Novel bridge multi-species ELISA for detection of SARS-CoV-2 antibodies

Abstract

Considering the course of the current SARS-CoV-2 pandemic, it is important to have serological tests for monitoring humoral immune response against SARS-CoV-2 infection and vaccination. Herein we describe a novel bridge enzyme-linked immunosorbent assay (b-ELISA) for SARS-CoV-2 antibodies detection in human and other species, employing recombinant Spike protein as a unique antigen, which is produced at high scale in insect larvae.

Methods: Eighty two human control sera/plasmas and 169 COVID-19 patients' sera/plasmas, confirmed by rRT-PCR, were analyzed by the b-ELISA assay. In addition, a total of 27 animal sera (5 horses, 13 rats, 2 cats and 7 dogs) were employed in order to evaluate the b-ELISA in other animal species.

Results: Out of the 169 patient samples, 129 were positive for IgG anti-SARS-CoV-2 and 40 were negative when they were tested by ELISA COVIDAR® IgG. When a cut-off value of 5.0 SDs was established, 124 out of the 129 COVID-19 positive samples were also positive by our developed b-ELISA (sensitivity: 96.12%). Moreover, the test was able to evaluate the humoral immune response in animal models and also detected as positive a naturally infected cat and two dogs with symptoms, whose owners had suffered the COVID-19 disease.

Conclusion: The obtained results demonstrate that the method developed herein is versatile, as it is able to detect antibodies against SARS-CoV-2 in different animal species without the need to perform and optimize a new assay for each species.

Keywords: Bridge multi-species ELISA; Immune response; SARS-CoV-2; Spike protein.

Fig. 1

Schematic representation of b-ELISA protocol for detection of anti-S protein antibodies.

Fig. 2

Analysis of the performance of b-ELISA resulting from the study of 82 sera/plasmas from normal control individuals and 129 sera from patients with COVID-19 IgG+ by COVIDAR® IgG ELISA test. (A) Sensitivity curve (o) and specificity (x) as function of the possible cut-off values. The vertical dashed line indicates the cut-off value with the optimized sensitivity and specificity parameters (cut-off = 5.0). (B) ROC curve analysis of b-ELISA, AUC is included.

Fig. 3

SARS-CoV-2 antibody test results obtained by b-ELISA from pre-pandemic samples and samples obtained from seropositive (COVID-19 IgG Pos) and seronegative (COVID-19 IgG Neg) individuals as determined by indirect ELISA (COVIDAR® IgG). Results are expressed as Standard Deviation score (SDs). The cut-off value (SDs > 5.0) is indicated by a dotted line and median SDs for each group is indicated by a solid line (*p < 0.0001).

Fig. 4

Correlation between b-ELISA and Indirect ELISA results. The regression slope was 4.22 ± 0.29 and the correlation coefficient (r²) was 0.52. Dotted lines indicate the cut-off value for each assay (b-ELISA SDs > 5.0; indirect ELISA SDs > 3.0).

Fig. 5

Performance of the b-ELISA applied on animal sera or plasma (5 horses, 13 rats, 2 cats and 7 dogs). Serum samples from experimental immunization (horse and rat) or after RT-PCR-positive throat swab sample (cat) or with symptoms (dog) are indicated by black symbols. Negative control samples are shown by white symbols. Results are expressed as optical density (OD).