Dissociation rate constants and fractional binding of tracer estimated for three antibody populations in unstripped and stripped antiserum

Abstract

The dissociation rate constants of a high-affinity thyroxine antiserum were estimated on the basis of curves showing the dissociation of labelled thyroxine-antibody complexes as a function of time. The dissociation curves were fitted by computer to an equation with a sum of three exponentials. The analysis of the curves gave estimates of the dissociation rate constants for fast, medium and slowly dissociating antibody populations, and also estimates of the size of the fraction of tracer bound to each of the three populations of antibodies. The coefficients of variation for the estimates were of the order of 10%, and the estimates were reproducible. The dissociation rate constants for the thyroxine antibodies were 0.55, 0.045 and 0.0058 h^–1 at 23°C. When labelled thyroxine and antiserum were incubated for increasing periods of time, there was an increase in the fractional binding of tracer to the slow-dissociating antibodies. The use of stripped antiserum as against unstripped antiserum also improved the fractional binding of tracer to the slow-dissociating antibodies. The stripping effect was slight for three other antisera which contained relatively fast dissociating antibodies.

Key Words:

• curve analysis
• dissociation curve
• dissociation rate constant
• equilibrium constant
• fractional binding
• Scatchard plot
• stripping
• thyroxine