Several epitopes of p85 glycoprotein (CDw44) are dependent on intact disulphide bonds. Isolation of cDNA clones requires a polyclonal antibody raised against the reduced protein.
Monoclonal antibodies 50B4 and 50E6 recognize two distinct epitopes of human p85 glycoprotein (CDw44). Both epitopes are destroyed by reduction of the purified glycoprotein as demonstrated by inhibition of cellular radioimmunoassay and Western blot analysis. Endoglycosidase F treated p85 glycoprotein, with an apparent molecular weight of 73,000, is still reactive with both monoclonal antibodies. Thus both epitopes are conformational determinants of the polypeptide chain. A rabbit antibody produced against purified native p85 glycoprotein also reacted only with the non-reduced form of p85. Repeated immunizations with SDS-dissociated and reduced p85 yielded a polyclonal antibody reactive by Western blot analysis with reduced and non-reduced forms of p85 glycoprotein. When a HOON leukemia cell line cDNA expression library was screened with this polyclonal antibody, two cDNA clones were isolated which reacted specifically with the antiserum and not with the control non-immune serum. Preliminary characterization of these clones indicates that they are p85-related.
Rogers
I
I
Department of Immunology, University of Toronto.
D'Agostaro
G
G
Vera
S
S
Letarte
M
M
D000906:Antibodies; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004220:Disulfides / Q000032:analysis* / Q000378:metabolism; D000939:Epitopes / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D009844:Oligosaccharides / Q000378:metabolism; D010084:Oxidation-Reduction; D016201:Receptors, Lymphocyte Homing
D000906:Antibodies; D004220:Disulfides; D000939:Epitopes; D006023:Glycoproteins; D009844:Oligosaccharides; D016201:Receptors, Lymphocyte Homing; D004247:DNA