purification of OKT3

Mike Clark mrc7 at cam.ac.uk
Tue Aug 27 05:39:16 EST 1996


In article <32218B1A.2643 at ulg.ac.be>, Nathalie Jacobs
<mailto:N.Jacobs at ulg.ac.be> 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

Nathalie,
I don't know if you have checked this but one of the earliest lessons
learnt about hybridomas is that during culture you frequently observe
the levels of secreted Ig decline in line with an accumulation of 
chain loss clones. Before trying to produce large quantities of antibody
it is always worth the time to clone the cell line and to check for clones
producing the highest level of antibody. This can be done using an ELISA
and testing the supernatants at several dilutions.
The frequency with which chain loss variants accumulate does depend on the
cell line in question but in my experience mouse X mouse hybridomas should
be cloned frequently because they are less stable than rat x rat.
see Clark & Milstein, Somatic Cell Genetics 7: 657-666 (1981)


 

Mike Clark, mrc7 at cam.ac.uk          http://www.path.cam.ac.uk/~mrc7/
-- 
  o/ \\    //            ||  ,_ o   Dr. M.R. Clark, Division of Immunology
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