Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients

Abstract

The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; antibody; immunity; immunoglobin; infection; neutralization; serology.

FIG 1

Demographics of convalescent plasma (CP) donors. (A) Distribution of convalescent plasma donor ages (left, blue bars) compared to U.S. population (right, red bars). Dotted lines represent Gaussian distribution curve fits. n = 263; Pearson’s correlation coefficient. (B) Distribution of convalescent plasma donor sexes (blue bars) compared to U.S. population (red bars). n = 354; binomial test for discrepancy versus U.S. population; n.s., not significant. (C) Distribution of convalescent plasma donor blood group antigens (blue bars) compared to U.S. population (red bars). n = 370; binomial test for discrepancy versus U.S. population; * P < 0.05. (D) Distribution of convalescent plasma donor ethnicities (blue bars) compared to U.S. population (red bars). n = 204; binomial test for discrepancy versus U.S. population; *, P < 0.05; n.s., not significant.

FIG 2

Neutralizing activity analysis of convalescent plasma (CP) donors. (A) Distribution of neutralization 50% inhibitory concentration (IC₅₀) values (NT₅₀, reciprocal plasma dilution) of convalescent donor plasma samples using HIV-1 (red) or VSV (blue) pseudovirus overexpressing the SARS-CoV-2 spike protein (S) (HIV-S and VSV-S, respectively). (B) Frequencies of convalescent plasma donor NT₅₀ values within indicated groups using HIV-S (top) or VSV-S (bottom) pseudovirus constructs. (C) Frequency distribution of convalescent plasma HIV-S NT₅₀ values versus age groups (years). n = 5 to 38; Kruskal-Wallis test. (D) Frequencies of convalescent plasma donor NT₅₀ values versus sex. n = 190; Mann-Whitney test; **, P < 0.01. (E) Frequencies of convalescent plasma donor NT₅₀ values versus blood group antigens. n = 15 to 82; Kruskal-Wallis test. (F) Frequencies of convalescent plasma donor NT₅₀ values versus time (days) since last reported symptom. n = 19 to 33; Mann-Whitney t test; *, P < 0.05.

FIG 3

Serological analysis of convalescent plasma donors. (A) Frequencies of densitometric IgG (left) or IgM (right) results from LFA bands relative to control bands. (B) Frequencies of HTSA results using the total Ig or IgG assays derived from the Ortho Diagnostics platform (left) or Abbott IgG assay platform (right). Results from fresh frozen plasma (FFP) units collected before COVID-19 are shown as healthy controls. AU, arbitrary units. (C) Frequencies of S1 spike protein (left), nucleocapsid protein (NP) (center), and RBD spike protein (right) ELISA titer results. Titers reflect concentrations calculated using an MAb standard curve and not absolute plasma concentrations.

FIG 4

Correlation of serology assays versus neutralization activities for convalescent plasma donors. (A) Linear regression of HIV-S NT₅₀ values (abscissa) versus serological assay values (ordinate). Number of samples is indicated in each graph; r² = goodness of fit; AU, arbitrary units; NP, nucleocapsid protein. (B) Spearman correlation coefficients, r, of neutralization and serological assays. n = 137 samples; NP, nucleocapsid protein. (C) Distributions of CP donor sample HTSA scores within indicated HIV-S NT₅₀ groups using Ortho total Ig (left), Ortho IgG (center), or Abbott IgG (right) assay. (D) Frequencies of convalescent donor S1 protein and nucleocapsid protein (NP) ELISA values defined in Fig. S4B in the supplemental material. n = 241 samples; DP, double positive. (E) Distribution of NT₅₀ values corresponding to populations defined in Fig. S4B in the supplemental material. n = 4 to 51; Kruskall-Wallis test; *, P < 0.05; **, P < 0.01.