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Sandwich ELISA Troubleshooting

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Hello all,

Hopefully someone might have some words of wisdom for me about my problem. 

I am trying to develop a sandwich ELISA assay for antigen detection purposes.  I will give the protocol that I have been using to try to get it to work at this point:

1. Coat 1ug/well of capture antibody (this antibody has been purified from serum using a specific N-terminus peptide sequence that is unique to the pathogen I am studying) with commercial carbonate/bicarbonate (pH 9.6)
2. Incubate 2 hours at 4 degrees celsius
3. Wash (using pipettor) each well 3 times with 200ul of PBS with 0.1% Tween20 (pH 7.4).
4. Add 200ul of blocking buffer (PBS-Tween20 0.1% + 1% BSA (pH 7.4)) to each well.
5. Incubate 2 hours at 4 degrees celsius
6. Wash (using pipettor) each well 3 times with 200ul of PBS with 0.1% Tween20
7. Add 1ug of recombinant protein of interest to each well (the protein is diluted in blocking buffer) 
8. Incubate 2 hours at 4 degrees celsius
9. Wash (using pipettor) each well 3 times with 200ul of PBS with 0.1% Tween20.
10. Add 1ug/well of commercial polyclonal antibody (conjugated to HRP) recognizing a specific sequence on the C-terminus (this antibody is prepared in blocking buffer as well)
11. Incubate 2 hours at 4 degrees celsius
12. Wash (using pipettor) each well 5 times with 200ul of PBS with 0.1% Tween20.
13. Add 100ul per well of TMB substrate
14. Wait to see color change.

The problem is that there is never any color change in my sandwich ELISA. I have tried the 2 hour incubations at room temp, incubating overnight at 4 degrees celcius, incubating at 37 degrees celcius without any success. My buffers are all at the correct pH. I am using concentrations that should be sufficient for the color change to occur. etc.

I know that these components intereact because I have run the recombinent protein on a gel and transfered to a membrane for western blotting. I have probed the membrane with both the capture and, at another time, with the detection antibodies. They were amazingly well on the western blot. 

I have also done direct ELISAs where I coated the recombinant protein in the wells of a 96 well plate, then added the capture antibody, followed by a secondary antibody with an HRP. This works well.

Likewise, I have coated with recombinent protein and added the commercial detection antibody that is already conjugated to an HRP. That also worked very well.

It seems like all the components interact, and everything works very well except when I try to do the full snadwich ELISA. Any ideas or advise for troubleshooting that you can give me would be very much appreciated!
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