The six equilibrium binding parameters of two antibodies in competitive radioimmunoassays.
A procedure is described for measuring the six parameters that theoretically determine the equilibrium bindings of two ligands in two-antibody antisera used in competitive radioimmunoassays (RIA). Four intensity parameters and two quantities are identified. They are the dissociation constants of the complexes formed by the bindings of the tracer and the specific ligand of two antibodies and the two corresponding binding capacities of a certain volume of the antiserum. Four series of assays, runs, are required to give enough information. Two very different amounts of serum are used, either with various doses of the tracer only or with one tracer dose and suitable dose ranges of the specific ligand. The corresponding equations and their use are presented. The amount of information is sufficient to offer several controls of consistency of the parameters with the two-antibody model. The RIA antisera of two prostaglandins, PGF2 alpha and PGE2, with the heavily tritiated analogues as tracers were found to be very different with regard to the ratios of the binding capacities and the affinities of the two antibodies. The intensity parameters of the two sera were stable over the period of the present study, almost a year. To interpret assay results of a run of unknowns it is necessary to calibrate precisely the amount of antiserum used as well as the residual non-specifically bound tracer fraction and the tracer dose. The random analytical error of an assay is then also predictable when the amounts of tracer or antiserum vary between assays of the same run.
Bojesen
E
E
D000906:Antibodies / Q000378:metabolism*; D000915:Antibody Affinity; D001667:Binding, Competitive; D015237:Dinoprost; D015232:Dinoprostone; D007106:Immune Sera / Q000276:immunology*; D008024:Ligands / Q000378:metabolism; D011458:Prostaglandins E / Q000032:analysis / Q000276:immunology; D011460:Prostaglandins F / Q000032:analysis / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods*
D000906:Antibodies; D007106:Immune Sera; D008024:Ligands; D011458:Prostaglandins E; D011460:Prostaglandins F; D015237:Dinoprost; D015232:Dinoprostone