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

Abstract

Background: Serologic 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 bloodspot (DBS) testing offers significant advantages; as it is minimally invasive, avoids venepuncture with specimens being mailed to the laboratory. Methods: A pathway utilising a newborn screening laboratory infrastructure was developed using an Enzyme-Linked Immunosorbent assay (ELISA) to detect IgG antibodies against the receptor-binding domain (RBD) 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 PCR positive subjects were tested. DBS specimen stability, effect of blood volume and punch location were also evaluated. Results: DBS from antibody-negative (n=85) and positive (n=35) subjects and PCR 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 was >99%. DBS were stable for at least 28 days at ambient room temperature and humidity. Conclusions: SARS-CoV-2 IgG RBD antibodies can be reliably detected in DBS. 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.

Evaluation of Dried Blood Spot Testing for SARS-CoV-2 Serology Using a Quantitative Commercial Assay

“…Several parameters affect the amount of predicted elution from the filter cards [ 26 , 28 , 49 ]. The number of circles spotted with blood, the punch size, and the number of punches will determine the size of the surface area exposed to elution buffer.…”

“…We found modifications of the standard procedure had limited impact on the performance of DBS, but overall, we found that 10 spots from 5 cards eluted in 750 µL of PBS gave the most consistent results. A recent study also showed the effect of DBS size and punch location on the observed DBS signal [ 26 ]. DBS specimens have been shown to be stable at room temperature, 4 °C and −20 °C, at ambient or low humidity conditions for at least 28 days [ 26 ].…”

“…These assays have demonstrated high specificity and sensitivity using serum/plasma. Although there have been preliminary studies evaluating DBS collection for anti-SARS-CoV-2 immunoassays, DBS serology tests have yet to be used on a widespread scale [ 2 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. Importantly, the majority of platforms evaluated have been for qualitative assays for antibodies against SARS-CoV-2, a potential gap should a quantitative option prove beneficial.…”

Whole blood-based measurement of SARS-CoV-2-specific T cell responses reveals asymptomatic infection and vaccine efficacy in healthy subjects and patients with solid organ cancers

“…An in-house direct ELISA was developed as previously described (16)(17)(18)(19), with some modifications. Maxisorp (Nunc, Loughborough, UK) 96-well plates were coated with RBD protein (recombinantly generated in a mammalian expression system, in house) at 2 µg/ml in bicarbonate buffer, pH 9.6 at 4°C overnight; wells were blocked for 1 hour at room temperature with 3% w/v non-fat dried milk powder (Sigma Aldrich, # 70166-500G) in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T), washed in PBS-T. Dilutions of patient sera (1 in 50 in 1% Milk PBS-T), were added in duplicate to wells coated with RBD protein and incubated for 2 hours at room temperature.…”

Comparison of dried blood spots and venous blood for the detection of SARS-CoV-2 antibodies in a population of nursing home residents

“…Several other studies in different populations, such as health care workers, key workers, athletes and children have evaluated DBS sampling for SAR-CoV-2 antibody testing (6)(7)(8)(9)(10)(11)(12)(13)(14). In these studies, similar test characteristics were reported with a sensitivity ranging from 89 up to 100% and a specificity of 100% (7,10,11), or, reported a nearly perfect agreement in SARS-CoV-2 antibody detection between DBS samples and paired venous blood samples (6,9,(12)(13)(14). However, some studies were limited by the lack of serum serology as a reference test (7,8) or the rather limited sample size (i.e., <50 positives as assessed by the reference test) (6,7,11,12).…”