B-087 Sars-cov-2 Nt Chip test performance: a novel microarray-based enzymatic immunoassay for in vitro identification of neutralizing antibodies against emerging variants

Abstract

Abstract Background The COVID-19 pandemic has created a need for reliable and adaptable diagnostic tools to detect exposure and neutralizing antibodies to emerging SARS-CoV-2 variants. To address this need, we evaluated the performance of the SARS-CoV-2 NT Chip Test, an ELISA-like immunoblot assay that incorporates a customizable panel of purified antigens from the variants of interest. This assay detects antibodies that neutralize the interaction between the receptor-binding domain (RBD) of the Wuhan strain and the Omicron BA1 variant with the cellular ACE-2 receptor and provides a marker of prior infection through the nucleocapsid protein. Methods We analyzed 73 clinical samples from six distinct groups: GROUP 1: Pre-pandemic samples (33); GROUP 2: Samples from children naive to Wuhan and infected with the Omicron variant (10); GROUP 3: Samples from individuals infected only with the original Wuhan strain (10); GROUP 4: Samples from individuals vaccinated and/or infected with the Wuhan strain and the Omicron variant (10); GROUP 5: Samples collected from individuals with 0-2 days after Omicron BA.1 infection (5) and GROUP 6: Samples collected from the same individuals 10 days after Omicron BA.1 infection (5). Evaluation utilized a proprietary non-numerical (qualitative) validation spreadsheet. Performance metrics included sensitivity, specificity, observed agreement (≥90%), and kappa index (≥0.70). Results were compared against the manufacturer's validation using the PRNT viral neutralization assay. Results Our comparative evaluation showed the following results for each parameter: 1) Wuhan neutralizing antibodies (sensitivity and specificity of 92%, agreement of 92%, and kappa index of 0.84); 2) Omicron neutralizing antibodies (sensitivity and specificity of 100%, agreement of 100%, and kappa index of 1.00); 3) Anti-nucleocapsid antibodies (sensitivity of 97%, specificity of 98%, agreement of 97%, and kappa index of 0.94). Three samples from GROUP 1 showed positive results for Wuhan RBD and one for Nucleocapsid, all close to the cutoff value. Four samples from GROUP 2 showed positive results for Wuhan RBD, which may be attributed to maternal antibodies. Three samples from GROUP 3 and three samples from GROUP 4, showed negative results for nucleocapsid, agreeing with the manufacturer's validation. Samples from GROUP 5 and GROUP 6 showed expected results, with Omicron RDB negative in GROUP 5, becoming positive in GROUP 6. Conclusions The test showed excellent performance in the verification of the three parameters in the populations studied, being approved in our laboratory. Due to its ability to incorporate antigens from emerging variants, it enables sensitive tracking of changes in neutralizing antibody profiles over time. The automated and customizable nature of the test provides an important tool for both public health epidemiological studies, monitoring changes in humoral immunity as this virus evolves, and for individual diagnostics, revealing the level of humoral protection of a patient against circulating strains in a timely manner to guide care. Although further studies are still warranted, the assay's ability to map evolving strain-specific serological immunity may offer valuable insights.