Immunoglobulin G genetic variation can confound assessment of antibody levels via altered binding to detection reagents

Abstract Objectives Amino acid variations across more than 30 immunoglobulin (Ig) allotypes may introduce structural changes that influence recognition by anti‐Ig detection reagents, consequently confounding interpretation of antibody responses, particularly in genetically diverse cohorts. Here, we assessed a panel of commercial monoclonal anti‐IgG1 clones for capacity to universally recognise two dominant IgG1 haplotypes (G1m‐1,3 and G1m1,17). Methods Four commercial monoclonal anti‐human IgG1 clones were assessed via ELISAs and multiplex bead‐based assays for their ability to bind G1m‐1,3 and G1m1,17 IgG1 variants. Detection antibodies were validated against monoclonal IgG1 allotype standards and tested for capacity to recognise antigen‐specific plasma IgG1 from G1m‐1,3 and G1m1,17 homozygous and heterozygous SARS‐CoV‐2 BNT162b2 vaccinated (n = 28) and COVID‐19 convalescent (n = 44) individuals. An Fc‐specific pan‐IgG detection antibody corroborated differences between hinge‐ and Fc‐specific anti‐IgG1 responses. Results Hinge‐specific anti‐IgG1 clone 4E3 preferentially bound G1m1,17 compared to G1m‐1,3 IgG1. Consequently, SARS‐CoV‐2 Spike‐specific IgG1 levels detected in G1m1,17/G1m1,17 BNT162b2 vaccinees appeared 9‐ to 17‐fold higher than in G1m‐1,3/G1m‐1,3 vaccinees. Fc‐specific IgG1 and pan‐IgG detection antibodies equivalently bound G1m‐1,3 and G1m1,17 IgG1 variants, and detected comparable Spike‐specific IgG1 levels between haplotypes. IgG1 responses against other human coronaviruses and influenza were similarly poorly detected by 4E3 anti‐IgG1 in G1m‐1,3/G1m‐1,3 subjects. Conclusion Anti‐IgG1 clone 4E3 confounds assessment of antibody responses in clinical cohorts owing to bias towards detection of G1m1,17 IgG1 variants. Validation of anti‐Ig clones should include evaluation of binding to relevant antibody variants, particularly as the role of immunogenetics upon humoral immunity is increasingly explored in diverse populations.