Abstract: TH-PO1079
Development of a Bead-Based Assay for Detection of PDZ-Domain-Specific Anti-HTRA1 Autoantibodies in HTRA1-Associated Membranous Nephropathy
Session Information
- Pathology and Lab Medicine
October 22, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Pathology and Lab Medicine
- 1700 Pathology and Lab Medicine
Authors
- Al-Rabadi, Laith, University of Utah Health, Salt Lake City, Utah, United States
- Takayama, Suguru, University of Utah Health, Salt Lake City, Utah, United States
- Abuelsamen, Tamer Rami, University of Utah Health, Salt Lake City, Utah, United States
- Sameh, Ahmed, University of Utah Health, Salt Lake City, Utah, United States
- Hoxha, Elion, Universitatsklinikum Wurzburg, Würzburg, BY, Germany
- Beck, Laurence H., Boston University, Boston, Massachusetts, United States
- Borza, Dorin-Bogdan, Meharry Medical College, Nashville, Tennessee, United States
Background
HTRA1-associated membranous nephropathy (HTRA1-MN) is an autoimmune glomerular disease in which circulating antibodies target HTRA1. Epitope mapping suggests that the PDZ domain is a major antigenic region, but assays to measure PDZ-specific antibodies are lacking. We developed a bead-based assay using recombinant HTRA1 PDZ-coupled beads to measure serum reactivity and assess specificity by soluble antigen competition
Methods
Recombinant HTRA1 PDZ was conjugated to fluorescent beads and incubated with HTRA1-MN and control sera. Bound human IgG was measured by flow cytometry-based mean fluorescence intensity (MFI), reported as raw and arcsinh-transformed MFI. A humanized anti-PDZ monoclonal antibody was used to optimize performance. A highly reactive patient serum was serially diluted across eight dilutions to define assay range. Thirteen sera were tested at 1:100 and 1:1,000 with PBS controls. Interpretation focused on 1:1,000, which fell within the log-linear range. Selected sera were pre-incubated with soluble PDZ.
Results
The humanized anti-PDZ monoclonal antibody produced a dose-dependent signal, supporting antigen coupling and assay performance. Serial dilution of a highly reactive serum showed an approximately 4-log dynamic range, with signal nearly three-fold above PBS background at 1:6,400. The curve demonstrated a hook effect below 1:400, consistent with saturation at lower dilutions. At 1:1,000, western blot-positive sera showed strong anti-PDZ reactivity, while sera with weak western blot signal showed low but detectable reactivity. In competition experiments, one highly reactive control sample did not show signal reduction with soluble PDZ, indicating that the signal was not competition-confirmed as PDZ-specific. In contrast, a highly reactive patient sample showed an approximately 50% decrease in MFI with 13 µg/mL soluble PDZ, supporting PDZ-specific binding.
Conclusion
We established a PDZ-focused bead assay for anti-HTRA1 autoantibodies. Serial dilution defined the working range and identified 1:1,000 as the analytical dilution. Soluble antigen competition distinguished PDZ-specific binding from high-signal reactivity that did not inhibit. This assay may provide a scalable approach for detecting and monitoring PDZ-directed anti-HTRA1 immunity in HTRA1-MN
Funding
- NIDDK Support