Application of a Biomimetic Nanoparticle-Based Mock Virus to Determine SARS-CoV-2 Neutralizing Antibody Levels in Blood Samples Using a Lateral Flow Assay

The presence of neutralizing antibodies against SARS-CoV-2 in blood, acquired through previous infection or vaccination, is known to prevent the (re)occurrence of outbreaks unless the virus mutates. Therefore, the measurement of neutralizing antibodies constitutes an indispensable tool in assessing an individual’s and a population’s immunity against SARS-CoV-2. For this reason, we have developed an innovative lateral flow assay (LFA) capable of detecting blood-derived neutralizing antibodies using a biomimetic SARS-CoV-2 mock virus system. Here, functionalized gold nanoparticles (AuNPs) featuring the trimeric spike (S) protein at its surface imitate the virus’s structure and are applied to monitor the presence and efficacy of neutralizing antibodies in blood samples. The detection principle relies on the interaction between mock virus and the immobilized angiotensin-converting enzyme 2 (ACE2) receptor, which is inhibited when neutralizing antibodies are present. To further enhance the sensitivity of our competitive assay and identify low titers of neutralizing antibodies, an additional mixing pad is embedded into the device to increase the interaction time between mock virus and neutralizing antibodies. The developed LFA is benchmarked against the WHO International Standard (21/338) and demonstrated reliable quantification of neutralizing antibodies that inhibit ACE2 binding events down to a detection limit of an antibody titer of 59 IU/mL. Additional validation using whole blood and plasma samples showed reproducible results and good comparability to a laboratory-based reference test, thus highlighting its applicability for point-of-care testing.


Figure S1 :
Figure S1: Electrophoretic mobility of AuNP Figure S2: Transmission electron microscopy images of AuNP Figure S3: Integration of mixing pad Figure S4: Example of images used for quantification using FIJI Figure S5: Comparison of 6 and 25 mm long sample pad by testing a serial dilution of WHO International Standard (21/338) Figure S6: Testing of samples with commercially available LFA detecting neutralizing antibodies.Figure S7: Comparison of RBD and S protein Figure S8: Sample #5 tested with LFA utilizing mock virus or AuNP-RBD Figure S9: Testing of lower antibody titer

Figure S7 :
Figure S1: Electrophoretic mobility of AuNP Figure S2: Transmission electron microscopy images of AuNP Figure S3: Integration of mixing pad Figure S4: Example of images used for quantification using FIJI Figure S5: Comparison of 6 and 25 mm long sample pad by testing a serial dilution of WHO International Standard (21/338) Figure S6: Testing of samples with commercially available LFA detecting neutralizing antibodies.Figure S7: Comparison of RBD and S protein Figure S8: Sample #5 tested with LFA utilizing mock virus or AuNP-RBD Figure S9: Testing of lower antibody titer

Figure S3 :
Figure S3: Integration of mixing pad (MP).A) Comparison of full-stick and full-stick with MP.B) Background signal of fullstick and full-stick + MP indicates that the full-stick + MP has a higher background due to the increased length of the strip.C) Neutralization of 199 U/mL with full-stick and full-stick + MP after increasing the volume of buffer from 10 to 15 µl.

Figure S4 :
Figure S4: Example of images used for quantification using FIJI.

Figure S5 :
Figure S5: Comparison of 6 and 25 mm long sample pad by testing a serial dilution of WHO International Standard (21/338).

Figure S7 :
Figure S7: Comparison of RBD and S protein.A) ELISA to study the binding to immobilized ACE2; B) AuNP modified with RBD or S protein tested in LFA; C) Serial dilution of WHO International Standard (21/338) in LFA utilizing trimeric S protein (mock virus)-or RBD-modified AuNP.D) Plasma samples tested with AuNP-RBD and AuNP-SPV-S protein.

Table S1 :
AuNP mixtures analyzed regarding the intensity ratio between test (T) and control (C) line.The control line binds mouse IgG AuNP and the test line binds the mock virus, consisting of AuNP functionalized with trimeric S protein.