Savinase ELISA with high background!

[Guest NathanBurley]

Hey,

Was wondering if anyone had any knowledge that could help me sort out a problem with an ELISA I'm working on. I'm using a savinase sandwich ELISA, with a BSA blocker, and H2O2 / Orthophenylene Diamene Dihydrochloride to colour the substrate. I'm recieving very high absorbance readings which is making my results difficult to analyse. I have also used this method to test for puradax, termamyl and alcalase with no excessive background. Does anyone have any ideas as to why savinase is the only one with high background, or any ideas as to how the problme might be remedied?

 

Thanks for any help you could give me

 

Nate

[daisy]

Is it possible there are high levels of savinase in BSA or something? Could you try milk as a blocker. Maybe you could explain a bit further about what you mean by background....how do you detect that in ELISA absorbance readings unless it was in the blanks, which you zero anyway. Or do you mean there are very high readings in your protein standards...sorry but a bit more info might help sort it out.

[badchad]

Yeah, I'd also look at your anitbody. Perhaps it is lacky in specificity or something. Check some recent publications and see what kinds of antibodies other laboratories are using. Also, there are some biotech companies who would help you if you call and explaint he problem. Good luck!