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Alpha-Gal (Gal) epitopes present in animal tissues are known to be the key xenoantigens that elicit xenorejection. However, a standardized method to determine Gal epitope in animal tissue-derived biomaterials does not exist. Herein, a standardized method for quantitative detection of Gal antigen was established based on an ELISA inhibition assay with Gal antibody. In this method, the key optimized experimental conditions were: (1) Gal-antigen positive and negative reference materials were developed, and used as positive and negative control in the test system, respectively; (2) A mixture of artificial Gal-BSA antigen plus Gal-negative matrix was used as the calibration standard sample, making it has similar composition with test sample; and (3) The lysis buffer was combined with the homogenate to expose the Gal antigen as much as possible. The results from validation and application experiments showed that the standardized method had good reproducibility (RSD = 12.48%), and the lower detection limit (LDL) is ~7.1 × 10 11  Gal epitopes/reaction. This method has been further developed into a detection Kit (Meitan 70101, China), and it has been developed as a standard method for detecting remnant immunogen of animal tissue derived medical devices, and as the industry standard has been released in China. (YY/T 1561–2017).

A standardized quantitative method for detecting remnant alpha-Gal antigen in animal tissues or animal tissue-derived biomaterials and its application