separat. hi-low affin Ab

haoxiao at CC.UManitoba.CA haoxiao at CC.UManitoba.CA
Sun Sep 4 03:41:34 EST 1994


Approach one:
Try to exploit the kinetic behavior of Ab-Ag reactions involved polyclonal 
antibodies.The hi-affinity population of Ab should react faster with Ag. 
eg: t1/2 for Ab1=5 min.  t1/2 for Ab2=30 min.
In a period of 10 min. 75% of Ab1 should have bound to Ag (immobilized)
while the majority of Ab2 remain free (not bound to Ag). 
So, you can selectively separate the polyclonal antibodies by 

a)	control the incubation time in a ELISA.

b)	control the flow rate in a affinity chromatography.

Problem: a hi-affinity Ab may not be neccessary to be the fast reacted
one (but it usually does). Since the total (intrinsic) energy can divided
into energy for reaction, energy for stabilzation of Ab-Ag complex (affinity).

Approach two:
Gradient chaotropic detachment of bound Ab (pH 4.5 to 2, salt conc. low to
high, %organic sohlvent low to high). Low affin Ab should be more readily
detached than the high affin one. 




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