Evaluation of a multiplexed coronavirus antigen array for detection of SARS-CoV-2 specific IgG in COVID-19 convalescent plasma

Abstract

Mitigation of the COVID-19 pandemic requires an understanding of the antibody response to SARS-CoV-2. However, throughout the development of SARS-CoV-2 IgG antibody assays during the past year, cross-reactivity to other coronaviruses remained a question. To address these issues, we evaluated IgG in COVID-19 convalescent plasma samples for reactivity against three SARS-CoV-2 antigens including full-length spike, receptor binding domain, and the proximal extracellular fusion domain, and spike antigens from other coronaviruses (SARS-CoV, MERS-CoV, hCoV-HKU1, hCoV-OC43, hCoV-229E and hCoV-NL63) using the VaxArray Coronavirus SeroAssay which is a multiplexed antigen assay developed by InDevR Inc. These results were compared to two commercial SARS-CoV-2 IgG ELISAs targeting either the SARS-CoV-2 nucleocapsid or spike antigens and a live virus focus reduction neutralizing antibody test (FRNT). The VaxArray platform showed high specificity for detection of SARS-CoV-2 IgG, evident from lack of reactivity to SARS-CoV-2 antigens despite significant reactivity to endemic coronavirus antigens in pre-pandemic samples. SARS-CoV-2 IgG positive samples reacted weakly to SARS-CoV spike but not to MERS-CoV. While the VaxArray platform had overall comparable results to the spike and nucleocapsid IgG ELISAs, results were more similar to the spike antigen ELISA and the platform displayed a higher sensitivity and specificity than both ELISAs. Samples with FRNT titers below 1/23 reported negative on VaxArray, while positive samples on VaxArray had significantly higher neutralizing antibody titers. These results suggest that the VaxArray Coronavirus SeroAssay performs with high sensitivity and specificity for the detection of SARS-CoV-2 IgG, and positive results on the platform indicate SARS-CoV-2 neutralizing activity.

Keywords: Antigen array; COVID-19; SARS-CoV-2; SARS-CoV-2 IgG; Spike antigen.

Fig. 1

Representative slide samples for the VaxArray Coronavirus SeroAssay. The spike antigens of SARS-CoV, MERS-COV, hCoV-HKU1, hCoV-OC43, hCoV-229E and hCoV-NL63, and the full spike (S, nCoV(i)), receptor binding domain (RBD, nCoV(ii)) and extracellular domain of S (S2, nCoV(iii)) are spotted onto a glass slide (A). Comprised in the slide's 9 × 9 grid, each antigen is represented in a 3 × 3 grid, yielding 9 replicate sets of data for each of the 9 antigens. Representative pre-pandemic sample slides (B) and representative VaxArray positive SARS-CoV-2 PCR positive sample slides (C).

Fig. 2

Antibody response to coronavirus antigens on the VaxArray Coronavirus SeroAssay. Ninety-six SARS-CoV-2 PCR positive samples and 30 presumed negative samples were evaluated for IgG antibody response to SARS-CoV-2 antigens (A and C, respectively) and to other hCoVs (B and D, respectively). The normalized fluorescence signals are displayed in two different heat maps; panels A and C with the SARS-CoV-2 antigens displaying a negative, low, medium and high response and panels B and D with the other hCoVs displaying a spectrum with blue indicating a low response and yellow representing a high response. In panel A, an asterisk beside the sample indicates a VaxArray negative sample and the samples highlighted in red indicate that while the nCoV(i) signal was greater than 1.5, the sum of the three antigens was less than 6.18 so the sample was categorized as negative. Representative slides of the SARS-CoV-2 PCR positive samples that were negative on the VaxArray platform (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Comparison of qualitative results from EDI, EUROIMMUN, and VaxArray to FRNT neutralizing results. Thirty SARS-CoV-2 PCR positive samples were evaluated on EDI, EUROIMMUN, and VaxArray assays. Qualitative results, based on kit-specific cut-off values were compared to FRNT50 titer. The mean FRNT50 titer for VaxArray-classified positive and negative samples is shown as a horizontal bar and the error bars on the negative samples represent one standard deviation. The mean FRNT50 titer plus one standard deviation was used as the negative cut-off for each assay. The three negative samples on the VaxArray platform are highlighted in green across each assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)