Inversing Ligand / Analyte

I have immobilized a membrane protein (approx 65 kDa) as ligand via biotin on an avidin chip (target level approx 800 pg/mm2) and measured the binding of an antibody to it. The Kd is around 500 pM. When inverting the setup (i.e. ligand: antibody immobilized via amine-coupling; analyte: membrane protein), the Kd increases to approx 120 nM. Has anyone observed such an effect and has an explanation for it?
Thank you very much for advice.

Hi,
Without sensorgrams we can't say much about your experiments. So, show them, tells us more what you did and read www.sprpages.nl/equilibrium-dissociation-constant .
Kind regards
Arnoud Marquart