoring. Conventional LFAs are typically performed by examg. the color changes in the test bands by naked eye. However, for crit. biochem. markers that are present in extremely small amts. in the clin. specimens, this readout method is not quant., and does not provide sufficient sensitivity or suitable detection limit for a reliable assay. Diverse technologies for high-sensitivity LFA detection have been developed and commercialization efforts are underway. In this review, we aim to provide a crit. and objective overview of the recent progress in high-sensitivity LFA detection technologies, which involve the exploitation of the phys. and chem. responses of transducing particles. The features and biomedical applications of the technologies, along with future prospects and challenges, are also discussed.
- 18Urusov, A. E.; Zherdev, A. V.; Dzantiev, B. B. Towards Lateral Flow Quantitative Assays: Detection Approaches. Biosensors 2019, 9, 89, DOI: 10.3390/bios9030089Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtF2msrrN&md5=8dd5eb6a2ea0944f9d903da0924324e3Towards lateral flow quantitative assays: detection approachesUrusov, Alexandr E.; Zherdev, Anatoly V.; Dzantiev, Boris B.Biosensors (2019), 9 (3), 89CODEN: BIOSHU; ISSN:2079-6374. (MDPI AG)Point-of-care (POC) or bedside anal. is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufg. technol. for lateral flow (immunochromatog.) test strips. They are widely used to rapidly and easily control a variety of biomarkers of infectious diseases and metabolic and functional disorders, as well as in consumer protection and environmental monitoring. However, traditional lateral flow tests rely on visual assessment and qual. conclusion, which limit the objectivity and information output of the assays. Therefore, there is a need for approaches that retain the advantages of lateral flow assays and provide reliable quant. information about the content of a target compd. in a sample mixt. This review describes the main options for detecting, processing, and interpreting immunochromatog. anal. results. The possibilities of modern portable detectors that register colored, fluorescent, magnetic, and conductive labels are discussed. Prospects for further development in this direction are also examd.
- 19Lan, T.; Zhang, J.; Lu, Y. Transforming the Blood Glucose Meter into a General Healthcare Meter for in Vitro Diagnostics in Mobile Health. Biotechnol. Adv. 2016, 34, 331– 341, DOI: 10.1016/j.biotechadv.2016.03.002Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktVOnurY%253D&md5=5aeaf550daf477c4d8fc706c212208ceTransforming the blood glucose meter into a general healthcare meter for in vitro diagnostics in mobile healthLan, Tian; Zhang, Jingjing; Lu, YiBiotechnology Advances (2016), 34 (3), 331-341CODEN: BIADDD; ISSN:0734-9750. (Elsevier)Recent advances in mobile network and smartphones have provided an enormous opportunity for transforming in vitro diagnostics (IVD) from central labs to home or other points of care (POC). A major challenge to achieving the goal is a long time and high costs assocd. with developing POC IVD devices in mobile Health (mHealth). Instead of developing a new POC device for every new IVD target, we and others are taking advantage of decades of research, development, engineering and continuous improvement of the blood glucose meter (BGM), including those already integrated with smartphones, and transforming the BGM into a general healthcare meter for POC IVDs of a wide range of biomarkers, therapeutic drugs and other anal. targets. In this review, we summarize methods to transduce and amplify selective binding of targets by antibodies, DNA/RNA aptamers, DNAzyme/ribozymes and protein enzymes into signals such as glucose or NADH that can be measured by com. available BGM, making it possible to adapt many clin. assays performed in central labs, such as immunoassays, aptamer/DNAzyme assays, mol. diagnostic assays, and enzymic activity assays onto BGM platform for quantification of non-glucose targets for a wide variety of IVDs in mHealth.
- 20Xiang, Y.; Lu, Y. Using Personal Glucose Meters and Functional DNA Sensors to Quantify a Variety of Analytical Targets. Nature Chem 2011, 3, 697– 703, DOI: 10.1038/nchem.1092Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptlamu70%253D&md5=4340300b8a4bdb89f7e72dedb90784acUsing personal glucose meters and functional DNA sensors to quantify a variety of analytical targetsXiang, Yu; Lu, YiNature Chemistry (2011), 3 (9), 697-703CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Portable, low-cost and quant. detection of a broad range of targets at home and in the field has the potential to revolutionize medical diagnostics and environmental monitoring. Despite many years of research, very few such devices are com. available. Taking advantage of the wide availability and low cost of the pocket-sized personal glucose meter - used worldwide by diabetes sufferers - the authors demonstrate a method to use such meters to quantify nonglucose targets, ranging from a recreational drug (cocaine, 3.4 μM detection limit) to an important biol. cofactor (adenosine, 18 μM detection limit), to a disease marker (interferon-gamma of tuberculosis, 2.6 nM detection limit) and a toxic metal ion (uranium, 9.1 nM detection limit). The method is based on the target-induced release of invertase from a functional-DNA-invertase conjugate. The released invertase converts sucrose into glucose, which is detectable using the meter. The approach should be easily applicable to the detection of many other targets through the use of suitable functional-DNA partners (aptamers, DNAzymes or aptazymes).
- 21Chávez, F. P.; Rodriguez, L.; Díaz, J.; Delgado, J. M.; Cremata, J. A. Purification and Characterization of an Invertase from Candida Utilis: Comparison with Natural and Recombinant Yeast Invertases. J. Biotechnol. 1997, 53, 67– 74, DOI: 10.1016/S0168-1656(97)01663-5Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjtlKnsbg%253D&md5=80cab2b4f800f4002c7f8d6744393330Purification and characterization of an invertase from Candida utilis: comparison with natural and recombinant yeast invertasesChavez, Francisco P.; Rodriguez, Luis; Diaz, Joaquin; Delgado, Julio M.; Cremata, Jose A.Journal of Biotechnology (1997), 53 (1), 67-74CODEN: JBITD4; ISSN:0168-1656. (Elsevier)A periplasmic invertase (I) from C. utilis was purified to homogeneity from cells fully derepressed for I synthesis. I was purified by successive Sephacryl S-300 and affinity chromatogs. and shown to be a dimeric glycoprotein composed of 2 identical monomer subunits with an apparent mol. wt. of 150 kDa. After Endo H treatment, the deglycosylated protein showed an apparent mol. wt. of 60 kDa. The apparent Km values for sucrose and raffinose were 11 and 150 mM, resp., similar to those reported for I from Saccharomyces cerevisiae. The range of optimum temp. was 60-75°. The optimum pH was 5.5, and I was stable over the pH range of 3-6.
- 22Gangadhara; Ramesh Kumar, P.; Prakash, V. Influence of Polyols on the Stability and Kinetic Parameters of Invertase from Candida Utilis: Correlation with the Conformational Stability and Activity. Protein J. 2008, 27, 440– 449, DOI: 10.1007/s10930-008-9154-zGoogle Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtl2lsLbL&md5=bd6480ad2d04e54c5928a4b4b21dff17Influence of polyols on the stability and kinetic parameters of invertase from Candida utilis: Correlation with the conformational stability and activityGangadhara; Kumar, Parigi Ramesh; Prakash, VishweshwaraiahProtein Journal (2008), 27 (7-8), 440-449CODEN: PJROAH; ISSN:1572-3887. (Springer)Invertase (EC 3.2.1.26) (I) is a sucrose-hydrolyzing enzyme found in microbial, plant, and animal sources. I of C. utilis is a dimeric glycoprotein composed of 2 identical monomer subunits with an apparent mol. wt. of 150 kDa. Here, the authors investigated the mechanism of stabilization of I with polyols (glycerol, xylitol, and sorbitol). Enzyme activity, thermodn. and kinetic measurements of I were performed as a function of polyol concn. and showed that polyols acted as very effective stabilizing agents. The result from the I-solvent interaction showed preferential exclusion of the polyols from the protein domain leading to preferential hydration of protein. The apparent thermal denaturation temp. of the protein (Tm) rose from 75° to a max. of 85° in 30% glycerol. The stabilization was attributed to preferential hydration of the enzyme.
- 23Joo, J.; Kwon, D.; Shin, H. H.; Park, K.-H.; Cha, H. J.; Jeon, S. A Facile and Sensitive Method for Detecting Pathogenic Bacteria Using Personal Glucose Meters. Sens. Actuators, B 2013, 188, 1250– 1254, DOI: 10.1016/j.snb.2013.08.027Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Sns7zN&md5=04f1cf2e1e3644f104b9e8c7302a7db4A facile and sensitive method for detecting pathogenic bacteria using personal glucose metersJoo, Jinmyoung; Kwon, Donghoon; Shin, Hwa Hui; Park, Ki-Hwan; Cha, Hyung Joon; Jeon, SangminSensors and Actuators, B: Chemical (2013), 188 (), 1250-1254CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)The authors report a facile and sensitive method for the detection of Salmonella bacteria in milk using a personal glucose meter (PGM). Monoclonal antibody-functionalized magnetic nanoparticle clusters (MNCs) were used to capture Salmonella bacteria in milk, and MNC-Salmonella complexes were magnetically sepd. from the sample using a permanent magnet. The complexes were further conjugated to polyclonal antibody-functionalized invertase and dispersed in a 0.5 M sucrose soln. After the hydrolysis of sucrose to glucose and fructose, the concn. of glucose was measured using the PGM. The hydrolysis reaction was conducted at various temps., and the optimal temp. and activation energy were detd. The detection limit of Salmonella in milk was found to be 10 cfu/mL.
- 24Li, L.; Liang, D.; Guo, W.; Tang, D.; Zeng, Y. Antibody-invertase Cross-linkage Nanoparticles: A New Signal Tag for Point-of-care Immunoassay of Alpha-fetoprotein for Hepatocellular Carcinoma with Personal Glucometer. Electroanalysis 2021, 34, 246– 251, DOI: 10.1002/elan.202100212Google ScholarThere is no corresponding record for this reference.
- 25Lin, J.; Tang, D. Glucometer-Based Signal Readout for a Portable Low-Cost Electrochemical Immunoassay Using Branched Platinum Nanowires. Anal. Methods 2016, 8, 4069– 4074, DOI: 10.1039/C6AY00897FGoogle Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsF2isLw%253D&md5=b5bd20b8c4a1f1db4391d59e2cbb7373Glucometer-based signal readout for a portable low-cost electrochemical immunoassay using branched platinum nanowiresLin, Jiashi; Tang, DianpingAnalytical Methods (2016), 8 (20), 4069-4074CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)A simple and low-cost electrochem. immunosensing platform with a personal glucometer (PGM)-based signal readout device was developed for the quant. detection of human carbohydrate antigen 125 (CA 125) using invertase for the hydrolysis of sucrose. To achieve a high sensitivity, branched platinum nanowires (BPtNWs) synthesized by a wet-chem. method were utilized for the conjugation of invertase and anti-CA 125 secondary antibody. The immuno-reaction was carried out on an anti-CA 125 capture antibody-coated polystyrene microtiter plate with a sandwich-type assay mode, using the biofunctionalized BPtNWs as signal-generation tags. Accompanying the formation of the sandwiched immuno complexes, the carried invertase with the BPtNWs hydrolyzed sucrose into glucose and fructose. The glucose produced could be detd. for quantification using a portable personal glucometer. By integrating the secondary antibody and invertase with branched platinum nanowires, this method displayed an excellent sensitivity with a detection limit of 23 mU mL-1 CA 125. The selective expts. revealed that the developed system was specific for the target CA 125, even coexisting with a high concn. of other biomarkers. Finally, anal. of human serum samples was also performed, showing that our strategy was reliable and had great potential application in early clin. diagnostics.
- 26Xiang, Y.; Lu, Y. Portable and Quantitative Detection of Protein Biomarkers and Small Molecular Toxins Using Antibodies and Ubiquitous Personal Glucose Meters. Anal. Chem. 2012, 84, 4174– 4178, DOI: 10.1021/ac300517nGoogle Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xkt1Omtrs%253D&md5=c64a075e32840ddb83cb2310711640bdPortable and Quantitative Detection of Protein Biomarkers and Small Molecular Toxins Using Antibodies and Ubiquitous Personal Glucose MetersXiang, Yu; Lu, YiAnalytical Chemistry (Washington, DC, United States) (2012), 84 (9), 4174-4178CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Developing portable and low-cost methods for quant. detection of large protein biomarkers and small mol. toxins can play a significant role in controlling and preventing diseases or toxins outbreaks. Despite years of research, most current methods still require lab.-based or customized devices that are not widely available to the general public for quant. anal. The authors have previously demonstrated the use of personal glucose meters (PGMs) and functional DNAs for the detection of many nonglucose targets. However, the range of targets detectable by functional DNAs is limited at the current stage. To expand the range of targets that can be detected by PGMs, the authors report here the use of antibodies in combination with sandwich and competitive assays for quant. detection of protein biomarkers (PSA, with a detection limit of 0.4 ng/mL) and small mol. toxins (Ochratoxin A, with a detection limit of 6.8 ng/mL), resp. In both assay methods, with invertase conjugates as the link, quant. detection is achieved via the dependence between the concns. of the targets in the sample and the glucose measured by PGMs. Given the wide availability of antibodies for numerous targets, the methods demonstrated here can expand the range of target detection by PGMs significantly.
- 27Wang, Q.; Liu, F.; Yang, X.; Wang, K.; Wang, H.; Deng, X. Sensitive Point-of-Care Monitoring of Cardiac Biomarker Myoglobin Using Aptamer and Ubiquitous Personal Glucose Meter. Biosens. Bioelectron. 2015, 64, 161– 164, DOI: 10.1016/j.bios.2014.08.079Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFagsrnE&md5=1092cfb77db8538a60fa5e06e6e24512Sensitive point-of-care monitoring of cardiac biomarker myoglobin using aptamer and ubiquitous personal glucose meterWang, Qing; Liu, Fang; Yang, Xiaohai; Wang, Kemin; Wang, Hui; Deng, XinBiosensors & Bioelectronics (2015), 64 (), 161-164CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Myoglobin (Myo), which is one of the early markers to increase after acute myocardial infarction (AMI), plays a major role in urgent diagnosis of cardiovascular diseases. Hence, monitoring of Myo in point-of-care is fundamental. Here, a novel assay for sensitive and selective detection of Myo was introduced using a personal glucose meter (PGM) as readout. In the presence of Myo, the anti-Myo antibody immobilized on the surface of polystyrene microplate could capture the target Myo. Then the selected aptamer against Myo, which was obtained using our screening process, was conjugated with invertase, and such aptamer-invertase conjugates bound to the immobilized Myo due to the Myo/aptamer interaction. Subsequently, the resulting "antibody-Myo-aptamer sandwich" complex contg. invertase conjugates hydrolyzed sucrose into glucose, thus establishing direct correlation between the Myo concn. and the amt. of glucose measured by PGM. By employing the enzyme amplification, as low as 50 pM Myo could be detected. This assay also showed high selectivity for Myo and was successfully used for Myo detection in serum samples. This work may provide a simple but reliable tool for early diagnosis of AMI in the world, esp. in developing countries.
- 28Zhang, S.; Luan, Y.; Xiong, M.; Zhang, J.; Lake, R.; Lu, Y. DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose Meter. ACS Appl. Mater. Interfaces 2021, 13, 9472– 9481, DOI: 10.1021/acsami.0c20417Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjtFSmtbY%253D&md5=389bd24fc583d0a5c5f907fd0d50df59DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose MeterZhang, Songbai; Luan, Yunxia; Xiong, Mengyi; Zhang, Jingjing; Lake, Ryan; Lu, YiACS Applied Materials & Interfaces (2021), 13 (8), 9472-9481CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Aptamer-based sensors have emerged as a major platform for detecting small-mol. targets, because aptamers can be selected to bind these small mols. with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-mol. targets such as toxins and a biotin-labeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-mol. target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small mols. of the toxin is successfully transformed into the measurement of glucose, and the sensitive anal. of small mols. such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quant. anal. of small mols., at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the anal. of other analytes.
- 29Ismail, N. F.; Lim, T. S. Site-Specific ScFv Labelling with Invertase via Sortase A Mechanism as a Platform for Antibody-Antigen Detection Using the Personal Glucose Meter. Sci. Rep. 2016, 6, 19338, DOI: 10.1038/srep19338Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1elsL0%253D&md5=a0ebe96d0a498517ac36aef728478eb5Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meterIsmail, Nur Faezee; Lim, Theam SoonScientific Reports (2016), 6 (), 19338CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Antibody labeling to reporter mols. is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addn., conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform.
- 30Jefferis, R.; Reimer, C. B.; Skvaril, F.; de Lange, G.; Ling, N. R.; Lowe, J.; Walker, M. R.; Phillips, D. J.; Aloisio, C. H.; Wells, T. W. Evaluation of Monoclonal Antibodies Having Specificity for Human IgG Sub-Classes: Results of an IUIS/WHO Collaborative Study. Immunol. Lett. 1985, 10, 223– 252, DOI: 10.1016/0165-2478(85)90082-3Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXltVeitLo%253D&md5=47294f2cb4add5b136853eb7066c4d02Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative studyJefferis, R.; Reimer, C. B.; Skvaril, F.; De Lange, G.; Ling, N. R.; Lowe, J.; Walker, M. R.; Phillips, D.; Aloisio, C. H.; et al.Immunology Letters (1985), 10 (3-4), 223-52CODEN: IMLED6; ISSN:0165-2478.Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG subclasses (59) or Gm allotypes (4) were evaluated for reactivity and specificity in 8 labs. employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities, McAb were produced that can be satisfactorily applied in most methodologies employed and which have potential as ref. reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated, demonstrating apparent assay restriction, and, while performing well in some assays, proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable cause of variability rather than capricious behavior by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the phys. and chem. procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.
- 31Evaluation of Thirty-One Mouse Monoclonal Antibodies to Human IgG Epitopes|Hybridoma. https://www.liebertpub.com/doi/10.1089/hyb.1984.3.263 (accessed Feb 19, 2022).Google ScholarThere is no corresponding record for this reference.
- 32Sabaté del Río, J.; Henry, O. Y. F.; Jolly, P.; Ingber, D. E. An Antifouling Coating That Enables Affinity-Based Electrochemical Biosensing in Complex Biological Fluids. Nat. Nanotechnol. 2019, 14, 1143– 1149, DOI: 10.1038/s41565-019-0566-zGoogle Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFelsrvK&md5=41d756f23312eeaa167a09857dd13716An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluidsSabate del Rio, Jonathan; Henry, Olivier Y. F.; Jolly, Pawan; Ingber, Donald E.Nature Nanotechnology (2019), 14 (12), 1143-1149CODEN: NNAABX; ISSN:1748-3387. (Nature Research)Affinity-based electrochem. detection in complex biol. fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 mo of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy prepn., stability and simplicity of this nanocomposite allow the generation of electrochem. biosensors that can operate in complex biol. fluids such as blood plasma or serum.
- 33Patel, E. U.; Bloch, E. M.; Clarke, W.; Hsieh, Y.-H.; Boon, D.; Eby, Y.; Fernandez, R. E.; Baker, O. R.; Keruly, M.; Kirby, C. S.; Klock, E.; Littlefield, K.; Miller, J.; Schmidt, H. A.; Sullivan, P.; Piwowar-Manning, E.; Shrestha, R.; Redd, A. D.; Rothman, R. E.; Sullivan, D.; Shoham, S.; Casadevall, A.; Quinn, T. C.; Pekosz, A.; Tobian, A. A. R.; Laeyendecker, O. Comparative Performance of Five Commercially Available Serologic Assays To Detect Antibodies to SARS-CoV-2 and Identify Individuals with High Neutralizing Titers. J. Clin. Microbiol. 2021, 59, e02257– 20, DOI: 10.1128/JCM.02257-20Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsVWqsr0%253D&md5=ba95a4502908ded74eca8a891efc2469Comparative performance of five commercially available serologic assays to detect antibodies to SARS-CoV-2 and identify individuals with high neutralizing titersPatel, Eshan U.; Bloch, Evan M.; Clarke, William; Hsieh, Yu-Hsiang; Boon, Denali; Eby, Yolanda; Fernandez, Reinaldo E.; Baker, Owen R.; Keruly, Morgan; Kirby, Charles S.; Klock, Ethan; Littlefield, Kirsten; Miller, Jernelle; Schmidt, Haley A.; Sullivan, Philip; Piwowar-Manning, Estelle; Shrestha, Ruchee; Redd, Andrew D.; Rothman, Richard E.; Sullivan, David; Shoham, Shmuel; Casadevall, Arturo; Quinn, Thomas C.; Pekosz, Andrew; Tobian, Aaron A. R.; Laeyendecker, OliverJournal of Clinical Microbiology (2021), 59 (2), e02257CODEN: JCMIDW; ISSN:1098-660X. (American Society for Microbiology)Accurate serol. assays to detect antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to characterize the epidemiol. of SARS-CoV-2 infection and identify potential candidates for coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donation. This study compared the performances of com. enzyme immunoassays (EIAs) with respect to detection of IgG or total antibodies to SARS-CoV-2 and neutralizing antibodies (nAbs). The diagnostic accuracy of five com. available EIAs (Abbott, Euroimmun, EDI, ImmunoDiagnostics, and Roche) for detection of IgG or total antibodies to SARS-CoV-2 was evaluated using cross-sectional samples from potential CCP donors who had prior mol. confirmation of SARS-CoV-2 infection (n = 214) and samples from prepandemic emergency department patients without SARS-CoV-2 infection (n = 1,099). Of the 214 potential CCP donors, all were sampled >14 days since symptom onset and only a minority (n = 16 [7.5%]) had been hospitalized due to COVID-19; 140 potential CCP donors were tested by all five EIAs and a microneutralization assay. Performed according to the protocols of the manufacturers to detect IgG or total antibodies to SARS-CoV-2, the sensitivity of each EIA ranged from 76.4% to 93.9%, and the specificity of each EIA ranged from 87.0% to 99.6%. Using a nAb titer cutoff value of ≥160 as the ref. representing a pos. test result (n = 140 CCP donors), the empirical area under the receiver operating curve for each EIA ranged from 0.66 (Roche) to 0.90 (Euroimmun). Com. EIAs with high diagnostic accuracy to detect SARS-CoV-2 antibodies did not necessarily have high diagnostic accuracy to detect high nAb titers. Some but not all com. EIAs may be useful in the identification of individuals with high nAb titers among convalescent individuals.
- 34Conklin, S. E.; Martin, K.; Manabe, Y. C.; Schmidt, H. A.; Miller, J.; Keruly, M.; Klock, E.; Kirby, C. S.; Baker, O. R.; Fernandez, R. E.; Eby, Y. J.; Hardick, J.; Shaw-Saliba, K.; Rothman, R. E.; Caturegli, P. P.; Redd, A. D.; Tobian, A. A. R.; Bloch, E. M.; Larman, H. B.; Quinn, T. C.; Clarke, W.; Laeyendecker, O. Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point-of-Care Testing. J. Clin. Microbiol. 2021, 59, e02020-20, DOI: 10.1128/JCM.02020-20Google ScholarThere is no corresponding record for this reference.
- 35Thermo Scientific ELISA Technical Guide and Protocols; Tech Tip; 65Thermo Scientific, 2010.Google ScholarThere is no corresponding record for this reference.
- 36Pratt, R. P.; Roser, B. Comparison of Blocking Agents for ELISA; Application NoteThermo Scientific, 2014.Google ScholarThere is no corresponding record for this reference.
- 37Whitman, J. D.; Hiatt, J.; Mowery, C. T.; Shy, B. R.; Yu, R.; Yamamoto, T. N.; Rathore, U.; Goldgof, G. M.; Whitty, C.; Woo, J. M.; Gallman, A. E.; Miller, T. E.; Levine, A. G.; Nguyen, D. N.; Bapat, S. P.; Balcerek, J.; Bylsma, S. A.; Lyons, A. M.; Li, S.; Wong, A. W.; Gillis-Buck, E. M.; Steinhart, Z. B.; Lee, Y.; Apathy, R.; Lipke, M. J.; Smith, J. A.; Zheng, T.; Boothby, I. C.; Isaza, E.; Chan, J.; Acenas, D. D.; Lee, J.; Macrae, T. A.; Kyaw, T. S.; Wu, D.; Ng, D. L.; Gu, W.; York, V. A.; Eskandarian, H. A.; Callaway, P. C.; Warrier, L.; Moreno, M. E.; Levan, J.; Torres, L.; Farrington, L. A.; Loudermilk, R.; Koshal, K.; Zorn, K. C.; Garcia-Beltran, W. F.; Yang, D.; Astudillo, M. G.; Bernstein, B. E.; Gelfand, J. A.; Ryan, E. T.; Charles, R. C.; Iafrate, A. J.; Lennerz, J. K.; Miller, S.; Chiu, C. Y.; Stramer, S. L.; Wilson, M. R.; Manglik, A.; Ye, C. J.; Krogan, N. J.; Anderson, M. S.; Cyster, J. G.; Ernst, J. D.; Wu, A. H. B.; Lynch, K. L.; Bern, C.; Hsu, P. D.; Marson, A. Test Performance Evaluation of SARS-CoV-2 Serological Assays, medRxiv , 2020, DOI: 10.1101/2020.04.25.20074856 (accessed Nov 02, 2021).Google ScholarThere is no corresponding record for this reference.
- 38Bangdiwala, S. I.; Haedo, A. S.; Natal, M. L.; Villaveces, A. The Agreement Chart as an Alternative to the Receiver-Operating Characteristic Curve for Diagnostic Tests. Journal of Clinical Epidemiology 2008, 61, 866– 874, DOI: 10.1016/j.jclinepi.2008.04.002Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1crgsFKltw%253D%253D&md5=c5dfa07c97e14378356c6bbbcfe9c473The agreement chart as an alternative to the receiver-operating characteristic curve for diagnostic testsBangdiwala Shrikant I; Haedo Ana S; Natal Marcela L; Villaveces AndresJournal of clinical epidemiology (2008), 61 (9), 866-74 ISSN:0895-4356.OBJECTIVE: For diagnostic tests, the most common graphical representation of the information is the receiver-operating characteristic (ROC) curve. The "agreement chart" displays the information of two observers independently classifying the same n items into the same k categories, and can be used if one considers one of the "observers" as the diagnostic test and the other as the known outcome. This study compares the two charts and their ability to visually portray the various relevant summary statistics that assess how good a diagnostic test may be, such as sensitivity, specificity, predictive values, and likelihood ratios. STUDY DESIGN AND SETTING: The geometric relationships displayed in the charts are first described. The relationship between the two graphical representations and various summary statistics is illustrated using data from three common epidemiologically relevant health issues: coronary heart disease, screening for breast cancer, and screening for tuberculosis. RESULTS: Whereas the ROC curve incorporates information on sensitivity and specificity, the agreement chart includes information on the positive and negative predictive values of the diagnostic test. CONCLUSION: The agreement chart should be considered as an alternative visual representation to the ROC for diagnostic tests.
- 39Center for Devices and Radiological Health, United States Food and Drug Administration. Self-Monitoring Blood Glucose Test Systems for Over-the-Counter Use: Guidance for Industry and Food and Drug Administration Staff. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/self-monitoring-blood-glucose-test-systems-over-counter-use (accessed April 27, 2022).Google ScholarThere is no corresponding record for this reference.
- 40Li, L.; Tan, C.; Zeng, J.; Luo, C.; Hu, S.; Peng, Y.; Li, W.; Xie, Z.; Ling, Y.; Zhang, X.; Deng, E.; Xu, H.; Wang, J.; Xie, Y.; Zhou, Y.; Zhang, W.; Guo, Y.; Liu, Z. Analysis of Viral Load in Different Specimen Types and Serum Antibody Levels of COVID-19 Patients. J. Transl. Med. 2021, 19, 30, DOI: 10.1186/s12967-020-02693-2Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhslSqsrk%253D&md5=fb3f8f7b4f3ba222657f797b1bb492adAnalysis of viral load in different specimen types and serum antibody levels of COVID-19 patientsLi, Ling; Tan, Chianru; Zeng, Jia; Luo, Chen; Hu, Shi; Peng, Yanke; Li, Wenjuan; Xie, Zhixiong; Ling, Yueming; Zhang, Xuejun; Deng, E.; Xu, Haixia; Wang, Jue; Xie, Yudi; Zhou, Yaling; Zhang, Wei; Guo, Yong; Liu, ZhongJournal of Translational Medicine (2021), 19 (1), 30CODEN: JTMOBV; ISSN:1479-5876. (BioMed Central Ltd.)Abstr.: Background: COVID-19 has caused a global pandemic and the death toll is increasing. However, there is no definitive information regarding the type of clin. specimens that is the best for SARS-CoV-2 detection, the antibody levels in patients with different duration of disease, and the relationship between antibody level and viral load. Methods: Nasopharyngeal swabs, anal swabs, saliva, blood, and urine specimens were collected from patients with a course of disease ranging from 7 to 69 days. Viral load in different specimen types was measured using droplet digital PCR (ddPCR). Meanwhile, anti-nucleocapsid protein (anti-N) IgM and IgG antibodies and anti-spike protein receptor-binding domain (anti-S-RBD) IgG antibody in all serum samples were tested using ELISA. Results: The pos. detection rate in nasopharyngeal swab was the highest (54.05%), followed by anal swab (24.32%), and the pos. detection rate in saliva, blood, and urine was 16.22%, 10.81%, and 5.41%, resp. However, some patients with neg. nasopharyngeal swabs had other specimens tested pos. There was no significant correlation between antibody level and days after symptoms onset or viral load. Conclusions: Other specimens could be pos. in patients with neg. nasopharyngeal swabs, suggesting that for patients in the recovery period, specimens other than nasopharyngeal swabs should also be tested to avoid false neg. results, and anal swabs are recommended. The antibody level had no correlation with days after symptoms onset or the viral load of nasopharyngeal swabs, suggesting that the antibody level may also be affected by other factors.
- 41Yang, W.; Zhou, Y.-F.; Dai, H.-P.; Bi, L.-J.; Zhang, Z.-P.; Zhang, X.-H.; Leng, Y.; Zhang, X.-E. Application of Methyl Parathion Hydrolase (MPH) as a Labeling Enzyme. Anal. Bioanal. Chem. 2008, 390, 2133– 2140, DOI: 10.1007/s00216-008-1987-yGoogle Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktlahu7w%253D&md5=600386374df256069502706c8113d2e8Application of methyl parathion hydrolase (MPH) as a labeling enzymeYang, Wei; Zhou, Ya-Feng; Dai, He-Ping; Bi, Li-Jun; Zhang, Zhi-Ping; Zhang, Xiao-Hua; Leng, Yan; Zhang, Xian-EnAnalytical and Bioanalytical Chemistry (2008), 390 (8), 2133-2140CODEN: ABCNBP; ISSN:1618-2642. (Springer)Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degrdn. of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alk. phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in soln. is the monomer, with a mol. wt. of 37 kDa; (3) its turnover no. is 114.70±13.19 s-1, which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for ELISA. To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)5-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp.
- 42Kobayashi, N.; Iwakami, K.; Kotoshiba, S.; Niwa, T.; Kato, Y.; Mano, N.; Goto, J. Immunoenzymometric Assay for a Small Molecule, 11-Deoxycortisol, with Attomole-Range Sensitivity Employing an ScFv–Enzyme Fusion Protein and Anti-Idiotype Antibodies. Anal. Chem. 2006, 78, 2244– 2253, DOI: 10.1021/ac051858fGoogle Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhsl2nurY%253D&md5=f805ace252feb2c662d5531ef246a748Immunoenzymometric Assay for a Small Molecule, 11-Deoxycortisol, with Attomole-Range Sensitivity Employing an scFv-Enzyme Fusion Protein and Anti-Idiotype AntibodiesKobayashi, Norihiro; Iwakami, Keiichi; Kotoshiba, Shuhei; Niwa, Toshifumi; Kato, Yoshinori; Mano, Nariyasu; Goto, JunichiAnalytical Chemistry (2006), 78 (7), 2244-2253CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)To overcome the sensitivity limit in immunoassays for small mols. (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten mols. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alk. phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chem. linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amts. of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse β-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic sepn. using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized α-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels detd. for healthy subjects were validated as reliable.
- 43Erdag, B.; Balcioglu, K. B.; Bahadir, A. O.; Hinc, D.; Ibrahimoglu, O.; Bahar, A.; Basalp, A.; Yucel, F. Cloning of Anti-HBsAg Single-Chain Variable Fragments from Hybridoma Cells for One-Step ELISA. Biotechnol. Biotechnol. Equip. 2017, 31, 964– 973, DOI: 10.1080/13102818.2017.1348256Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntlymtLk%253D&md5=e813e3fdf71f3d7f758574158914241eCloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISAErdag, Berrin; Balcioglu, Koray Bertan; Bahadir, Aylin Ozdemir; Hin, Duygu; Ibrahimoglu, Ozlem; Bahar, Aydin; Basalp, Aynur; Yucel, FatimaBiotechnology & Biotechnological Equipment (2017), 31 (5), 964-973CODEN: BTTEEJ; ISSN:1314-3530. (Taylor & Francis Ltd.)Hepatitis B virus (HBV) infection is a worldwide health problem. More than 400 million people are chronic HBV carriers in the world. Infected individuals are at a high risk of developing liver cirrhosis and hepatocellular carcinoma as the main consequences of HBV. The discoveries of fast diagnostic systems and new therapeutic applications are crucial in the fight against viral hepatitis. In this paper we present the generation of a single-chain variable fragment (scFv) from a mouse monoclonal antibody specific to the HBV surface antigen (HBsAg) and demonstrate its expression as a bacterial alk. phosphatase (AP) fusion protein. In this study, we constructed scFvs from hybridoma cells expressing HBsAg-specific antibody using phage display technol. and expressed them in Escherichia coli. The anti-HBsAg scFvs were inserted into pQE-2 vector to produce scFv antibody genetically fused to bacterial AP. Reproducibility of the recombinant HBsAg-scFv fusion protein was tested using ELISA (ELISA). Present preliminary findings indicate that the anti-HBsAg-scFv AP conjugate could be further used for the development of one-step ELISA for the detection of HBV.
- 44Sasajima, Y.; Iwasaki, R.; Tsumoto, K.; Kumagai, I.; Ihara, M.; Ueda, H. Expression of Antibody Variable Region-Human Alkaline Phosphatase Fusion Proteins in Mammalian Cells. J. Immunol. Methods 2010, 361, 57– 63, DOI: 10.1016/j.jim.2010.07.012Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFygu7zK&md5=bcc2a59a6e3b9d80169d4b4fb34b5d3bExpression of antibody variable region-human alkaline phosphatase fusion proteins in mammalian cellsSasajima, Yoshiyuki; Iwasaki, Ryohei; Tsumoto, Kouhei; Kumagai, Izumi; Ihara, Masaki; Ueda, HiroshiJournal of Immunological Methods (2010), 361 (1-2), 57-63CODEN: JIMMBG; ISSN:0022-1759. (Elsevier B.V.)Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and mol./cellular biotechnol. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addn., frequently used bacterial alk. phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here the authors tried to express the fusion proteins of antibody variable region(s) and secreted human placental alk. phosphatase (SEAP) using mammalian cell culture. As a result, functional VH-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid prodn. of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent VH/VL interaction for antigen quantitation.
- 45Venisnik, K. M. Bifunctional Antibody-Renilla Luciferase Fusion Protein for in Vivo Optical Detection of Tumors. Protein Eng., Des. Sel. 2006, 19, 453– 460, DOI: 10.1093/protein/gzl030Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xps1yhurs%253D&md5=f79e987bda28d346c2cbb571c1700ea8Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumorsVenisnik, Katy M.; Olafsen, Tove; Loening, Andreas M.; Iyer, Meera; Gambhir, Sanjiv S.; Wu, Anna M.Protein Engineering, Design & Selection (2006), 19 (10), 453-460CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-pos. xenografts, with a tumor:background ratio of 6.0 at 6 h after i.v. injection, compared with antigen-neg. tumors at 1.0. Targeting and distribution was also evaluated by microPET imaging using 124I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.
- 46Kerschbaumer, R. J.; Hirschl, S.; Schwager, C.; Ibl, M.; Himmler, G. PDAP2: A Vector for Construction of Alkaline Phosphatase Fusion-Proteins. Immunotechnology 1996, 2, 145– 150, DOI: 10.1016/1380-2933(96)00040-1Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XkslGrs7c%253D&md5=7b9e3b6830eb0bbca97035537e3832dbpDAP2: a vector for construction of alkaline phosphatase fusion-proteinsKerschbaumer, Randolf J.; Hirschl, Sonja; Schwager, Cornelia; Ibl, Martin; Himmler, GottfriedImmunotechnology (1996), 2 (2), 145-150CODEN: IOTEER; ISSN:1380-2933. (Elsevier)Expression of enzymically active protein fusions in Escherichia coli could facilitate the anal. of proteins and even replace some reagents frequently used in immunol. such as chem. produced antibody-enzyme conjugates. For this purpose there is up to now no system of general utility available. He vector pDAP2 was designed for simplified fusion of single-chain Fv fragments to the N-terminus of E. coli alk. phosphatase. The resulting immunoconjugates can be produced by expression in E. coli and purified in a single step via metal affinity chromatog. Several different single-chain Fv genes as well as peptide-coding oligonucleotides have been cloned into pDAP2 and tested for expression levels, purifn. properties and usefulness in ELISA and immunowestern blotting. The fusion proteins from pDAP2 can be prepd. at levels of several milligrams per L culture from the periplasm of the cells. The proteins work well in different immunoassay formats such as ELISA or western blotting. Thus, pDAP2 is compatible to phage display vectors such as pHEN1, pCOCK and the pCANTAB-series (Pharmacia, Sweden). Genes of single-chain antibody fragments can be simply swapped from these vectors into pDAP2. Furthermore, we demonstrate that it is possible to produce alk. phosphatase-active peptides with this vector by inserting synthetic coding oligonucleotides. This allows simple anal. of the binding properties of peptides and may have some advantages over other systems such as fusion to glutathione-S-transferase.
- 47Ritthisan, P.; Ojima-Kato, T.; Damnjanović, J.; Kojima, T.; Nakano, H. SKIK-Zipbody-Alkaline Phosphatase, a Novel Antibody Fusion Protein Expressed in Escherichia coli Cytoplasm. J. Biosci. Bioeng. 2018, 126, 705– 709, DOI: 10.1016/j.jbiosc.2018.06.009Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2ltrfL&md5=8245998ac9872836fe3d6c791a673cb3SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasmRitthisan, Panwad; Ojima-Kato, Teruyo; Damnjanovic, Jasmina; Kojima, Takaaki; Nakano, HideoJournal of Bioscience and Bioengineering (2018), 126 (6), 705-709CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)Antibody-enzyme fusion proteins have been used for various immunol. detection techniques, such as ELISA, Western blotting and so on. The use of genetically-fused antibody-enzyme complexes has advantages over conventional chem. conjugation methods, as they require no complex chem. reactions and allow for the strict control of the no. of enzymes fused with antibodies, resulting in a more stable performance of the bifunctional protein. Here, we describe efficient cytoplasmic sol. expression of an antigen-binding fragment (Fab) fused with Escherichia coli alk. phosphatase (AP), N-terminal Ser-Lys-Ile-Lys (SKIK) tag that can improve the synthesis of the tagged protein, as well as leucine zipper (LZ) to enhance the assocn. of the light chain and the heavy chain of Fab. Our results demonstrated that the SKIK-Fab-LZ-AP fusion was well expressed in E. coli oxidative cytoplasm in sol. form having both antigen antigen-binding and AP activity, and was purified to homogeneity by two step column chromatog., suggesting that the combination of the SKIK tag and AP fusion can greatly increase the productivity and soly. of the Fab-enzyme fusion in an E. coli cytoplasmic expression system.
- 48Mori, A.; Ojima-Kato, T.; Kojima, T.; Nakano, H. Zipbodyzyme: Development of New Antibody-Enzyme Fusion Proteins. J. Biosci. Bioeng. 2018, 125, 637– 643, DOI: 10.1016/j.jbiosc.2017.12.021Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslKgsg%253D%253D&md5=a87c8a9338e605509306af72c9d1473dZipbodyzyme: Development of new antibody-enzyme fusion proteinsMori, Akihiro; Ojima-Kato, Teruyo; Kojima, Takaaki; Nakano, HideoJournal of Bioscience and Bioengineering (2018), 125 (6), 637-643CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)A new antibody-enzyme fusion protein, named Zipbodyzyme, composed of a Fab antibody (i.e., an antigen-binding fragment of an antibody) and an enzyme, has been successfully produced in the cytoplasm of Escherichia coli. Zipbodyzymes have a leucine zipper (LZ) pair at the C-termini of the heavy chain (Hc) and the light chain (Lc) of Fab, to promote the assocn. of the Hc and the Lc in E. coli cytoplasm, adjoining a fused enzyme. A Zipbodyzyme contg. mouse-derived anti-E. coli O157 Fab and a luciferase or a green fluorescent protein retained both the antigen-binding and an enzymic activity/fluorescence. The bifunctional proteins were applicable in direct ELISA (ELISA) without the need for a secondary antibody, indicating that the utility of the antibody enzyme bifunctional fusion protein will be expanded.
- 49Han, C.; Ihara, M.; Ueda, H. Expression of an Antibody-Enzyme Complex by the L-Chain Fusion Method. J. Biosci. Bioeng. 2013, 116, 17– 21, DOI: 10.1016/j.jbiosc.2013.01.012Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXis1Oms7k%253D&md5=9e6423dfc5b8dd01ff766bd831dab257Expression of an antibody-enzyme complex by the L-chain fusion methodHan, Chungyong; Ihara, Masaki; Ueda, HiroshiJournal of Bioscience and Bioengineering (2013), 116 (1), 17-21CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)In this report, the authors describe a novel method for directly prepg. enzyme-labeled antibodies harvested from IgM-producing hybridoma cells. They constructed expression vectors for antibody light (L) chain-enzyme fusion proteins by linking either the genes for the murine lambda L chain or its const. region (CL) with one of two proteins, either the secreted placental alk. phosphatase or Gaussia luciferase (Gluc). When the vectors were transfected into anti-NP (4-hydroxy-3-nitrophenacetyl) IgM-producing myeloma cells, secretion of the IgM-enzyme complex from the gene-transfected cells was confirmed by a direct ELISA with an immobilized antigen. Furthermore, when human hybridoma HF10B4, a cell line that produces anti-human lung cancer IgM, was transfected with the vector contg. L-Gluc, a significantly stronger signal was obtained for the human lung carcinoma SBC-1 cells than for cervical HeLa cells. Because successful prodn. of an active IgM-enzyme complex contg. a heterologous L chain-enzyme fusion was obsd., the L-chain fusion method will be a generally applicable method for prepg. various IgM-enzyme complexes.
- 50Mader, A.; Chromikova, V.; Kunert, R. Recombinant IgM Expression in Mammalian Cells: A Target Protein Challenging Biotechnological Production. ABB 2013, 04, 38– 43, DOI: 10.4236/abb.2013.44A006Google ScholarThere is no corresponding record for this reference.
- 51Chromikova, V.; Mader, A.; Steinfellner, W.; Kunert, R. Evaluating the Bottlenecks of Recombinant IgM Production in Mammalian Cells. Cytotechnology 2015, 67, 343– 356, DOI: 10.1007/s10616-014-9693-4Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtVemsb8%253D&md5=52add8077ece64c9a4bd460b3c31e6cfEvaluating the bottlenecks of recombinant IgM production in mammalian cellsChromikova, Veronika; Mader, Alexander; Steinfellner, Willibald; Kunert, RenateCytotechnology (2015), 67 (2), 343-356CODEN: CYTOER; ISSN:0920-9069. (Springer)Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant prodn. is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM mols. (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy nos. and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy nos. as well as in transcription levels were obsd., they did not seem to be a limitation. Levels of relevant endoplasmic reticulum-stress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously obsd. on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.
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Abstract
Figure 1
Figure 1. Schematic overview of the detection assay to quantify COVID-19-specific antibodies using a commercially available glucometer. A strip coated with SARS-CoV-2 spike protein RBD is incubated with patient serum, and RBD-specific antibodies bind to the strip. The strip is rinsed to remove any nonspecific antibodies and transferred to a solution containing Ab+Inv fusion protein, which binds to the patient antibody captured on the strip. The strip is again rinsed, transferred to a solution of sucrose, and incubated. The amount of sucrose converted to glucose is measured using a commercial glucometer and is proportional to the amount of the patient’s antibodies in the serum.
Figure 2
Figure 2. Design and purification of antibody–invertase (Ab+Inv) fusion proteins. (A) Schematic of Ab+Inv fusion proteins containing an antihuman IgG antibody. The C-terminus of the antibody light chain (LC) was tethered to the N-terminus of invertase via a flexible linker 15 or 25 amino acids in length (LC15 and LC25, respectively) or the C-terminus of the heavy chain (HC) is tethered to the N-terminus of invertase via a flexible linker 17 or 27 amino acids in length (HC17 and HC27, respectively). HC and LC variable and constant domains are labeled. (B) Ab+Inv fusion proteins migrate at slightly larger molecular weights (MW) than expected by SDS-PAGE. The unfused antibody (Ab) and invertase (Inv) alone run at the expected sizes under nonreducing (146 kDa for Ab and 62 kDa for Inv) and reducing (49 kDa for Ab HC, 24 kDa for Ab LC, and 62 kDa for Inv) conditions. Note that the samples for both SDS-PAGE gels were boiled to minimize the divergent migration of invertase and the fusion proteins relative to their expected molecular weights (62 kDa for invertase and ∼266 kDa for the Ab+Inv fusion proteins). The Ab+Inv fusion proteins run somewhat larger than their expected MW (∼266 kDa) under nonreducing conditions, as do the Inv-fused LC (∼84 kDa) and HC (∼110 kDa) under reducing conditions. (C) Yield per liter of transfected cells for each Ab+Inv fusion protein is shown compared to the yield of the unfused antibody (Ab). For LC15, HC17, and HC27, the yield from the pooled peak 1 is shown in a lighter shade and the yield from the pooled peak 2 is shown in the darker shade. Bar height reflects the total yield for both peaks. (D) Representative size-exclusion chromatography (SEC) traces for the four Ab+Inv fusion proteins are shown. The separately pooled fractions that were tested for LC15, HC17, and HC27 (peak 1 and peak 2) are indicated.
Figure 3
Figure 3. Ab+Inv fusion proteins bind hIgG with the same affinity as the unfused antibody. (A) Equilibrium BLI titrations of the soluble HP6017 antibody, Ab+Inv LC15, LC25, HC17, and HC27 against immobilized hIgG. Invertase (Inv) is included as a negative control. (B) KD derived from the three-parameter curve fit of the equilibrium binding data in panel (A) is shown. (C) Association rate (kon) generated from the kinetic BLI curve fit of the data in Figure S2, assuming a 1:1 binding model, is shown.
Figure 4
Figure 4. Ab+Inv fusion proteins exhibit equivalent catalytic activity to unfused invertase. (A) Ab+Inv fusion proteins or invertase (Inv) were incubated with 250 mM sucrose for 6 to 24 min. Concentrations of 166 nM Ab+Inv fusion proteins and 330 nM for Inv were used to achieve a molar equivalent amount of the enzyme, n = 1. (B) Various concentrations of unfused Ab, Ab+Inv fusion proteins, or Inv were incubated with 250 mM sucrose for 8 min. The concentration of the Ab+Inv fusion proteins is one-half of the invertase concentration shown on the x-axis, as each fusion protein contains two copies of invertase, n = 2. (C) Unfused Ab, Ab+Inv fusion proteins, or Inv were incubated with various concentrations of sucrose for 15 min. Concentrations of 166 nM Ab+Inv fusion proteins and 330 nM for Inv were used to achieve a molar equivalent amount of the enzyme, n = 2. (D) Unfused Ab, Ab+Inv fusion proteins, or Inv were incubated with 250 mM sucrose for 15 min. Concentrations of 166 nM Ab+Inv fusion proteins and 330 nM for Inv were used to achieve a molar equivalent amount of the enzyme, n = 3. The dotted lines in (A)–(D) indicate the limit of detection for the glucometer (10 mg/dL). Error bars indicate standard deviation in all panels.
Figure 5
Figure 5. Glucometer-based immunoassay using Ab+Inv fusion protein detects the presence of SARS-CoV-2-targeted antibodies in patient samples. (A) Schematic of the detection workflow for the glucometer-based strip immunoassay, which detects sucrose conversion by Ab+Inv fusion protein LC15 in proportion to SARS-CoV-2 spike protein RBD-targeted antibodies in a test sample. (B) Platform shown in (A) was used to build calibration curves in 10% serum, detecting glucose readouts for various concentrations of the spiked anti-SARS-CoV-2 spike protein RBD antibody. The fitted single curve yields EC50 = 35 nM, with n = 3. The limit of detection (LOD) is 1.2 ± 0.2 nM. (C) This platform was used to measure anti-SARS-CoV-2 antibody titers in patient samples with known seroconversion status (TS1), with n = 3. (D) Schematic of the detection workflow for a benchmark spectrophotometric strip immunoassay, which detects tetramethylbenzidine (TMB) oxidation by horseradish peroxidase (HRP)-labeled anti-hIgG antibodies in proportion to SARS-CoV-2 spike protein RBD-targeted antibodies in a test sample. (E) The strategy shown in (D) was used to build calibration curves in 10% serum, detecting glucose readouts for various concentrations of spiked anti-SARS-CoV-2 spike protein RBD antibodies. The fitted single curve yields EC50 = 2 nM, with n = 3. The LOD is 0.8 ± 0.1 nM. (F) This strategy was used to measure anti-SARS-CoV-2 antibody titers in patient samples with known seroconversion status (TS1), with n = 3. Discrimination between positive and negative samples matched that of the glucometer-based assay. Error bars indicate standard deviation in all panels.
Figure 6
Figure 6. Glucometer-based immunoassay using Ab+Inv fusion protein enables monitoring of anti-SARS-CoV-2 antibody titers in hospitalized patients. A glucometer-based strip immunoassay was used to measure SARS-CoV-2 spike protein RBD-targeted antibody levels in samples from hospitalized patients with COVID-19 (TS2), n = 1. The glucometer assay consistently determined the emergence of anti-SARS-CoV-2 antibody responses at ∼10 days post symptom onset, and responses plateaued at ∼20 days post symptom onset in patients A, B, F, and G. Solid lines are included for visual clarity to highlight IgG titer trends.
Figure 7
Figure 7. Robust clinical agreement observed between glucometer-based and commercial spectrophotometric immunoassays. (A) Longitudinal time course of SARS-CoV-2 spike protein RBD-targeted antibody levels in the hospitalized patient with COVID-19 (patient B from the TS2 cohort), as detected by the glucometer-based immunoassay using Ab+Inv fusion protein, with n = 1. (B) Longitudinal time-course of SARS-CoV-2 spike protein RBD-targeted antibody levels in the hospitalized patient with COVID-19 (patient B from the TS2 cohort), as detected by a commercial spectrophotometric assay (Epitope Diagnostics), with n = 1. (C) To establish a seroconversion threshold for the glucometer-based assay, we computed the average and standard deviation (gray shaded areas) of the glucose output generated by five confirmed SARS-CoV-2 negative samples, obtaining a seroconversion cutoff of 39 ± 32 ng/mL. Extrapolating this threshold to all the data from the TS2 cohort, we were able to accurately stratify by seroconversion status. The short horizontal lines represent the mean and ± standard errors for the two groups. (D) Agreement chart generated using the Bangdiwala method, (38) comparing glucometer-based measurements with a commercial spectrophotometric assay (Coronacheck). A 95% positive and a 96% negative percent agreement were observed.
References
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- 4Feng, S.; Phillips, D. J.; White, T.; Sayal, H.; Aley, P. K.; Bibi, S.; Dold, C.; Fuskova, M.; Gilbert, S. C.; Hirsch, I.; Humphries, H. E.; Jepson, B.; Kelly, E. J.; Plested, E.; Shoemaker, K.; Thomas, K. M.; Vekemans, J.; Villafana, T. L.; Lambe, T.; Pollard, A. J.; Voysey, M.; the Oxford COVID Vaccine Trial Group Correlates of Protection against Symptomatic and Asymptomatic SARS-CoV-2 Infection. Nat. Med. 2021, 27, 2032– 2040, DOI: 10.1038/s41591-021-01540-14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFCit7%252FL&md5=a71a2f4eabb9c8e4100ec2a4e60c2cc9Correlates of protection against symptomatic and asymptomatic SARS-CoV-2 infectionFeng, Shuo; Phillips, Daniel J.; White, Thomas; Sayal, Homesh; Aley, Parvinder K.; Bibi, Sagida; Dold, Christina; Fuskova, Michelle; Gilbert, Sarah C.; Hirsch, Ian; Humphries, Holly E.; Jepson, Brett; Kelly, Elizabeth J.; Plested, Emma; Shoemaker, Kathryn; Thomas, Kelly M.; Vekemans, Johan; Villafana, Tonya L.; Lambe, Teresa; Pollard, Andrew J.; Voysey, Merryn; tNature Medicine (New York, NY, United States) (2021), 27 (11), 2032-2040CODEN: NAMEFI; ISSN:1078-8956. (Nature Portfolio)The global supply of COVID-19 vaccines remains limited. An understanding of the immune response that is predictive of protection could facilitate rapid licensure of new vaccines. Data from a randomized efficacy trial of the ChAdOx1 nCoV-19 (AZD1222) vaccine in the United Kingdom was analyzed to det. the antibody levels assocd. with protection against SARS-CoV-2. Binding and neutralizing antibodies at 28 days after the second dose were measured in infected and noninfected vaccine recipients. Higher levels of all immune markers were correlated with a reduced risk of symptomatic infection. A vaccine efficacy of 80% against symptomatic infection with majority Alpha (B.1.1.7) variant of SARS-CoV-2 was achieved with 264 (95% CI: 108, 806) binding antibody units (BAU)/mL: and 506 (95% CI: 135, not computed (beyond data range) (NC)) BAU/mL for anti-spike and anti-RBD antibodies, and 26 (95% CI: NC, NC) IU (IU)/mL and 247 (95% CI: 101, NC) normalized neutralization titers (NF50) for pseudovirus and live-virus neutralization, resp. Immune markers were not correlated with asymptomatic infections at the 5% significance level. These data can be used to bridge to new populations using validated assays, and allow extrapolation of efficacy ests. to new COVID-19 vaccines.
- 5Chen, Y.; Tong, P.; Whiteman, N. B.; Moghaddam, A. S.; Zuiani, A.; Habibi, S.; Gautam, A.; Xiao, T.; Cai, Y.; Chen, B.; Wesemann, D. R. Differential Antibody Dynamics to SARS-CoV-2 Infection and Vaccination bioRxiv, DOI: 10.1101/2021.09.09.459504 .There is no corresponding record for this reference.
- 6Khoury, D. S.; Cromer, D.; Reynaldi, A.; Schlub, T. E.; Wheatley, A. K.; Juno, J. A.; Subbarao, K.; Kent, S. J.; Triccas, J. A.; Davenport, M. P. Neutralizing Antibody Levels Are Highly Predictive of Immune Protection from Symptomatic SARS-CoV-2 Infection. Nat. Med. 2021, 27, 1205– 1211, DOI: 10.1038/s41591-021-01377-86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFSktLrP&md5=b960e523ac74485ee699933e77c97043Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infectionKhoury, David S.; Cromer, Deborah; Reynaldi, Arnold; Schlub, Timothy E.; Wheatley, Adam K.; Juno, Jennifer A.; Subbarao, Kanta; Kent, Stephen J.; Triccas, James A.; Davenport, Miles P.Nature Medicine (New York, NY, United States) (2021), 27 (7), 1205-1211CODEN: NAMEFI; ISSN:1078-8956. (Nature Portfolio)Predictive models of immune protection from COVID-19 are urgently needed to identify correlates of protection to assist in the future deployment of vaccines. To address this, we analyzed the relationship between in vitro neutralization levels and the obsd. protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using data from seven current vaccines and from convalescent cohorts. We estd. the neutralization level for 50% protection against detectable SARS-CoV-2 infection to be 20.2% of the mean convalescent level (95% confidence interval (CI) = 14.4-28.4%). The estd. neutralization level required for 50% protection from severe infection was significantly lower (3% of the mean convalescent level; 95% CI = 0.7-13%, P = 0.0004). Modeling of the decay of the neutralization titer over the first 250 d after immunization predicts that a significant loss in protection from SARS-CoV-2 infection will occur, although protection from severe disease should be largely retained. Neutralization titers against some SARS-CoV-2 variants of concern are reduced compared with the vaccine strain, and our model predicts the relationship between neutralization and efficacy against viral variants. Here, we show that neutralization level is highly predictive of immune protection, and provide an evidence-based model of SARS-CoV-2 immune protection that will assist in developing vaccine strategies to control the future trajectory of the pandemic.
- 7Wei, J.; Stoesser, N.; Matthews, P. C.; Ayoubkhani, D.; Studley, R.; Bell, I.; Bell, J. I.; Newton, J. N.; Farrar, J.; Diamond, I.; Rourke, E.; Howarth, A.; Marsden, B. D.; Hoosdally, S.; Jones, E. Y.; Stuart, D. I.; Crook, D. W.; Peto, T. E. A.; Pouwels, K. B.; Eyre, D. W.; Walker, A. S.; the COVID-19 Infection Survey team Antibody Responses to SARS-CoV-2 Vaccines in 45,965 Adults from the General Population of the United Kingdom. Nat Microbiol 2021, 6, 1140– 1149, DOI: 10.1038/s41564-021-00947-37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1Skt7%252FO&md5=11cc3fc7307c8f79193b7c917f62bd64Antibody responses to SARS-CoV-2 vaccines in 45,965 adults from the general population of the United KingdomWei, Jia; Stoesser, Nicole; Matthews, Philippa C.; Ayoubkhani, Daniel; Studley, Ruth; Bell, Iain; Bell, John I.; Newton, John N.; Farrar, Jeremy; Diamond, Ian; Rourke, Emma; Howarth, Alison; Marsden, Brian D.; Hoosdally, Sarah; Jones, E. Yvonne; Stuart, David I.; Crook, Derrick W.; Peto, Tim E. A.; Pouwels, Koen B.; Eyre, David W.; Walker, A. SarahNature Microbiology (2021), 6 (9), 1140-1149CODEN: NMAICH; ISSN:2058-5276. (Nature Portfolio)Abstr.: We report that in a cohort of 45,965 adults, who were receiving either the ChAdOx1 or the BNT162b2 SARS-CoV-2 vaccines, in those who had no prior infection with SARS-CoV-2, seroconversion rates and quant. antibody levels after a single dose were lower in older individuals, esp. in those aged >60 years. Two vaccine doses achieved high responses across all ages. Antibody levels increased more slowly and to lower levels with a single dose of ChAdOx1 compared with a single dose of BNT162b2, but waned following a single dose of BNT162b2 in older individuals. In descriptive latent class models, we identified four responder subgroups, including a 'low responder' group that more commonly consisted of people aged >75 years, males and individuals with long-term health conditions. Given our findings, we propose that available vaccines should be prioritized for those not previously infected and that second doses should be prioritized for individuals aged >60 years. Further data are needed to better understand the extent to which quant. antibody responses are assocd. with vaccine-mediated protection.
- 8Amanat, F.; Stadlbauer, D.; Strohmeier, S.; Nguyen, T. H. O.; Chromikova, V.; McMahon, M.; Jiang, K.; Arunkumar, G. A.; Jurczyszak, D.; Polanco, J.; Bermudez-Gonzalez, M.; Kleiner, G.; Aydillo, T.; Miorin, L.; Fierer, D. S.; Lugo, L. A.; Kojic, E. M.; Stoever, J.; Liu, S. T. H.; Cunningham-Rundles, C.; Felgner, P. L.; Moran, T.; García-Sastre, A.; Caplivski, D.; Cheng, A. C.; Kedzierska, K.; Vapalahti, O.; Hepojoki, J. M.; Simon, V.; Krammer, F. A Serological Assay to Detect SARS-CoV-2 Seroconversion in Humans. Nat. Med. 2020, 26, 1033– 1036, DOI: 10.1038/s41591-020-0913-58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptFyqsbY%253D&md5=6f5df9742dec5a6bdb94ac5ccaad043dA serological assay to detect SARS-CoV-2 seroconversion in humansAmanat, Fatima; Stadlbauer, Daniel; Strohmeier, Shirin; Nguyen, Thi H. O.; Chromikova, Veronika; McMahon, Meagan; Jiang, Kaijun; Arunkumar, Guha Asthagiri; Jurczyszak, Denise; Polanco, Jose; Bermudez-Gonzalez, Maria; Kleiner, Giulio; Aydillo, Teresa; Miorin, Lisa; Fierer, Daniel S.; Lugo, Luz Amarilis; Kojic, Erna Milunka; Stoever, Jonathan; Liu, Sean T. H.; Cunningham-Rundles, Charlotte; Felgner, Philip L.; Moran, Thomas; Garcia-Sastre, Adolfo; Caplivski, Daniel; Cheng, Allen C.; Kedzierska, Katherine; Vapalahti, Olli; Hepojoki, Jussi M.; Simon, Viviana; Krammer, FlorianNature Medicine (New York, NY, United States) (2020), 26 (7), 1033-1036CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)We describe a serol. ELISA for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma, and is amenable to scaling. Serol. assays are of crit. importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy, and investigate correlates of protection.
- 9Mendrone-Junior, A.; Dinardo, C. L.; Ferreira, S. C.; Nishya, A.; Salles, N. A.; Almeida Neto, C.; Hamasaki, D. T.; Facincani, T.; Oliveira Alves, L. B.; Machado, R. R. G.; Araujo, D. B.; Durigon, E. L.; Rocha, V.; Sabino, E. C. Correlation between SARS-COV-2 Antibody Screening by Immunoassay and Neutralizing Antibody Testing. Transfusion 2021, 61, 1181– 1190, DOI: 10.1111/trf.162689https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovVKltro%253D&md5=6e7b8dedfc4e77bc7513f9587e7ad7caCorrelation between SARS-COV-2 antibody screening by immunoassay and neutralizing antibody testingMendrone-Junior, Alfredo; Dinardo, Carla Luana; Ferreira, Suzete Cleuza; Nishya, Anna; Salles, Nanci Alves; de Almeida Neto, Cesar; Hamasaki, Debora Toshei; Facincani, Tila; de Oliveira Alves, Lucas Bassolli; Machado, Rafael Rahal Guaragna; Araujo, Danielle Bastos; Durigon, Edison Luiz; Rocha, Vanderson; Sabino, Ester CerdeiraTransfusion (Malden, MA, United States) (2021), 61 (4), 1181-1190CODEN: TRANAT; ISSN:0041-1132. (Wiley-Blackwell)The efficacy of convalescent plasma (CP), an alternative for the treatment of COVID-19, depends on high titers of neutralizing antibodies (nAbs), but assays for quantifying nAbs are not widely available. Our goal was to develop a strategy to predict high titers of nAbs based on the results of anti-SARS-CoV-2 immunoassays and the clin. characteristics of CP donors. A total of 214 CP donors were enrolled and tested for the presence of anti-SARS-CoV-2 antibodies (IgG) using two com. immunoassays: EUROIMMUN (ELISA) and Abbott (Chemiluminescence). Quantification of nAbs was performed using the Cytopathic Effect-based Virus Neutralization test. Three criteria for identifying donors with nAbs ≥ 1:160 were tested: - C1: Curve ROC; - C2: Conditional decision tree considering only the IA results and - C3: Conditional decision tree including both the IA results and the clin. variables. The performance of the immunoassays was similar referring to both S/CO and predictive value for identifying nAbs titers ≥1:160. Regarding the studied criteria for identifying CP donors with high nAbs titers: (a) C1 showed 76.1% accuracy if S/CO = 4.65, (b) C2 presented 76.1% accuracy if S/CO ≥4.57 and (c) C3 had 71.6% accuracy if S/CO was ≥4.57 or if S/CO was between 2.68-4.57 and the last COVID-19-related symptoms were recent (within 19 days). SARS-CoV-2 IgG immunoassays (S/CO) can be used to predict high anti-SARS-CoV-2 nAbs titers. This study has proposed different criteria for identifying donors with ≥1:160 nAbs titers, all with high efficacy.
- 10Oh, H.; Ahn, H.; Tripathi, A. A Closer Look into FDA-EUA Approved Diagnostic Techniques of Covid-19. ACS Infect. Dis. 2021, 7, 2787– 2800, DOI: 10.1021/acsinfecdis.1c0026810https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitV2rsbnO&md5=193523ad54754b65632f0b9d5c03e2c1A Closer Look into FDA-EUA Approved Diagnostic Techniques of Covid-19Oh, Hyunju; Ahn, Hyunjeong; Tripathi, AnubhavACS Infectious Diseases (2021), 7 (10), 2787-2800CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)A review. The 2019 coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 virus, caused a worldwide pandemic in 2020 and is the most urgent health issue worldwide. In this review, we highlight the details of Food and Drug Administration-Emergency Use Authorizations approved diagnostics kits, focusing on the similarities and differences. It is essential to understand the currently available options and the advantages and disadvantages each provides to select the appropriate products that maximize the testing efficiency. We believe this work will provide a holistic evaluation of the current COVID-19 diagnostic resources, including variations across the countries, and guide developing novel diagnostic techniques to improve and optimize the current testing options.
- 11Lee, C. Y.-P.; Lin, R. T. P.; Renia, L.; Ng, L. F. P. Serological Approaches for COVID-19: Epidemiologic Perspective on Surveillance and Control. Front. Immunol. 2020, 11, 879, DOI: 10.3389/fimmu.2020.0087911https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVWntrvN&md5=eafcca8c0e6b2cb576101ccc604d8384Serological approaches for COVID-19: epidemiologic perspective on surveillance and controlLee, Cheryl Yi-Pin; Lin, Raymond T. P.; Renia, Laurent; Ng, Lisa F. P.Frontiers in Immunology (2020), 11 (), 879CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)A review. Since Dec. 2019, the novel coronavirus, SARS-CoV-2, has garnered global attention due to its rapid transmission, which has infected more than two million people worldwide. Early detection of SARS-CoV-2 is one of the crucial interventions to control virus spread and dissemination. Mol. assays have been the gold std. to directly detect for the presence of viral genetic material in infected individuals. However, insufficient viral RNA at the point of detection may lead to false neg. results. As such, it is important to also employ immune-based assays to det. one's exposure to SARS-CoV-2, as well as to assist in the surveillance of individuals with prior exposure to SARS-CoV-2. Within a span of 4 mo, extensive studies have been done to develop serol. systems to characterize the antibody profiles, as well as to identify and generate potentially neutralizing antibodies during SARS-CoV-2 infection. The vast diversity of novel findings has added value to coronavirus research, and a strategic consolidation is crucial to encompass the latest advances and developments. This review aims to provide a concise yet extensive collation of current immunoassays for SARS-CoV-2, while discussing the strengths, limitations and applications of antibody detection in SARS-CoV-2 research and control.
- 12Porstmann, B.; Porstmann, T.; Nugel, E.; Evers, U. Which of the Commonly Used Marker Enzymes Gives the Best Results in Colorimetric and Fluorimetric Enzyme Immunoassays: Horseradish Peroxidase, Alkaline Phosphatase or β-Galactosidase?. J. Immunol. Methods 1985, 79, 27– 37, DOI: 10.1016/0022-1759(85)90388-612https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXksFagsbk%253D&md5=29951f3bc50194081427a6febbfde135Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or β-galactosidase?Porstmann, Baerbel; Porstmann, T.; Nugel, E.; Evers, U.Journal of Immunological Methods (1985), 79 (1), 27-37CODEN: JIMMBG; ISSN:0022-1759.Comparing the marker enzymes horseradish peroxidase (HRP), alk. phosphatase (AP) and β-galactosidase (βGal) in IgG-coupled form with respect to their temp.-dependent kinetics over a period of 22 h, the temp. of 37° warrants highest substrate turnover of all enzymes at all reaction times using fluorogens. Also, applying chromogens, the optimum temp. for βGal is 37°; for HRP and AP, the optimum temp.depends on the reaction time. The substrate turnover of HRP using ABTS as chromogen is much higher compared to the other enzymes, with respect to both mol enzyme (molar activity) and to gram enzyme (specific activity). The turnover decreases for all enzymes in different degrees after coupling to IgG. The turnover of fluorogenic substrates is lower for all enzymes than the turnover of chromogenic substrates but due to the more sensitive detection of fluorogenic products the detection limits for all conjugates were lowered too; in particular, detection limits for βGal-IgG were lower by a factor of 333 compared to the colorimetric procedure. In a 2-site binding EIA for α1-fetoprotein (AFP), the detection limit for AFP was reduced only by a factor of 2 with all 3 marker enzymes when the fluorimetry assay was used rather than the colorimetry assay. The HRP-IgG conjugates allowed for lowest detection limits for AFP (0.5-1 μg/1), highest anal. sensitivity (slope of std. curves) at shortest periods of substrate reaction compared to the other enzymes.
- 13Gundinger, T.; Spadiut, O. A Comparative Approach to Recombinantly Produce the Plant Enzyme Horseradish Peroxidase in Escherichia coli. J. Biotechnol. 2017, 248, 15– 24, DOI: 10.1016/j.jbiotec.2017.03.00313https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXksFChu7s%253D&md5=da0e0766b0c03e31cfdfff6a549d45d9A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coliGundinger, Thomas; Spadiut, OliverJournal of Biotechnology (2017), 248 (), 15-24CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)Horseradish peroxidase (HRP) is used in various biotechnol. and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither prodn. from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titers of 10 mg per L cultivation broth. Thus, the traditional way of prodn. of HRP from plant still prevails. In this study, we revisited the recombinant prodn. of HRP in E. coli and investigated and compared both strategies, (a) the prodn. of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the prodn. of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titer of 100 mg L-1 cultivation broth, and were able to produce 48 mg active HRP per L cultivation broth in the periplasm. In terms of biochem. properties, sol. HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and prodn. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amts., which are then refolded.
- 14Lewis, T. L.; Roth, K. A. Immunohistochemical Detection Methods. In Pathobiology of Human Disease, Elsevier, 2014; pp https://doi.org/10.1016/B978-0-12-386456-7.07405-0 pp 3829– 3840.There is no corresponding record for this reference.
- 15Vashist, S. K.; Luong, J. H. T. Enzyme-Linked Immunoassays. In Handbook of Immunoassay Technologies; Elsevier, 2018; pp 97– 127.There is no corresponding record for this reference.
- 16Moshe, M.; Daunt, A.; Flower, B.; Simmons, B.; Brown, J. C.; Frise, R.; Penn, R.; Kugathasan, R.; Petersen, C.; Stockmann, H.; Ashby, D.; Riley, S.; Atchison, C.; Taylor, G. P.; Satkunarajah, S.; Naar, L.; Klaber, R.; Badhan, A.; Rosadas, C.; Marchesin, F.; Fernandez, N.; Sureda-Vives, M.; Cheeseman, H.; O’Hara, J.; Shattock, R.; Fontana, G.; Pallett, S. J. C.; Rayment, M.; Jones, R.; Moore, L. S. P.; Ashrafian, H.; Cherapanov, P.; Tedder, R.; McClure, M.; Ward, H.; Darzi, A.; Elliott, P.; Cooke, G. S.; Barclay, W. S. SARS-CoV-2 Lateral Flow Assays for Possible Use in National Covid-19 Seroprevalence Surveys (React 2): Diagnostic Accuracy Study. BMJ 2021, n423, DOI: 10.1136/bmj.n423There is no corresponding record for this reference.
- 17Nguyen, V.-T.; Song, S.; Park, S.; Joo, C. Recent Advances in High-Sensitivity Detection Methods for Paper-Based Lateral-Flow Assay. Biosens. Bioelectron. 2020, 152, 112015 DOI: 10.1016/j.bios.2020.11201517https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFOjt7w%253D&md5=636c5c3be6ce03fc803cd92598430209Recent advances in high-sensitivity detection methods for paper-based lateral-flow assayNguyen, Van-Thuan; Song, Seungri; Park, Seungkyung; Joo, ChulminBiosensors & Bioelectronics (2020), 152 (), 112015CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Paper-based lateral-flow assays (LFAs) have achieved considerable com. success and continue to have a significant impact on medical diagnostics and environmental monitoring. Conventional LFAs are typically performed by examg. the color changes in the test bands by naked eye. However, for crit. biochem. markers that are present in extremely small amts. in the clin. specimens, this readout method is not quant., and does not provide sufficient sensitivity or suitable detection limit for a reliable assay. Diverse technologies for high-sensitivity LFA detection have been developed and commercialization efforts are underway. In this review, we aim to provide a crit. and objective overview of the recent progress in high-sensitivity LFA detection technologies, which involve the exploitation of the phys. and chem. responses of transducing particles. The features and biomedical applications of the technologies, along with future prospects and challenges, are also discussed.
- 18Urusov, A. E.; Zherdev, A. V.; Dzantiev, B. B. Towards Lateral Flow Quantitative Assays: Detection Approaches. Biosensors 2019, 9, 89, DOI: 10.3390/bios903008918https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtF2msrrN&md5=8dd5eb6a2ea0944f9d903da0924324e3Towards lateral flow quantitative assays: detection approachesUrusov, Alexandr E.; Zherdev, Anatoly V.; Dzantiev, Boris B.Biosensors (2019), 9 (3), 89CODEN: BIOSHU; ISSN:2079-6374. (MDPI AG)Point-of-care (POC) or bedside anal. is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufg. technol. for lateral flow (immunochromatog.) test strips. They are widely used to rapidly and easily control a variety of biomarkers of infectious diseases and metabolic and functional disorders, as well as in consumer protection and environmental monitoring. However, traditional lateral flow tests rely on visual assessment and qual. conclusion, which limit the objectivity and information output of the assays. Therefore, there is a need for approaches that retain the advantages of lateral flow assays and provide reliable quant. information about the content of a target compd. in a sample mixt. This review describes the main options for detecting, processing, and interpreting immunochromatog. anal. results. The possibilities of modern portable detectors that register colored, fluorescent, magnetic, and conductive labels are discussed. Prospects for further development in this direction are also examd.
- 19Lan, T.; Zhang, J.; Lu, Y. Transforming the Blood Glucose Meter into a General Healthcare Meter for in Vitro Diagnostics in Mobile Health. Biotechnol. Adv. 2016, 34, 331– 341, DOI: 10.1016/j.biotechadv.2016.03.00219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktVOnurY%253D&md5=5aeaf550daf477c4d8fc706c212208ceTransforming the blood glucose meter into a general healthcare meter for in vitro diagnostics in mobile healthLan, Tian; Zhang, Jingjing; Lu, YiBiotechnology Advances (2016), 34 (3), 331-341CODEN: BIADDD; ISSN:0734-9750. (Elsevier)Recent advances in mobile network and smartphones have provided an enormous opportunity for transforming in vitro diagnostics (IVD) from central labs to home or other points of care (POC). A major challenge to achieving the goal is a long time and high costs assocd. with developing POC IVD devices in mobile Health (mHealth). Instead of developing a new POC device for every new IVD target, we and others are taking advantage of decades of research, development, engineering and continuous improvement of the blood glucose meter (BGM), including those already integrated with smartphones, and transforming the BGM into a general healthcare meter for POC IVDs of a wide range of biomarkers, therapeutic drugs and other anal. targets. In this review, we summarize methods to transduce and amplify selective binding of targets by antibodies, DNA/RNA aptamers, DNAzyme/ribozymes and protein enzymes into signals such as glucose or NADH that can be measured by com. available BGM, making it possible to adapt many clin. assays performed in central labs, such as immunoassays, aptamer/DNAzyme assays, mol. diagnostic assays, and enzymic activity assays onto BGM platform for quantification of non-glucose targets for a wide variety of IVDs in mHealth.
- 20Xiang, Y.; Lu, Y. Using Personal Glucose Meters and Functional DNA Sensors to Quantify a Variety of Analytical Targets. Nature Chem 2011, 3, 697– 703, DOI: 10.1038/nchem.109220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptlamu70%253D&md5=4340300b8a4bdb89f7e72dedb90784acUsing personal glucose meters and functional DNA sensors to quantify a variety of analytical targetsXiang, Yu; Lu, YiNature Chemistry (2011), 3 (9), 697-703CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Portable, low-cost and quant. detection of a broad range of targets at home and in the field has the potential to revolutionize medical diagnostics and environmental monitoring. Despite many years of research, very few such devices are com. available. Taking advantage of the wide availability and low cost of the pocket-sized personal glucose meter - used worldwide by diabetes sufferers - the authors demonstrate a method to use such meters to quantify nonglucose targets, ranging from a recreational drug (cocaine, 3.4 μM detection limit) to an important biol. cofactor (adenosine, 18 μM detection limit), to a disease marker (interferon-gamma of tuberculosis, 2.6 nM detection limit) and a toxic metal ion (uranium, 9.1 nM detection limit). The method is based on the target-induced release of invertase from a functional-DNA-invertase conjugate. The released invertase converts sucrose into glucose, which is detectable using the meter. The approach should be easily applicable to the detection of many other targets through the use of suitable functional-DNA partners (aptamers, DNAzymes or aptazymes).
- 21Chávez, F. P.; Rodriguez, L.; Díaz, J.; Delgado, J. M.; Cremata, J. A. Purification and Characterization of an Invertase from Candida Utilis: Comparison with Natural and Recombinant Yeast Invertases. J. Biotechnol. 1997, 53, 67– 74, DOI: 10.1016/S0168-1656(97)01663-521https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjtlKnsbg%253D&md5=80cab2b4f800f4002c7f8d6744393330Purification and characterization of an invertase from Candida utilis: comparison with natural and recombinant yeast invertasesChavez, Francisco P.; Rodriguez, Luis; Diaz, Joaquin; Delgado, Julio M.; Cremata, Jose A.Journal of Biotechnology (1997), 53 (1), 67-74CODEN: JBITD4; ISSN:0168-1656. (Elsevier)A periplasmic invertase (I) from C. utilis was purified to homogeneity from cells fully derepressed for I synthesis. I was purified by successive Sephacryl S-300 and affinity chromatogs. and shown to be a dimeric glycoprotein composed of 2 identical monomer subunits with an apparent mol. wt. of 150 kDa. After Endo H treatment, the deglycosylated protein showed an apparent mol. wt. of 60 kDa. The apparent Km values for sucrose and raffinose were 11 and 150 mM, resp., similar to those reported for I from Saccharomyces cerevisiae. The range of optimum temp. was 60-75°. The optimum pH was 5.5, and I was stable over the pH range of 3-6.
- 22Gangadhara; Ramesh Kumar, P.; Prakash, V. Influence of Polyols on the Stability and Kinetic Parameters of Invertase from Candida Utilis: Correlation with the Conformational Stability and Activity. Protein J. 2008, 27, 440– 449, DOI: 10.1007/s10930-008-9154-z22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtl2lsLbL&md5=bd6480ad2d04e54c5928a4b4b21dff17Influence of polyols on the stability and kinetic parameters of invertase from Candida utilis: Correlation with the conformational stability and activityGangadhara; Kumar, Parigi Ramesh; Prakash, VishweshwaraiahProtein Journal (2008), 27 (7-8), 440-449CODEN: PJROAH; ISSN:1572-3887. (Springer)Invertase (EC 3.2.1.26) (I) is a sucrose-hydrolyzing enzyme found in microbial, plant, and animal sources. I of C. utilis is a dimeric glycoprotein composed of 2 identical monomer subunits with an apparent mol. wt. of 150 kDa. Here, the authors investigated the mechanism of stabilization of I with polyols (glycerol, xylitol, and sorbitol). Enzyme activity, thermodn. and kinetic measurements of I were performed as a function of polyol concn. and showed that polyols acted as very effective stabilizing agents. The result from the I-solvent interaction showed preferential exclusion of the polyols from the protein domain leading to preferential hydration of protein. The apparent thermal denaturation temp. of the protein (Tm) rose from 75° to a max. of 85° in 30% glycerol. The stabilization was attributed to preferential hydration of the enzyme.
- 23Joo, J.; Kwon, D.; Shin, H. H.; Park, K.-H.; Cha, H. J.; Jeon, S. A Facile and Sensitive Method for Detecting Pathogenic Bacteria Using Personal Glucose Meters. Sens. Actuators, B 2013, 188, 1250– 1254, DOI: 10.1016/j.snb.2013.08.02723https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Sns7zN&md5=04f1cf2e1e3644f104b9e8c7302a7db4A facile and sensitive method for detecting pathogenic bacteria using personal glucose metersJoo, Jinmyoung; Kwon, Donghoon; Shin, Hwa Hui; Park, Ki-Hwan; Cha, Hyung Joon; Jeon, SangminSensors and Actuators, B: Chemical (2013), 188 (), 1250-1254CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)The authors report a facile and sensitive method for the detection of Salmonella bacteria in milk using a personal glucose meter (PGM). Monoclonal antibody-functionalized magnetic nanoparticle clusters (MNCs) were used to capture Salmonella bacteria in milk, and MNC-Salmonella complexes were magnetically sepd. from the sample using a permanent magnet. The complexes were further conjugated to polyclonal antibody-functionalized invertase and dispersed in a 0.5 M sucrose soln. After the hydrolysis of sucrose to glucose and fructose, the concn. of glucose was measured using the PGM. The hydrolysis reaction was conducted at various temps., and the optimal temp. and activation energy were detd. The detection limit of Salmonella in milk was found to be 10 cfu/mL.
- 24Li, L.; Liang, D.; Guo, W.; Tang, D.; Zeng, Y. Antibody-invertase Cross-linkage Nanoparticles: A New Signal Tag for Point-of-care Immunoassay of Alpha-fetoprotein for Hepatocellular Carcinoma with Personal Glucometer. Electroanalysis 2021, 34, 246– 251, DOI: 10.1002/elan.202100212There is no corresponding record for this reference.
- 25Lin, J.; Tang, D. Glucometer-Based Signal Readout for a Portable Low-Cost Electrochemical Immunoassay Using Branched Platinum Nanowires. Anal. Methods 2016, 8, 4069– 4074, DOI: 10.1039/C6AY00897F25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsF2isLw%253D&md5=b5bd20b8c4a1f1db4391d59e2cbb7373Glucometer-based signal readout for a portable low-cost electrochemical immunoassay using branched platinum nanowiresLin, Jiashi; Tang, DianpingAnalytical Methods (2016), 8 (20), 4069-4074CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)A simple and low-cost electrochem. immunosensing platform with a personal glucometer (PGM)-based signal readout device was developed for the quant. detection of human carbohydrate antigen 125 (CA 125) using invertase for the hydrolysis of sucrose. To achieve a high sensitivity, branched platinum nanowires (BPtNWs) synthesized by a wet-chem. method were utilized for the conjugation of invertase and anti-CA 125 secondary antibody. The immuno-reaction was carried out on an anti-CA 125 capture antibody-coated polystyrene microtiter plate with a sandwich-type assay mode, using the biofunctionalized BPtNWs as signal-generation tags. Accompanying the formation of the sandwiched immuno complexes, the carried invertase with the BPtNWs hydrolyzed sucrose into glucose and fructose. The glucose produced could be detd. for quantification using a portable personal glucometer. By integrating the secondary antibody and invertase with branched platinum nanowires, this method displayed an excellent sensitivity with a detection limit of 23 mU mL-1 CA 125. The selective expts. revealed that the developed system was specific for the target CA 125, even coexisting with a high concn. of other biomarkers. Finally, anal. of human serum samples was also performed, showing that our strategy was reliable and had great potential application in early clin. diagnostics.
- 26Xiang, Y.; Lu, Y. Portable and Quantitative Detection of Protein Biomarkers and Small Molecular Toxins Using Antibodies and Ubiquitous Personal Glucose Meters. Anal. Chem. 2012, 84, 4174– 4178, DOI: 10.1021/ac300517n26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xkt1Omtrs%253D&md5=c64a075e32840ddb83cb2310711640bdPortable and Quantitative Detection of Protein Biomarkers and Small Molecular Toxins Using Antibodies and Ubiquitous Personal Glucose MetersXiang, Yu; Lu, YiAnalytical Chemistry (Washington, DC, United States) (2012), 84 (9), 4174-4178CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Developing portable and low-cost methods for quant. detection of large protein biomarkers and small mol. toxins can play a significant role in controlling and preventing diseases or toxins outbreaks. Despite years of research, most current methods still require lab.-based or customized devices that are not widely available to the general public for quant. anal. The authors have previously demonstrated the use of personal glucose meters (PGMs) and functional DNAs for the detection of many nonglucose targets. However, the range of targets detectable by functional DNAs is limited at the current stage. To expand the range of targets that can be detected by PGMs, the authors report here the use of antibodies in combination with sandwich and competitive assays for quant. detection of protein biomarkers (PSA, with a detection limit of 0.4 ng/mL) and small mol. toxins (Ochratoxin A, with a detection limit of 6.8 ng/mL), resp. In both assay methods, with invertase conjugates as the link, quant. detection is achieved via the dependence between the concns. of the targets in the sample and the glucose measured by PGMs. Given the wide availability of antibodies for numerous targets, the methods demonstrated here can expand the range of target detection by PGMs significantly.
- 27Wang, Q.; Liu, F.; Yang, X.; Wang, K.; Wang, H.; Deng, X. Sensitive Point-of-Care Monitoring of Cardiac Biomarker Myoglobin Using Aptamer and Ubiquitous Personal Glucose Meter. Biosens. Bioelectron. 2015, 64, 161– 164, DOI: 10.1016/j.bios.2014.08.07927https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFagsrnE&md5=1092cfb77db8538a60fa5e06e6e24512Sensitive point-of-care monitoring of cardiac biomarker myoglobin using aptamer and ubiquitous personal glucose meterWang, Qing; Liu, Fang; Yang, Xiaohai; Wang, Kemin; Wang, Hui; Deng, XinBiosensors & Bioelectronics (2015), 64 (), 161-164CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Myoglobin (Myo), which is one of the early markers to increase after acute myocardial infarction (AMI), plays a major role in urgent diagnosis of cardiovascular diseases. Hence, monitoring of Myo in point-of-care is fundamental. Here, a novel assay for sensitive and selective detection of Myo was introduced using a personal glucose meter (PGM) as readout. In the presence of Myo, the anti-Myo antibody immobilized on the surface of polystyrene microplate could capture the target Myo. Then the selected aptamer against Myo, which was obtained using our screening process, was conjugated with invertase, and such aptamer-invertase conjugates bound to the immobilized Myo due to the Myo/aptamer interaction. Subsequently, the resulting "antibody-Myo-aptamer sandwich" complex contg. invertase conjugates hydrolyzed sucrose into glucose, thus establishing direct correlation between the Myo concn. and the amt. of glucose measured by PGM. By employing the enzyme amplification, as low as 50 pM Myo could be detected. This assay also showed high selectivity for Myo and was successfully used for Myo detection in serum samples. This work may provide a simple but reliable tool for early diagnosis of AMI in the world, esp. in developing countries.
- 28Zhang, S.; Luan, Y.; Xiong, M.; Zhang, J.; Lake, R.; Lu, Y. DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose Meter. ACS Appl. Mater. Interfaces 2021, 13, 9472– 9481, DOI: 10.1021/acsami.0c2041728https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjtFSmtbY%253D&md5=389bd24fc583d0a5c5f907fd0d50df59DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose MeterZhang, Songbai; Luan, Yunxia; Xiong, Mengyi; Zhang, Jingjing; Lake, Ryan; Lu, YiACS Applied Materials & Interfaces (2021), 13 (8), 9472-9481CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Aptamer-based sensors have emerged as a major platform for detecting small-mol. targets, because aptamers can be selected to bind these small mols. with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-mol. targets such as toxins and a biotin-labeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-mol. target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small mols. of the toxin is successfully transformed into the measurement of glucose, and the sensitive anal. of small mols. such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quant. anal. of small mols., at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the anal. of other analytes.
- 29Ismail, N. F.; Lim, T. S. Site-Specific ScFv Labelling with Invertase via Sortase A Mechanism as a Platform for Antibody-Antigen Detection Using the Personal Glucose Meter. Sci. Rep. 2016, 6, 19338, DOI: 10.1038/srep1933829https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1elsL0%253D&md5=a0ebe96d0a498517ac36aef728478eb5Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meterIsmail, Nur Faezee; Lim, Theam SoonScientific Reports (2016), 6 (), 19338CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Antibody labeling to reporter mols. is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addn., conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform.
- 30Jefferis, R.; Reimer, C. B.; Skvaril, F.; de Lange, G.; Ling, N. R.; Lowe, J.; Walker, M. R.; Phillips, D. J.; Aloisio, C. H.; Wells, T. W. Evaluation of Monoclonal Antibodies Having Specificity for Human IgG Sub-Classes: Results of an IUIS/WHO Collaborative Study. Immunol. Lett. 1985, 10, 223– 252, DOI: 10.1016/0165-2478(85)90082-330https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXltVeitLo%253D&md5=47294f2cb4add5b136853eb7066c4d02Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative studyJefferis, R.; Reimer, C. B.; Skvaril, F.; De Lange, G.; Ling, N. R.; Lowe, J.; Walker, M. R.; Phillips, D.; Aloisio, C. H.; et al.Immunology Letters (1985), 10 (3-4), 223-52CODEN: IMLED6; ISSN:0165-2478.Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG subclasses (59) or Gm allotypes (4) were evaluated for reactivity and specificity in 8 labs. employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities, McAb were produced that can be satisfactorily applied in most methodologies employed and which have potential as ref. reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated, demonstrating apparent assay restriction, and, while performing well in some assays, proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable cause of variability rather than capricious behavior by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the phys. and chem. procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.
- 31Evaluation of Thirty-One Mouse Monoclonal Antibodies to Human IgG Epitopes|Hybridoma. https://www.liebertpub.com/doi/10.1089/hyb.1984.3.263 (accessed Feb 19, 2022).There is no corresponding record for this reference.
- 32Sabaté del Río, J.; Henry, O. Y. F.; Jolly, P.; Ingber, D. E. An Antifouling Coating That Enables Affinity-Based Electrochemical Biosensing in Complex Biological Fluids. Nat. Nanotechnol. 2019, 14, 1143– 1149, DOI: 10.1038/s41565-019-0566-z32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFelsrvK&md5=41d756f23312eeaa167a09857dd13716An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluidsSabate del Rio, Jonathan; Henry, Olivier Y. F.; Jolly, Pawan; Ingber, Donald E.Nature Nanotechnology (2019), 14 (12), 1143-1149CODEN: NNAABX; ISSN:1748-3387. (Nature Research)Affinity-based electrochem. detection in complex biol. fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 mo of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy prepn., stability and simplicity of this nanocomposite allow the generation of electrochem. biosensors that can operate in complex biol. fluids such as blood plasma or serum.
- 33Patel, E. U.; Bloch, E. M.; Clarke, W.; Hsieh, Y.-H.; Boon, D.; Eby, Y.; Fernandez, R. E.; Baker, O. R.; Keruly, M.; Kirby, C. S.; Klock, E.; Littlefield, K.; Miller, J.; Schmidt, H. A.; Sullivan, P.; Piwowar-Manning, E.; Shrestha, R.; Redd, A. D.; Rothman, R. E.; Sullivan, D.; Shoham, S.; Casadevall, A.; Quinn, T. C.; Pekosz, A.; Tobian, A. A. R.; Laeyendecker, O. Comparative Performance of Five Commercially Available Serologic Assays To Detect Antibodies to SARS-CoV-2 and Identify Individuals with High Neutralizing Titers. J. Clin. Microbiol. 2021, 59, e02257– 20, DOI: 10.1128/JCM.02257-2033https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsVWqsr0%253D&md5=ba95a4502908ded74eca8a891efc2469Comparative performance of five commercially available serologic assays to detect antibodies to SARS-CoV-2 and identify individuals with high neutralizing titersPatel, Eshan U.; Bloch, Evan M.; Clarke, William; Hsieh, Yu-Hsiang; Boon, Denali; Eby, Yolanda; Fernandez, Reinaldo E.; Baker, Owen R.; Keruly, Morgan; Kirby, Charles S.; Klock, Ethan; Littlefield, Kirsten; Miller, Jernelle; Schmidt, Haley A.; Sullivan, Philip; Piwowar-Manning, Estelle; Shrestha, Ruchee; Redd, Andrew D.; Rothman, Richard E.; Sullivan, David; Shoham, Shmuel; Casadevall, Arturo; Quinn, Thomas C.; Pekosz, Andrew; Tobian, Aaron A. R.; Laeyendecker, OliverJournal of Clinical Microbiology (2021), 59 (2), e02257CODEN: JCMIDW; ISSN:1098-660X. (American Society for Microbiology)Accurate serol. assays to detect antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to characterize the epidemiol. of SARS-CoV-2 infection and identify potential candidates for coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donation. This study compared the performances of com. enzyme immunoassays (EIAs) with respect to detection of IgG or total antibodies to SARS-CoV-2 and neutralizing antibodies (nAbs). The diagnostic accuracy of five com. available EIAs (Abbott, Euroimmun, EDI, ImmunoDiagnostics, and Roche) for detection of IgG or total antibodies to SARS-CoV-2 was evaluated using cross-sectional samples from potential CCP donors who had prior mol. confirmation of SARS-CoV-2 infection (n = 214) and samples from prepandemic emergency department patients without SARS-CoV-2 infection (n = 1,099). Of the 214 potential CCP donors, all were sampled >14 days since symptom onset and only a minority (n = 16 [7.5%]) had been hospitalized due to COVID-19; 140 potential CCP donors were tested by all five EIAs and a microneutralization assay. Performed according to the protocols of the manufacturers to detect IgG or total antibodies to SARS-CoV-2, the sensitivity of each EIA ranged from 76.4% to 93.9%, and the specificity of each EIA ranged from 87.0% to 99.6%. Using a nAb titer cutoff value of ≥160 as the ref. representing a pos. test result (n = 140 CCP donors), the empirical area under the receiver operating curve for each EIA ranged from 0.66 (Roche) to 0.90 (Euroimmun). Com. EIAs with high diagnostic accuracy to detect SARS-CoV-2 antibodies did not necessarily have high diagnostic accuracy to detect high nAb titers. Some but not all com. EIAs may be useful in the identification of individuals with high nAb titers among convalescent individuals.
- 34Conklin, S. E.; Martin, K.; Manabe, Y. C.; Schmidt, H. A.; Miller, J.; Keruly, M.; Klock, E.; Kirby, C. S.; Baker, O. R.; Fernandez, R. E.; Eby, Y. J.; Hardick, J.; Shaw-Saliba, K.; Rothman, R. E.; Caturegli, P. P.; Redd, A. D.; Tobian, A. A. R.; Bloch, E. M.; Larman, H. B.; Quinn, T. C.; Clarke, W.; Laeyendecker, O. Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point-of-Care Testing. J. Clin. Microbiol. 2021, 59, e02020-20, DOI: 10.1128/JCM.02020-20There is no corresponding record for this reference.
- 35Thermo Scientific ELISA Technical Guide and Protocols; Tech Tip; 65Thermo Scientific, 2010.There is no corresponding record for this reference.
- 36Pratt, R. P.; Roser, B. Comparison of Blocking Agents for ELISA; Application NoteThermo Scientific, 2014.There is no corresponding record for this reference.
- 37Whitman, J. D.; Hiatt, J.; Mowery, C. T.; Shy, B. R.; Yu, R.; Yamamoto, T. N.; Rathore, U.; Goldgof, G. M.; Whitty, C.; Woo, J. M.; Gallman, A. E.; Miller, T. E.; Levine, A. G.; Nguyen, D. N.; Bapat, S. P.; Balcerek, J.; Bylsma, S. A.; Lyons, A. M.; Li, S.; Wong, A. W.; Gillis-Buck, E. M.; Steinhart, Z. B.; Lee, Y.; Apathy, R.; Lipke, M. J.; Smith, J. A.; Zheng, T.; Boothby, I. C.; Isaza, E.; Chan, J.; Acenas, D. D.; Lee, J.; Macrae, T. A.; Kyaw, T. S.; Wu, D.; Ng, D. L.; Gu, W.; York, V. A.; Eskandarian, H. A.; Callaway, P. C.; Warrier, L.; Moreno, M. E.; Levan, J.; Torres, L.; Farrington, L. A.; Loudermilk, R.; Koshal, K.; Zorn, K. C.; Garcia-Beltran, W. F.; Yang, D.; Astudillo, M. G.; Bernstein, B. E.; Gelfand, J. A.; Ryan, E. T.; Charles, R. C.; Iafrate, A. J.; Lennerz, J. K.; Miller, S.; Chiu, C. Y.; Stramer, S. L.; Wilson, M. R.; Manglik, A.; Ye, C. J.; Krogan, N. J.; Anderson, M. S.; Cyster, J. G.; Ernst, J. D.; Wu, A. H. B.; Lynch, K. L.; Bern, C.; Hsu, P. D.; Marson, A. Test Performance Evaluation of SARS-CoV-2 Serological Assays, medRxiv , 2020, DOI: 10.1101/2020.04.25.20074856 (accessed Nov 02, 2021).There is no corresponding record for this reference.
- 38Bangdiwala, S. I.; Haedo, A. S.; Natal, M. L.; Villaveces, A. The Agreement Chart as an Alternative to the Receiver-Operating Characteristic Curve for Diagnostic Tests. Journal of Clinical Epidemiology 2008, 61, 866– 874, DOI: 10.1016/j.jclinepi.2008.04.00238https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1crgsFKltw%253D%253D&md5=c5dfa07c97e14378356c6bbbcfe9c473The agreement chart as an alternative to the receiver-operating characteristic curve for diagnostic testsBangdiwala Shrikant I; Haedo Ana S; Natal Marcela L; Villaveces AndresJournal of clinical epidemiology (2008), 61 (9), 866-74 ISSN:0895-4356.OBJECTIVE: For diagnostic tests, the most common graphical representation of the information is the receiver-operating characteristic (ROC) curve. The "agreement chart" displays the information of two observers independently classifying the same n items into the same k categories, and can be used if one considers one of the "observers" as the diagnostic test and the other as the known outcome. This study compares the two charts and their ability to visually portray the various relevant summary statistics that assess how good a diagnostic test may be, such as sensitivity, specificity, predictive values, and likelihood ratios. STUDY DESIGN AND SETTING: The geometric relationships displayed in the charts are first described. The relationship between the two graphical representations and various summary statistics is illustrated using data from three common epidemiologically relevant health issues: coronary heart disease, screening for breast cancer, and screening for tuberculosis. RESULTS: Whereas the ROC curve incorporates information on sensitivity and specificity, the agreement chart includes information on the positive and negative predictive values of the diagnostic test. CONCLUSION: The agreement chart should be considered as an alternative visual representation to the ROC for diagnostic tests.
- 39Center for Devices and Radiological Health, United States Food and Drug Administration. Self-Monitoring Blood Glucose Test Systems for Over-the-Counter Use: Guidance for Industry and Food and Drug Administration Staff. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/self-monitoring-blood-glucose-test-systems-over-counter-use (accessed April 27, 2022).There is no corresponding record for this reference.
- 40Li, L.; Tan, C.; Zeng, J.; Luo, C.; Hu, S.; Peng, Y.; Li, W.; Xie, Z.; Ling, Y.; Zhang, X.; Deng, E.; Xu, H.; Wang, J.; Xie, Y.; Zhou, Y.; Zhang, W.; Guo, Y.; Liu, Z. Analysis of Viral Load in Different Specimen Types and Serum Antibody Levels of COVID-19 Patients. J. Transl. Med. 2021, 19, 30, DOI: 10.1186/s12967-020-02693-240https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhslSqsrk%253D&md5=fb3f8f7b4f3ba222657f797b1bb492adAnalysis of viral load in different specimen types and serum antibody levels of COVID-19 patientsLi, Ling; Tan, Chianru; Zeng, Jia; Luo, Chen; Hu, Shi; Peng, Yanke; Li, Wenjuan; Xie, Zhixiong; Ling, Yueming; Zhang, Xuejun; Deng, E.; Xu, Haixia; Wang, Jue; Xie, Yudi; Zhou, Yaling; Zhang, Wei; Guo, Yong; Liu, ZhongJournal of Translational Medicine (2021), 19 (1), 30CODEN: JTMOBV; ISSN:1479-5876. (BioMed Central Ltd.)Abstr.: Background: COVID-19 has caused a global pandemic and the death toll is increasing. However, there is no definitive information regarding the type of clin. specimens that is the best for SARS-CoV-2 detection, the antibody levels in patients with different duration of disease, and the relationship between antibody level and viral load. Methods: Nasopharyngeal swabs, anal swabs, saliva, blood, and urine specimens were collected from patients with a course of disease ranging from 7 to 69 days. Viral load in different specimen types was measured using droplet digital PCR (ddPCR). Meanwhile, anti-nucleocapsid protein (anti-N) IgM and IgG antibodies and anti-spike protein receptor-binding domain (anti-S-RBD) IgG antibody in all serum samples were tested using ELISA. Results: The pos. detection rate in nasopharyngeal swab was the highest (54.05%), followed by anal swab (24.32%), and the pos. detection rate in saliva, blood, and urine was 16.22%, 10.81%, and 5.41%, resp. However, some patients with neg. nasopharyngeal swabs had other specimens tested pos. There was no significant correlation between antibody level and days after symptoms onset or viral load. Conclusions: Other specimens could be pos. in patients with neg. nasopharyngeal swabs, suggesting that for patients in the recovery period, specimens other than nasopharyngeal swabs should also be tested to avoid false neg. results, and anal swabs are recommended. The antibody level had no correlation with days after symptoms onset or the viral load of nasopharyngeal swabs, suggesting that the antibody level may also be affected by other factors.
- 41Yang, W.; Zhou, Y.-F.; Dai, H.-P.; Bi, L.-J.; Zhang, Z.-P.; Zhang, X.-H.; Leng, Y.; Zhang, X.-E. Application of Methyl Parathion Hydrolase (MPH) as a Labeling Enzyme. Anal. Bioanal. Chem. 2008, 390, 2133– 2140, DOI: 10.1007/s00216-008-1987-y41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktlahu7w%253D&md5=600386374df256069502706c8113d2e8Application of methyl parathion hydrolase (MPH) as a labeling enzymeYang, Wei; Zhou, Ya-Feng; Dai, He-Ping; Bi, Li-Jun; Zhang, Zhi-Ping; Zhang, Xiao-Hua; Leng, Yan; Zhang, Xian-EnAnalytical and Bioanalytical Chemistry (2008), 390 (8), 2133-2140CODEN: ABCNBP; ISSN:1618-2642. (Springer)Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degrdn. of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alk. phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in soln. is the monomer, with a mol. wt. of 37 kDa; (3) its turnover no. is 114.70±13.19 s-1, which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for ELISA. To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)5-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp.
- 42Kobayashi, N.; Iwakami, K.; Kotoshiba, S.; Niwa, T.; Kato, Y.; Mano, N.; Goto, J. Immunoenzymometric Assay for a Small Molecule, 11-Deoxycortisol, with Attomole-Range Sensitivity Employing an ScFv–Enzyme Fusion Protein and Anti-Idiotype Antibodies. Anal. Chem. 2006, 78, 2244– 2253, DOI: 10.1021/ac051858f42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhsl2nurY%253D&md5=f805ace252feb2c662d5531ef246a748Immunoenzymometric Assay for a Small Molecule, 11-Deoxycortisol, with Attomole-Range Sensitivity Employing an scFv-Enzyme Fusion Protein and Anti-Idiotype AntibodiesKobayashi, Norihiro; Iwakami, Keiichi; Kotoshiba, Shuhei; Niwa, Toshifumi; Kato, Yoshinori; Mano, Nariyasu; Goto, JunichiAnalytical Chemistry (2006), 78 (7), 2244-2253CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)To overcome the sensitivity limit in immunoassays for small mols. (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten mols. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alk. phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chem. linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amts. of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse β-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic sepn. using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized α-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels detd. for healthy subjects were validated as reliable.
- 43Erdag, B.; Balcioglu, K. B.; Bahadir, A. O.; Hinc, D.; Ibrahimoglu, O.; Bahar, A.; Basalp, A.; Yucel, F. Cloning of Anti-HBsAg Single-Chain Variable Fragments from Hybridoma Cells for One-Step ELISA. Biotechnol. Biotechnol. Equip. 2017, 31, 964– 973, DOI: 10.1080/13102818.2017.134825643https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntlymtLk%253D&md5=e813e3fdf71f3d7f758574158914241eCloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISAErdag, Berrin; Balcioglu, Koray Bertan; Bahadir, Aylin Ozdemir; Hin, Duygu; Ibrahimoglu, Ozlem; Bahar, Aydin; Basalp, Aynur; Yucel, FatimaBiotechnology & Biotechnological Equipment (2017), 31 (5), 964-973CODEN: BTTEEJ; ISSN:1314-3530. (Taylor & Francis Ltd.)Hepatitis B virus (HBV) infection is a worldwide health problem. More than 400 million people are chronic HBV carriers in the world. Infected individuals are at a high risk of developing liver cirrhosis and hepatocellular carcinoma as the main consequences of HBV. The discoveries of fast diagnostic systems and new therapeutic applications are crucial in the fight against viral hepatitis. In this paper we present the generation of a single-chain variable fragment (scFv) from a mouse monoclonal antibody specific to the HBV surface antigen (HBsAg) and demonstrate its expression as a bacterial alk. phosphatase (AP) fusion protein. In this study, we constructed scFvs from hybridoma cells expressing HBsAg-specific antibody using phage display technol. and expressed them in Escherichia coli. The anti-HBsAg scFvs were inserted into pQE-2 vector to produce scFv antibody genetically fused to bacterial AP. Reproducibility of the recombinant HBsAg-scFv fusion protein was tested using ELISA (ELISA). Present preliminary findings indicate that the anti-HBsAg-scFv AP conjugate could be further used for the development of one-step ELISA for the detection of HBV.
- 44Sasajima, Y.; Iwasaki, R.; Tsumoto, K.; Kumagai, I.; Ihara, M.; Ueda, H. Expression of Antibody Variable Region-Human Alkaline Phosphatase Fusion Proteins in Mammalian Cells. J. Immunol. Methods 2010, 361, 57– 63, DOI: 10.1016/j.jim.2010.07.01244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFygu7zK&md5=bcc2a59a6e3b9d80169d4b4fb34b5d3bExpression of antibody variable region-human alkaline phosphatase fusion proteins in mammalian cellsSasajima, Yoshiyuki; Iwasaki, Ryohei; Tsumoto, Kouhei; Kumagai, Izumi; Ihara, Masaki; Ueda, HiroshiJournal of Immunological Methods (2010), 361 (1-2), 57-63CODEN: JIMMBG; ISSN:0022-1759. (Elsevier B.V.)Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and mol./cellular biotechnol. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addn., frequently used bacterial alk. phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here the authors tried to express the fusion proteins of antibody variable region(s) and secreted human placental alk. phosphatase (SEAP) using mammalian cell culture. As a result, functional VH-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid prodn. of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent VH/VL interaction for antigen quantitation.
- 45Venisnik, K. M. Bifunctional Antibody-Renilla Luciferase Fusion Protein for in Vivo Optical Detection of Tumors. Protein Eng., Des. Sel. 2006, 19, 453– 460, DOI: 10.1093/protein/gzl03045https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xps1yhurs%253D&md5=f79e987bda28d346c2cbb571c1700ea8Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumorsVenisnik, Katy M.; Olafsen, Tove; Loening, Andreas M.; Iyer, Meera; Gambhir, Sanjiv S.; Wu, Anna M.Protein Engineering, Design & Selection (2006), 19 (10), 453-460CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-pos. xenografts, with a tumor:background ratio of 6.0 at 6 h after i.v. injection, compared with antigen-neg. tumors at 1.0. Targeting and distribution was also evaluated by microPET imaging using 124I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.
- 46Kerschbaumer, R. J.; Hirschl, S.; Schwager, C.; Ibl, M.; Himmler, G. PDAP2: A Vector for Construction of Alkaline Phosphatase Fusion-Proteins. Immunotechnology 1996, 2, 145– 150, DOI: 10.1016/1380-2933(96)00040-146https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XkslGrs7c%253D&md5=7b9e3b6830eb0bbca97035537e3832dbpDAP2: a vector for construction of alkaline phosphatase fusion-proteinsKerschbaumer, Randolf J.; Hirschl, Sonja; Schwager, Cornelia; Ibl, Martin; Himmler, GottfriedImmunotechnology (1996), 2 (2), 145-150CODEN: IOTEER; ISSN:1380-2933. (Elsevier)Expression of enzymically active protein fusions in Escherichia coli could facilitate the anal. of proteins and even replace some reagents frequently used in immunol. such as chem. produced antibody-enzyme conjugates. For this purpose there is up to now no system of general utility available. He vector pDAP2 was designed for simplified fusion of single-chain Fv fragments to the N-terminus of E. coli alk. phosphatase. The resulting immunoconjugates can be produced by expression in E. coli and purified in a single step via metal affinity chromatog. Several different single-chain Fv genes as well as peptide-coding oligonucleotides have been cloned into pDAP2 and tested for expression levels, purifn. properties and usefulness in ELISA and immunowestern blotting. The fusion proteins from pDAP2 can be prepd. at levels of several milligrams per L culture from the periplasm of the cells. The proteins work well in different immunoassay formats such as ELISA or western blotting. Thus, pDAP2 is compatible to phage display vectors such as pHEN1, pCOCK and the pCANTAB-series (Pharmacia, Sweden). Genes of single-chain antibody fragments can be simply swapped from these vectors into pDAP2. Furthermore, we demonstrate that it is possible to produce alk. phosphatase-active peptides with this vector by inserting synthetic coding oligonucleotides. This allows simple anal. of the binding properties of peptides and may have some advantages over other systems such as fusion to glutathione-S-transferase.
- 47Ritthisan, P.; Ojima-Kato, T.; Damnjanović, J.; Kojima, T.; Nakano, H. SKIK-Zipbody-Alkaline Phosphatase, a Novel Antibody Fusion Protein Expressed in Escherichia coli Cytoplasm. J. Biosci. Bioeng. 2018, 126, 705– 709, DOI: 10.1016/j.jbiosc.2018.06.00947https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2ltrfL&md5=8245998ac9872836fe3d6c791a673cb3SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasmRitthisan, Panwad; Ojima-Kato, Teruyo; Damnjanovic, Jasmina; Kojima, Takaaki; Nakano, HideoJournal of Bioscience and Bioengineering (2018), 126 (6), 705-709CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)Antibody-enzyme fusion proteins have been used for various immunol. detection techniques, such as ELISA, Western blotting and so on. The use of genetically-fused antibody-enzyme complexes has advantages over conventional chem. conjugation methods, as they require no complex chem. reactions and allow for the strict control of the no. of enzymes fused with antibodies, resulting in a more stable performance of the bifunctional protein. Here, we describe efficient cytoplasmic sol. expression of an antigen-binding fragment (Fab) fused with Escherichia coli alk. phosphatase (AP), N-terminal Ser-Lys-Ile-Lys (SKIK) tag that can improve the synthesis of the tagged protein, as well as leucine zipper (LZ) to enhance the assocn. of the light chain and the heavy chain of Fab. Our results demonstrated that the SKIK-Fab-LZ-AP fusion was well expressed in E. coli oxidative cytoplasm in sol. form having both antigen antigen-binding and AP activity, and was purified to homogeneity by two step column chromatog., suggesting that the combination of the SKIK tag and AP fusion can greatly increase the productivity and soly. of the Fab-enzyme fusion in an E. coli cytoplasmic expression system.
- 48Mori, A.; Ojima-Kato, T.; Kojima, T.; Nakano, H. Zipbodyzyme: Development of New Antibody-Enzyme Fusion Proteins. J. Biosci. Bioeng. 2018, 125, 637– 643, DOI: 10.1016/j.jbiosc.2017.12.02148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslKgsg%253D%253D&md5=a87c8a9338e605509306af72c9d1473dZipbodyzyme: Development of new antibody-enzyme fusion proteinsMori, Akihiro; Ojima-Kato, Teruyo; Kojima, Takaaki; Nakano, HideoJournal of Bioscience and Bioengineering (2018), 125 (6), 637-643CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)A new antibody-enzyme fusion protein, named Zipbodyzyme, composed of a Fab antibody (i.e., an antigen-binding fragment of an antibody) and an enzyme, has been successfully produced in the cytoplasm of Escherichia coli. Zipbodyzymes have a leucine zipper (LZ) pair at the C-termini of the heavy chain (Hc) and the light chain (Lc) of Fab, to promote the assocn. of the Hc and the Lc in E. coli cytoplasm, adjoining a fused enzyme. A Zipbodyzyme contg. mouse-derived anti-E. coli O157 Fab and a luciferase or a green fluorescent protein retained both the antigen-binding and an enzymic activity/fluorescence. The bifunctional proteins were applicable in direct ELISA (ELISA) without the need for a secondary antibody, indicating that the utility of the antibody enzyme bifunctional fusion protein will be expanded.
- 49Han, C.; Ihara, M.; Ueda, H. Expression of an Antibody-Enzyme Complex by the L-Chain Fusion Method. J. Biosci. Bioeng. 2013, 116, 17– 21, DOI: 10.1016/j.jbiosc.2013.01.01249https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXis1Oms7k%253D&md5=9e6423dfc5b8dd01ff766bd831dab257Expression of an antibody-enzyme complex by the L-chain fusion methodHan, Chungyong; Ihara, Masaki; Ueda, HiroshiJournal of Bioscience and Bioengineering (2013), 116 (1), 17-21CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)In this report, the authors describe a novel method for directly prepg. enzyme-labeled antibodies harvested from IgM-producing hybridoma cells. They constructed expression vectors for antibody light (L) chain-enzyme fusion proteins by linking either the genes for the murine lambda L chain or its const. region (CL) with one of two proteins, either the secreted placental alk. phosphatase or Gaussia luciferase (Gluc). When the vectors were transfected into anti-NP (4-hydroxy-3-nitrophenacetyl) IgM-producing myeloma cells, secretion of the IgM-enzyme complex from the gene-transfected cells was confirmed by a direct ELISA with an immobilized antigen. Furthermore, when human hybridoma HF10B4, a cell line that produces anti-human lung cancer IgM, was transfected with the vector contg. L-Gluc, a significantly stronger signal was obtained for the human lung carcinoma SBC-1 cells than for cervical HeLa cells. Because successful prodn. of an active IgM-enzyme complex contg. a heterologous L chain-enzyme fusion was obsd., the L-chain fusion method will be a generally applicable method for prepg. various IgM-enzyme complexes.
- 50Mader, A.; Chromikova, V.; Kunert, R. Recombinant IgM Expression in Mammalian Cells: A Target Protein Challenging Biotechnological Production. ABB 2013, 04, 38– 43, DOI: 10.4236/abb.2013.44A006There is no corresponding record for this reference.
- 51Chromikova, V.; Mader, A.; Steinfellner, W.; Kunert, R. Evaluating the Bottlenecks of Recombinant IgM Production in Mammalian Cells. Cytotechnology 2015, 67, 343– 356, DOI: 10.1007/s10616-014-9693-451https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtVemsb8%253D&md5=52add8077ece64c9a4bd460b3c31e6cfEvaluating the bottlenecks of recombinant IgM production in mammalian cellsChromikova, Veronika; Mader, Alexander; Steinfellner, Willibald; Kunert, RenateCytotechnology (2015), 67 (2), 343-356CODEN: CYTOER; ISSN:0920-9069. (Springer)Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant prodn. is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM mols. (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy nos. and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy nos. as well as in transcription levels were obsd., they did not seem to be a limitation. Levels of relevant endoplasmic reticulum-stress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously obsd. on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.
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