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______________________________ Reply Separator _________________________________
Subject: Antibody concentrations
Author: Roederer@Beadle.Stanford.EDU at INTERNET
Date: 19/06/96 13:36
Gerhard Nebe-von-Caron writes:
> The reason for the importance of volume / concentration
> are collision statistics and affinity / avidity of your
> antibody. Therefore you should have approx. 10^3 fold
> molecular excess of your antibody if you want to saturate
> your binding sites within 30 minutes.
Not true:
In a situation of antibody excess, the rate at which a given antigen will
collide with an antibody will depend only on the concentration of the antibody
(i.e., there is no cooperation/inhibition between antigens). Therefore, the
rate at which
antibody binds to antigen is dependent ONLY on the concentration of antibody.
Thus, the antigen concentration is essentially irrelevant.
Of course, when antibody is not in excess, this is no longer true, because the
concentration of free antibody changes significantly as the binding reaction
proceeds. In general, as long as the concentration of free antibody does not
change during
a binding reaction, the concentration of antigen is not relevant. The
difference in binding kinetics between a situation where antibody is in 10-fold
excess and one where Ab is in 100-fold excess is less than 10%.
This is borne out experimentally: many antibodies show identical titration
curves at 5x10^4 cells, 10^6 cells, and 2x10^7 cells (and some even at 10^8).
Indeed, most of these titrations saturate within 5 minutes on ice!
This topic is dealt with in detail in a chapter that Aaron Kantor and I wrote
for the Handbook of Experimental Immunology (fifth edition, due out this
fall)--for many of you who previously downloaded an electronic version of this
chapter, see Figure 3 and its discussion.
mr
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