polyconal ab 95%+ pure

Pfaust pfaust at aol.com
Fri Jan 20 20:29:04 EST 1995


The antibody is a rabbit anti- estradiol-B-6-BSA conjugate. I have
purified it on protein A.  In the next step I further purify it on an
analog to estradiol, one that it cross reacts with at about 1%.. The
analog is NHS activated then coupled  to BGG. And then coupled to CPG (
contorted pour glass). This is what I use to affinity purify the anti E2
AB. The elution buffer is 10uM estron also a cross reactant. After elution
I then dialyze it against PBS to remove the estron from the AB. I then
label the AB for use as a tracer in a noncompetitive duel solid phase
assay for estradiol.  If the AB tracer is not 95%+  pure the back round is
way to high for the assay to work. The best iv been able to do is 87%
pure. Even trying to do two purification the % activity dose not
increases. What I have discovered is that there is about 1800 pg/ml of
endogenous estradiol in the AB after the protein A. This I’m assuming is
bound to the AB in the polyconal with the highest affinity. . I am now in
the process of attempting to remove the endogenous estradiol hopefully
this will solve my problem. What I would do for a good monoclonal AB their
a peice of cake to get to 95% pure too bad all the Mab i'v screened are
way to cross reactive to use. If you can think of anything that might work
I would be more then happy to try it. 

Thanks. peter faust



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