Thibaudeau K, Borche L, Soulillou J P, Blanchard D
Centre Régional de Transfusion Sanguine, Nantes, France.
Blood. 1996 Jun 1;87(11):4636-42.
Human natural "anti-Gal" antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180-, 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha-Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147-7B6; IgG1) specific for its beta-chain.
人类天然的“抗Gal”抗体特异性地针对非灵长类哺乳动物和新大陆猴细胞上表达的Galα1-3Galβ1-4GlcNAc残基。我们研究了纯化的IgG和IgM抗Gal作为异种反应性天然抗体（XNA）的相对作用。从人血浆中分离出IgG和IgM，并通过在Synsorb-14柱（加拿大艾伯塔省埃德蒙顿的Chembiomed公司）上进行亲和层析纯化抗Gal抗体。在酶联免疫吸附测定（ELISA）中，IgM和IgG类别的抗Gal代表了与猪血小板结合的大部分人XNA。在免疫印迹上，正常人血清以及纯化的IgM和IgG组分与115、125、135、150、180、210和240kd的猪血小板蛋白发生反应，而IgM和IgG类别的纯化抗Gal抗体主要与135、150、180和210kD的糖蛋白结合。在ELISA中观察到抗Gal游离的IgM和IgG反应性较低，表明异种抗体并非仅针对半乳糖基表位。这些抗体显示出115、125和240kD的条带，经亲和层析富集的猪血小板糖蛋白（gp）用α-半乳糖苷酶处理后，消除了135和210kD蛋白的反应性。N-和O-糖苷酶处理表明α-半乳糖基残基位于135kD组分的O-聚糖上。最后，通过氨基酸测序将90和135kD的糖蛋白分别鉴定为人糖蛋白IIIa和IIb的猪类似物，而使用一种针对其β链的新型鼠单克隆抗体（naM147-7B6；IgG1）将240kD的组分鉴定为猪纤维蛋白原。
