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ELISA not working - (Aug/07/2009 )

I am having troubles developing a sandwich ELISA for a secreted extracellular protein, size 262 kDa, for human serum and other bodily fluids.

I coat with a Chicken pAb and detect with rabbit pAb. I see very high background with an OD of around 0.4 in the blanks and the standard curve seems to saturate with an OD over the entire curve of 1.6-1.5. The samples vary from 1.5 to 0.6 depending on the dilution.

My protocol is:
Coat at 4 ug/ml in 10mM carbonate buffer, pH 9.6 overnight at 4C
Wash 4 times with 250ul of PBS w/ Tween20 0.05%
Block with 3% BSA in PBS, 2hours, RT
Wash 5 times 250ul PBST
Add samples/standard diluted in 1% BSA
Wash 7 times 250ul PBST
Add biotinylated detection Ab diluted in 1% BSA
Wash 7 times 250ul PBST
Add strep-HRP diluted in 1% BSA
Wash 9 times 300ul PBST
Detect with TMB, stop with H2SO4

I use an automatic plate washer and I have cleaned the entire system and made all new buffers in case their was contamination. The Abs do not contain sodium azide so as to interfere with the HRP. This is a very similar protocol to what I used in a previous lab with great results. Maybe its the antibodies?
Any suggestions would be most helpful! Thank you in advance!

-alexanam-

What concentrations of detecting antibody and step-HRP are you using? You might want to play (titrate down) with these variables to cut down on your variable

Jay
CellSpecific
www.cellspecific.com

-Jay Dela Cruz PhD-