monoclonal antibody - use of HT (Aug/05/2006 )
I am producing monoclonal antibodies to a mycobacterial protein. In the course of my work i came to know that HT medium should be maintained until the single cell cloning stage and removing HT even before single cell cloning could cause loss of antibody secretion. Why does removing HT from the medium cause loss of antibody secretion?
Any suggestions are grately welcomed
I am not sure wht u mean by keeping HT until single cell cloning stage.. or which protocol u are refering to.. I work with monoclones too but i normally change my HT medium to HAT the day after fusion and keep it tht way until some degree of selection.. If i am not wrong.. the aminopterin will work to allow the survival of hybridomas only.. it will work to stop the mechanism for normal cell growth.. so therefore if u keep HT medium before selection this would allow the non-mab secreting cells which are cells that was not fused (non-hybridomas) to grow and may over grow the hybrids. I cant remember the framework of the entire cell cycle.. but i hope the explanation is suffice..
It's me again. Thank you for your nice advice.
The question I asked was whether HT medium is required until the limiting dilution step.
I use HAT medium during the selection stage until one week after fusion and aliquoting into wells.
Later feedings are done with HT medium to dilute off the aminopterin which could be toxic to cells at high concentrations. This protocol is followed routinely in many labs.
But the question haunting me is when I removed the HT medium before limiting dilution of the clones, the cells stopped secreting. Why is this so?
Thanking you in advance and hoping an early reply