Spike vs nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens

Abstract

Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many "antigen" detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false positive results. In this work, we developed a novel alternative spike-based antigen assay, comprising four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results. Schematic representation of sample collection and analysis. The figure was created using BioRender.com.

Keywords: Antigen; Nasopharyngeal swab specimens; Nucleocapsid; SARS-CoV-2; Spike; TRF-ELISA.

Schematic representation of sample collection and analysis. The figure was created using BioRender.com

Fig. 1

Characterization of the antibodies incorporated in the SARS-CoV-2 TRF-based S-assay. a Concomitant binding of anti-SARS-CoV-2 antibodies. The ability of the assay’s antibodies to bind simultaneously to SARS-CoV-2 was tested using the Octet Red biolayer interferometry (BLI) system. Biotinylated BL11 was immobilized to a streptavidin sensor and interacted with the spike’s S1 subunit. The complex was then immersed in a well containing the indicated antibody (pointed by an arrow), washed again (dashed vertical line), and immersed with the next antibody. b Schematic representation of the interaction of the assay’s antibodies with the spike protein. c Affinity of the anti RBD/NTD antibodies as determined by BLI analysis

Fig. 2

Specificity and sensitivity of TRF-based ELISA assays. Recombinant antigens a spike, b nucleocapsid, or c SARS-CoV-2 virus were diluted in PBS (0.5–1000 ng/ml or 2 × 10³–1 × 10⁶ pfu/ml) and analyzed with the NC-assay (red) or S-assay (blue). Signal-to-noise (S/N) ratios were calculated as described in “Materials and methods.” The dotted line represents the assay’s LOD. Nonlinear regression (performed using GraphPad Prism 6) yielded R square values of 0.99 for the curves presented in panels a, b, and c. d Schematic representation of TRF ELISA-based assays

Fig. 3

S/N ratios vs Ct values. a S-assay. b NC-assay. The assay’s LOD is indicated by the dashed line. Ct value >41 represents negative qRT-PCR samples. The gray area, enclosed by the dotted line, represents the difference in sensitivity required for enhanced specificity

Fig. 4

Comparison of viral loads (Ct values<25) among positive (P) and false negative (FN) results. a For the S- (blue) vs NC- (red) assays (for all qRT-PCR positive samples). For qRT-PCR positive samples from emergency departments (ED) (cyan) and nursing homes (homes) (pink) utilizing b S-assay; c NC-assay. Statistical analysis was performed using GraphPad Prism 6, applying one-way ANOVA test followed by Kruskal-Wallis multiple comparison tests. ns, not significant; *p < 0.05; ***p < 0.001

Fig. 5

Logic flowchart for possible applications of immune-detection tests. a For screening of symptomatic patients. b For routine screening of healthy/asymptomatic individuals