B-221 Development of a prototype Bridge ELISA for detection of anti-nephrin autoantibodies

Abstract

Background Anti-nephrin autoantibodies bind at the slit diaphragm of podocytes in the kidney, leading to failure of the glomerular filtration barrier and inducing podocyte dysfunction leading to nephrotic syndrome [1]. Circulating anti-nephrin autoantibodies are common in patients with podocytopathies such as minimal change disease (MCD),primary focal segmental glomerulosclerosis (FSGS), and childhood idiopathic nephrotic syndrome, and their levels correlate with disease activity [2]. The current gold standard for anti-nephrin antibody detection is immunoprecipitation (IP) [2,3]. Conventional ELISAs have been ineffective in detecting these antibodies, possibly because of the low abundance or affinity of these autoantibodies, showing the need for a highly sensitive assay [2, 3]. Here we report on the development of a new prototype Bridge ELISA for the detection of anti-nephrin autoantibodies. The study evaluates the performance of the prototype assay. Methods The detection principle of the prototype Anti-Nephrin Bridge ELISA (Euroimmun) involves capturing bivalent anti-nephrin antibodies between two antigens, one capture and one detection antigen, forming a "bridge". The utilization of both antibody valences helps to increase the specificity that allows to apply serum samples at a significantly lower dilution. This in turn finally is the key to enable the detection of the low-abundance antibodies while still ensuring proper discrimination between anti-nephrin-positive and anti-nephrin-negative samples. The performance of the prototype Anti-Nephrin Bridge ELISA was evaluated using a panel (n=40) comprising 8 anti-nephrin-positive serum samples from MCD patients precharacterized by IP (using the recombinant human nephrin ectodomain [2]), 29 sera from healthy adult blood donors, and 3 anti-nephrin IgG positive control substances. Results were compared with those obtained with an in-house Anti-Nephrin ELISA with a conventional detection principle. From the blood donors, 9 samples were additionally tested for anti-nephrin autoantibodies using IP. Agreement of qualitative results (n=17, 8 patients + 9 blood donors) between ELISAs and IP was calculated using Cohen’s kappa (?). Results The prototype Anti-Nephrin Bridge ELISA detected all 40 samples correctly, which corresponds to an accuracy of 100%. The agreement between qualitative results of IP and the Bridge ELISA were perfect (?=1), but slight (?=0) between IP and the conventional in-house ELISA. All patient sera had higher optical densities when analyzed with the prototype Anti-Nephrin Bridge ELISA than with the conventional Anti-Nephrin ELISA, providing tentative initial evidence for a very high sensitivity of the prototype Anti-Nephrin Bridge ELISA. Conclusion The prototype Anti-Nephrin Bridge ELISA could be a valuable tool for supporting precise and sensitive detection of anti-nephrin autoantibodies. Autoantibody quantification can support treatment monitoring of glomerular diseases, enhance treatment evaluation, and improve kidney-transplantation strategies in FSGS patients [4].