We are developing in-house assays to measure quantitatively the amount of polyclonal antiserum produced in rabbits against a specific antigen.
To do this we have an elisa where we coat with the antigen, use an affinity purified (in house) preparation of polyclonal antibody from a representative pool of rabbit serum (approx 20 rabbits), to prepare a standard curve using known amounts of antibody determined by A280. Then the amount of antibody bound is measured by an anti rabbit HRP conjugate.
The assay works really well. However I have always maintained that the theory is flawed, but my boss disagrees with me.
If each rabbit makes a seperate population of polyclonal antibodies with different affinities, then each rabbit serum will react differently with the antigen coated on the plate. the only antibody population that can be measured accurately with the standard curve is the one from which the standard curve was made, with each affinity of antibodies in the same ratios. This is because what you measure depends not just on the amount of antibody present but also the affinities of the antibodies present.
We do indeed see a differnece in the quantitation that the most recent batch of affinity purified antibody (from different rabbits to that the standard curve is made up with ) measured in the ELISA and by A280.
So anybody any thoughts? I now have to justify why my ELISA reds different results to the A280 and she does not accept my argument...