I am trying to do indirect ELISA to detect a specific antibody in mouse serum from this group of mice we immunized. During ELISA, even for the plate without any antigen coating, after we added mouse serum (1:100 dilution), it gives a high background.
We suspected that it was the blocking buffer (2%BSA in PBS) that wasn't doing a good job in blocking the wells, so we tried different blocking buffers such as (2%BSA + tween 20 0.05%, tween 20% 0.05% in water, super block solution...) for overnight blocking, yet they all gave high background values.
What could be the cause for the high background?
Perhaps your secondary antibody step is the problem. Maybe the concentration is too high, or the washing is insufficient.
Hi Zhilin, try immunodepleted anti-mouse secondaries from Jax labs.
Hi Zhilin,
There are some questions I thought:
(1) What's your immunogen? Is that BSA-conjugated protein or compound? Is it possible a part of induced mouse antibodies recognize BSA in your blocking solution? How about dilute mouse serum by blocking buffer to subtract anti-BSA antibody?
(2) What do you mean about the indirect ELISA? Is it sandwich ELISA or others? Is the capture antibody you used not cross-reacted by secondary antibody?
(3) As Dr. Adam B Shapiro says, perhaps secondary antibody is a problem.
Good luck,
Thanks for all the answers!
Weber Lin, thanks for your questions! The immunogen is a small peptide, and it is not related to BSA, presumably. Therefore, we are assuming the mice would not generate anti-BSA antibody. It is sandwich ELISA we are doing here, first peptide coating on plate, then use mouse serum, then use 2nd antibody- HRP for mouse IgG, finally use TMB substrate.
Hi Zhilin, coating small peptides onto plastic will always cause problems because coating small peptides onto plastic is not very efficient and plastic coated peptides may not expose the epitopes.
I am assuming you are dealing with polyclonal Abs?
If so, you can get around this by purifying your Ab (by protein A or G).
Use ~2-5 ug/ml of that antibody to coat wells to capture the peptide.
Label some of the same Ab with HRP (or AP) and use that as the secondary. If it is a poly, they will have different epitopes.
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