Automated chemiluminescence immunoassay for the detection of anti-nephrin autoantibodies.

INTRODUCTION: Circulating anti-nephrin autoantibodies are being increasingly recognized in autoimmune podocytopathies, particularly minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). However, circulating anti-nephrin reactivity is often low, and currently available assays differ substantially in design, antigen production, and analytical performance. This methodological heterogeneity highlights the need for standardized and quantitative assays for clinical application. METHODS: Recombinant human nephrin ectodomain-coated magnetic microparticles were used to establish an automated chemiluminescence immunoassay (CLIA). Serum anti-nephrin IgG was measured in 121 children with biopsy-confirmed MCD (n=103) or primary FSGS (n=18), together with disease controls (genetically confirmed MCD/FSGS, IgA nephropathy, IgA vasculitis with nephritis, lupus nephritis, and ANCA-associated glomerulonephritis) and healthy individuals. Samples were tested in parallel by immunoprecipitation-Western blot (IP-WB). Antigen inhibition assays and cohort-based clinical analyses were performed. RESULTS: The CLIA met predefined analytical performance criteria and showed substantial agreement with IP-WB, with an overall concordance of 86.8% and Cohen's kappa coefficient of 0.714. CLIA detected anti-nephrin IgG more frequently than IP-WB in patients with MCD or FSGS (41.3% vs. 28.1%), including additional low-level positive samples. Antigen inhibition assays showed dose-dependent inhibition in CLIA-positive samples. Among patients with baseline nephrotic-range proteinuria, antibody positivity was associated with greater proteinuria, lower serum albumin, and steroid-sensitive nephrotic syndrome. Patients with elevated anti-nephrin IgG levels had significantly shorter relapse-free survival. During complete remission, anti-nephrin IgG positivity was associated with relapse within three months, with relapse risk increasing according to antibody level. CONCLUSIONS: Together, our automated CLIA may help move anti-nephrin IgG testing from specialized research workflows toward standardized clinical application, supporting future use in disease stratification and longitudinal monitoring.