Biotin
A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.
W6/32 is a mouse anti-HLA class I monoclonal antibody (MoAb) of BALB/c (H-2d) origin with a monomorphic reaction pattern on human cells. In this study, we explain that the previously reported (Ivanyi D et al., Immunogenetics 20:6gg, 1984) cross-reactions of W6/32 with the H-2Db antigen are completely dependent on the formation of a complex between the H-2Db heavy chain with bovine beta 2-microglobulin (beta 2m) from the culture medium. M0Ab W6/32 cross-reacted with various H-2 class I antigens only in the presence of bovine or human beta 2m but not in the presence of beta 2m from other species (goat, sheep, rabbit) or syngeneic mouse beta 2m. The exposure of the W6/32 determinant on mouse cells was dependent on the concentration of human or bovine beta 2m and was influenced by the temperature and time of incubation. The reaction pattern of W6/32 on a large panel of mouse strains showed that the binding is due to at least two critical factors: (i) the H-2 haplotype of the target cells; and (ii) the substitution of murine beta 2m for bovine or human beta 2m. These results show that exposure of a polymorphic class I determinant is dependent on the species origin of beta 2m with which the heavy chain is complexed. Comparison of beta 2m amino acid sequences from various species does not give a clear answer about the shared quality of human and bovine beta 2m. One amino acid position (89) was identified at which human and bovine beta 2m are identical but differ from all other known beta 2m sequences.
