Evaluation of SARS-CoV-2 prototype serologic test in hospitalized patients

Abstract

Objectives: Humoral immune response to SARS-CoV-2 infection has been reported in several patient cohorts with results that vary by method and population studied due to the lack of reliable commercial assays available as the pandemic initially spread. We sought to clinically assess commercial prototype SARS-CoV-2 IgG and IgA assays for use in screening for prior infection and convalescent plasma donation.

Design and methods: Prototype SARS-CoV-2 IgG and IgA assays from Euroimmun were assessed utilizing remnant specimens. Specificity testing used specimens in their convalescent window for the common coronaviruses and other infectious diseases known to be associated with increased non-specificity in serologic assays. Sensitivity testing utilized serial specimens from molecularly confirmed SARS-CoV-2 critically ill patients to assess seroconversion. Utilizing recombinant spike protein we also developed a competitive confirmation procedure to increase assay specificity.

Results: We determined specificity to be 97% and 81%, respectively, when indeterminate samples were considered positive and 99% and 86% when indeterminate samples were considered negative. We developed a new confirmation methodology to enhance the specificity of the assays with an anticipated specificity of 98% for IgA. Valuation of hospitalized COVID-19 patients determined median IgA seroconversion to be 8 days and IgG 10 days. Neither level nor timing of antibody response correlated with days on ventilation. End titer measurements indicate that validated improved assays may be capable of semi-quantitative measurement.

Conclusions: We found these assays to be clinically acceptable for the high prevalence population tested, for instance, for convalescent plasma donation.

Keywords: Antibody; COVID; SARS CoV2; Serology.

Fig. 1

Validation of specificity testing for SARS-CoV-2 IgA and IgG antibodies in human serum samples. A. Patients tested nucleic acid positive for common coronaviruses prior to COVID-19 presence in the geographic region and had a subsequent blood sample available. X-axis: Days from nucleic acid positive test. B. Extinction ratio for all patients from (A) are plotted for comparison (n = 19). C. Patients presenting in the geographic region before COVID-19 with respiratory symptoms that prompted a nucleic acid respiratory pathogens panel test. X-axis: Days from testing indicated. D. Extinction ratio for all patients from (C) are plotted (n = 37). E. Patient cohorts positive for listed diseases prior to COVID-19 presence in the geographic region were tested for SARS-CoV-2 IgA (red dots) and IgG (blue dots) (n = 170). Vol, healthy volunteers; Syph, syphilis. Horizonal dashed/dotted lines indicate indeterminate interpretive region. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Seroconversion of hospitalized nucleic acid-confirmed COVID-19 patients in the United States. A. Results of six COVID-19 patients (one per panel) with serial specimen collection during hospitalization. Green shading – days intubated: Vertical green lines indicate day of intubation (left) and day of extubation or continued intubation (right). Horizontal dashed lines indicate indeterminate interpretive region. B. Three samples for each IgG (blue) and IgA (red) were diluted to end titer to determine the correlation between extinction ratio and end titer. Left panels demonstrate the full dilution series. Right panel plots initial extinction ratio to final titer of each sample with 95% confidence intervals (green dotted lines) and predictive interval (purple dashed lines). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Competitive spike proteins significantly improve assay specificity. A. Nucleic acid confirmed COVID-19 patient serum specimens (n = 3) were assayed for SARS-CoV-2 IgG and IgA antibodies. Pretreatment of the sample with SARS-CoV S1 subunit protein (SARS-S1), MERS spike protein (MERS-S), SARS-CoV-2 S1 subunit protein (S1), SARS-CoV-2 S2 subunit protein (S2), and SARS-CoV-2 S1 subunit and S2 subunit (S1 + S2) was performed to assess IgG and IgA antibody reaction specificity. Results are mean ± SEM, percent of inhibition from non-treated control sample (n = 3). B. SARS-CoV-2 IgA antibodies were assessed in nucleic acid confirmed COVID-19 patients (n = 9) and non-COVID-19 samples that displayed cross-reactivity (n = 12) with and without S1 + S2. Horizonal dashed lines indicate indeterminate interpretive region. *, p < 0.01 (paired t-test). C. SARS-CoV-2 IgG antibodies were assessed in nucleic acid confirmed COVID-19 patients (n = 19) with and without S1 + S2. Horizonal dashed lines indicate indeterminate interpretive region. *, p < 0.01 (paired t-test). Positive control(s) from the kit were used as additional controls (both in B and C) to confirm spike protein specificity.