Affinity, what is it?

[Dr. Mike Clark]

In article <47uqrl$r95 at biovax.biobase.dk> , Troels Wind
<mailto:wind at biobase.dk> wrote:
> 
> Hi all,
> 
> Heres a little theoretical question for you immunologists. Imagine, that two
> monoclonal Abs towards the SAME antigen are available. For simplicity, lets
> assume the antigen is monomeric, i.e. no epitopes exist in duplicate.
> 
> If one uses these two Abs in ELISA towards the antigen under the same 
> conditions (same coating, same concentration of Ab, same detection Abs etc.),
> and one of them gives a higher signal than the other, is it then fair to say 
> that it has a higher affinity than the other? If not, then WHAT is higher or
> better, yhat results in the better ELISA-signals?
> 
> I would really appreciate any input on this!
> 
> Thanks in advance,
> Troels Wind
> PhD-student
> University of Aarhus
> Denmark
There are many variables in the system which could give rise to a
higher apparent binding of one antibody than another.

A rough and ready way to estimate the "avidity" of a monoclonal
antibody is to measure it's immunoglobulin concentration and then
titrate it's binding in any suitable assay. The concentration which
gives 50% maximal binding is the dissociation constant with Molar
units ie the recipricol of the association constant. It is not necessary
for the plateau values of two antibodies to be the same for this
method to work.

So why do plateau values vary? Well if the two antibodies are of
different isotypes, or if there are not an equal number of antigen
epitopes available for binding you would possibly see this effect.

Mike Clark, mrc7 at cam.ac.uk          http://www.path.cam.ac.uk/~mrc7/
--
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