antibodies from hybridoma - (Mar/02/2007 )

I am hoping someone here can offer a tip?

I am going to attempt purification of mAb from a hybridoma line, using supernatant. I know I want late-log-phase to ensure a good yield, but how late? should the cells be stressed and croaking, or just at very high numbers in the flask? what is a good point at which to do the harvest?

thanks to anyone who can help me

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When i'm purifying antibodies from hybridomas, i dont grow in flasks, i do a roller bottle culture. I add about 1 flasks worth of cell culture in a roller bottle containing about 800 mls of medium and let it grow for upto a week max . At this point you'd have some cell death i would think, but you would also have a good yeild of antibody. If your hybridoma is a really slow growing one you could also leave it for maybe 3-4 more days.

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The highest possible numbers of cells with minimal cell death (<10% cell death)

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May be it will be useful to estimate viability by trypan Blue to get some experience. Persons who highly skilled in this technology can estimate this visually or intuitively

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see this one hybridoma cell viability test

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circlepoint, I know how to assess viability I am all over that one

I just wanted a general guideline for which phase of growth was the best for ab production...thank you very much, Minnie

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hi,
when you grow cells in flask for several days, i m not qute sure how u can decide end log phase?
how about increasing the serum % with the time (over incubation period), so that cells will have enough nutrients and can secret antibodies till the end of the week (if you grow cells for 1 week).

i hope this hint helps!
payeli

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