A monoclonal anti-peptide antibody reacting with the insulin receptor beta-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors.
A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D002462:Cell Membrane / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007163:Immunosorbent Techniques; D008099:Liver / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D010766:Phosphorylation; D019000:Phosphotyrosine; D010920:Placenta / Q000737:chemistry; D051381:Rats; D011972:Receptor, Insulin / Q000276:immunology* / Q000302:isolation & purification*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin