IgM capture/competitive ELISA
ANDREI.POPOV at bbsrc.ac.uk
Mon Aug 28 08:28:27 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?
I always wandered where this idea of alcaline carbonate buffer came from.
In fact most of the commercially available ELISA plates are have a
negative charge on the their surface, so even theoretically one should look
for a neutral of slightly acidic buffer to absorb proteins with pK
close to 7 or less. In practice I tried several buffers for coating
of purified mABs (IgG) and always found that carbonate buffer worked
much worse than 10 mM phosphate buffer. Any salt (eg, saline) would only
increase the solubility of your IgM.
Another trick: treat your AB breifly (1-5 min) with Glycin buffer
pH 2.2-2.5, neutralise with phosphate and use after dilution for coating.
Fc fragment denatures first and you get orientated coating with Fc fragment
sticking to the plastic and Fab looking upwards.
Bottom line: look for low salt buffer with pH close to the pK of your
protein. IgM is not soluble in low salt buffers when present in high
concentrations, but should by OK at 1-10 mkg/ml.
If still not satisfied try NUNC's MAXISORB- sometimes it makes a difference.
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