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- Title
Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species.
- Authors
Mladenovic Stokanic, Maja; Simovic, Ana; Jovanovic, Vesna; Radomirovic, Mirjana; Udovicki, Bozidar; Krstic Ristivojevic, Maja; Djukic, Teodora; Vasovic, Tamara; Acimovic, Jelena; Sabljic, Ljiljana; Lukic, Ivana; Kovacevic, Ana; Cujic, Danica; Gnjatovic, Marija; Smiljanic, Katarina; Stojadinovic, Marija; Radosavljevic, Jelena; Stanic-Vucinic, Dragana; Stojanovic, Marijana; Rajkovic, Andreja
- 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.
- Subjects
WUHAN (China); RECOMBINANT proteins; SARS-CoV-2; ANTIGEN analysis; TRITON X-100; SARS-CoV-2 Omicron variant; MONOCLONAL antibodies; IMMUNOGLOBULINS
- Publication
International Journal of Molecular Sciences, 2024, Vol 25, Issue 1, p333
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms25010333