Gates, Katherine Yvonne

American Heart Association estimates the prevalence of valvular heart disease to be 2.5% in the United States. Biological heart valve replacements, such as glutaraldehyde‐fixed bovine pericardium (BP), are considered the current standard of care. The NHLBI xenotransplantation working group identified xenoantigenicity as the major barrier to expanding use of such xenogeneic tissues. Previous research has utilized an immunoproteomic approach based on two‐dimensional western blots to identify a small number of BP xenoantigens, which is time consuming and technically challenging. These limitations have resulted in an ever increasing use of shotgun proteomic approaches due to their superior throughput and reproducibility. However, traditional Liquid Chromatography‐Mass Spectrometry (LC‐MS) fails to separate proteins based on their antigenicity, and therefore it is impossible to determine which identified proteins are antigenic versus not. We hypothesized that an affinity chromatography method utilizing poly‐polyclonal serum would bridge this gap and represent an innovative shotgun proteomic antigen identification approach. Using affinity chromatography rabbit IgG antibodies were isolated from whole serum with Protein A HP SpinTrap (GE Healthcare). IgG capture was confirmed by serum protein electrophoresis and ELISA. Two types of antibody affinity columns were generated, with naive serum (D0) or serum from rabbits immunized with bovine pericardium (D84). Bovine pericardial protein extracts were run through both affinity columns, allowing for the specific capture of antigens, which were later eluted and collected. To confirm specificity of antigen capture, eluants were subjected to western blot, and probed with D84 serum. Affinity enriched antigenic proteins were identified by LC‐MS/MS, with statistical comparisons made between D0 and D84. This approach identified 133 putative antigenic proteins that were enriched on D84 compared to D0 columns. Ten of these antigens corresponded to previously published BP xenoantigens. Of the remaining 123 previously unidentified antigens, 30 have been indicated to be immunogenic in other fields such as cancer and autoimmunity. This method was then applied to three humans who had been implanted with bovine pericardial heart valves. Surprisingly, 300 to 500 proteins were identified from patient columns. Between 57 to 79% of the identified proteins were shared among the three individuals, as well as almost all of the proteins identified in the rabbit model were also present in the human. This work therefore represents the first high throughput method of antigen identification that can now be used to monitor currently implanted patients as well as inform the development of BP‐based biomaterials in the future. Support or Funding Information Research supported by grant number R01HL115205 from the National Heart Lung and Blood Institute (NHLBI) at the National Institutes of Health (NIH) and grant number 5T32OD010931‐10 from the NIH.