A monoclonal anti-peptide antibody reacting with the insulin receptor β-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors

Abstract

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.

CSF-1 Receptor/Insulin Receptor Chimera Permits CSF-1-dependent Differentiation of 3T3-L1 Preadipocytes

“…1). Furthermore, tyrosine-phosphorylated CSF1R/IR chimera was precipitated by an antibody directed against the extracellular ligand binding domain of the CSF1R or by monoclonal antibody CT-1, which is specific for the carboxyl termi-nus of the human insulin receptor (18) (Fig. 1).…”

In IGF-I receptor-deficient leiomyosarcoma cells autocrine IGF-II induces cell invasion and protection from apoptosis via the insulin receptor isoform A

“…The following antibodies were employed: (a) anti-IR antibodies: MA-20 monoclonal antibody that recognizes IR a subunit (ID Goldfine, San Francisco, CA, USA) (Forsayeth et al, 1987;Roth et al, 1982); a rabbit polyclonal antibody that recognizes the IR b subunit (Transduction Laboratories, Lexington, KY, USA) and CT-1 monoclonal antibody that recognizes the IR b subunit (Ganderton et al, 1992). (b) Anti-IGF-IR antibodies: aIR3 monoclonal antibody that recognizes IGF-IR a subunit (Kull et al, 1983) (Oncogene Research, Cambridge, MA, USA); chicken polyclonal antibody that recognizes IGF-IR a subunit (UBI, Lake Placid, NY, USA); 17 -69 monoclonal antibody that recognizes IGF-IR a subunit in both IGF-IR and Hybrid-R and 24 -60 monoclonal antibody that recognizes IGF-IR a subunit (K Siddle, Cambridge, UK).…”

“…IRS were captured with anti-IR MA-20 antibody and detected with biotinylated anti-IR CT-1 antibody (Ganderton et al, 1992;Sciacca et al, 1999). IGF-IRs were captured with anti-IGF-IR aIR-3 antibody and detected with biotinylated 17 -69 antibody (Kull et al, 1983).…”

Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling

“…Receptors were captured by incubating lysates (0.5-60 mg/well) in Maxisorp immunoplates (Nunc Int., Rochester, NY, USA) precoated with anti-IR antibody MA-20 (2 mg/ml) and detected with biotinylated anti-IR antibody CT-1 (Forsayeth et al, 1987;Ganderton et al, 1992) or with anti-IGF-1R antibody aIR-3 and detected with biotinylated antibody 17-69 (Kull et al, 1983;Soos et al, 1992). Reaction was amplified with peroxidase-conjugated streptavidin.…”