Abstract
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 × 1011 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).
Highlights
A shortage of organ donors has led humans to explore the field of xenotransplantation by using the tissue or organ of animals
When xenotransplants or animal tissue-derived biomaterials containing Gal antigens are transplanted into the human body, they likely contribute to hyperacute rejection (HAR) or chronic immune rejection, and further delay tissue repair and re-construction[9,10]
Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolian 1 isolectin B4 (IB4), which shows high affinity to galactose
Summary
A shortage of organ donors has led humans to explore the field of xenotransplantation by using the tissue or organ of animals. Myeloma cells expressed about 1.2 × 106 Gal epitopes per cell when detected by IB41, and the sensitivity of rabbit red blood cells was found to be 2-fold compared to myeloma cells, suggesting that the Gal epitope expressed on rabbit red blood cells was about 2 × 106,17 Based on these data, rabbit red blood cells along with standard materials have been utilized to produce calibration curves for detecting Gal antigen in animal tissues in the test system of ELISA inhibition assay with monoclonal M86 antibody[17,19]. Rabbit red blood cells along with standard materials have been utilized to produce calibration curves for detecting Gal antigen in animal tissues in the test system of ELISA inhibition assay with monoclonal M86 antibody[17,19] These measurements allow for relative quantitative determination of Gal epitopes, but do not provide an accurate calculation of the number of Gal epitopes per cell or per biomaterials (e.g. per mg). The results to be detected do not represent the actual results of animal tissues or animal tissue derived biomaterials when using mouse myeloma cells or rabbit red blood cells as standard samples due to the Gal antigen-antibody reaction system were quite different
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