Preparation of galactose alpha 1-3 galactose xenoantigen for the removal of anti-alpha-GAL ABs

摘要

Xenotranphlant hyperacute rejection is through to be intitated by a materianl non-reducing disaccharide, galactose alpha 1-3 galactose (G alpha G), on the implant to which G alpha G-specific IgM and IgG bind. The subsequent complement activatioon then leads to coagulation and inflammatory reeactions. The objective of this work was top prepare an immobilized ligand containing G alpha G for prophylactic reduction of G alpha G specific circulating Abs. To produce subh ligands it was necessary to find ologometric forms of G alpha G that would provide both the antigan and a binding site for immobiliztion. Carrageenan-lambda was acetylated, phydolyzed, and then deacetyl ated. The resulting syrup was chromatographed on Sephadex G15 to produce a series of sized fractions. The presence of anti-G alpha G Abs in sera was mesured by a dot blot procedure using bovine thyroglobulin (BThy), a glycorpotein expressing th G alpha G marker, immobilized non PVDF transfer membrane. This procedure was sued to determine the ability of the G-15 fractions to compete for Ig G alpha G binding sites. Increasng amounts of these fractons added to plasma resulted in progressive reduction in Ig binding to BThy; at less than 0.6 mg carbohydrate/ml plasma complete binding inhibition was observed with the more active fractions. Comparative measurements showed that Melibiose (6-O-alpha-D-galactoryranosyl-D-glucose), an analogue which is also recognized by anti G alpha G Abs, could not inhibit completely at 7.5 mg/ml plasma. Methyl-alpha- glucopyranoside and alpha-D-glucose used as negative controls showed no inhibition of anti- G alphas G Ab activity. The G alpha G fractions produced here contain adiditional glycosidic rings suitable for immobilization on hydrazide-modified matrices. We prepared hydrazide terminated leashes on microposours poly(amde) hollw fibers and immobilized several oligomeric fractions. Thee showed high selectivity for anti-G alpha G Abs in sera and indicated the feasibility of producing an affinity membrane capable of preventing hyperacture rejection of xenotransplants.