title: Quantification of α-Gal Antigen Removal in the Porcine Dermal Tissue by α-Galactosidase.
abstract: The α-Gal (Galα1,3-Galβ1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 (n = 10) to 6.1 ± 2.6 (n = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 (n = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼ 84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a nonhuman primate model.
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Gao
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Su-Bo
authors.initials: SB
authors.lastname: Li
authors.affiliation: 2 School of Medical Instruments and Food Engineering, University of Shanghai for Science and Technology , Shanghai, China .
authors.forename: Wendell Q
authors.initials: WQ
authors.lastname: Sun
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Zhi-Min
authors.initials: ZM
authors.lastname: Yun
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Xue
authors.initials: X
authors.lastname: Zhang
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Jin-Wen
authors.initials: JW
authors.lastname: Song
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Shi-Kun
authors.initials: SK
authors.lastname: Zhang
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Ling
authors.initials: L
authors.lastname: Leng
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Shou-Ping
authors.initials: SP
authors.lastname: Ji
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Ying-Xia
authors.initials: YX
authors.lastname: Tan
authors.affiliation: 1 Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine , Beijing, China .
authors.forename: Feng
authors.initials: F
authors.lastname: Gong
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000941:Antigens / Q000378:metabolism*; D002417:Cattle; D020405:Dermis / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D005690:Galactose / Q000378:metabolism*; D034421:Sus scrofa; D000519:alpha-Galactosidase / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D000519:alpha-Galactosidase; D005690:Galactose