class switching M hybridomas

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Wed Oct 25 19:39:47 EST 1995


>Hi all,
>       does anybody out there have experience of class switching M
>hybridomas to G. I don't have access to a FACS for sorting, so it
>would have to be done manually by cloning. I have heard of people
>doing it by this method and wondered how feasible it really was.
>Also I know that highly specific isotyping reagents would be needed
>to identify the class-switched clones. Many of the reagents we have
>looked at across the years give some cross reactivity with some
>other isotypes, does anybody know of a good source?
>
>
>
>Thanks
>
>Rob

Hi Rob,

you can try as well a plaque-forming assay in agar
(look at some of M.Shulman's papers on homologous recombination
within IgM locus, you can probably do the same with IgG)
Several years ago (1991-2-3?) in the J Immun Methods was a paper on detection
of specific clones after embedding cells into agar, making a n/c
replica and probing the filter with Abs. If I do not mind one of the authors
was C.Milstein.
We use a lot of Abs from The Binding Site and Amersham, all of the so-called
absorbed antisera against human IgM are cross-reactive with mouse IgM,
but I had no problems with IgM/IgG descrimination/background when
using absorbed sera.

           
Best wishes

Andrei Popov





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