Comparing the results of two ELISAs
We are working with a solution of purified egg antibodies (IgY), both specific (polyclonal) for certain microorganism and non-specific. The problem is, we want to measure the proportion of specific antibodies we have in the mix and had thought of running two parallel ELISAs.
In the first one, we would fix to the bottom of the well the microorganism, let react our solution of IgY and finally use a commercial goat IgG (anti-IgY) conjugated. We intend to measure the amount of specific IgY.
In the other ELISA, we would fix polyclonal goat IgG (anti-IgY) (not conjugated). Again, we would let react our solution of IgY, and quantify with goat-antibody against IgY conjugated. Our intention here is to measure the total amount of IgY in our solution (specific and non-specific).
We would use that data to obtain the proportion of specific IgY (against the microorganism) in the total IgY solution, but we are not sure if this approach is appropriate to our purpose. Or maybe is there a more suitable method? Any suggestions will be very appreciated!
If you have a larger amount of your antibodies, which might be the case with IgY, you might use affinity chromatography. Your antigen is immobilized on an activated Sephadex column and the antibodies are applied to this column. The specific antibodies are eluted by low pH (e.g. pH 2.3) and quantified by a protein test (UV or BCA).
Hi Antonio, I think your first option can’t give you the amount of specific IgY. By this option you will calculate specific titers of IgY which depends both on concentration and avidity of antibodies.
I think you could go for your second option (sandwich Elisa) to quantify total IgY and then going as suggested by Michael G. Weller to obtain the specific IgY fraction.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies