A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

Abstract

The spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose-response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

Figure 1

Neutralization of SARS-CoV-2 Spike-ACE2 interaction in DBS samples from convalescent COVID-19 cases and pre-pandemic negative samples. Reduction in neutralization with dilution of 12 DBS samples (1:4, 1:16, 1:64, 1:256) after initial elution of one 5 mm punch in 100 μL assay buffer. Negative samples are indicated with dashed lines.

Figure 2

Agreement in neutralization results for matched serum and DBS samples. Scatterplot and Passing-Bablok regression line (95% CI) demonstrating agreement in % neutralization of SARS-CoV-2 Spike-ACE2 interaction in DBS and serum samples. PCR positive samples are indicated with open circles; negative samples are indicated with black circles. The concordance correlation of absolute agreement = 0.991.

Figure 3

Neutralization of SARS-CoV-2 Spike-ACE2 interaction in DBS samples. Negative samples were collected in 2019 or were seronegative for anti-RBD IgG; PCR positive samples are from convalescent COVID-19 cases. Mild/asymptomatic samples are from individuals who were seropositive for anti-RBD IgG but did not get a PCR test for acute infection. The black line represents median neutralization for each group. Wilcoxon rank-sum tests were used to evaluate differences across the groups. *p < 0.05; ***p < 0.001.