The conjugate was blocked by glycine and purified by gel filtration, stored at 2C8?C. Assay process In CL2-SN-38 the first step, sample (10 L), sample treatment solution, RBD-coated magnetic beads, and ACE2-ALP conjugate are added into a reaction cuvette. (pVNT) and immunoassays detecting anti-SARS-CoV-2 binding antibodies was performed. Receiver operating characteristic (ROC) curve analysis was generated to assess the optimal threshold for detecting nAbs by each assay. All three sVNTs showed an excellent performance in terms of specificity (100%) and sensitivity (100%, 97.0%, and 97.1% for GenScript, Dynamiker, and Mindray, respectively) in samples collected from vaccinated subjects. GenScript demonstrated the strongest correlation with pVNT (interquartile range. Serological testing The initial serological screening was done on the automated analyzer CL-900i? from Mindray Bio-Medical Electronics8,9,16 using three chemiluminescence immunoassays to detect anti-SARS-CoV-2 bAbs targeting either the S and N proteins or exclusively the RBD: (i) Anti-SARS-CoV-2 IgG against S/N protein (Cat. No. SARS-CoV-2 IgG121) with a cutoff index of??10?IU/ml, (ii) Anti-SARS-CoV-2 S-receptor binding domain (S-RBD) IgG (Cat. No. SARS-CoV-2 S-RBD IgG122, Mindray, China) with a cutoff index of??10C1000 BAU/ml, and (iii) Anti-S-RBD SARS-CoV-2 total antibodies (IgG, Rabbit Polyclonal to IL4 IgA, and IgM) CL2-SN-38 (Cat. No. SARS-CoV-2 Total Antibodies 122, Mindray, China) with a cutoff index of??10C2000 AU/ml. All plasma samples were tested with these assays following the manufacturer’s instructions. ELISA-based surrogate virus neutralization tests (sVNT) Two different SARS-CoV-2 sVNTs were assessed for detecting nAbs that block the interaction between SARS-CoV-2 RBD and human angiotensin-converting enzyme 2 (ACE2) receptors. The first assay is an ELISA-based inhibition test developed by GenScript (Cat. No. L00847, GenScript Biotech, NJ, USA)7,8,17. This assay utilizes the same principle as ELISA, using a 96-well microplate to serologically screen for nAbs targeting SARS-CoV-2 RBD (Fig.?1A). The performance of this assay was previously assessed by several studies demonstrating high sensitivity, specificity, and correlation with MNA and pVNT7,18,19. Absorbance was measured at 450?nm, and the percentage of inhibition was calculated using the following formula: % inhibition?=?(1???(OD450 sample/OD450 of negative control))??100. Result interpretation was as follows: percent inhibition??30% is positive (detectable nAbs), and?CL2-SN-38 on the magnetic beads. (C) Mechanism of pseudovirus neutralization test (pVNT) where anti-SARS-CoV-2 nAbs block the binding of SARS-CoV-2 spike CL2-SN-38 (S) protein to human ACE2 receptor on the host cell surface. CL2-SN-38 All illustrations were created using BioRender. Figures (A) and (C) were adapted from Wang et al.7. SARS-CoV-2 NTAb assay The third assay is an automated competitive binding immuno-enzymatic assay (anti-SARS-CoV-2 NTAb assay) developed by Mindray (catalog No. SARS-CoV-2 Neutralizing Antibody 121) for quantitative detection of nAbs against SARS-CoV-2.