I've tried increasing the concentration of both capture and reporter agents to increase binding capacity on both ends of the sandwich, however the peak of the 'hook effect' hasn't shifted. However, the hook effect is now more of a plateau. Perhaps the secondary ab is limiting. Either way, I'm wondering if anyone has feedback on this as I investigate.
There will be a maximum amount of capture attainable. Perhaps you are at that maximum? Not knowing what your antibodies are, I would suggest that the coating Ab be polyclonal And the detecting Ab monoclonal, or if poly, then a different species of course. The polyclonal coat will give you the best coat coverage with the highest epitopic hold of analyte. I would also suggest running a multi-row test of different coating antibody dilutions, with a range of analyte that goes as high as you think you'd ever want to go, while also having a low end that is solidly in the linear range of what you have now. That way you can tell if the higher attainable analyte is destroying your ability to see changes at the lower end.
The most direct method of getting a higher range in a sandwich will be getting the highest coat possible. But at some point that is going to prevent you from seeing the dilution effects at the low end of the dilution profile.
I would doubt the secondary Ab is limiting anything. As long as it is a normal secondary, and you're not into the 1:100,000 range or something like that.
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