Affinity, what is it?

Troels Wind wind at biobase.dk
Sat Nov 11 01:21:52 EST 1995


(Sorry about the messy layout, I havent really understood this editor yet)

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?

Troels Wind

Dr. Mike Clark (mrc7 at cam.ac.uk) wrote:
: 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/
: --
:   o/ \\    //            ||  ,_ o   Dr. M.R. Clark, Division of Immunology
:  <\__,\\  //   __o       || /  /\,  Cambridge University, Dept. Pathology
:   ">    ||   _`\<,_    //  \\ \> |  Tennis Court Rd., Cambridge CB2 1QP
:    `    ||  (_)/ (_)  //    \\ \_   Tel. 01223 333705  Fax. 01223 333875




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