Dear all,

I am working on a competitive ELISA against a biochemical parameter for which I use streptavidin-coated plates, commercial Mab (biotinylated inhouse) and Mab-HRP. My sample type is blood. Initially I prepared sub-stock of both these antibodies to 1:100 and then at final concentrations of 0.1ug/ml-1:5000, 0.05ug/ml-1:2500 and 0.075ug/ml-1:3000 of biotinylated Mab and Mab-HRP in the assay. I am using incubation temperature

of 30 deg cel with first step of incubation with biotinylated Mab for 60 min and later after

washing, Mab-HRP for 35 min. I am getting flat curve for Marquardt 4PL and R2 of 0.967, d(asymptotic top) of 0.322. But the calibrators or the standards used have never given correct values. The competitor kit has 25 deg incubation steps with 90 min of biotinylated antibody incubation step and 60 min incubation with HRP antibody step. Somebody please give me some hints of proceeding with any other dilutions combinations. Thanks