Big Problems with ELISA

Thorsten Schmidt Thorsten.Schmidt at rz.ruhr-uni-bochum.de
Fri Jan 24 17:29:14 EST 1997


Dear Reader!

I have big problems with ELISA.
The main problem is that the color does not develop satisfactorally.

I use as second antibody horse-radish peroxidase (HRP) coupled
anti-rabbit antibodies from donkey (Amersham).
To develop the color I use a solution of OPD.
(ortho-phenylenediamin-dihydrochloride)
in 10 ml 0,05 M Citrate pH 5,2: 
+ 40 mg OPD
+ 15 µl 30 % H2O2

Some of my experiments might have shown that H2O2 might be an important
factor for color development.

Just for a test, I add 1 µl of the HRP-antibodies in 1 ml of my
OPD-solution.
This should give a concentration of HRP-antibody which will never be
reached
in an ELISA experiment!
But after 30 min. at room temperature, I got only a extinction at 450 nm
of 0,3 ! ! !
I´ve been told that 3,0 (!!!) in ELISA is not unusual. 

Where is the problem? What can I do?
Why didn´t I get more color development?
Which OPD-solution do you use?

Another problem is that I want to make the ELISA by binding
a polypeptid to the microtiter-plate wells (Maxisorp, Nunc).
But I have the impression that the binding to the plate is extremely poor.
I did it in PBS and 0,2 M Carbonate-Buffer pH 9,3.
What can I do to increase the peptid´s binding?

It would be too kind of you if you´ll send me an answer!

Thank you in advance

Thorsten Schmidt




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