help in ELISA - (Nov/06/2012 )

Helo members ,
i am runing ELISA with normal serum contols and patholoical serums of tuberculosis patients....i am on optimization steps .....but i optimized only antigen concentration (2ug/ml) which i have expressed and purified in my lab.... regarding ELIS i have issue that my OD of normal and abnormal does not show any marked difference ....so that i can decid anthing .....followimg are my settings

antigen dilution 2ug/ml in PBS (PH 7.4), overnight incubation at 4 degrees
plasma dilution 1:100 in plasma dilutin buffer (2%BSA in 0.25%PBST)
secondary antibody dilution 1:3000 goat anti human Ig G conjugated with alkaline phosphatase
substrate p-nitropheny phosphate

i used to was my plates three times after ech step with 0.25%PBST
m also attaching a file which ...that might b helpful

So, from what I understand...you are trying to quantify antibodies specific for a particular protein expressed by TB in serum samples from TB patients. Your results suggest that you don't seen any difference between serum samples derived from individuals without TB, and serum samples from patients with TB.

You don't know if your assay is working, because you have no direct confirmation that your positive control samples (serum from TB patients) actually have the antibodies that you are trying to detect.

From what i can see, your background values for blank serum are very high.

Do you have a positive control serum sample with known binding activity to your antigen?

What is the quality of your detection reagent? Is your BSA IgG free? Does your detection reagent cross react with bovine IgG? If not, your detection reagent may be binding to a component of your blocker and contributing to your overall high ODs.

More complete details of your procedures and what you are trying to achieve may be useful in providing more suggestions

could it be the substrate ? i used to have problem with PNPP. just optimize the last step because the rest looks ok to me.