Development of autoimmune antibodies against human phospholipase A2 receptor and the application as diagnostic criteria for primary membranous nephropathy.

Abstract

Introduction: Membranous nephropathy (MN) is a severe chronic kidney disease (CKD) with ~ 30% of patients progressing to end-stage renal disease within 10 years. Phospholipase A2 receptor (PLA2R) is one of the major target antigens of MN. Despite the importance of PLA2R antibodies in the pathogenesis, diagnosis, and prognosis of MN, anti-human PLA2R monoclonal antibodies have not been obtained. This lack hinders the measurement of chemical PLA2R antibody titers, which are critical for drug development.

Purpose: The purpose of this study is to obtain standard anti-human PLA2R monoclonal antibodies by creating a phage antibody library to screen for antibodies.

Materials and methods: Our study used CHO cells expressing the human PLA2R CysR-FnII-CTLD1 structural domain (CC1h) and obtained candidate antibodies by screening a human phage display library. These candidate antibodies were expressed in CHO cells, and their binding affinity for CC1h was determined by enzyme-linked immunosorbent assay (ELISA) and biological layer interferometry (BLI). In addition, we calibrated the representative antibody names of A13 and A13SP to obtain transforming factors using a commercial PLA2R antibody ELISA kit.

Results: 43 high-affinity candidate antibodies were obtained by screening human phage display libraries, and 9 groups of IgG antibodies and their IgG4SP subtype antibodies showed high affinity by CHO cell expression, ELISA, and BLI experiments, and we screened 1 group of antibodies with high protein expression levels and high affinity names of A13 and A13SP subtypes from these 9 antibodies. A few of the remaining 8 groups of antibodies and subtypes also showed high protein expression levels and high affinity, and these antibodies can also be used as alternative antibodies. In addition, we calibrated the representative antibody A13 using a commercial PLA2R antibody ELISA kit and obtained an average conversion factor of 1 RU = 70 ng, and we found that the standard curves of the A13 and A13SP subtype antibody groups exhibited a high concordance with that of the commercial PLA2R antibody ELISA kit, which was higher than that of the other alternative antibodies, and therefore this also indicates that the A13 and A13SP subtype antibodies are the most preferred antibody groups in our antibody library.

Conclusion: The A13 and A13SP subtype antibodies showed high protein expression levels and high antibody affinity, and the standard curves showed high concordance with the commercial kits, and this conclusion suggests that the A13 and A13SP subtype antibodies can be used as standard antibodies in the ELISA kits for the quantitative detection of human PLA2R antibodies.