Control Peptide/Antigen -- Reverse Curve -- Help

[Ronnin]

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.

Thank you.

[luisillo]

This might be because your plate isn't properly blocked: without control peptide the assay gives signal because the primary and/or secondary antibody bind unspecificly to the unblocked part of the well. The blocking should be done 2 h at 37ºC not at RT.

On the other hand, the lack of signal at high control peptide conc, could be because your primary antibody does not recognize (properly at least) the control peptide. So, as you increase the peptide conc, the unblocked sites of the wells (as mentioned above) will become blocked with the peptide, leaving less and less binding sites for the detection antibodies, and since these detection antibodies do not recognize the peptide, the signal will be blocked.

I hope this could be of help.