Hi Josh

Thank you so much! the problem is, the amount of antigen will be changing, and I can only use 1 concentration of enzyme.

what about this: the challenge is that i want to ideally have all the antigen attached with a enzyme, and not waste additional reagents. so i should use excess amount of enzyme.

1) I find out the antigen concentration that would occupy all the primary antibody sites [ eg, find out the [antigen] at the point where the signal flattens out, so i know most of the antibody sites has bee occupied]

2) I use the [antigen] that occupies most antibody sites, and see which [enzyme] would result in the highest signal, (eg i try 10ng/mL - 1mg/mL of ALP, and lets say.. signal flattens out at 100ng/mL)

3) Then i can easily find out the optimal substrate concentration to use based on the report optimal time for the enzyme to process substrate.

I think the enzyme concentration determined following these 3 steps should have an excess amount of enzyme to bind with the antigen, Would this cause any other concerns?

dear Josh

Thank you so much!