Loss of binding

Hi,

I was wondering if anyone could help me! I was running a series of different inhibition assays with the same antibody and BSA-protein binding pair however saw differing results with two attempts. For example one drug that cross-reacted the first time, did not cross-react the second. Additionally odd binding was seen. To investigate this further we did a test injection of the binding pair followed by an injection of the drug. A test injection of the antibody was done again but there was a complete loss of binding implying the drug was somehow killing the chip or stuck in the system causing the antibody to bind to this rather than the BSA-protein which was immobolised onto the CM5 chip.

Does anyone have any suggestions on what could be happening?

Thanks!

Hi Carol,

I am a little confused about the description.
Could you give the reactions in this way:
1) ligand + analyte = result
2) ligand + .... etc

kind regards
Arnoud

The series of injections were:
1) BSA-MDMA (immobilised) + Anti-MDMA injected = binding seen
2) MDMA injected
3) BSA-MDMA (immobilised) + Anti-MDMA injected again = no binding seen anymore

The inhibition assays prior to this were drugs similar to MDMA incubated with Anti-MDMA prior to injection. However as with both attempts different effects were seen we did the above injection series to see if MDMA influenced the system at all. We tested a completely different binding pair also to try and understand if its just our system that is affected. We saw no loss in binding after the MDMA injection suggesting its a problem unique to our system.

Thanks!

Also forgot to mention when BSA-MDMA was reimmobilised onto the chip, binding was seen again.

I wonder what is the purpose of the MDMA injection over the BSA-MDMA surface?
Did you use a regeneration solution that could destroy the BSA-MDMA?

We injected MDMA over the chip because we were seeing strange results when we were conducting inhibition assays with MDMA and other drugs (which was repeated twice and both times different results were seen). Therefore we wanted to see if MDMA could be affecting the system somehow. We had managed to do binding studies previously simply with the antibody and BSA-MDMA and we saw that the regeneration solution did not affect the BSA-MDMA or chip.