Coating: Because limiting the capture antibody would also limit the capacity/sensitivity of the assay. Thus, for the maximum sensitivity you may coat the plates with maximum amount of capture antibody. Thus, depending on the type and capacity of the assay plate you may add in 25-50% excess amount so that the wells are coated with capture antibodies to their maximum capacity.

We generally coat with 50ul capture antibody at 5ug/ml diluted in carbonate buffer. This is equivalent to 250 ng/well.

Note: Low/medium binding plates have 100-200ng while high binding plates can bind upto 500ng, you may refer to the plate specifications.

Sample antigen: Depending on the amount available you may start with 1:1 or 1:10. Similarly, you can do a run with positive control samples with 6 or 10 point dilution.

Detection antibody: You may try detection antibody at 1:500, 1:1000, 1:2000, 1:5000, 1:10000, and 1:50000.

For more information you may refer to

https://www.seracare.com/globalassets/seracare-resources/tg-protocols-and-troubleshooting.pdf

good luck

S

I agree.

I read in a couple sources that a good concentration for coating is anywhere from 1-10 ug/ml. The first coating antibody I got is 7ug/20ul which comes out to 350ug/ml. So now I am thinking i will do a 6 point serial dilution from 1:50 to 1:1,600 adding 50ul to each well since I only have 20ul of antibody to work with and I need enough to coat 36 wells. For the 25-50% excess are you talking about in regards to the 50ul I would be adding to each well? Should I be adding 65ul instead?

It would be good but you should compare it with another test like Real Time PCR to calculate sensitive