Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts

Abstract

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.

Fig 1:. ELISA to detect and measure SARS-CoV-2 spike-specific IgG and IgA in COVID-19 convalescent sera.

Serially diluted convalescent patient sera (colored circles) and a pre-2020 negative control (gray diamonds) were added to recombinant SARS-CoV-2 spike protein-coated ELISA plates. Captured IgG (a) and IgA (b) were detected using Ig class-specific secondary antibody-HRP conjugates. Absorbance (A₄₅₀) values were fitted to a sigmoidal curve. Samples were re-analyzed at three dilutions that best characterized the extent of the antibody reactivity for IgG (c) and IgA (d). Averages +/− SD are shown, n=4 from two independent experiments. SD values smaller than the height of the symbols are not shown.

Fig 2:. Analysis of spike-specific IgG and IgA reactivity in convalescent and control cohorts.

Spike-specific IgG (a–b) and IgA (d–e) responses at the indicated serum dilutions were determined for convalescent (Conv, n=197) and control (Ctrl, n=216) cohorts. (c, f) Inter-assay reproducibility of independent IgG and IgA assays at serum dilutions of 1:1,000 and 1:200, respectively, was assessed by a linear regression analysis. (g–h) Spike-specific IgG and IgA reactivity for Conv and Ctrl cohorts at the selected test dilution (1:1,000 and 1:200 serum dilutions, respectively). Diagnostic thresholds for IgG and IgA tests are shown as dotted lines (A₄₅₀ values of 0.90 and 0.60, respectively). Ctrl cohort is separated into Pre-2020 (n=171) and Jan 2020 groups (n=45). (i) IgG and IgA reactivities of each sample in the Conv (orange circles) and Ctrl (green circles). Respective diagnostic thresholds are indicated as dotted lines. Percentages reflect the proportion of Ctrl and Conv in each quadrant.

Fig 3:. Selection of diagnostic thresholds for IgG and IgA tests using receiver-operator curve (ROC) analysis.

(a) ROC analyses for the IgG and IgA tests. AUC, area under the curve. Filled circles indicate the point on each ROC that corresponds to the selected diagnostic threshold. (b) The sum of assay sensitivity and specificity for each candidate diagnostic threshold was extracted from the ROCs for the IgG and IgA tests. Dotted lines indicate the selected thresholds (A₄₅₀ of 0.90 and 0.60 for IgG and IgA, respectively).

Fig 4:. Specificity of IgG and IgA tests for SARS-CoV-2 vs. commonly circulating human coronaviruses.

Serum samples from two cohorts of COVID-19–negative patients with RT-qPCR-confirmed exposure to one or more commonly circulating human coronavirus (hCoV) were analyzed in the SARS-CoV-2 IgG (a) and IgA (b) tests (SARS-2) and for IgG and IgA reactivity against recombinant spike proteins from the indicated alpha- and betacoronaviruses by ELISA. Dotted lines indicate diagnostic thresholds for the SARS-CoV-2 antibody tests only. Data from at least 2 independent experiments (n=4–8) are shown.

Fig 5:. Longitudinal analysis of hospitalized patients at two early time points with the IgG and IgA tests.

Serum samples from patients at two time points following hospital admission were analyzed for spike-specific IgG (a) and IgA (c). Individual patient samples (circles) and cumulative positive results (blue bars) are graphed as a function of days post-symptom onset (self-reported) for IgG (b) and IgA (d). Data from two independent experiments (n=4) are shown.

Fig 6:. Clinical translation of IgG test and cross-validation with New York State Department of Health test.

Heat maps comparing results from a subset of Conv and Ctrl samples obtained with the manual IgG test performed in the laboratory at Einstein (Research) (a–b) to those obtained with the Wadsworth Center microsphere immunoassay (NY State) (a) and the clinically translated test performed in the CLIA-certified laboratory at MMC (Clinical) (b). Asterisks denote discrepant results.

Fig 7:. IgG test affords quantitative assessment of serum IgG from a single measurement.

(a) The relationship between the log-transformed readout value (A₄₅₀ at 1/1,000 serum dilution) in the IgG antibody test and the endpoint IgG titer (determined from full ELISA curves), for each serum sample in the Conv cohort. Data were fit to a sigmoidal function through a nonlinear regression analysis. (b) A ten-fold cross-validation method was used to evaluate the predictive utility of this model. For each serum sample, the experimentally determined endpoint IgG titer was compared to that predicted from a single measurement with the antibody test using linear regression analysis. Shaded blue areas represent the 95% confidence intervals for the curve fits.