A Quantitative ELISA to Detect Anti-SARS-CoV-2 Spike IgG Antibodies in Infected Patients and Vaccinated Individuals

Abstract

There is an ongoing need for high-precision serological assays for the quantitation of anti-SARS-CoV-2 antibodies. Here, a trimeric SARS-CoV-2 spike (S) protein was used to develop an ELISA to quantify specific IgG antibodies present in serum, plasma, and dried blood spots (DBS) collected from infected patients or vaccine recipients. The quantitative S-ELISA was calibrated with international anti-SARS-CoV-2 immunoglobulin standards to provide test results in binding antibody units per mL (BAU/mL). The assay showed excellent linearity, precision, and accuracy. A sensitivity of 100% was shown for samples collected from 54 patients with confirmed SARS-CoV-2 infection more than 14 days after symptom onset or disease confirmation by RT-PCR and 58 vaccine recipients more than 14 days after vaccination. The assay specificity was 98.3%. Furthermore, antibody responses were measured in follow-up samples from vaccine recipients and infected patients. Most mRNA vaccine recipients had a similar response, with antibody generation starting 2-3 weeks after the first vaccination and maintaining positive for at least six months after a second vaccination. For most infected patients, the antibody titers increased during the second week after PCR confirmation. This S-ELISA can be used to quantify the seroprevalence of SARS-CoV-2 in the population exposed to the virus or vaccinated.

Keywords: COVID-19; ELISA; SARS-CoV-2; serological test; spike protein; vaccination.

Figure 1

Accuracy of in-house SARS-CoV-2 S-IgG ELISA (blue) determined (A) in comparison to Euroimmun, Anti-SARS-CoV-2-QuantiVac-ELISA (red) using NIBSC 20/162 in five replicates and four dilutions (1:1000, 1:2000, 1:3000, 1:4000); standard deviations are shown as error bars (blue) and (B) using a standard with known concentration (NIBSC 21/234, 832 BAU/mL) analyzed in twelve replicates on four different days. The grey area indicates the anti-S titer of NIBSC 21/234 of 746–929 BAU/mL (95% CI).

Figure 2

Anti-S antibody titers (BAU/mL) of samples from SARS-CoV-2-infected patients, COVID-19 vaccine recipients, and negative controls. (A) A total of 54 serum samples from persons infected with SARS-CoV-2 collected at least 14 days after a positive PCR-test, 58 samples collected from vaccinated donors 14 days or longer after first vaccine (Pfizer-BioNTech, Moderna, and AstraZeneca), and 173 serum samples collected before 2018. (B) Serum samples (36) collected from persons infected with SARS-CoV-2 (>14 days after positive PCR test) were grouped by severity of symptoms based on their WHO score (2 to 4). Results were converted to binding antibody units (BAU/mL) using the absorbances recorded at 450 nm of six calibrators. The highest positive (red) have a titer of >4280 BAU/mL because the analysis was performed up to this value. The cut-off was 22 BAU/mL for the in-house ELISA. The grey zone above the cut-off (22–44 BAU/mL) indicates a borderline range.

Figure 3

Equivalence of sample matrices. (A) Comparison of serum (black) and EDTA plasma (red) samples from two patients collected >14 days after symptom onset and two negative patients. (B) Serum (black), EDTA plasma (green), citrate plasma (blue), and NaF plasma (orange) were spiked with anti-S1 antibody. (C) Correlations between 32 positive serum and plasma samples collected from vaccinated donors (R = 0.9810). (D) Correlations between 32 positive serum and capillary blood DBS collected from vaccinated donors (R = 0.9494). (E) Correlations between in-house ELISA to EUROIMMUN quantitative ELISA using 16 whole-blood DBS samples collected from vaccinated donors (R = 0.9733).

Figure 4

Time course of antibody titers (BAU/mL) determined following four different types of COVID-19 vaccination. Serum or DBS samples collected from donors vaccinated with (A) Pfizer-BioNTech (n = 13), (B) Moderna (n = 13), (C) AstraZeneca (n = 4), and (D) Johnson & Johnson (n = 4) vaccines. Blood samples were collected from five to seven time points for each vaccine before or after vaccination. Results were converted to binding antibody units (BAU/mL) using the absorbances recorded at 450 nm of six calibrators. The cutoff was 22 BAU/mL for the in-house ELISA. The grey zone above the cutoff (22–44 BAU/mL) indicates a borderline range.

Figure 5

Time course of antibody titers (BAU/mL) during SARS-CoV-2 infection. (A) Serum samples collected from nine unvaccinated individual patients. The second week after PCR diagnosis is highlighted in orange. (B) DBS samples collected from a patient who was infected six months after the second vaccinations (Pfizer-BioNTech). The grey zone above the cutoff (22 BAU/mL) indicates the range of 22–44 BAU/mL, which is considered as positive.