Hi netters,
I'm currently attempting to develop a quick assay for determinetaion of
monclonal antibody binding/affinity constants for some molecular
stability testing. I'm using a monoclonal, IgG1, and a rather precious
antigen.
I've read some of the papers on the topic and have been playing with the
calculations from Friguet, et.al, who wrote a neat paper on a quick ELISA
method (which I am partial to) and a later paper by Stevens regarding the
need to add valency considerations to the calculations.
So, my question is:
Part of the assay involves a preincubation step so that the antigen and
antibody can reach an equilibrium in solution:
Ag + Ab <-> AgAb
and that you can determine the binding constant using Scatchard or double
reciprocal analysis.
Are the antibody sites considered to be equivalent? If so, can one make
the assumption that at equilibrium 1/2 of the binding sites are tied up
with antigen? And, if this is the case, do valency calculations
necessarily need to be included to determine the K(binding)?
Thanks in advance for the help...please E-mail to
hydex004 at gold.tc.umn.edu to save bandwidth.
Fred Hyde
