Methods and Techniques Discussion => Enzyme Linked ImmunoSorbentAssay (ELISA) => Topic started by: Grazi on January 20, 2004, 06:01:35 AM

Title: ELISA
Post by: Grazi on January 20, 2004, 06:01:35 AM
Hi everybody!!!   :D

I am working with ELISA method which use Strepdavidin-HRP. Last week the chromogen(TMB) finished.... :(  but I still need to keep working with ELISA...
So, I would like to know if somebody has a protocol which teaches how to make TMB!
Maybe, somebody has another idea to improve an adaptation to this protocol....maybe another chromogen that works with  Strepdavidin-HRP...

Any help will be apreciated!!!
Thanks a lot!!!
Title: ELISA
Post by: Frank on February 02, 2004, 08:11:52 AM
I have a protocol that we used some years ago. The original comes from an article by M.Mesulam in J.Histochem&Cytochem,26,(2),pp106-117,1978.
1.Rinse in dem. water.
2.Pre-incubate for 20 min at RT in a mixture of 100 mg sodiumnitroferricyanide, 5 ml 0.2 M acetatebuffer pH 3.3, 92.5 ml dem. water and 5 mg TMB in 2.5 ml abs. ethanol.
3. Add 2.0-4.0 ml 0.3 % hydrogenperoxide and incubate for 20 min at RT.
4. Stabilize reaction for 20 min at 4 C in a mixture of 9 g sodiumnitroferricyanide in 45 ml dem.water, 50 ml abs. ethanol and 5 ml 0.2 M acetatebuffer pH 3.3.
5. Rinse.
I must say we didn't use it for ELISA but maybe it could give you some idea's about the concentration, solubility and pH of the TMB solution.