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

Autor: Mladenovic Stokanic M; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Simovic A; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Jovanovic V; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Radomirovic M; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Udovicki B; Department of Food Safety and Quality Management, Faculty of Agriculture, University of Belgrade, Nemanjina 6, Zemun, 11080 Belgrade, Serbia., Krstic Ristivojevic M; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Djukic T; Institute of Medical Chemistry, Faculty of Medicine, University of Belgrade, Višegradska 26, 11000 Belgrade, Serbia., Vasovic T; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Acimovic J; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Sabljic L; Institute for the Application of Nuclear Energy-INEP, University of Belgrade, Banatska 31b, Zemun, 11080 Belgrade, Serbia., Lukic I; Institute of Virology, Vaccines, and Sera-TORLAK, Vojvode Stepe 458, 11152 Belgrade, Serbia., Kovacevic A; Institute of Virology, Vaccines, and Sera-TORLAK, Vojvode Stepe 458, 11152 Belgrade, Serbia., Cujic D; Institute for the Application of Nuclear Energy-INEP, University of Belgrade, Banatska 31b, Zemun, 11080 Belgrade, Serbia., Gnjatovic M; Institute for the Application of Nuclear Energy-INEP, University of Belgrade, Banatska 31b, Zemun, 11080 Belgrade, Serbia., Smiljanic K; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Stojadinovic M; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Radosavljevic J; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Stanic-Vucinic D; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia., Stojanovic M; Department of Molecular Biology, Institute for Biological Research 'Siniša Stanković', University of Belgrade, 142 Despot Stefan Blvd., 11000 Belgrade, Serbia., Rajkovic A; Department of Food Safety and Quality Management, Faculty of Agriculture, University of Belgrade, Nemanjina 6, Zemun, 11080 Belgrade, Serbia.; Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, geb. A, B-9000 Ghent, Belgium., Cirkovic Velickovic T; Centre of Excellence for Molecular Food Sciences, Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia.; Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, geb. A, B-9000 Ghent, Belgium.; Serbian Academy of Sciences and Arts, Kneza Mihaila 35, 11000 Belgrade, Serbia.; Global Campus, Ghent University, 119-5 Songdomunwha-ro, Yeonsu-gu, Incheon 21985, Republic of Korea.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2023 Dec 26; Vol. 25 (1). Date of Electronic Publication: 2023 Dec 26.
DOI: 10.3390/ijms25010333
Abstrakt: 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.
Databáze: MEDLINE
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