Adrian, had the same problem with a murine IgG1. You will need to go through a lengthy
trouble shooting procedure, and may find no improvement. First check actual yeild of
antibody, you may find you are getting less than expected because of high affinity
for protein G, or due to ppt in column. Try alternative elution at pH 10, with water
(neutral pH), MgCl2. Any more chaotropic and you will probably not get any usable
antibody. The other alterantive is to use ionic chromatography, but you'll need to
concentrate your antibody by NH4 ppt, and I found that wasn't helpful as the mIgG
still ppt.
All I can say is good luck. I ended up using mine as was in tissue culture medium.
Craig
----------
From: A.Smith@centenary.usyd.edu.AU
To: cyto-inbox
Subject: Problems with monoclonal antibodies
Date: Wednesday, August 30, 2000 20:58
Hi all,
We have been having some problems with some of our
monoclonals and would appreciate any advice or comments...
Bascially we have two problems which may be related: after
purification on Protein A or G and elution (glycine pH2.5 then tris
pH8 neutralisation and dialysis against PBS), the antibody either
precipitates spontaneously at 4 degrees, or the biotinylated
conjugates rapidly lose activity (which may also be due to
precipitation - we haven't checked yet). We suspect low levels of
acid denaturation may cause ongoing spontaneous formation of
aggregates. Does anyone have any experience of this and how to get
around it (most mAbs are fine after purification this way and only
some mAbs seem to be affected)?
Thanks,
Adrian
--
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Adrian Smith (PhD Student) T CELL BIOLOGY GROUP
Centenary Institute of Cancer Medicine & Cell Biology
Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA.
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