I am trying to assess binding activity of a His-tagged recombinant to ganglioside GT1b via a sandwich assay (ELISA). I have performed the experiment numerous times with 2 different gangliosides and 3 different positive control proteins as well as performing with and without GT1b bound to the plate, and EVERY time my test and negative control His-tagged proteins bind to the plate regardless of whether GT1b is present or not, and my positive control proteins show baseline readings. I'm starting to go a bit crazy. I don't know what I'm doing wrong. I am following a published protocol. Does anyone have any ideas?

Assay:

GT1b in MetOH coated overnight (or just MetOH), (have also tried GT1b in PBS/BSA or PBS/skim)

Block (have tried BSA and skim milk) 1 h @ 37C

Proteins 2 h @ 37C

Primary antibody 1 h @ 37C

Secondary antibody 1 h @ 37C

Develop (HRP)

Any help would greatly appreciated, as this is one of the last experiments I need to do to finish my PhD.

Natalie