Aggregation of IgG during purification

Mike Clark mrc7 at cam.ac.uk
Wed Jun 28 04:26:41 EST 2000


In article <8jc99o$pfg$1 at nnrp1.deja.com>, Tarmo Humppi
<URL:mailto:tarmo.humppi at pvtt.mil.fi> wrote:
> We have tried to purify polyclonal rabbit antibodies from serum using
> affinity column that has the peptide antigen attached to it. The
> problem is that the eluted IgG aggregates immediately after elution. We
> have used Tris buffer pH 7.5 as starting buffer and glysine pH 3.0 for
> elution. Is there a way to get the aggregated IgG back to solution? We
> have tried increasing ionic strenght but it hasn´t helped. All tips are
> appreciated!
> 

The 'aggregated immunoglobulin' is quite likely to include denatured
protein which is unlikely to completely renature. Firstly do you you need
to get it all back into solution? Have you estimated the activity of the
starting and the eluted material (that is the total activity e.g. titre x
volume not the specific activity e.g. titre) ? Perhaps you have recovered
enough anyway. There will always be some losses in any purification
process. It may also be that you have eluted at too low a pH to start with.
The way low pH works is that you are partially dentaturing the antibody Fab
to elute it. As your antigen is a peptide I would be surprised if the
affinity is so high to require pH3. Try a stepwise elution using pH3.5,
pH3.2, pH3.0 and perhaps finish with pH2.5 to clean up the column. Check
the activity in each eluate after dialysis and pool the appropriate
fractions.


Mike Clark,                        <URL:http://www.path.cam.ac.uk/~mrc7/>
-- 
M.R. Clark, PhD. Division of Immunology
Cambridge University, Dept. Pathology
Tennis Court Rd., Cambridge CB2 1QP
Tel.+44 1223 333705  Fax.+44 1223 333875






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