Figure 3.
Development of antigen-capture ELISA
(A) Schematic representation of sandwich ELISA. Each mAb was labeled with horseradish peroxidase (HRP) and subjected to ELISA analysis. 9 pairs of antibodies were tested.
(B) Determination of the optimal combination of capturing and detection mAbs. S/N ratios for antigen detection by each of the 9 combinations were calculated in the presence of 2 ng/mL antigen versus blank (two technical replicates).
(C) Detection limit of ELISA. Serially diluted recombinant NP and inactivated SARS-CoV-2 were subjected to ELISA. Graph data are presented as mean ± SD (six technical replicates). The error bars represent SD. The detection limits of both recombinant NP protein and SARS-CoV-2 were determined according to the cutoff value, which was calculated by the formula (average +3SD).
(D) Specificity of ELISA. Simulated specimens positive for indicated viruses prepared by adding recombinant protein or common respiratory viruses to pooled COVID-19 RT-PCR-negative specimens were analyzed by ELISA. “Virus” indicates inactivated virus (at least 106 copies/mL for HCoV-229E and HCoV-OC43 or at least 105 TCID50/mL [50% tissue culture infectious dose] for other viruses). “Recombinant protein” indicates recombinant NP antigen of human coronavirus (200 ng/mL). Graph data are presented as mean ± SD (three technical replicates). HRV, human rhinovirus; IFAV, influenza A virus; IFBV, influenza B virus; RSV, respiratory syncytial virus.
(E) Detection of variant of concerns strains by ELISA. All the three major variants were detected efficiently. Graph data are presented as mean ± SD (three technical replicates).
(F) Clinical performance of ELISA assay for nasal swab samples from RT-PCR negative (n = 72) and positive (n = 72). Boxplots of index values at ELISA assay were depicted. The p value was calculated using Welch’s t test (two-tailed).
(G) Receiver operating characteristic curves for antigen-capture ELISA.
(H and I) Sensitivity and specificity of ELISA results according to real-time PCR cycle threshold (Ct) values group based on NIID-N2 primer set.
(J) Relation between RNA copy number in PCR-positive specimens and reactivity of antigen-capture ELISA (n = 72; Spearman’s correlation).