monoclonal antibody production - cell death in few wells (Aug/05/2006 )
Hi everybody,
I am producing monoclonal antibodies against a mycobacterial antigen. During the course of my work, after fusing the cells with PEG and aliquoting into 96 well plates, in the initial screening by ELISA, i observed that few of the wells were showing very high O.D. but to my surprise there were no cells at all. When I collected the supernatants of such kind of wells only and added to the normal myeloma cells which was used as a fusion partner, all the cells died after 2-3 days. I just wanted to know whether this kind of behaviour is common in monoclonal antibody production or specific only to my protein?
I will be very grateful to know your kind suggestions
Thanking you in advance
Leelaram
Ok.. i am not sure at which stage of work u are in.. but a high od without any cells visible could be contaminants... are u sure tht ur well is not contaminated.. Does the media in the well is yellow in color with a cloudy look.. if so then when u observe under microscope.. do you see like debris like.. look closer.. u may see contaminants.. the high OD could be due to interferences frm contaminants.... if u are screening using ELISA.. Anyway i hope tht ur wells are not contaminated.. but if they are.. better quickly wash them out.. best of luck..!!! 
Hi Microkiller,
Thank you for your for you suggestion. Ofcourse, it is the first doubt whether the wells were contaminated but the supernatants collected from those wells when i chekced on myeloma cells, it showed death of the cells and they look more like apoptotic bodies. The supernatants i checked by ELISA and showed very high O.D. but they also showed a nice specific band to the antigen on western too. So I don't think there is any contamination in the wells. But, in the rest of the wells i got few clones which are secreting. Hence, I am skeptical about the death of the cells in few wells.
Hoping an early reply
Thank you once again
Leelaram