Development and Optimization of In-house ELISA for Detection of Human IgG Antibody to SARS-CoV-2 Full Length Spike Protein

Abstract

The ongoing coronavirus disease 19 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to human health. Despite this, many affected countries are now in the process of gradual lifting of COVID-19 restrictions that were initially implemented in response to the pandemic. The success of the so-called "exit strategy" requires continued surveillance of virus circulation in the community and evaluation of the prevalence of protective immunity among population. Serology tests are valuable tools for these purposes. Herein, SARS-CoV-2 full-length spike (S) recombinant protein was utilized to develop and optimize an indirect enzyme-linked immunoassay (ELISA) that enables a reliable detection of virus-specific IgG antibody in human sera. Importantly, the performance of this assay was evaluated utilizing micro-neutralization (MN) assay as a reference test. Our developed ELISA offers 100% sensitivity, 98.4% specificity, 98.8% agreement, and high overall accuracy. Moreover, the optical density (OD) values of positive samples significantly correlated with their MN titers. The assay specifically detects human IgG antibodies directed against SARS-CoV-2, but not those to Middle East respiratory syndrome coronavirus (MERS-CoV) or human coronavirus HKU1 (HCoV-HKU1). The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications.

Keywords: COVID-19; ELISA; SARS-CoV-2; immunoassay.

Figure 1

Optimization of an indirect ELISA utilizing SARS-CoV-2 full-length S (S1 + S2) recombinant protein. (A) and (B) Plates were coated overnight at 4 ℃ with different concentrations of either SARS-CoV-2 full-length S (S1 + S2) extracellular domain with a polyhistidine tag (ECD-His) recombinant protein, or bovine serum albumin (BSA) as a coating control. Following three washes with PBS containing 0.1% Tween 20 (PBST), positive (green) and negative (red) samples based on micro-neutralization assay were serially diluted (as indicated) and added. PBST was used as blank (empty black circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with conjugate (peroxidase-labelled anti-human IgG secondary antibody) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD₄₅₀). (C) Plates were coated with 100 ng/well of viral antigen, samples were used at 1:100 dilution, and conjugate dilutions ranged from 1:1000 to 1:128,000. The protocol was performed as described above.

Figure 2

The cut-off value of the developed SARS-CoV-2 full-length S (S1 + S2)-based indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBS containing 0.1% Tween 20 (PBST), positive (green) and negative (red) samples based on micro-neutralization assay were diluted 1:100 and added. PBST was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD₄₅₀). The actual values for each sample is shown. Dashed lines represent the cut-off value (Mean + 3 x standard deviation).

Figure 3

Evaluation of the developed SARS-CoV-2 full-length S (S1 + S2)-based indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBST, positive (green) and negative (red) samples based on micro-neutralization assay were diluted 1:100 and added. PBS containing 0.1% Tween 20 (PBST) was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST was performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD₄₅₀). Dashed lines represent the cut-off value (Mean + 3 x standard deviation). (A) shows the actual values for each sample, and (B) demonstrates the correlation between OD₄₅₀ readings and micro-neutralization (MN) titer. Boxes show the 25th–75th percentile range, the black line represents median, and whiskers are the minimum and maximum values.

Figure 4

Assessment of the cross-reactivity of the developed SARS-CoV-2 S1 + S2 indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBST, Serum samples containing anti-SARS-CoV-2 antibodies (green), anti-Middle East respiratory syndrome coronavirus (MERS-CoV) antibodies (purple), and anti-human coronavirus HKU1 (HCoV HKU1) antibodies (blue) were diluted 1:100 and added. Serum from healthy donors were used as a negative control (red). PBS containing 0.1% Tween 20 (PBST) was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD₄₅₀).

Figure 5

Receiver operating characteristics (ROC) analysis of the developed ELISA. ROC analysis was performed for positive samples based on results obtained from micro-neutralization assay. (A) shows the distribution of data utilized for ROS analysis, (B) demonstrates ROC curve, and (C) presents a range of cut-off values and their associated sensitivity and specificity with 95% confidence interval (CI) as obtained from ROC analysis.