I am having a problem with ELISA. I am using homemade ELISA to detect a protein. I started with checker board titration to find out the optimal concentration. However, I am finding a similar OD values in wells with or without antigen. I have attached a sheet with my results as well as the procedure. Please suggest.
Thanks
Gaju
Hi,
There are three main probable issue for colored blanks: 1) either cross reactivity exists between the capture and the revealing antibodies, 2) the blocking step is not as thorough as it should or there is some kind of cross reactivity between the revealing antibody and the blocking solution, 3) or the washing steps aren't thorough enough. I don't have all the details on the antibodies used (reactivity, polyclonal or monoclonal...) but from what I saw on the sheet it is likely that your inespecificities are linked to the blocking step. Why using 1% BSA in serum?
For troubleshooting I would recommend for you to try with a 5% BSA disolved in coating buffer NOT serum. You can also try blocking with milk to compare.
I would also strongly recommend adding PBS instead of BSA-serum as blank. I'm not aware of the nature of the samples, but even if in some assays they may coincide, there are differences between a negative control (a true negative sample e.g. negative serum if you are doing a serological test) and the blanks (checking for inespecificities within the reagents of the system).
Good luck!
Check if you are using polyclonal abs instead of monoclonal abs. If you are using polyclonal abs, there might be color development in blank wells due to background noises. In this case, you have to use monoclonal abs to clear the background noises.
As mentioned above, washing and blocking steps are also important.
Thanks for your suggestion. Just to correct myself, 1% BSA in PBST not serum. Both capture and detection antibodies are monoclonal. We wash our plate 3 times with 280 ul of PBST/well.
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