I want to know that why blocking is not effective when we increase the concentration of detection antibody. Is the blocking only effective for a certain range of antibody? or is there any other reason?
Regardless of your blocking method, using too much antibody can cause background. What dilution are you using with your antibody? Have you tried using the manufacturer's recommended concentration?
Actually, I already finished my study. I am asking just to clarify my concept that why blocking is not effective when we increase the concentration of detection? I mean what happen to that blocking? how the higher concentration of detection antibody non-specifically adsorb on the surface in the presence of blocker?
Dear Om Parkash;
You can read my article published in "Scandinavian J of Laboratory and Invest; 2012, 72;531-39).
It would help uou to find some reason for Non-specific binding in immunoassays requiring long incubations, and recommendations to circumvent the these problems.
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