Comparison of alloantisera and hybridoma antibody for purification of the H-2Db murine histocompatibility antigen and preliminary molecular characterization of this antigen

Abstract

Previous studies on the determination of the complete amino acid sequence of H-2 molecules have relied on alloantisera to purify the H-2 molecules by immunoprecipitation. In this study, directed toward the determination of the complete amino acid sequence of the H-2Db molecule, it was found that all H-2.2 alloantisera examined had very low antibody titers when analyzed by immunoprecipitation and that most of these antisera reacted with not only the H-2Db heavy chain but also with a 75,000 dalton virus-associated glycoprotein (gp 75). The problems encountered with conventional alloantisera were surmounted by utilizing a monoclonal antibody from hybridoma B22-249R1 obtained as described by Hämmerling et al. (1979) and maintained in ascites form in (CBA x Balb/c) F1 mice. This antibody reacted with only 45,000 molecular weight material and had the added advantage of requiring only 0.1 ml of hybridoma ascites fluid to bind all of the H-2Db antigen from 3 x 10(8) EK-4 tumor cells. Biochemical examination of the purified H-2Db molecule showed that it possessed overall structural properties similar to other previously characterized H-2 molecules. An NH2-terminal amino acid sequence was determined for the H-2Db molecule for 28 residues and this sequence was compared to the complete NH2-terminal sequences of the H-2Kb and H-2Dd molecules, and the partial NH2-terminal sequence for the H-2Ld molecule; these comparisons indicated 82%, 75% and 100% homology respectively. In addition, these preliminary studies indicated that there was little or no beta-2-microglobulin (beta 2 mu) in the immunoprecipitates with the H2-2Db heavy chain. This lack of beta 2 mu in the immunoprecipitates was found to be true for alloantisera directed against private or public specificities, a xenoantiserum (sheep anti-H-2b), or hybridoma antibody used in the immunoprecipitation.