Actually I don't know how to calculate that, but I can suggest a textbook which may help you;

" Calculations for Molecular Biology and Biotechnology, A Guide to Mathematics in the Laboratory "

by Frank H. Stephenson

Thank you very much for your guidance. I have read that book and it does not any section in respect with affinity calculation.

There are two ways to do this and both require you to first purify the antibody from serum (I don't know of anyway to do it from serum). To purify the antibody first by immobilizing the antigen and elutie the antibody with a low pH buffer (elute into a tube with a neutral pH buffer, something like 10uL of 1M Tris, pH 8).

1) The easy way:

Use Biacore with the antibody as your stationary phase. And inject different concentrations of your purified antigen for you mobile phase. This is the preferred method because it's so much easier. But Biacore instruments are sometimes difficult to come by. And if you don't have access, you can always:

2) The hard way:

Once you have the purified antibodies, you want to use a single concentration of antibodies and add different concentrations of purified antigen. Then, pull down your antibody (using protein A or G sepharose). Elute with SDS ad load onto your gel. Do a western blot (ideally blot for a purification epitope on the antigen and the antibody needs to be from a species different from what you raised the new antibody in) and you will want to plot fraction bound as a function of antigen concentration. If you have a typhoon or other digital western blot imaging, you should be able to quantitate the blot easily. If you only have film, then it will be much more work scanning your blot, quantitating with ImageJ (or similar software) and needing to be in the linear range of detection, etc. Once you have your plot of fraction bound as a function of antigen concentration, the kd is at 1/2 the maximum bound. This method has several limitations including your analyte cannot be near the size of the light chain or heavy chain of the antibody. Another major concern is that you will probably have to use very low concentrations of antigen so you may need to add BSA to prevent the antigen from adsorbing to the plastic tube. This way will work if you're careful and think through your experiment.

If either of these don't work, there are a few other ideas I have that will give you an estimation of the affinity. But these require much more work and time.

Thanks. I have access to Biacore, but as far as I know we can’t calculate the mixed and unpurified polyclonal in sera by biacore, because the K-on is associate to concentration of your Abs. In the case of sera sample we don’t know how much Abs we have in sera.

This is why you need to purify the antibodies from sera first. And you will need to purify the antibody using the antigen so you will have the number of molecules of YOUR antibodies not total antibodies. Take detailed notes about how much antigen you've immobilized, and bradford your purified (polyclonal) antibody. This will give you number of molecules for your concentration measurements. If you know how much sera you put in, and you know how many antibodies you get out, then you can back-calculate your concentration of bulk antibodies in serum.