Antibody contamination during immunoaffinity purification

Bernard Murray bernard at
Tue Aug 20 17:26:20 EST 1996

In article <4vbog8$di6 at>, dykim at says...

>Hello, I need some help  
>I purified the connexin 43 using polyclonal antibodies
>which were crosslinked to Sepharose CN4B. My problem is that the eluted
>connexin 43 contains the antibodies. I suspect that this antibodies were
>dissociated during the acidic elution steps(pH 2.4). In the case of using
>monoclonal antibody, no antibody contaminat were shown. I want to 
> prevent the dissociation of antibodies during elution steps. 
>Is there anyone to meet similiar problems like me?  Any suggestions will
>be appriciated.              

Why can't you simply use the monoclonal antibody? - just curious.

Are you sure that it is really immunoglobulin that is being eluted
from the polyclonal antibody column?  If so I have some suggestions;

I assume that the antibody was linked to the Sepharose using CNBr.  If
this is indeed the case I can only suggest that the Sepharose was not
washed sufficiently after the procedure.  I've used such resins even years
after they were made and never seen any elution of the antibody even at
pH 2.0  I suggest that you wash your immobilised antibody with elution
buffer then return it to the start condition before binding the antigen.
If the elution buffer contains reducing agents (mercaptoethanol or DTT
etc.) as well as strong denaturants you could be dissociating the
antibody into its component chains - usually only 1/4 chains is immobilised
so the other three would be "eluted".  The only way to prevent this would
be to change the buffer (leave out the reducing agents).
	As a rescue procedure I can only suggest back-absorbing your
partially purified extract with either protein-A, protein-G or immobilised
anti (H+L)-Ig.  This should remove the contaminating antibody.
	Lastly, if you did not couple your antibody to the Sepharose with
CNBr I strongly suggest trying this as the stability is much greater than
that of binding to protein-A.  Alternatively, there have been posts in the
past which indicated that it is possible to bind antibody to protein A
agarose/Sepharose and then treat the complex with a protein cross-linking
agent to more permanently immobilise the complex.
	Good luck,

Bernard Murray, Ph.D.
bernard at  (National Cancer Institute, NIH, Bethesda MD, USA)

More information about the Methods mailing list