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
