Rapid Generation of In-House Serological Assays Is Comparable to Commercial Kits Critical for Early Response to Pandemics: A Case With SARS-CoV-2

Abstract

Introduction: Accurate and sensitive measurement of antibodies is critical to assess the prevalence of infection, especially asymptomatic infection, and to analyze the immune response to vaccination during outbreaks and pandemics. A broad variety of commercial and in-house serological assays are available to cater to different laboratory requirements; however direct comparison is necessary to understand utility.

Materials and methods: We investigate the performance of six serological methods against SARS-CoV-2 to determine the antibody profile of 250 serum samples, including 234 RT-PCR-confirmed SARS-CoV-2 cases, the majority with asymptomatic presentation (87.2%) at 1-51 days post laboratory diagnosis. First, we compare to the performance of two in-house antibody assays: (i) an in-house IgG ELISA, utilizing UV-inactivated virus, and (ii) a live-virus neutralization assay (PRNT) using the same Cambodian isolate as the ELISA. In-house assays are then compared to standardized commercial anti-SARS-CoV-2 electrochemiluminescence immunoassays (Elecsys ECLIAs, Roche Diagnostics; targeting anti-N and anti-S antibodies) along with a flow cytometry based assay (FACS) that measures IgM and IgG against spike (S) protein and a multiplex microsphere-based immunoassay (MIA) determining the antibodies against various spike and nucleoprotein (N) antigens of SARS-CoV-2 and other coronaviruses (SARS-CoV-1, MERS-CoV, hCoVs 229E, NL63, HKU1).

Results: Overall, specificity of assays was 100%, except for the anti-S IgM flow cytometry based assay (96.2%), and the in-house IgG ELISA (94.2%). Sensitivity ranged from 97.3% for the anti-S ECLIA down to 76.3% for the anti-S IgG flow cytometry based assay. PRNT and in-house IgG ELISA performed similarly well when compared to the commercial ECLIA: sensitivity of ELISA and PRNT was 94.7 and 91.1%, respectively, compared to S- and N-targeting ECLIA with 97.3 and 96.8%, respectively. The MIA revealed cross-reactivity of antibodies from SARS-CoV-2-infected patients to the nucleocapsid of SARS-CoV-1, and the spike S1 domain of HKU1.

Conclusion: In-house serological assays, especially ELISA and PRNT, perform similarly to commercial assays, a critical factor in pandemic response. Selection of suitable immunoassays should be made based on available resources and diagnostic needs.

Keywords: ELISA; PRNT; SARS-CoV-2; immunoassay; serology.

Figure 1

Results of in-house ELISA and PRNT. (A) Correlation matrix with Spearman r values of all investigated serology assay: anti-S IgM and anti-S IgG determined by flow cytometry (IgM FACS and IgG FACS, respectively), N- and S-targeting CLIA (N and S ECLIA), in-house IgG ELISA and PRNT. Individual result of each sample (total 250) for (B) in-house ELISA, and (C) in-house PRNT. Lines represent median and interquartile range. The respective thresholds (dotted line) are for in-house IgG ELISA OD₄₀₅ ≥ 1, and for PRNT ≥ 1PRNT₅₀ titer. The samples were categorized based on their SARS-CoV-2 RT-PCR result in PCR negative (n = 16; gray), and in 3 groups of SARS-CoV-2 confirmed cases: seronegative samples (n = 36; blue) with negative results in the flow cytometry based assay and ECLIAs, early seropositive samples (n = 8; green) that are positive for anti-S IgM, and seropositive samples (n = 190; red) that are positive for anti-S IgG determined by flow cytometry and/or in one or both ECLIAs. Multiple comparison was performed by Kruskal-Wallis test with α = 0.05.