An indirect ELISA for detecting anti-SARS-CoV-2 antibodies in human sera using a baculovirus-expressed recombinant nucleocapsid antigen

• Published in: Biologicals Vol. 86; p. 101769
• Main Authors: Fogaça, Matheus Bernardes Torres, Crispim, Gildemar José Bezerra, Saavedra, Djairo Pastor, Lopes-Luz, Leonardo, da Silva, Leonardo Assis, de Camargo, Brenda Rabello, Guimarães, Rafael Alves, Nagata, Tatsuya, Ribeiro, Bergmann Morais, Bührer-Sékula, Samira
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-05-2024
• Subjects: Animals; Antibodies, Viral - blood; Antibodies, Viral - immunology; Antigens, Viral - genetics; Antigens, Viral - immunology; Baculoviridae - genetics; Baculovirus; Coronavirus Nucleocapsid Proteins - genetics; Coronavirus Nucleocapsid Proteins - immunology; COVID-19; COVID-19 - blood; COVID-19 - diagnosis; COVID-19 - immunology; COVID-19 Serological Testing - methods; ELISA; Enzyme-Linked Immunosorbent Assay - methods; Humans; Immunoglobulin G - blood; Immunoglobulin G - immunology; Nucleocapsid Proteins - genetics; Nucleocapsid Proteins - immunology; Phosphoproteins - genetics; Phosphoproteins - immunology; Recombinant Proteins - genetics; Recombinant Proteins - immunology; SARS-CoV-2; SARS-CoV-2 - genetics; SARS-CoV-2 - immunology; Sensitivity and Specificity; Sf9 Cells; COVID-19; SARS-CoV-2; Baculovirus; ELISA
• ISSN: 1045-1056; 1095-8320
• DOI: 10.1016/j.biologicals.2024.101769

This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.