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.
--
Paul J Travers phone : +44-(0)171-631-6862 (office)
ICRF Structural Biology Unit " " " 6868 (lab)
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England or : paul at histo.cryst.bbk.ac.uk
