IgM capture/competitive ELISA

RATKITY at ktc.com RATKITY at ktc.com
Wed Aug 30 15:10:23 EST 1995

> >I have a panel of IgM monoclonal antibodies which I have been trying
> >to use in a competitive capture ELISA as a lead-in to epitope mapping.
> >However, at least half of these antibodies do not capture antibody when
> >they are coated onto a flexible 96-well ELISA plate (I think that they
> >are probably not coating properly).  I have tried pH 9.0 borate saline
> >and two separate carbonate buffers as my coating buffer without success.
> >Does anyone have any suggestions for a coating buffer or system which
> >may be more successful with IgMs?
> >
> >Thanks,
> >
> >David B.

Dear David,

Don't forget that an IgM molecule has more steric hinderance than
an IgG molecule.  Keep that in mind when formulating protein concentration
in your coating buffer.  Some methods that have proven successful is 
pre-treating the plate with gluteraldehyde before coating (if you're 
interested, e-mail me and I'll get you the ref).  Also, a coating buffer
of 0.1 M phosphate buffer, pH 6.5 works well for peptides.  The lower pH
shouldn't be a pbm with your antibody.  I have also found the NUNC plates
to be superior for antigen binding.

Patricia Beetham
ratkity at ktc.com or a03pbeetham at attmail.com

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