stability of hybridoma cell lines - how to produce totally stable hybridoma cel line? (Jul/01/2006 )

I have done two fusions using spleen cells of mice immunized with crude Fasciola hepatica antigen and excretory secretory product of adult Fasciola worms and myeloma cell Px A63, after screening for posistively secreting hybridomas and several cloning and subcloning steps I found that antibody secretion of selected cell lines decreased consistently... I'm so frustrated ... I need to know is there an explanation for this instability of my cell lines? .. is there any solution or advice to avoid this happening again?

I also faced this similar complain from my lab mates abt their hybridomas.. but the fact that for them the activity drops to very low activity sometimes lost totally...In theory the affinity should increase after subculturing because the cells secreting the abs are more selective and would multiply in higher amounts. After several passages of sub-culturing it is possible tht the DNA of the cells may mutate and alter to form not ab secreting cells.. also ... it is possible tht the non-ab secreting cells are higher in population in comparison to the ab secreting ones.. therefore the amount of interfering proteins secreted by the non-ab secreting cells may cause the affinity of the antibodies to drop or masked by them.. I am not sure how u conduct ur sub-culturing but if u are doing sub-culturing by means of equal volumes u can try a dilution pattern if you wnt.. let me know how it goes.. Im also interested to know cause my lab-mates are in a dilemma here..

hi friends,
even i faced the same problem but i could sort out why it is so
perhaps what microkiller has told is right in the sense that there may be mixture of secretors and non-secretors in your hybridoma and with time the non-secretors are outgrowing the secretors.
hence you see a fall inthe O.D.
i tried to solve this problem by doing a 0.1cell/well limiting dilution of my clones in HT medium.
one of the clone which i followed the same way gave only 4% secretors of all the surviving clones
and the 4% clones are stably secreting till now
hope this could solve your problem.
all the best
Leelaram

There are many instances where stability of clones is an issue - but if you are having this issue across a whole panel of Mabs- check your antigen - if you have been storing it at 4 degrees the antigen may be slowly degrading - unless you have a reference positive control that is giving a consistently high OD - I would bet this is your actual issue....