Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species

Abstract

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52-48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19-100.00%) and sensitivity was 52.94% (95% CI, 35.13-70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67-65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

Keywords: COVID-19 diagnosis; ELISA; antigen test; nucleocapsid protein; polyclonal antibodies.

Figure 1

Western blot analysis of N protein probed with purified mouse and rabbit antiserum, followed by incubation with either biotinylated goat anti-mouse IgG antibodies and streptavidin conjugated with alkaline phosphatase (AP) or goat anti-rabbit IgG-AP conjugate (+). Secondary antibody controls (−) were also included.

Figure 2

Representative calibration curve for quantification of N protein by developed prototype ELISA.

Figure 3

N protein quantification in five rtRT-PCR-positive samples after different chemical and thermal sample pretreatments: no treatment (without Triton X-100, 1 h at room temperature), chemical treatment (with Triton X-100, 1 h at room temperature); thermal treatment (without Triton X-100, 1 h at 56 °C) and combined chemical and thermal treatment (with Triton X-100, 1 h at 56 °C). Statistical significance (one-way ANOVA with Tukey’s multiple comparisons at p < 0.05) was determined for the effects of different treatments within one sample, and the same letter annotations mean no statistically significant difference while different letter annotations represent statistically significant results.

Figure 4

Distribution of N protein concentration in rtRT-PCR-positive samples plotted against Ct values. Lines: Ct = 40, Ct = 30, Ct = 25, and Ct = 20—blue; LOD for N protein concentration (1.00 ng/mL)—red. (A) all rtRT-PCR-positive samples with Triton X-100 and (B) without Triton X-100; (C) rtRT-PCR-positive samples mostly containing Wuhan-similar variants with Triton X-100 and (D) without Triton X-100; (E) rtRT-PCR-positive samples mostly containing Wuhan-distant variants with Triton X-100 and (F) without Triton X-100 t.