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VOL. 75 (1), 25-41, 2009 MONO AND BIEXPONENTIAL IN...

antibody sites. Because the antibody is immobilized to the wall of a
polypropylene tube, simply decanting the supernatant suffices to ter-
minate the competition and to isolate the antibody-bound fraction of
the radiolabeled insulin. Counting the tube in a gamma counter then
yields a number, which converts by way of a calibration curve to a
measure of the insulin present in the patient sample. Rodbard,
Munson, Ekins (2-4) and others suggested some models for the ad-
justment of calibration curves, the most widespread and used in cur-
rent software is that of the four parameters.

Kinetics and equilibrium in antigen-antibody reactions are deter-
mining factors in the sensitiveness and accuracy of immunoanalyti-
cal techniques (5-7). A diffusion-controlled process must meet some
typical requirements such as a considerable reaction rate decrease
when medium viscosity is greater, and scarce temperature influence
with a reduced energy demand with regards activation, this causing
activation enthalpy values to be the same order as the solvent’s vis-
cous flow energy (19000 J mol-1 for water) (8). Diffusion control for
this type of processes has been theoretically studied by Nigren,
Stenberg et al (9-13). They proposed an application model for reac-
tions produced in the solid-liquid interphase which provided an equa-
tion containing four diffusion influence parameters. Raman (14) also
observed diffusion control for monoclonal antibody binding to cy-
tochrome C. Xavier and Wilson (15) studied the association and dis-
sociation reactions of Anti-Hen Egg Lysozyme (HEL) with two of its
specific antibodies (HyHEL-5 and HyHEL-10) under pseudo first or-
der conditions for the association, and found diffusion control. The
decrease in the reaction rate constants as a result of viscosity turned
out to be more drastic than theoretically expected, this aspect being
put down to potential osmotic effects. In addition, rate constants were
found to approximately double when ionic strength goes down from
500 mM to 27 mM, which indicates that the process occurs between
species with opposite charges that affect the orientational require-
ments of association.

Equilibrium data analysis is largely used in determining the ca-
pacity of a substance to bind to one or several receptor populations.
Nonetheless, as pointed out by Weber (16), detecting two binding sites
through such an assay requires the ligand to have very different affin-
ity for the two binding sites.

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