I am developping an ELISA, which seems to work, although I am puzzled by the titration of the capture AB.

The Assay:

All samples were in dublicates, volume used 50ul, except 150 for blocking, reagent diluent was 0.1%BSA with 0.05% PBS-T, 0.05% PBS-T for washing (3x each), Blocking with 1%BSA and Strep-HRP also in 1%BSA. A previous trial showed that there is not a difference between PBS (pH 7.4) and bicarb. buffer (pH 9.6) during capturing, therefore I use the PBS.

I used for this small trial three concentrations of my recombinant protein which I will also use for the standard curve, 0.0ng, 0.1 and 1.0ng/ml. (The samples are expected to be between 0.2 and 0.5ng/ml).

As capture mAB concentration I have used 4ug/ml, 6ug/ml and 8ug/ml, and incubated at 4°C overnight. As a substrate for the HRP-Streptavidin (1:2'000), which binds to the biotynilated detection pAB, I used TMB(eBioscience).

The questionmark:

I got almost similar values after wavelenght substraction for the 0.1ng/ml value, i.e. all between 0.028 and 0.030 (blank for all between 0.011-0.012), but the value for 1ng/ml rec. protein increased with higher concentrations, i.e.

4ug/ml => 0.213 +/-0.008

6ug/ml => 0.297 +/-0.009

8ug/ml => 0.375 +/-0.012

So which capture concentration, regarding the expected amount of protein in our samples, would be suited? (also considering the costs)

In case someone of you has further tips/hints how to improve the method/reagents, please inform me.