Determination of specific activity of labeled ligands by nonlinear regression analysis

The specific radioactivity (SA) of 125I-lysine vasopressin (LVP) was determined by analyzing the binding B (cpm/tube) of variable amounts of tracer T (cpm/tube) to a constant amount of an LVP antibody, in the presence of known quantities L (mol/tube) of LVP standards. The parameters of the equations B = f(F) and B/F = g(T), describing B as a function of free F (cpm/tube) tracer or the ratios B/F as a function of T, were first calculated by nonlinear regression analysis of the results obtained with tracer alone. Then the dependent variables B or B/F were measured in the presence of LVP and analyzed with the same equations by substituting the independent variables F or T with (F + alpha FL) and (T + alpha L), respectively, where alpha (cpm/mol) represents a measure of the SA and FL (FL = L.F/T), free LVP, respectively. The SA was thus treated as an unknown parameter to be calculated by nonlinear regression. This method was compared with the traditional interpolation of the SA from the self-displacement and standard curves. Tracer and ligand were found to have the same affinity for the binding sites, since the set of equations B = f(F + alpha FL) and B/F = g(T + alpha L), describing the binding of the tracer in the presence of LVP and equations B = f(F) and B/F = g(T) to which these equations reduce in the absence of LVP (L = 0), had identical binding parameters. To be valid, any method based on self-displacement requires that the tracer and standards have the same affinity for the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)