You need some reference. For example, you may compare serum samples collected at different times (ideally frozen in aliquots) at the same plate to demonstrate that the immunization and boosting processes have worked and the animals have developed antibodies.

For standardization and possibly antigen affinity purification, you'll need some antigen.

Hi, Christopher Mayhugh . The method we follow is to set up a plate with increasing dilutions of antibody in the horizontal direction and antigen vertically. Then at the end of the ELISA, we make a line graph of the absorbance value (y-axis) vs. the respective dilution of antibody (x-axis) at each antigen concentration. The dilution at which the line plot drops below the trend is assigned as the titer value at that particular antigen concentration (dilution up to which the antibody can detect the antibody efficiently). Since you do not have standards for your raised antibody this would be the preferred method. Good luck with your experiment.

Hi Christopher Mayhugh,

This is in addition to Akhil Baby's answer.

Speaking from experience, we use 10^8 CFU/mL (for bacteria) and 1 mcg/mL (virus) as the concentration of the antigen. Then for the serum, we start with a 1000x dilution and we increase the dilution 3-fold. We usually have at least 10 different concentrations, excluding a negative control (buffer).

Hope this helps.

Best regards,

Shya

Hi Christopher,

if you are developing in-house ELISA. You need standardize the condition using known positive and negative samples. Then choose best antigen concentration with optimum sample and conjugate dilutions to test your samples.