A novel highly specific biotinylated MAC-ELISA for detection of anti-SARS-CoV-2 nucleocapsid antigen IgM antibodies during the acute phase of COVID-19

• Published in: Brazilian journal of microbiology Vol. 54; no. 4; pp. 2893 - 2901
• Main Authors: Lopes-Luz, Leonardo, Fogaça, Matheus Bernardes Torres, Bentivoglio-Silva, Brenda Garcia, Saavedra, Djairo Pastor, Alves, Luana Michele, Franca, Luísa Valério, Crispim, Gildemar José Bezerra, de Andrade, Ikaro Alves, Ribeiro, Bergmann Morais, Nagata, Tatsuya, Bührer-Sékula, Samira
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.12.2023; Springer Nature B.V
• Subjects: Antibodies; Antigens; Autoantibodies; Biomedical and Life Sciences; Clinical Microbiology - Short Communication; COVID-19; Cross-reactivity; Enzyme-linked immunosorbent assay; Food Microbiology; Immunoglobulin M; Life Sciences; Medical Microbiology; Microbial Ecology; Microbial Genetics and Genomics; Microbiology; Mycology; Nucleocapsids; Pandemics; Performance evaluation; Peroxidase; Rheumatoid factor; Sensitivity analysis; Severe acute respiratory syndrome coronavirus 2; Signs and symptoms; Viral diseases; Viremia; Biotinylated antigen; Rheumatoid factor; IgM; Streptavidin; MAC-ELISA
• ISSN: 1517-8382; 1678-4405; 1678-4405
• DOI: 10.1007/s42770-023-01160-6

The gold standard for diagnosing COVID-19 in the acute phase is RT-qPCR. However, this molecular technique can yield false-negative results when nasopharyngeal swab collection is not conducted during viremia. To mitigate this challenge, the enzyme-linked immunosorbent assay (ELISA) identifies anti-SARS-CoV-2 IgM antibodies in the initial weeks after symptom onset, facilitating early COVID-19 diagnosis. This study introduces a novel and highly specific IgM antibody capture ELISA (MAC-ELISA), which utilizes biotinylated recombinant SARS-CoV-2 nucleocapsid (N) antigen produced in plants. Our biotinylated approach streamlines the procedure by eliminating the requirement for an anti-N-conjugated antibody, circumventing the need for peroxidase-labeled antigens, and preventing cross-reactivity with IgM autoantibodies such as rheumatoid factor. Performance evaluation of the assay involved assessing sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy using 682 RT-qPCR-positive samples, categorized by weeks relative to symptoms onset. Negative controls included 205 pre-pandemic serum samples and 46 serum samples from patients diagnosed with other diseases. Based on a cut-off of 0.087 and ROC curve analysis, the highest sensitivity of 81.2% was observed in the 8–14 days post-symptom (dps) group (2nd week), followed by sensitivities of 73.8% and 68.37% for the 1–7 dps (1st week) and 15-21 dps groups (3rd week), respectively. Specificity was consistently 100% across all groups. This newly developed biotinylated N-MAC-ELISA offers a more streamlined and cost-effective alternative to molecular diagnostics. It enables simultaneous testing of multiple samples and effectively identifies individuals with false-negative results.