Hi Claire,

ELISA is much more sensitive for quanitifcation, and there are lots of commercial kits available for human Cathepsin G. Whether you buy a kit or develop an in-house ELISA depends on how often you will do the ELISA and how much money you have, and/or whether you can be bothered spending the time to optimise your in-house ELISA. Where possible we prefer to develop in-house because once you have the antibodies and the system working you can spin out as many assays as you want and it's more cost effective (it is also internally validated)

To develop an in-house ELISA, you can follow the same sandwich technique that kits use. Basically, the most attractive approach would be to coat the plate with anti-human cathepsin G antibody in (usually) carbonate buffer, block the plate, add your plasma samples and then use a secondary anti-human cathepsin G antibody that is conjugated to biotin. You'll need to also purchase a human cathepsin G standard to use at known concentrations (over a range, dependent on physiological levels of cathepsin G) in order to quantify your unknown concentration in plasma.

Attached is a guide to help you develop your ELISA, the principles are fairly straightforward.

Something to think about might be whether you can use plasma in your ELISA at all - a lot of assays recommend serum. Sometimes the sample matrix makes a difference, but is relatively straightforward to determine by using serum and plasma from the same source and seeing if the amount of cathepsin G differs. Plasma can be preferred because you can get more back from whole blood than with serum.

Good luck!

As Jeremy said, there are a number of commercial ELISAs available for human Cathepsin G. If you need help locating sources, you can search here  http://www.linscottsdirectory.com/search/products . Just follow "More Info" to the suppliers' data pages.

Good luck.

Hi,

thank you very much for your help!

I have tried WB again with plasma protein elimination (cut-off 50 000kDa). For now is not convincing... So I will try  ELISA, I think you are right, it will be more appropriate.

Thanks.