>>I think that maybe I have misled people a bit with my first message.
>Which after reading it again probably isn't all that clear. What I am
>after is are buffers that will increase the signal : noise ratio in
the
>actual ELISA. My actual Alkaline Phosphatase conjugates Pabs or mabs
are
>quite stable enough. I don't really have any problems, I'm just
trying
>to increase the sensitivity of the ELISA. By changing the buffer I
>dilute my conjugates in, I have managed a 1.5 - 2 times increase in
>senstivity. Not spectacular I know but it gives me the encouragement
to go
>on and find better buffers if they exist. What I'd really like is a
well
>tested commercial buffer, that I can use to see how my various
mixtures
>compare.
> Thanks
> David
>
I really don't know much about commercial diluents. There are quite
a few out there. I've always made my own. I do know that the source
and purity of stabilizing protein can have a great effect on S/N.
You might want to try using fatty acid free BSA, or fetal calf serum
in your diluent. Concentration and variations from lot to lot could
have an effect. Variations in buffer ionic strength and pH could also
have an effect.
I guess a commercial supplier may be the best choice if you don't want
to worry about variations. You may want to try Kirkengard and Perry
Labs, Pierce Chemical, BioRad, BioWhittaker, Sigma BioScience, to name
just a few. There is also a new biotechnology suppliers Web site at
http:/www.iscpubs.com. However, even commercial buffers may differ
depending on your antibody-antigen interactions and kinetics. Most
suppliers are willing to provide samples for evaluation without charge
(if you're kind to the rep and say please).
Another possibility is changing washing buffers. Is non-specific
binding a problem that reduces sensitivity? If so, you may want to
try increasing wash cycle times, increase NaCl concentration, or try
an alternative surfactant. These all are interdependant, so be
careful. Also, have you thought about going to an avidin-biotin
conjugate. This can significantly increase sensitivity.
This is all I can really say without more specific information and
hard data. Please E-Mail me if you have any questions.
Best regards,
Les Confer
lconfer at moose.erie.net
