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Sandwich ELISA - Sample values increases on dilution of coating antibody (Feb/11/2010 )

I am developing SANDWICH elisa to detect the amount of antigen in CSF samples. i coated different concentration of coating antibody and also prepared recombinnat standards. The result i get is as coating antibody concentration decreases the standard values decreases but CSF samples values increases as coating antibody concentration decreases.
The background is low.
Please let me know what is the problem.


What happens when you put your recombinate protein in "normal" CSF? Do you see the same response?

If your assay is working: antigen in buffer and in your matrix should produce parallel curves. If there is no matrix effect then the dose response curves will be on top of one another.

There are many many steps before you can go from just coating a plate with different ab coating concentrations to analysis of actual patient specimens.