TMB, (3,3′,5,5′-tetramethybenzidine) has been shown to be a safe-sensitive substrate for the assay of HRP. Initially, in the presence of HRP and hydrogen peroxide, a one-electron oxidation product is formed. This compound, a cation free radical, is blue in color with an adsorption maximum of 653 nm. Further reaction with HRP/H2O2 or acidification of the radical with acid yields the diimine terminal oxidation product adsorbing light at 450 nm. The extinction coefficient of the radical (E653nm=3.9 x 104 mol-1 cm-1) and diimine (E450nm=5.9 x 104 mol-1 cm-1) provide a remarkably sensitive system for the assay of HRP and HRP labeled probes. ES001 is a single component reagent stable at room temperature and not sensitive to normal laboratory light. It is optimized with respect to TMB and hydrogen peroxide concentrations and yields a linear response with the concentrations of HRP usually employed in immunological assays.
Application: METHOD SYNOPSIS: After completion of analyte binding to a solid phase and reaction with a HRP labeled probe, ES001 is added. Oxidation of TMB produces a blue reaction product that is measured at 650 nm. The color formation as a function of time can be recorded or the reaction stopped with sodium fluoride after a fixed interval. Increased sensitivity can be achieved by converting the blue radical to the diimine by addition of acid. The resulting yellow color is measured immediately at 450 nm. SAMPLE PROCEDURE 1. Complete all required incubations with antibodies, probes, HRP labeled reagents. 2. Wash plate at least 4 times with PBS or Tris buffered saline containing 0.1% Tween-20. 3. After the final wash, shake and blot all residual buffer from plate wells. 4. Add 0.1 mL of ES001 (TMBE Solution) to appropriate wells and incubate 5-30 minutes. Note: The reaction time will depend upon the activity of the HRP probe. If color develops too briskly, zero order kinetics will not prevail. Dilution of the probe, antibody or HRP labeled reagent may be required. 5. The reaction can be monitored as a function of time for kinetic assays or stopped with 0.1 mL of 0.1% sodium fluoride and read at 650 nm. 6. If the procedure demands conversion to the yellow diimine, add 0.1 mL of either acid described in the reagent section above and record the absorbance within 5 minutes. Variations of time, reagent volume and temperature require standardization by the user.
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