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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
cfp 10 with 2% plasma diluting buffer 31-10-12.xlsx 9.89K
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