In article <travers-1111951107560001 at macmhc.cryst.bbk.ac.uk> , Paul J Travers
<mailto:travers at europa.lif.icnet.uk> wrote:
>> In article <481fe0$bpo at biovax.biobase.dk>, wind at biobase.dk (Troels Wind) wrote:
>>> > Lets assume there cannot be more than one epitope for any of the Abs
> > on the antigen. Furthermore, I am dealing with scFv-fragments, so the
> > Abs are monovalent. Now, what else than affinity could cause a difference
> > in the ELISA signal? Especially if difference is evident with any
> > concentration of Ab?
> >
>> Binding will only reflect affinity if the system is at equilibrium for
> both of the antibodies you are comparing. Otherwise the level of binding
> primarily reflects the on-rate for the Fv. If Fv1 has both an xfold
> faster on-rate and a greater than xfold faster off-rate than Fv2, then an
> initial binding measurement will show Fv1 having higher binding even
> though the affinity will be lower than for Fv2. So how long does it take
> to reach equilibrium in your system? And how long are your incubations?
> I have a recollection that Don Mason did some experiments on the kinetics
> of binding assays in about 1978/79 and concluded that with his monoclonals
> (w3/35 perhaps?) they didn't reach equilibrium till about 18 hours (at
> 4degC). Someone reading this group may have done this more recently - and
> you could always do the experiment yourself with your Fv's.
>> Paul T.
>I agree with Paul's statement but there are other explanations even when
the antibodies are at equilibrium.
You say that your antigen only has one epitope for each antibody? How do
you know that every antigen molecule in your system has the same number
of epitopes for each antibody ie 1:1 and not say 1:2 for example?
You may wonder how this might come about? Well what if your antigen is
partly degraded or denatured in such a way as to lose a greater proportion
of one epitope? Also if you bind your antigen to a solid phase such as
an ELISA plate, are all epitopes randomly available or does the molecule
tend to bind to the plate in one orientation in preference to another?
There are so many different variables involved that what would suprise me
would be to find that two different antibodies or phage antibodes
always gave the same plateau under all circumstances!
Bye the way Paul, I think Don Mason's experiments were with W3/25, W3/13
and W6/32 if I recall correctly.
Mike Clark, mrc7 at cam.ac.ukhttp://www.path.cam.ac.uk/~mrc7/
--
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