Transplantation in miniature swine. IX. Swine histocompatibility antigens: isolation and purification of papain-solubilized SLA antigens.
Histocompatibility antigens have been purified by papain treatment of crude cellular membranes obtained from spleen and mesenteric lymph nodes of individual miniature swine that are homozygous at the major histocompatibility complex. The purification protocol included ultracentrifugation, gel filtration, ion-exchange chromatography, and in some instances preparative isoelectric focusing. This procedure was applied to the preparation of sLA antigens from the 3 haplotypes available in our partially inbred miniature swine herd. The purity of the SLAdd molecules was evidenced by: 1) a single activity peak of approximately 50,000 daltons on gel-permeation chromatography; 2) the antigen elution as a symmetrical peak from ion-exchange resins and from isoelectric focusing columns; 3) the presence of 2 predominant polypeptide chains on SDS-PAGE at apparent m.w. 43,000 and 13,000 daltons; and 4) specific immunoprecipitations by alloantisera and by anti-beta 1 microglobulin antisera. The antigenic activity of SLA products was analyzed by inhibition of complement-mediated cytotoxicity and by removal on anti-beta 2-microglobulin affinity columns. Heavy and light chains were separated by gel-filtration on Sephacryl S-200 in 6 molar guanidine. These SLA products, which have thus been shown to be pure by a variety of criteria, are now readily available for structural analysis and for analysis of biologic activity.
D000818:Animals; D002852:Chromatography, Ion Exchange; D006041:Goats; D006649:Histocompatibility Antigens / Q000302:isolation & purification*; D006650:Histocompatibility Testing; D007460:Iodoacetamide / Q000494:pharmacology; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010206:Papain / Q000494:pharmacology; D010455:Peptides / Q000302:isolation & purification; D012995:Solubility; D013552:Swine; D014461:Ultracentrifugation
D006649:Histocompatibility Antigens; D010455:Peptides; D010206:Papain; D007460:Iodoacetamide