In the presence of hydrogen peroxide and HRP, ABM’s ELISA ABTS Substrate for HRP is oxidized to a radical cation showing adsorption maxima at 820nm, 734nm, 650nm and 405nm. Oxidation of ABTS produces a blue-green reaction product that can be measured from 405-410nm. The colour formation can be recorded as a function of time or the reaction can be stopped by the addition of an acid. Stopping the reaction with acid does not alter the 405nm spectrum allowing kinetic and endpoint methods to be measured at the same wavelength.
1. Complete all required incubations with antibodies, probes and HRP labeled reagents. 2. Wash plates, at least 4 times, with phosphate buffered saline or Tris buffered saline containing 0.1% Tween-20. 3. After the final wash, shake and blot all residual buffers from plate wells. 4. Add 100ul of ABTS solution to each well and incubate at room temperature for 30 minutes. Readings at 405nm can be taken at predetermined intervals if a kinetic assay is desired. 5. After 30 minutes, 100ul of acid can be added as stop solution and read at 405nm. The colour will be stable for at least 1 hour if 0.625M oxalic acid is used.
Suitable for kinetic and endpoint assays using Horseradish peroxidase (HRP) labeled probes.
Store at room temperature (17-28ºC). Storage at refrigerator temperatures (2-4ºC) will not inhibit product performance but warm the product to assay temperatures prior to use. Protect from exposure to direct sunlight and discard the solution if it becomes yellow and turbid.
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 a probe, antibody, HRP labeled reagent may be required. In addition, time, reagent volume and temperature variations may require standardization by the user.