problem with an ELISA
jmcvicke at midlandbio.com
Thu Jan 13 19:59:12 EST 2000
High background absorbance may not be due any "clutter" from the plasma
sample. It is possible that you are experiencing non-specific binding of
either your primary or secondary antibody and the milk that you use for
blocking. I would suggest the following:
1. Change your blocking buffer. Try using a 1% fish gelatin solution.
2. Include the milk in the diluent that you use for the primary and
secondary antibody. After you make the dilutions, let them mix for around
15 minutes so that if there is non-specific binding it will occur in the
mixing tube prior to applying to the plate. This of course will reduce your
overall signal, so you may need to change the dilutions of the antibodies.
3. Reduce the concentration of the milk that you use for block. This would
need to be titrated.
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