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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunology forum and cytometry forum
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Hybridoma stability
Posted by: wouter bruin (IP Hidden, Unregistered user, )
Date: February 11, 2005 03:57AM
Hello,
Can somebody please give me some advise about how to keep my hybridoma stabile?? I`m doing fusions with sp2/0 to make a hybridoma! If i test the fusion there are ussually 5 clones positive (pretty low but I cann`t make it more). These Primairy clone are directly subclone and then after 7-10 tested again but then it has lost all it`s abbilaty to produce the right antibody!!! How is that possible and can somebody help me with this problem?? Many Thanks already Wouter Bruin
Re: Hybridoma stability
Posted by: izolda (IP Hidden, Unregistered user, )
Date: February 16, 2005 04:14PM
To: Wouter Bruin
Hi, I hope it will help. First, find the best FBS (try Fetal Bovine Serum Certified, Gibco, Cat # 16000-044). It is the most important thing to get your hybrids to grow healthy and happy, and secrete Ig. Second, pay more attention to changing culture medium before primary screening - do it twice to avoid false positive signal that is the result of remaining B cells secreting the same Ab (if you leave even 10-20ul of medium in a well). Check early the possible overgrowth - at this stage the hybrids very much sensitive, if the growth conditions becomes a bit tense the hybrids will lose the Ig producing chromosome. Third, be sure of the optimal conditions of your screening technique (Ag, plates and coating buffer (if ELISA), secondary antibody, make titration of your antigen. If possible coat the plates with an excess of Ag - you might be underestimated the hybridoma activity. Good luck
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