Human Ia molecules carrying MT1 determinants: purification with a monoclonal antibody.
A monoclonal antibody 77.34, reactive with polymorphic HLA class II molecules, was produced. The allotype specificity of this IgG2a antibody was analyzed by cytotoxicity, flow cytometry, and cellular radioimmunoassay. Cytotoxic reactivity on a panel of B cells from 88 unrelated individuals was concordant with the MT1 (DC1) allospecificity (r = 0.83). Immunoanalysis by flow cytometry showed that cells from MT1+ homozygous cell lines were reactive, whereas MT2+ and MT3+ homozygous cells were not. A cellular radioimmunoassay performed under saturating conditions indicated that three MT1+ cell lines bound 14-45 X 10(5) molecules of antibody per cell representing 30-40% of the amount detected with monomorphic anti-DR monoclonal antibody 21w4. The subset of molecules bearing the MT1 allospecificity was purified with a 77.34 IgG immunoadsorbent. The purified molecules were antigenically reactive with several antibodies directed at DQw1 molecules but were devoid of reactivity to monomorphic anti-DR antibodies. Two-dimensional gel electrophoresis showed that the alpha subunit is composed of several acidic spots of Mr 32,000 whereas the beta subunit was seen as a single spot of Mr 25,000, corresponding to DQw1 molecules. DR molecules purified by monoclonal antibody affinity were unreactive with 77.34 antibody. All of the 77.34 reactivity was observed with the fractions depleted of DR molecules. Two-dimensional gel analysis showed marked differences between the purified DR and DQw1 molecules. The presence of the MT1 determinant on Ia molecules referred to as the DQw1 molecules and distinct from those bearing the DR epitopes was confirmed on two DR1, MT1 homozygous cell lines. Thus, DQw1 molecules can be purified away from DR1 molecules by affinity chromatography to 77.34 IgG, specifically reactive with the MT1 (DQw1) allospecificity. The binding of 77.34 IgG to MT1+ cells was not inhibited by all monoclonal antibodies reported to be correlated with the MT1 allospecificity suggesting that the latter might be comprised of more than a single epitope.
J A
JA
Falk
W H
WH
Lewis
M
M
Letarte
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D005434:Flow Cytometry; D006680:HLA Antigens / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000302:isolation & purification*; D006720:Homozygote; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D006680:HLA Antigens; D000949:Histocompatibility Antigens Class II