A radioimmunoassay to screen for antibodies to native conformational antigens and analyse ligand-induced structural states of antigenic proteins.
A radioimmunoassay is described in which antigenic protein was immobilized by incubating nitrocellulose filters of defined diameter with antigen-containing solutions. The amount of adsorbed antigen increased in a linear fashion over a wide range of antigen concentrations. The antigen-antibody reactions and the indicator reactions were performed by incubating the filters with appropriate solutions. During the test any contact of the antigen with air was avoided. Antigenic sites which are sensitive to protein denaturation by drying could be detected with the assay. The assay was also used to screen hybridoma supernatants for antibodies directed against Na+ cotransport proteins from renal brush-border membranes. Monoclonal antibodies were selected which showed different binding characteristics depending on whether or not substrates of Na+ cotransporters were present. Since binding of several antibodies was altered by two different substrates and not by non-transported control substances, these monoclonal antibodies were believed to interact with more than one transport system. One of the antibodies, which showed different antibody binding after addition of D-glucose or L-lactate, bound to a polypeptide component of the renal Na+-D-glucose cotransporter and was able to inhibit Na+ gradient-dependent D-glucose uptake in brush-border membrane vesicles (Koepsell, Korn, Raszeja-Specht, Bernotat-Danielowski, Ollig, 1988, J. Biol. Chem., in press). To investigate the effects of D-glucose and L-lactate on the binding of this antibody concentration dependence was measured. High and low affinity binding sites for D-glucose and L-lactate were characterized thereby demonstrating that the radioimmunoassay permits investigations of the properties of high and low affinity substrate binding sites.
Bernotat-Danielowski
S
S
Max-Planck Institut für Biophysik, Frankfurt, F.R.G.
Koepsell
H
H
D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D001666:Binding Sites, Antibody; D003429:Cross Reactions; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008871:Microvilli / Q000276:immunology; D009004:Monosaccharide Transport Proteins / Q000276:immunology*; D011487:Protein Conformation; D011863:Radioimmunoassay / Q000379:methods; D013329:Structure-Activity Relationship
D000906:Antibodies; D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins