Ig and Protein A question

wetsel_r at wums.wustl.edu wetsel_r at wums.wustl.edu
Sun Aug 16 04:59:29 EST 1992


Hello Immuno-Netters:

    I have a question that has been bugging me for quite some time.

    Is there a pH optimum for the binding of Ig to Staph protein-A?  If so, 
does it vary between species of Ig (i.e., goat, rabbit, mouse, human, etc.)

****  Background for my Question  ****

    In grad school and since then, I had always purified MAbs by affinity 
isolation.  Specifically, murine MAbs were purified by column chromatography 
using a rat anti-mouse kappa Ig conjugated to Sepharose CL-4B.  Although 
somewhat time consuming, I can proudly boast that I was able to get 98%+ pure 
Ig as determined by a silver stained SDS-PAGE gel (one of the few things in 
life I ever could brag about! ::grin:: ). 
    Well, one day we obtained the OKT3 MAb from ATCC.  OKT3 is a lambda MAb 
and thus our affinity column could not be used.  My mentor suggested I use a 
Sepharose protein A column, so I did.  I passed the liter and a half of 
supernatant over the column only to recover about 100 ugs (as in 
*micrograms*!)   I went back to my mentor, explained the problem to which he 
quipped, "OH! I forgot to tell you (sound familiar anyone?), do it again but 
bring up the pH of the supernatant to over 8-8.1, it's commonly known that Ig 
binds Staph-A better at higher pH's." - That was news to me! 
    Dumbfounded, I did what he suggested and sure enough I obtained about 2 
milligrams of OKT3 Ig!!!  BTW - the starting pH of the supernatant was about 
7.2-7.3. 

    In my own hands this demonstrated to me that there was, in fact, a pH 
optimum for protein-A binding but I have *never* seen it referenced anywhere.
Does anyone have any comments or could provide a reference?

    A side note: Recently the question posed itself again to me and I had a 
rare strike of inspiration as I decided to conduct side by side 
immunoprecipitations of metabolically labeled complement proteins using immune 
goat serum and protein-A at pH's of 7.4 and 8.2.  I had myself psyched up that 
I'd see a marked qualitative difference between the two pH conditions with the 
higher being the better....  no such luck!  The two pH conditions were 
effectively identical.  I was crest fallen... so much for my pH optimum 
hypothesis... 

    These two pieces of information would suggest that a pH optimum holds for 
murine immunoglobulins but not goat.  Is this a fair conclusion?  Did I re-
invent a commonly known wheel????

    Can anyone shed any light on this mystery?  

    Thanks in advance...

David L. Haviland, Ph.D.
haviland at kids.wustl.edu

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+  David L. Haviland, Ph.D.	     Internet:"haviland at kids.wustl.edu"   +
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