In article <3896D799.237755F1 at gengenp.rug.ac.be>, Esbjorn Fiers
<URL:mailto:esfie at gengenp.rug.ac.be> wrote:
> Dear all,
> I am working with scFv phage display libraries. In some articles I come
> across the words on- and off-rate, being the rate at which an antibody
> binds its antigen and the rate at which an antibody dissociates from its
> antigen. Furthermore, they say that when antibody affinities become
> higher this is mostly due to a lower off-rate, with the on-rate being
> less important. I kind of have problems to completely understand this.
> Is there anybody who could explain this to me, or has anyone a reference
> in which this is clearly explained?
> Thanks!
>> Esbjorn Fiers
>
I don't know if it will help but I can give you a URL to an acrobat file to
download from my website. The file is some lecture notes on Ab/Ag affinity
which I use for my undergraduate teaching.
The URL is
<http://www.path.cam.ac.uk/~mrc7/affinity/abaffinity.pdf>
There are a couple of points I would like to add.
In text books and papers you frequently read that monoclonal antibodies
usually have affinities of 100 mM or above. However you can turn this
around and ask the following question.
Most hybridomas in the early stages of a fusion make antibodies at
concentrations of 0.001 mg/ml or less. Thus what affinity would an antibody
have to have in order to detect it's binding at that level? You could do
the same thing for concentration of Fv from a phage although I'm not
familiar with the usual observed ranges.
Regarding the on and off rates of antibodies. How many people screen their
monoclonal antibodies after incubating with antigen for a few seconds or
alternatively for a few days? Most researchers use incubation times of
hours followed by wash times of minutes. Now plug in the concentration you
expect the antibodies to be at in the assay and you can estimate what the
range of affinities and kinetic rates you expect to get. Not surprisingly
it tends to agree with what people find!
It's kind of obvious but it's not the way most researchers think of the
problem.
If you want to find antibodies with 'unusual' kinetics what you have to do
is change the conditions of the selection procedure you use for screening
and isolation.
Mike <http://www.path.cam.ac.uk/~mrc7/>
--
o/ \\ // || ,_ o M.R. Clark, PhD. Division of Immunology
<\__,\\ // __o || / /\, Cambridge University, Dept. Pathology
"> || _`\<,_ // \\ \> | Tennis Court Rd., Cambridge CB2 1QP
` || (_)/ (_) // \\ \_ Tel.+44 1223 333705 Fax.+44 1223 333875
