I am trying to develop an ELISA assay for the quantitation of antigen-specific IgG or IgA production. The antigen I am using is only 2.2 kDa. It seems that it may not bind properly to a general ELISA plate due to its relatively small molecular weight. So I wonder whether there is a method that I can determine whether the antigen binds to the plate or not?
If anyone could have any suggestions, it would be grateful.
Coat with your antigen overnight in fridge, wash plate, then coat with capture antibodies for another elisa. Additionally, coat another plate with just the capture antibodies. Then perform the Elisa on both plates as normal. If your 2.2kda protein does not bind, then there will be no difference in performance between the 2 plates.
For example: coat plate 1 with antigen-a, rinse, then coat plates 1 & 2(new plate) with monoclonal antibodies to IL-6. Wash, add Il-6 standard, incubate, wash, add detection, etc. etc..
Coat with 5-10ug/ml of your 2.2 kda protein to adequately saturate binding sites on the plate. Some proteins bind poorly if the coating buffer has a pH close to the isoelectric point of the protein. try coating with both PBS-pH7.4 and carbonate-pH~9.xx
This answer is slim on details, so let me know if you need further clarification.
Good luck
Thank you Evan, I think I get it. These are pretty good suggestions!
The only question I have is, if I perform two "coatings" (one is the peptide, then the other is the IL-6 capture antibody), will they "stack" together so I that I may not see a difference in ELISA performance (but the peptide actually binds already)? There may be some hydrophobic interaction between the peptide and the capture antibody?
Also, thanks for reminding me of the pI. The theoretical pI of my peptide is about 8.5 (calculated by ProtParam) so I think it would be reasonable to try using PBS pH 7.4 for the coating. Thus, it may leave my peptide positively charged and better for my high binding ELISA plate (ionic interaction).
It's certainly possible that they will interact (igg/peptide), but there is only one way to find out... (Try it.) I think it will be fine.
Alternatively, many antibody production projects conjugate peptides to a larger protein carrier (i.e.: Klh). If your peptide was conjugated to klh for immunization, then conjugate your peptide to BSA (or something similarly innocuous). Coat half your plate with BSA:Peptide, the other half with just BSA, then check the titers of your immune serum. The BSA:peptide will certainly bind to your plate, eliminating your worry for peptide-coating efficiency.
Another idea: Coat your plate with the full sized protein to which your peptides belong. The antibodies should bind the protein. To check for specificity, run a competitive binding ELISA, where you block the antibodies from binding the native protein (coated on plate) by titrating in your peptides to which the antibodies were raised.
I have never performed a Competitive ELISA myself, so I would ask others for technical tips on those.
You are using concentrations between 1 µg/mL to 10 µg/mL for coating. It's a huge excess. In old references (1975/78) you will find numbers of about 200 - 400 pg/cm² protein bound on a polystyrol surface.
So you will be not able to detect the loss of protein from your solution you prepared for coating. In a simple experiment you can reuse the coating solution. You can do this several times. The only problem: proteins will start to denature at low concentrations or small amounts of proteases will cleave to protein ....
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