Low response during secondary binding using NHS/EDC

Hi, I'm using homemade SPR system. I have a general question on surface functionalization that somebody might have similar experience and solution: I'm trying to do antigen-antibody capture using NHS/EDC . I saw a response during the binding of antigen on NHS/EDC modified carboxylated surface but I have a negative response when I inject the corresponding antibody. The antigen and antibody pair are known to react with each other. I obtained a response for these antigen-antibody before and they just suddenly stopped working. Any guess on what is wrong with the chemistry? Thank you.

Hi,

Please look at www.sprpages.nl/experiments/troubleshooting.html for some trouble shooting.