Determinants of C1q binding in the single antigen bead assay

Background. A modified single antigen bead (SAB) assay measuring C1q binding to human leukocyte antigen antibodies has recently been introduced. The aim of this study was to investigate the determinants of C1q binding on SAB. Methods. Sera from 73 sensitized patients were analyzed by the generic IgG_pan as well as IgG subclass specific SAB assays and correlated with the standard and an anti-human globulin (AHG) enhanced C1q test. Results. Among 2,665 SABs with positive IgG_pan results (mean fluorescence intensity [MFI]<500), strong complementbinding IgG1 and IgG3 subclasses accounted for a median of 99% (interquartile range, 76%Y100%) of the total IgG amount. IgG_pan MFI alone showed a very strong association with standard C1q positivity (r²=0.72), which was superior to a model including all IgG subclass MFI (r²=0.62). Combining all IgG subclass MFI and IgG_pan MFI only marginally improved the prediction of standard C1q positivity compared with IgG_pan MFI alone ($r²=0.02). IgG_pan MFI greater than 14,154 predicted standard C1q positivity, with 92% sensitivity and 96% specificity. Notably, 1,840 (93%) of the 1,974 C1q-negative SABs contained human leukocyte antigen antibodies with strong complement-binding IgG1 and IgG3 subclasses. AntiYhuman globulin significantly enhanced the signal in the C1q assay, but the association of AHG C1q positivity with IgG_pan MFI was less strong (r²=0.51). Conclusion. C1q binding on SAB is strongly associated with IgG_pan MFI. IgG subclass information only marginally improves prediction of C1q binding likely because complement-binding IgG1 and IgG³ subclasses dominate regarding frequency and relative amounts. A negative C1q assay result does not indicate the absence of strong complement-binding IgG subclasses.