Make sure you heat- inactivate the fbs and then subclone and screen by elisa

Lee, the problem might be that high secretors are dividing slower than low secretors, because producing lots of antibodies is consuming resources (energy, amino acids). So the low secretors are outgrowing the high producers over time. Do you have backup clones you could use to re-start production?

Otherwise, you could try to rescue good cells by plating them in microplates at high dilution (seed about 2 cells per well), regrow clones and screen the supernatants for high titers. If there should be the option to do staining of living cells, you could sort them directly with FACS, of course.

Edit: As a third option, you might rescue the genetic information of your IgMs by PCR and express them in a suitable cell line as secreted proteins (needs some genetic engineering).

hi

IgM Production is more about the nature of your antigen. For example, most of the clones produced against "LPS " will be IgM. usually IgM clones do not have good stability and you will face difficulties in purification and other stages of work.

good luck

Thank you all for your suggestions. In this case we need specific IgM class antibody, so we're stuck with less stable clones. We selected for IgM clones after fusion. I have no doubt the clones are secreting antibody, only that the percentage of secretors has consistently decreased and the saves into liquid nitrogen are often recovered with 0-10% secretor cells. Also the staining for antibody finds patchy fluorescence, instead of the original "whole cell" fluorescence. They are used in co-culture, so we are still hoping to enrich the secretor cells, which probably divide slower than non-secretors. This is the fun of cell culture (problems to solve). Anyone else working with IgM clones?