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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