Evaluation of the chemical parameters of four systems of radioimmunological assay

An iterative indirect model fitting (IIMF) procedure has been designed to evaluate antibody binding capacities and ligand affinities of fairly simple RIA systems. The information is provided by one or more series of standard equilibrium assays, the mass of tracer ligand, the residual fraction of free ligand which contaminates measured bound fractions, and the chemical purity of the tracer. The procedure uses theoretical models which describe systems with one or two antibodies with identical or different affinities towards the hot and the cold ligands. The tracer may be the purified radioactive ligand or a mixture of the hot and the cold ligands with the cold species as the major component. The theoretical models corresponding to two antibody systems are simplified and only strictly valid within limited ranges of ligand concentrations. The chemical parameters of both antibodies may in suitable cases be estimated by two runs of assays with different doses of antiserum and tracer. The IIMF procedure involves the computation of the regression line of a dose function for which the dependent variable is the model derived bound fraction over the measured bound fraction multiplied by the dose. Two tallies are combined of which the statistics of means of replicates being on the regression line has a higher priority than the minimal deviation of the slope of the regression line from 1. The procedure is applied to three RIA systems using carbon adsorption of the free ligand: prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and angiotensin 1 (Ang. 1), and to one using antibody precipitation-arginine vasopressin (AVP). The consistency of the results was tested by repeated runs of standards or by varying the concentrations of the reactants and the temperature.