Novel antibodies and assays to ascertain the role of C4 protein variants in Schizophrenia risk

Schizophrenia is a chronic psychiatric disorder affecting over 21 million people worldwide and conferring huge economic/social costs. Schizophrenia is highly heritable (~80%) 1 ; GWAS identified strong genetic association in the major histocompatibility complex, later localised to the C4 gene 2 . C4 is duplicated; C4A and C4B encode C4 proteins that differ by four amino acids in a six amino acid stretch in C4d, conferring distinct chemical reactivities. Schizophrenia risk correlated with C4A copy number 2 . The capacity to measure C4A and C4B in fluids and tissues would aid diagnosis and management but antibodies that distinguish the isoforms are unavailable. Over 100 donors were screened by PCR to identify individuals lacking either C4A or C4B, one C4AQ0 donor was identified and a C4BQ0 donor was gifted by Prof Garred (University of Copenhagen). C4 protein was purified by classical methods and functionality confirmed by addback to methylamine-treated human sera. Novel monoclonal mouse antibodies were generated by hybridoma technology utilising recombinant C4d as the immunogen. Two mAbs, E8 and D5H3, showed preferential binding to C4B. Sandwich ELISAs using E8 or D5H3 as capture and polyclonal anti-C4 detect failed to detect C4 in C4BQ0 plasma with normal levels detected in C4AQ0 plasma, implying specificity for C4B. Furthermore, serum depleted on an E8/D5H3 column retained haemolytic activity and residual C4 in westerns and ELISAs utilising anti-C4 pAbs. We have obtained the necessary standard by elution of C4B from an E8/D5H3 column and quantified plasma C4B using the specific ELISA in a healthy cohort. Work is ongoing to produce C4A-specific mAbs and a C4A ELISA. We have generated C4B-specific mAbs, purified the necessary proteins and identified C4-isotype donors for mAb characterisation and C4 isotype quantification. The completed assays will enable analysis of C4 isoform protein levels in schizophrenia and other contexts. 1. PMID:10813800 1. PMID:26814963 PMID:10813800, PMID:26814963