Hello, Flow-ers! Thank you all so much for the responses I got regarding this question (see below) > I have a question that is perhaps a bit naive, but here goes...I have > some locally produced antibodies that are still in the spent > supernatant from hybridoma cells. My question is this, are all bets > off for the antibody and it's specificity if there has been some > growth, such as fungus, in the sup? My gut level feeling is that the > antibody is no longer useful. There were essentially two camps, can't trust the antibody and it might be able to be saved IF... But almost EVERYONE said, "don't use it on cells." Here is the compilation of the answers, (I include the e-mail address). Once again, thanks! simon.hunt@path.ox.ac.uk .It's 50:50, I'd say. Flip your dime, Nancy - or, better, just do a quick proper check. With 10 mM sodium azide your sups would have been as good as the day they were made, simply kept at 4 deg C. We've just re-assayed some 1987-vintage spent hybridoma culture sups preserved in this way, and they're fine (they're mostly mouse IgG1s). BARVP@msn.com How could you trust it rozenkov@netscape.net ust to put the actions in order, if this sup is really valuable and non-replaceable (and depending on what "some growth" means, as if the sup is "cloudy" then those billions of fungus may have absorbed all antibodies), you can try the following: 1. Filter the sup through a 0.2uM filter. Now it is sterile. 2. Check for antibody staining of desired specificity. Pay attention also to the level of antibody staining (if you have any previous data to compare to), as antibody concentration may be reduced, see whether you are happy with that concentration. Make sure also that the staining is specific. 3. If antibodies work, keep them further with 0.05-0.1% Sodium Azide dhewish@unimelb.edu.au You would have to be lucky. The specificity of the antibodies wouldn't be affected but the titre could be low if the bugs produced a protease. If the contamination was only at low level the serum proteins in the culture medium might have saved the immunoglobulin. Check the titre and purify the antibody ASAP if it still looks OK, maybe add a protease inhibitor cocktail. stephen.kwok@tufts.edu I would not use it if it was an important experiment. I've had some mixed success in the past when the antibody was not easy to make or readily available with mixed results. a.w.heath@sheffield.ac.uk This is mainly just anecdotal, but in my experience antibody is very resistant to destruction by proteases etc. As an example we once fed fleas with rabbit serum containing a specific antibody, using an artificial feeding system. When the dried flea feces were reconstituted in PBS, there was still a strong, specific, antibody signal in ELISA. So I'd give it a try, but of course if you're using it for in vitro stimulations etc remember there may be a load of LPS and similar nasty stuff in there. arubio@theranostech.com I purify antibodies all the time, sometimes under non-ideal circumstances, with great results. My recommendation, is to filter the supernatant through a sterile 0.22um filter, prefferably the bottletop variety. Add whatever preservative is appropriate for your use. Test the supernatant as it is in an indirect ELISA. If you get good results go ahead an purify it. My "gut" tells me that the fungus was probably living on media components and not necessarily on antibodies. But, freeze it as much of it as you can to prevent any proteases from acting on it. mmaciel@usp.br would say you have to test... try to concentrate or purify... I tend to believe the fungus there did not eat all the antibody... Use it with a control in paralel, of course! PS_ I would not use for in vivo application, though! jldow@unity.ncsu.edu I have a great deal of experience growing hybridomas and growing fungus in the supernatant. There is a good chance your antibodies are okay-the best thing to do is filter sterilize the sup and test it again. ken.mcdonald@mcgill.ca I don't think you want anything that's contaminated around your cells. Also with any results you get, you'll never know if they have been influenced by contaminated Abs. Best to work with a clean, reliable batch....Ken RJasman@ICOS.com I would give them a try. I have used precious antibodies in the past even after they had fuzzies growing in them and they worked fine. In your case, if these are not well characterized mAbs, then if they are negative, you won't necessarily know if it's a result of the mAb not binding the cells, or that the mAb is degraded from the contamination.Received on Thu Aug 24 14:38:00 2006
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