Abstract: TH-PO0439
Cell-Based Assays Enable Detection and Subclass Characterization of Nephrin Autoantibodies
Session Information
- Glomerular Diseases: Autoimmune Diseases
October 22, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Maier, Nastassia, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Hengel, Felicitas E., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Seifert, Larissa, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Schaffrath, Alessa Zoe, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Huang, Ming, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Berlin, Hannes, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Dehde, Silke, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Riecken, Kristoffer, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Fehse, Boris, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Huber, Tobias B., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Tomas, Nicola M., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
Background
Recent studies identified anti-nephrin antibodies (a-NA) in patients with primary podocytopathies as the key factor of podocyte injury. However, detection of these antibodies is assay- and conformation-dependent, as evidenced, for example, by failure of conventional ELISA techniques to yield reproducible results. We therefore aimed to develop native, cell-based assays for the detection and molecular characterization of a-NA.
Methods
11 a-NA-positive and 10 a-NA-negative patients with MCD and active nephrotic syndrome in comparison to 11 healthy controls, were analyzed with novel cell-based detection methods using immortalized, nephrin-overexpressing human podocytes. Stable overexpression of human nephrin was achieved by lentiviral transduction. Podocytes were incubated under native conditions with patient serum or purified IgG, and then staining for hIgG and hIgG subclasses was performed. Cells were analyzed using flow cytometry (FC) and immunocytochemistry (ICC).
Results
FC-based detection of a-NA antibodies in 11 a-NA-positive and 10 a-NA-negative nephrotic syndrome patients, as well as 11 healthy controls, was in high accordance with gold standard a-NA detection by immunoprecipitation (IP). Serum and purified IgG from a-NA positive patients (n=4) were further analyzed in ICC, showing a-NA-induced nephrin clustering and internalization of both nephrin and patient-derived hIgG. In contrast, serum or purified IgG from healthy donors (n=4) did not show specific binding and co-localization with nephrin. Larger cohort analysis and IgG subclass characterization are ongoing.
Conclusion
Newly developed cell-based a-NA detection assays show comparable sensitivity and specificity to the gold standard IP-based detection. In addition, the new methods offer several advantages, including reduced serum volume, time efficiency, simultaneous detection and quantification of antibodies, direct antibody visualization, and the ability to analyze antibody characteristics such as IgG subclass.
Funding
- Government Support – Non-U.S.