Multiplex assays to determine correlation of binding and neutralizing antibodies following SARS-CoV-2 infection and vaccination

Abstract The COVID-19 pandemic has led to the rapid development of many different therapeutics and vaccines to reduce the burden of the disease. Most vaccines target the highly immunogenic spike (S) protein for generating functional immune responses to block the entry of the virus into host cells. However, the mutations in the S-protein have led to the emergence of many variants that escape the immune response. We developed two multiplex assays using Luminex® xMAP® technology to assess antibody profiles after infection and vaccination; 1) to quantify the binding antibodies against SARS-CoV2 receptor binding protein (RBD), nucleocapsid protein (NP), S1-protein (S1), and Trimer having S1, S2, and extracellular domain protein; 2) a multiplex surrogate virus neutralization antibody assay targeting CoV2 wild type and various mutant viruses. The quantitative binding assay uses the WHO standard for determining the binding antibody unit (BAU)/mL. Surrogate virus neutralization assay measures the inhibition of ACE2 protein binding to wild-type and S protein variants coupled to beads in the presence of immune or convalescent sera. We determined the negative threshold using 81 pre-pandemic serum samples collected during 2017–18. We used 116 samples collected from 45 vaccinated and 15 infected subjects for antibody quantification. Binding antibodies to RBD, S1, and trimer were higher in vaccinated individuals with minimal reactivity to NP, whereas the NP antibody response was higher in the infected individuals. We observed a direct correlation between spike protein binding and neutralizing antibodies using the two assays. We saw the most elevated neutralization antibodies against variants from individuals with breakthrough infections.