Development of a recombinant membrane protein ELISA for analyzing antibody responses against SARS-CoV-2 envelope proteins

Recombinant surface antigens from enveloped viruses are commonly produced without the transmembrane domains to avoid difficulties associated with membrane protein expression and purification. Here, we overcame these challenges by fusing several SARS-CoV-2 envelope proteins to green fluorescent protein and a Strep-tag to facilitate protein detection and purification. This approach simplified the insect cell expression optimization and purification of recombinant prefusion stabilized full-length spike (rS-GFP), membrane (rM-GFP) and envelope (rE-GFP) proteins from the ancestral SARS-CoV-2 strain. We then used the isolated membrane proteins to develop an Enzyme-linked immunosorbent assay that leverages the Strep-tag for proper antigen display and GFP fluorescence for coating consistency. Initial tests with SARS-CoV-2 patient samples from 2020 (n = 68) and healthy adult volunteer samples from 2022-24 (n = 19) revealed a range of rS-GFP reactivity but minimal rM-GFP and rE-GFP reactivity. Approximately 55% of the 2020 patient samples were S-positive compared to ∼85% of the more recent healthy volunteer samples, suggesting the more recent healthy individuals had increased exposure to SARS-CoV-2 antigens by infection or vaccination. Recombinant S (rS) without the green fluorescent protein yielded similar results and detected more S-positive samples than the recombinant receptor binding domain alone. Finally, we confirmed all the S-positive samples neutralized S-pseudotyped particles and found that the enzyme-linked immunosorbent assay data from rS and recombinant receptor binding domain can be combined to identify high and low neutralization titer samples. The results from this proof of concept study suggest a similar strategy could be applied to rapidly develop assays for other enveloped virus surface antigens.