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Updated: Sep 14, 2025
Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis
Published on: October 15, 2021
Using MagSort to identify eplets in a high PRA patient serum and revealing a novel HLA-A*01:01 specific binding
Tri T M Vu1, Ronald Wang1, Shalu Mathew1
1Department of Research and Development, One Lambda, Inc. (A Part of Thermo Fisher Scientific Inc.), West Hills, CA, USA.
Sera from patients with previous transplants, transfusions, and pregnancies may contain multiple Human Leukocyte Antigen (HLA)-specific antibodies recognizing various functional epitopes or eplets. The identification and verification of eplets in these high Panel Reactive Antibody (PRA) cases are important for understanding HLA antigenicity and potentially improving transplantation outcomes. For a particular high PRA case, we utilized adsorption and elution techniques using cells and HLA-specific magnetic beads to identify and verify eplets. The resulting eluates confirmed seven established eplet patterns while also uncovering two novel binding patterns against A*01:01 and B*53:01. The isolation of the HLA-specific antibodies allows us to perform competition assays against monoclonal antibodies to confirm the epitope specificity. Using available HLA crystal structures, we proposed that the physicochemical properties of residues 163 and 167 are crucial for the binding of the A*01:01-targeted antibody identified in this study, as well as another A*01:01-targeted antibody previously discovered by Lima et al. [1]. Our studies demonstrate that isolating antibodies and analyzing the physicochemical properties of the eplet will lead to a more comprehensive understanding of HLA immunogenicity.
Sera from patients with previous transplants, transfusions, and pregnancies may contain multiple Human Leukocyte Antigen (HLA)-specific antibodies recognizing various functional epitopes or eplets. The identification and verification of eplets in these high Panel Reactive Antibody (PRA) cases are important for understanding HLA antigenicity and potentially improving transplantation outcomes. For a particular high PRA case, we utilized adsorption and elution techniques using cells and HLA-specific magnetic beads to identify and verify eplets. The resulting eluates confirmed seven established eplet patterns while also uncovering two novel binding patterns against A*01:01 and B*53:01. The isolation of the HLA-specific antibodies allows us to perform competition assays against monoclonal antibodies to confirm the epitope specificity. Using available HLA crystal structures, we proposed that the physicochemical properties of residues 163 and 167 are crucial for the binding of the A*01:01-targeted antibody identified in this study, as well as another A*01:01-targeted antibody previously discovered by Lima et al. [1]. Our studies demonstrate that isolating antibodies and analyzing the physicochemical properties of the eplet will lead to a more comprehensive understanding of HLA immunogenicity.
