purification of OKT3

Technical Assistance TechAssist at pierce.geis.com
Mon Aug 26 11:25:14 EST 1996


Nathalie Jacobs wrote:
> 
> Hi,
> we try to purify the mouse antibody OKT3 (IgG2a) from the hybridoma
> supernatant. We are using a home made sepharose A column (non HPLC).
> We have several problems:
> -a contamination with bovin IgG (the hybridoma is cultivated with RPMI
> 1640 + 20% SVF. Have somebody already try to cultivate them with
> hybridoma medium without serum?)
> -we don't obtain a lot of antibodies at the pH 4.5 (the pH for IgG2a).
> We obtain higher concentration at pH 5.5(certainly bovin IgG)! We try
> to concentrate the supernatant but everything have been detached at pH
> 5.5
> I would appreciate any suggestion to increase the concentration of
> purified OKT3.
> Thank you in advance.
> Nathalie Jacobs


You could try immobilizing some purified OKT3. That way you would not bind 
anything but your target antibodies.

Tom Brotcke



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