[Production of monoclonal antibodies to an insulin degrading enzyme and affinity purification of the enzyme].
Monoclonal antibodies to a cytosolic insulin-degrading enzyme (IDE) were produced by fusing spleen cells from mouse immunized highly purified human erythrocyte IDE with mouse myeloma cells. Four monoclonal antibodies were identified by their ability to bind to 125I-insulin covalently linked to a cytosolic IDE from human erythrocytes. All four antibodies were found to remove more than 90% of the insulin-degrading activity from erythrocytes extracts, demonstrating that these antibodies were directed against an enzyme which accounts for most of this activity. By immunoprecipitation from metabolically labelled cells and immunoblot procedure, the enzyme from a variety of tissue was shown to be composed of a single polypeptide chain of apparent Mr = 110 kDa. One of these antibodies; 31H7 was coupled to Affi-Gel 10 and used for the purification of this enzyme. Immobilized antigen was eluted at more than 85% efficiency with buffers consisting of either pH2.3, 2.5M MgCl2 or with 6M urea. However, the antigen eluted under 6M urea retained the highest antigenecity (44%) and biological activity (8%) and the yield of the enzyme obtained from this procedure increased up to 17 fold as compared with the conventional method. NaDodSO4/polyacrylamide gel electrophoresis showed a single band of this protein with apparent Mr 110 kDa. These monoclonal antibodies and the purified enzyme will be useful tools for a better understanding of this enzyme, so may lead to the design of specific inhibitors of this enzyme that may be used to treat patients with excessive insulin degradation.
Shii
K
K
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010447:Peptide Hydrolases / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D007328:Insulin; D010447:Peptide Hydrolases