Development of a high-throughput SARS-CoV-2 antibody testing pathway using dried blood spot specimens

Abstract

Background: Serological assays for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have roles in seroepidemiology, convalescent plasma-testing, antibody durability and vaccine studies. Currently, SARS-CoV-2 serology is performed using serum/plasma collected by venepuncture. Dried blood spot (DBS) testing offers significant advantages as it is minimally invasive, avoids venepuncture with specimens being mailed to the laboratory.

Methods: A pathway utilizing a newborn screening laboratory infrastructure was developed using an enzyme-linked immunosorbent assay to detect IgG antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein in DBS specimens. Paired plasma and DBS specimens from SARS-CoV-2 antibody-positive and -negative subjects and polymerase chain reaction positive subjects were tested. DBS specimen stability, effect of blood volume and punch location were also evaluated.

Results: DBS specimens from antibody-negative (n = 85) and -positive (n = 35) subjects and polymerase chain reaction positive subjects (n = 11) had a mean (SD; range) optical density (OD) of 0.14 (0.046; 0.03-0.27), 0.98 (0.41; 0.31-1.64) and 1.12 (0.37; 0.49-1.54), respectively. An action value OD >0.28 correctly assigned all cases. The weighted Deming regression for comparison of the DBS and the plasma assay yielded: y = 0.004041 + 1.005x, r = 0.991, Sy/x 0.171, n = 82. Extraction efficiency of antibodies from DBS specimens was >99%. DBS specimens were stable for at least 28 days at ambient room temperature and humidity.

Conclusions: SARS-CoV-2 IgG receptor-binding domain antibodies can be reliably detected in DBS specimens. DBS serological testing offers lower costs than either point of care or serum/plasma assays that require patient travel, phlebotomy and hospital/clinic resources; the development of a DBS assay may be particularly important for resource poor settings.

Keywords: COVID-19; Dried blood spots; SARS-CoV-2; antibodies; enzyme-linked immunosorbent assay.

Figure 1.

(a) Effect of sample volume on blood spot diameter. A whole blood EDTA sample was applied to filter paper collection devices at the volumes shown. Subpunches (6 mm) were taken from the central and peripheral locations. (b) Relationship between the volume of blood applied to filter paper collection device and the DBS area. Results are the mean of five replicates.

Figure 2.

Performance of the DBS SARS-CoV-2 IgG ELISA for specimens from SARS-CoV-2 antibody negative subjects (n = 85), SARS-CoV-2 antibody-positive subjects (n = 35) and SARS-CoV-2 PCR positive subjects (n = 11). The dotted line represents the action value for positivity on the DBS ELISA (OD > 0.28).

Figure 3.

Bland–Altman plot showing the absolute difference for SARS-CoV-2 IgG antibody OD’s between the plasma specimens analysed by the in-house ELISA and in paired DBS specimen eluates analysed by the in-house ELISA. The solid horizontal line denotes the mean bias between the two methods (–0.008 OD). The dashed horizontal lines indicate the 95% limits of agreement. The dashed vertical line denotes the action value for positivity on the DBS ELISA (>0.28). The weighted Deming plot yielded a slope of 1.005 (95% CI: 0.9573–1.053), intercept of 0.004 (95% CI: –0.007 to 0.015), an Sy/x of 0.171 and a correlation coefficient (r) of 0.991. Paired plasma and DBS specimens used were from SARS-CoV-2 negative subjects (n = 47) and positive antibody subjects (n = 35).

Figure 4.

Concordance between the DBS ELISA and the Healgen Scientific PoCT (IgG/IgM) lateral flow device using paired DBS and serum specimens from SARS-CoV-2 negative subjects (n = 47) and positive antibody subjects (n = 28). The dotted line represents the action value for positivity on the DBS ELISA (OD > 0.28).

Figure 5.

Concordance between the DBS ELISA and Roche commercial IgG serological assay using DBS specimen eluates from SARS-CoV-2 negative subjects (n = 44) and positive antibody subjects (n = 25). The dotted line represents the action value for positivity on the DBS ELISA (OD > 0.28).