IMMUNOCHEMICAL ANALYSIS OF A RECOMBINANT, GENETICALLY ENGINEERED, SECRETED HLA-A2/Q10B FUSION PROTEIN

We engineered a fusion gene which encodes the alpha-1 and alpha-2 domains of HLA-A2 with the alpha-3 and truncated transmembrane domains of the murine class I-like protein Q10b, and transferred it into mouse L cells along with the gene for human beta-2-microglobulin (beta-2-m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was non-covalently associated with the human beta-2-m light chain. Native detergent-solubilized HLA-A2 and secreted rA2/Q10b proteins were found to be similar by: (a) the binding to mouse monoclonal anti-HLA antibodies in an ELISA; (b) the blocking of lysis of HLA-A2+ cells by human anti-HLA-A2,-B17, anti-HLA-A2,9,28, and anti-HLA-A2,28 cross-reactive group (CREG) antisera in a complement-dependent cytotoxicity assay; and (c) the ability when coupled to Sepharose to selectively purify HLA-A2,9,28 and HLA-A2,28 CREG-specific antibodies. Mouse L cells expressing rA2/Q10b produced as much as 2.5-mu-g protein per 10(6) cells/day, or 50- to 100-fold more antigen on a per cell basis than the level of HLA-A2 expressed by B-lymphoblastoid cell line or spleen cells. Thus rA2/Q10b represents a viable alternative to detergent-solubilized HLA-A2 for purification of anti-HLA-A2 antibodies and analysis of anti-HLA-A2 immune responses.