I have 0.05 ml of an HRP conjugated antibody that is at a concentration of 1.0 mg/ml. The company reccomends starting at a 1:5,000 dilution, but proper working dilution needs to be optomized. Since I am developing a new ELISA I need to optimize the dilution and would like to start by only diluting half of the 0.05 ml (so 0.025 ml) to 1:5,000. What would be the correct calculation for this? Also, what would you dilute the antibody in?
For the actual application you are developing, you need to determine the optimum conditions which may not be the same as what the manufacturers suggest. I suggest you start with a higher concentration like 1:50 as your working stock solution and prepare lower dilutions from this working stock in case the lower concentrations end up to be too dilute for your application. Optimizing ELISA is a stepwise process that requires diligence and patience to be able to identify the best parameters for the best results.
For the dilution of the antibody, you need to check the certificate of analysis, COA, from the manufacturer because that COA tells you what buffer was used by the manufacturer. The buffer they used is usually the best buffer for the antibody giving it stability and optimum activity. If you use another buffer, the antibodies might precipitate and become unusable.
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