Abstract: FR-OR056
Multiplexed Detection of Membranous Antigens by Mass Spectrometry
Session Information
- Pathology: Novel Mechanisms and Modalities
November 07, 2025 | Location: Room 371A, Convention Center
Abstract Time: 04:40 PM - 04:50 PM
Category: Pathology and Lab Medicine
- 1800 Pathology and Lab Medicine
Authors
- Storey, Aaron J., Arkana Laboratories, Little Rock, Arkansas, United States
- Caza, Tiffany, Arkana Laboratories, Little Rock, Arkansas, United States
- Hassen, Samar, Arkana Laboratories, Little Rock, Arkansas, United States
- Edmondson, Ricky D., University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
- Larsen, Christopher Patrick, Arkana Laboratories, Little Rock, Arkansas, United States
Background
Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults, which is driven by an immune response against one of a series of target antigens. Over 20 target antigens have been described in MN to date, creating the need for multiplex approaches for detection. We developed a multiple reaction monitoring-based assay for the clinical mass spectrometry (MS) laboratory for determination of an antigen type in nephropathology practice.
Methods
Protein A/G immunoprecipitation was used to extract immune complexes from frozen kidney biopsy tissue remnants through an automated workflow on a King Fisher Flex immunoprecipitation system. Proteins were digested from magnetic beads, alkylated, and trypsin digested with an SP3-assisted sample preparation. Tryptic peptide quantities were measured by MS with internal standard heavy peptides. Peak area ratios were used in calling antigen type with thresholds set in TraceFinder software. Immunostaining was used as an independent method to confirm target antigen types identified by MS.
Results
Our IP-MS workflow demonstrated reliable quantitative detection of MN antigens. Our panel included 20 antigens, including PLA2R, THSD7A, EXT1/2, NELL1, NCAM1, MPO, NDNF, PCDH7, HTRA1, SEMA3B, TGFBR3, FAT1, PCSK6, CNTN1, NTNG1, CRIM1, FRAS1, NLGN3, RECK, and VASN. Analytical performance of our assay was assessed through adapting the Clinical Proteomic Tumor Analysis Consortium (CPTAC) guidelines for response curves with assessment of the limits of detection and quantitation, repeatability, selectivity, stability, and detection of endogenous analytes. Of a series of 114 cases of PLA2R/THSD7A/NELL1/EXT-negative MN, 56 had a rare antigen detected by this assay (49.1%). There was high concordance between antigen type with comparison of MS to immunostaining (70/79, 88.6%).
Conclusion
Multiple reaction monitoring MS following protein A/G immunoprecipitation of MN biopsy tissue can be effectively used for antigen typing in clinical practice.
Funding
- NIDDK Support