hi ~ I am working with protein A ELISA, but there is no commercial ELISA kits available. So I am making the kit by myself. I need to measure the amount of this Protein A which is secreted into supernatant by cells. Then I will use this supernatant as conditioned medium to do migration assay.
This is what I did:
1. coat 96-well plate with coating buffer + recombinant protein A for standard curve. which works perfectly.
2. coat sample wells with supernatant from culture medium (DMEM+10%FBS).
3. Add primary antibody for protein A.
4. Add secondary antibody.
5. TEM --read with plate reader.
The standard curve I got was really nice. But I barely detected any protein A in my samples. I think it is because of the FBS in the supernatant interfering with protein A. So I used DMEM+10%FBS as coating buffer, the OD value I got was really dim.
So in this case, what is the best way to measure protein A in the supernatant? Thank you in advance!
Dear Yan, do you mean Protein A from Staph. aureus? I'd assume that it will bind the IgG from the FBS. Can you resort to serum free medium or at least deplete your FBS from IgG (e.g. by passing it through a protein G column) ?
If "Protein A" is referring to your protein of interest, when you try to coat a microplate with a TC supernatant that has 10% FBS, you'll saturate your plates with the abundant serum proteins (BSA and IgG mostly), not much space is left for your protein of interest when it is there in only small amounts.
You need a sandwich ELISA instead: If your antibody is polyclonal, coat your plate with the antibody, block it with PBST/BSA (or FBS) and then add your supernatant. Detect with the same polyclonal (but e.g. biotinylated) and the a streptavidin conjugate (HRP etc) . If you have two different peptide antibodies or two monoclonals, you also may use them, too, of course.
The impact of the FBS will depend on the batch and how much Protein A your cells are producing. I would think it highly likely that endogenous IgG would bind to a great deal of what your cells produce. Whether this would render it unavailable to your primary antibody is another question. You should be able to check all this by comparing a standard curve or Protein A made up in DMEM alone versus one made in DMEM + 10% FBS. If the latter is indeed the problem, (rather than just very low production levels), try either FBS that has been stripped of IgG, (maybe at 1-3%), or better still DMEM alone or with Img/ml BSA just for the production period.
I would agree with Wolfgang, a sandwich ELISA is better choice. Moreover, try to use serum-reduced medium if it is possible.
Protein A ELISA kit is commercially available:
http://www.novateinbio.com/en/home/1156829-protein-a-elisa-kit.html
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