TY - JOUR
T1 - Immunochemical analysis of a recombinant, genetically engineered, secreted HLA-A2/Q10b fusion protein
AU - DeVito, Lynn D.
AU - Mason, Beth
AU - Schneck, Jonathan
AU - Margulies, David H.
AU - Sollinger, Hans W.
AU - Burlingham, William J.
PY - 1991/10
Y1 - 1991/10
N2 - We engineered a fusion gene which encodes the α1 and α2 domains of HLA-A2 with the α3 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 β2-microglobulin (β2m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was noncovalently associated with the human β2m 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 μg protein per 106 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.
AB - We engineered a fusion gene which encodes the α1 and α2 domains of HLA-A2 with the α3 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 β2-microglobulin (β2m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was noncovalently associated with the human β2m 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 μg protein per 106 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.
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U2 - 10.1016/0198-8859(91)90109-M
DO - 10.1016/0198-8859(91)90109-M
M3 - Article
C2 - 1744002
AN - SCOPUS:0026072122
SN - 0198-8859
VL - 32
SP - 125
EP - 133
JO - Human Immunology
JF - Human Immunology
IS - 2
ER -