THE SEPARATION AND IDENTIFICATION BY MONOCLONAL-ANTIBODIES OF DOG IGGFRACTIONS

Four fractions of IgG from normal dog serum have been successfully iso lated by gel filtration followed by protein A and protein G affinity c hromatography using the fast protein liquid chromatography (FPLC) syst em. Protein A chromatography produced three peaks: peak 1 was fallthro ugh material consisting of components which did not bind to protein A, peak 2 consisted of bound material eluting at pH 6, and peak 3 contai ned bound material eluting at pH 3.5. The three peaks were then subjec ted individually to protein G affinity chromatography. Peak 1 from pro tein A chromatography produced a fallthrough peak followed by a weakly binding component which eluted at pH 8, and was called peak w. Peak 2 from protein A chromatography bound to protein G and eluted as a sing le peak at pH 3.8, and was called peak x. Peak 3 from protein A chroma tography emerged as two separate peaks (y and z) off the protein G col umn; peak y bound and eluted at pH 4.1, and peak z bound weakly to pro tein G and emerged as a broad band at pH 8. Peaks w, x, y and z have b een named gamma(w), gamma(x), gamma(y) and gamma(z), respectively, and there purified IgG fractions were used to immunize mice for the prepa ration of monoclonal antibodies (McAbs). To date, two sets of McAbs ha ve been produced: one which recognizes an epitope present in both gamm a(w) and gamma(z) fractions and another set of McAbs which recognizes an epitope in the gamma(x) and gamma(y) fractions.