This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Immunology and Immunochemistry” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.
How can I get my antibody to stick to the plate? (Thread 837)Q I am trying to do a Sandwich-ELISA for TNF in fish. I think that my antibody is not linking to the plate since the standard curve is always blank.
The antibody was homemade and purified. I tried all of the usual coating buffers and none worked. I am using an ImmunoLink® plate from NUNC. How can I solve this problem?
A It is highly unlikely that your antibodies are not sticking to the plate. Try using an anti-IgG-HRP conjugate specific for the species where your antibody was raised to confirm binding.
It is more likely that the sandwich is not working. What are you using for the detecting antibody? You can biotinylate some of the antibody and use that for your second antibody. As long as the protein target has more than one epitope and is big enough to allow more than one antibody to bind, it should work.
Have you tried a checkerboard to optimize the antibody concentrations? Some antibodies are sensitive enough to need only ng/ml concentrations while others require as much as 50 µg/ml or more.
I assume that you have confirmed that the antibodies bind your target? A positive response by Western blot does not always mean that the antibody will work for ELISA. Coat a plate with antigen and try a direct ELISA to confirm.
How similar is fish TNF to other species’ TNF? You might find that a kit for another species will work for your protein.
Q My antibody pair includes anti-TNF IgG from rabbit and the same antibody with a biotin label. I performed a direct ELISA and detected as low as 1 ng in the standard curve. I could not detect the sample, which is why I decided to do a Sandwich-ELISA.
A Is your antibody polyclonal or monoclonal? If your antibody is monoclonal, the same epitope will be used for coating and detecting and you won't see anything in the ELISA.
Q My antibody is polyclonal. It was raised against a small peptide, not the whole molecule.
A If your antibody targets a small peptide, it is very unlikely to work in a Sandwich-ELISA format. Although your antibody is polyclonal, it may as well be monoclonal because the different antibodies will all be targeting the same area of the molecule. For the sandwich to work, you need two antibodies capable of binding two distinct and separate epitopes without steric hindrance. The recognition sites need to be on opposite sides of the target.
If you can't get another antibody, you could try competitive EIA. Coat the plate with your antibody and let the sample and a labeled standard compete for binding.
