I am trying to develop a sandwich ELISA for a 35kDa peptide. As a control, I used a 14KDa peptide with similar properties to my peptide of interest. Surprisingly, the binding curve for the control (non-specific to the antibody pair) is reverse. In other words, a very high signal were detected for very low concentration of the control peptide. The signal decreases as the concentration of the control peptide increase.
I am wondering how to explain this reverse bindning curve in order to troubleshoot my assay. I assume that ideally no signal should be detected for the control peptide.
A brief on my protocol:
1) 50uL/well-- capture antibody 1ng/uL overnight at 4 degree
2) Wash 3X PBS-0.05% Tween 20
3) Blocking 3%BSA-PBS for 2hrs RT
4) Antigen incubation (the peptide of interest or the control peptide) 2 hrs in the blocking buffer -- various dilutions for the standard curve
5) Wash 3X PBS-0.05% Tween 20
6) Primary antibody 1ng/uL incubation 1 hr RT
7) Wash 3X PBS-0.05% Tween 20
8) Secondary antibody 1 hr RT -- HRP conjugated
9) Wash 6X PBS-0.05% Tween 20
10) Chemiluminescence developing reagent 50uL/Well
11) Reading the plate
I really appreciate your help.