Hi,

ask what you need ;)

M.

Determination of an antibody binding affinity is an important step during the development of an ELISA. We have developed several ELISA kits for protein and steroid hormones wherein the determination of the antibody bonding affinity have been carried out. You can find the protocol and other relevant steps that could be sufficient to resolve your doubts(if any) and answer the questions regarding the assay and the raw data. I am giving below the links of our few papers wherein we have developed hCG, progesterone as well as testosterone ELISA:

Isolation of alpha- and beta-subunits of peak-I hCG and generation of highly specific polyclonal antisera. J Immunoassay Immunochem 2005;26(4):345-55. doi: 10.1080/15321810500220985. Article Isolation of α‐ and β‐Subunits of Peak‐I hCG and Generation ...

Heterologous enzyme linked immunosorbent assay for measurement of testosterone in serum. . 2012;33(3):252-68.J Immunoassay Immunochem

doi: 10.1080/15321819.2011.638525. Article Heterologous enzyme linked immunosorbent assay for measureme...

Influence of different length spacers containing enzyme conjugate on functional parameters of progesterone ELISA. J Immunoassay Immunochem 2013;34(1):94-108. doi: 10.1080/15321819.2012.686470. Article Influence of different length spacers containing enzyme conj...

Best

ELISAs are routinely used to quickly determine the dissociation constant (Kd) of antibodies, which is a quantitative measurement of antibody affinity.

To do this experimentally, first coat the target antigen on a plate (or buy a kit with plates provided), then perform the ELISA experiment at different dilutions of your antibody of interest. The concentration (nM) at which half maximal signal saturation is observed is the EC50 value or Kd for the molecule. Numerical values can be calculated by fitting curves to the data.

Hope that helps!