Detection of Anti-Toxoplasma gondii Antibodies in Human Sera Using Synthetic Glycosylphosphatidylinositol Glycans on a Bead-Based Multiplex Assay

Toxoplasmosis, while often an asymptomatic parasitic disease in healthy individuals, can cause severe complications in immunocompromised persons and during pregnancy. The most common method to diagnose Toxoplasma gondii infections is the serological determination of antibodies directed against parasite protein antigens. Here we report the use of a bead-based multiplex assay containing a synthetic phosphoglycan portion of the Toxoplasma gondii glycosylphosphatidylinositol (GPI1) for the detection of GPI1-specific antibodies in human sera. The glycan was conjugated to beads at the lipid site to retain its natural orientation and its immunogenic groups. We compared the response against GPI1 with that against the protein antigen SAG1, a common component of commercial serological assays, via the detection of parasite-specific human IgG and IgM antibodies, respectively. The GPI1-based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI1 test shows 97% specificity and 98% sensitivity for the assay. GPI1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.


Preparation of Mannose 3 conjugate and optimization of glycan loading
The mannose 3 unit was prepared following reported protocol. 4 Man 3 disulfide dimers (4 mg, 0.013 mmol) were dissolved in 2 mL of water and reduced using two equivalents (0.026 mmol) of resin bound Tris-(2-carboxyethyl)-phosphine (TCEP)). After 1 hour, the resin was removed and washed with water and the filtrates were lyophilized.
The maleimide (5) linker was installed on MagPlex® microspheres using the protocols described in the Luminex cookbook. 5 A permanent magnet was used as a magnetic separator

GPI-Loading determination by monosaccharide analysis using HPAE-PAD
The GPI1-Beads conjugates (5x10 5 beads ) were washed with deionized water, incubated with 200 L of 2 M TFA for 4 h at 100°C and lyophilized. The dry hydrolysis mixture was dissolved in 100 L of water and diluted 1:10 or 1:20.
with water. The monosaccharide content was analyzed using high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) (Dionex, Sunnyvale, CA, USA) equipped with a CarboPac™ PA20 column (3 × 150 mm) (Dionex) and a CarboPac™ PA20 guard column (3 × 30 mm) (Dionex). The separation was performed with isocratic concentration of 10 mM NaOH (J.T.Baker, Devneter, the Netherlands) at 0.5 mL/min flow rate for 15 min at 30°C (figure S1). The quantity of GPI was calculated based on the content of glucose in the injected samples. galactosamine, glucosamine and glucose ( Figure S2). The monosaccharides used in this study were purchased from Sigma.

Optimization of the BBMA with GPI1
To optimize the conditions for the detection of anti-GPI antibodies in sera, we analyzed a series of three positive and five negative sera under different BBMA conditions. Initial experiments were performed to establish the effect of the GPI1 loading on the beads and the type and amount of secondary antibody. The experiments were carried out using modified beads of the region 20 using 25 and 50 g of GPI1 for loading 2.5 million beads ( Figure S5). Figure S5. Each bar corresponds to the average of the MFI values obtained for the corresponding set of sera samples. The average of the standard deviation is also showed. The GPI-1 was conjugated either using 50 µg or 25 µg for 2.5 million beads. Sera samples were used in 1:100 dilution, a) Detection using a biotinylated secondary antibody and following detection using a 3 µg/mL of antibody (Streptavidin-phycoerythrin). b) Detection using phycoerythrin-coupled secondary antibody: Two concentrations of antibody were used 1.5 µg/mL or 1 µg/mL and two loading of GPI-1, 50 µg and 25 µg. c) Optimization of phycoerythrin-coupled secondary Ab concentration. d) Effect of buffer components EG: Ethylenglycol, Det: detergent.
From these experiments following parameter were selected: Amount of GPI1 for loading of 2.5x10 6 Beads: 50 g Detection with phycoerythrin-coupled secondary antibody at 1.5 g/mL concentration. A standard buffer without ethylenglycol (PBS pH 7.3, 1% BSA).

Optimization of sera dilution
Eight positive sera were diluted in ratios 1:100, 1:200, 1:500 and 1:1000 and evaluated by BBMA following the procedure in material and methods.
S7 Figure S6. Evaluation of serum sample dilution. The secondary antibody was 1.5 g/ml

Evaluation of two GPI-Beads conjugates
We evaluated the reproducibility of the GPI-bead conjugates for detection of anti-GPI IgM and IgG antibodies in 18 characterized sera. A correlation of the analysis with these two batches showed a great agreement between the conjugates for the detection of antibodies by BBMA. Figure S7. Correlation in the detection of anti-GPI antibodies by BBMA using two batches of the GPIbeads conjugate.

Analysis of the sera by Microarrays:
Twenty characterized sera were evaluated using GPI1 printed on microarrays. In agreement with our previous report, these sera contained anti-GPI antibodies binding GPI1.
Printing pattern: Figure S9. Printing pattern of the glycan array.
S9 Figure S10. Pictures of the glycanarray scans used for determination of IgG and IgM anti-GPI antibodies.
Each well correspond to one serum. Some sera also contain antibodies binding an 1-6-trimannose.