purification of OKT3

KLAUS mmm_klehnert at mednov1.aucland.ac.nz
Mon Aug 26 17:21:12 EST 1996


In article ,  says...
>
>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


Natalie,

We and the friends of mine had exactly the same problem: couldn't get reasonable 
amounts of murine mAb from the OKT3 hybridoma (maximum of approximately 
50micrograms/L) while there was plenty of bovine Abs. At the end, we found that the 
hybridoma didn't secrete very well, and we screened about 9 cell stocks from 
different sources. Only two produced decent amounts, and of those two, only one could 
stimulate T cell proliferation. Take home lesson is: Before trying to optimize your 
purififcation, make sure there is enough OKT3 present in your supernatant (eg. by 
ELISA with anti-mouse Ab, or by dot-blotting). I found that once there is enough mAb 
(any mAb, not only OKT3) in the supernatant, the bovine Ig and BSA-contaminations 
will be reduced drastically in the purified sample, even if we still way below the 
column capacity (we use perfusion chromatography with columns having a capacity of 
20mg mu IgG1 and at least 8mg muIgG2a. I believe the reduced contaminations are due 
to the dynamics in the column). However, note that OKT3 is one of the bad secreting 
hybridomas (We never got more than about 3mg/L, even in high-density cultivations), 
so it may be a good idea to produce some ascites (cruel, I know...)

Hope this helps, good luck

KLAUS






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