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 2m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was noncovalently associated with the human beta 2m 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 micrograms 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.
DeVito
L D
LD
Department of Surgery, University of Wisconsin, Madison.
Mason
B
B
Schneck
J
J
Margulies
D H
DH
Sollinger
H W
HW
Burlingham
W J
WJ
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line / Q000378:metabolism; D006183:H-2 Antigens / Q000096:biosynthesis* / Q000276:immunology; D015789:HLA-A2 Antigen / Q000096:biosynthesis* / Q000276:immunology; D064532:Histocompatibility Antigen H-2D; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007164:Immunosorbents; D007739:L Cells; D008285:Major Histocompatibility Complex; D051379:Mice; D009928:Organ Specificity / Q000276:immunology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D000906:Antibodies; D006183:H-2 Antigens; D015789:HLA-A2 Antigen; D064532:Histocompatibility Antigen H-2D; D007164:Immunosorbents; D011993:Recombinant Fusion Proteins