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Competitive protein binding radioimmunoassay (CPB-RIA) is a principal method for quantifying serum digoxin concentration. The accuracy of this method is critically dependent on factors that influence the substitution reaction between unlabelled (Q) antigen (digoxin) with 125I-labelled antigen (M) bound to anti-digoxin antibody (P). We studied the influence of initial concentration of M, ionic strength, and viscosity on the substitution reaction between M and Q. In addition, we propose a kinetic model for this reaction.We used a commercially available CPB-RIA for digoxin, a gamma counter, and a viscosimeter to study the effect of initial concentration of M, ionic strength, viscosity, and temperature on the substitution reaction between M and Q. Data were analyzed using Statistica software.The apparent rate constant for the reaction between M and Q in the formation of PM is dependent on the initial concentration of M, and the ionic strength, viscosity, and temperature of the reaction medium, and independent of the concentration of Q.A kinetic model for the displacement of the 125I-digoxin by the digoxin in its union to a specific antibody is proposed. Such model adjusts satisfactorily to the results and allows the prediction of the calibration curves of RIA (activity bound to the antibody vs. concentration of digoxin) showing the influence of the concentration of both species, the time of incubation, the viscosity and the ionic strength of the medium, on the sensitivity of the method of RIA on which the analytical determination of the digoxin is based.