Assessing the Impact of SARS-CoV-2 Spike Mutations on Antibody Binding: A Comparative Assessment of the Wuhan and JN.1 Variants' Full-Length Spikes in a Multiplex Luminex Assay

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to evolve, with mutations leading to the emergence of new variants. JN.1, a subvariant of omicron BA.2.86, has demonstrated marked immune escape and is now included in updated vaccine formulations. While reduced sensitivity has been reported for antibody assays using ancestral spike protein subunits to detect omicron-induced responses, the performance of full-length spike-based assays against omicron sublineages remains unclear. We aimed to compare the sensitivity of ELISA and Luminex assays using full-length spike proteins from the ancestral Wuhan strain and the JN.1 variant.

Methods: Wuhan and JN.1 full-length spike protein constructs were designed and expressed in Expi293F mammalian cells. In-house ELISAs based on previously validated protocols were used to measure anti-spike IgG levels. Additionally, a Luminex-based assay for anti-spike antibody detection was developed and validated. Both assays were applied to the following sample groups: pre-pandemic samples (designated "gold standard negatives"); PCR confirmed 2020 positives ("gold standard wildtype positives"); PCR confirmed 2024 positives ("gold standard omicron positives"); 2022 vaccinated individuals with verbal confirmed infection ("gold standard hybrid positives"); and 2024 household samples ("unknowns").

Results: Wuhan spike protein showed a sensitivity of 100% (95% CI: 0.88-1.0) in detecting omicron-specific antibodies using gold standard omicron positives with JN.1 spike protein as a reference assay. Overall, across all samples, in ELISA, the Wuhan antigen had a sensitivity of 0.93 (95% CI: 0.89-0.95) and a specificity of 0.98 (95% CI: 0.94-0.99). The JN.1 antigen showed a sensitivity of 0.91 (95% CI: 0.87-0.94) and a specificity of 0.97 (95% CI: 0.93-0.99). In Luminex, sensitivity was 0.95 (95% CI: 0.91-0.97) for Wuhan and 0.94 (95% CI: 0.91-0.96) for JN.1. Specificity for both antigens in Luminex was 0.98 (95% CI: 0.94-0.99).

Conclusions: Both ELISA and Luminex assays showed comparable sensitivity and specificity for both Wuhan and JN.1 antigens, indicating that mutations in the JN.1 variant do not significantly impact assay performance. This suggests preserved antigenic recognition across variants.