In article <5CAC1671BB5 at rna.bio.mq.edu.au>, Chris Weir
<URL:mailto:cweir at RNA.BIO.MQ.EDU.AU> wrote:
>> Hi all,
> Could someone please be of assistance!
> How do you determine the affinity constant of an antibody, when we
> are unable to purify the antigen (as it is expressed on the surface
> of the cell1)???
>> Thanks in advance
>> Chris Weir
> Macquarie University
>Hi Chris,
It really depends on how accurate you need to know the affinity constant?
For most antibodies I usually refer to the avidity of binding ie the
affinity constant for a whole antibody. The easiest way by far to get a very
reasonable estimate of this constant which works for almost any detection
system is to determine in a titration the concentration of antibody which
gives 50% of maximal binding to antigen. If you then assume that the total
input antibody is very nearly the same as free antibody (which is usually the
case for antibody affinities) the kd is equal to this concentration. If you
wish to express it as ka then just calculate the recipricol value.
To get the single site affinity you could repeat the experiment with Fab
fragments.
Using computer curve fitting it is very easy to determine the 50% value
with a high degree of accuracy and thus I rarely resort to transformations
such as Scatchard Analysis. In fact the biggest error in this process is
the determination of antibody concentration.
Mike Clark, mrc7 at cam.ac.ukhttp://www.path.cam.ac.uk/~mrc7/
--
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