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H2SO4 in ELISA - why do we use it (Aug/07/2005 )

In indirect ELISA we used Horse Radish Peroxidase as the conjugate enzyme and TMB(tri methyl benzidine) as the substrate. The reaction mixture turned blue. Then we added H2SO4 as a blocking reagent to stop the reaction. The reaction mixture turned yellow. I want to know that why did the colour change and what did H2SO4 exactly do in the reaction. It would really help if somebody could tell me the chemistry behind the reaction.


I can't give you the exact chemical reaction, but addition of H2SO4 leads to protonation of the chromophore (somewhere in the aromatic system of oxidised TMB - which to my knowledge is, to do so hair-splitting, tetra methyl benzidine) and stops the enzymatic reaction of HRP by dramatically changing the pH. By the way we onetime accidently used HCl instead of H2SO4, works almost all the same, just the absolute OD values were a little lower.....


Thank u so much jade.... and sorry for that tri..... rolleyes.gif