Quantitative serological detection of NELL1 autoantibodies in membranous nephropathy

Membranous nephropathy (MN) comprises distinct subtypes driven by autoantibodies targeting podocyte-associated proteins, one of which is neural EGFL-like 1 (NELL1). Currently, the diagnosis of NELL1-associated MN relies mainly on mass spectrometry or immunostaining of kidney biopsies. Serologic testing for anti-NELL1 antibodies is not yet commercially available, but research assays including Western blotting, indirect immunofluorescence, and ELISA have recently been developed. However, these are currently performed only in research settings, and ELISA may not be sufficiently specific for primary diagnostic purposes. High-throughput immunoassays are needed for the diagnosis and monitoring of NELL1 autoantibody-positive patients with MN.

Here, we developed a luciferase immunoprecipitation systems (LIPS) immunoassay to detect circulating NELL1 autoantibodies. After identifying the optimal antigenic region of NELL1, we evaluated the assay's diagnostic performance using serum samples derived from a biopsy-proven NELL1⁺ validation cohort. We then assessed NELL1 seropositivity in an independent NIH clinical evaluation cohort and examined antibody dynamics in longitudinal samples from selected positive cases.

Initial assay development using known NELL1 positive sera demonstrated an N-terminal fragment of NELL1 protein (amino acids 1-550) fused to Gaussia luciferase yielded superior diagnostic performance compared with full-length NELL1 (810 amino acids). In a validation cohort consisting of sera from 20 biopsy-proven NELL1-positive and 20 PLA2R⁺/NELL1-negative patients, this LIPS assay showed 90% sensitivity and 100% specificity and showed agreement with ELISA but with better specificity. Further testing of 63 MN cases and 20 disease controls from the NIH cohort identified six NELL1-positive MN cases. Longitudinal analysis of three NELL1 seropositive cases revealed that autoantibody levels tracked proteinuria, suggesting that monitoring antibody titers may be useful in clinical management like that with PLA2R antibodies.

Our NELL1 LIPS assay provides a noninvasive tool for diagnosing NELL1-associated MN, characterizing clinical subsets, and monitoring therapeutic response.