On 24 Jan 1997 22:29:14 GMT, "Thorsten Schmidt"
<Thorsten.Schmidt at rz.ruhr-uni-bochum.de> wrote:
>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
>Dear Torsten
If you do not get any enzymatic reaction by adding the HRP conjugate
directly I think that the peroxidase must be inactive or inactivated:
1)Check for the expirery date
2) Did you use any Sodium Azide for buffer preparation???- NaN3 is a
strong inhibitor of peroxidase(as well as cyanide) and therefore must
not be added to any buffer used for ELISA.
3) Peptide binding:
The buffer you use seems to be o.k.-if your peptide is very acidic it
might be that the binding is still poor-but this is not very probable
to my mind.
4) I read OD at 490nm and use only 4mg OPD/10ml citrate buffer
Hope this helps
martin
