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.
Can your capture antibody bind to protein A? Then you could precoat your plates. What is the concentration of the detecting antibody?
I understand, your EIA is a sandwich test with two monoclonals? Is it right?
Thank you both for your replies! The incubations are all (except TMB) performed on a horizontal shaker with slow speed (~30rpm), to increase the diffusion.
Dear Bernd
It is a sandwich ELISA, with a monoclonal capture and a polyclonal, biotinylated detection AB, which are a matched pair. Further, I also believe that the capture should bind to protein A, due to the fact that it is purified using protein A.
Dear Andrei
I use greiner high binding plates (600ng/cm2), the coating with the capture AB is performed overnight at 4°C, where the wells are covered with film to avoid evaporation & I use 340ul for the washing steps (each 1' incubation). The incubation with the detection AB is for 2h at room temperature (22-23°C).
Regarding the overnight incubation, what is the drawback of 4°C, i.e. the advantage of doing it at 37°C? Thanks for the link, I will have a look at it.
Best, Chris
Dear Bernd
It is not a membrane bound protein, however we don't know yet if it is excreated or bound to the surface, e.g. by GAGs, or stored in vesicles prior to release.
Dear Andrei
Before setting up the ELISA, I have read in a manual (from Licor if I am not mistaken) to use a shaker, and because it did not lead to any timeconsuming drawback or so I have not changed it.
Best, Chris
maybe, I would look at the reactivity of the biotinylated antibody in a western blot.
Dear Andrei
Regarding the 37°C incubation, did you simply put the plate onto a heatblock, or how did you achieve this?
Best, Chris
Dear Andrei
I used the suggested 37°C incubation, which increased the signal quite a bit, cheers :-)
Best, Chris
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