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I would like to the approximate time of incubation of TMB substrate used for ELISA. Is it within 30 minutes of addition of substrate or it vary anywhere between 15 to 50minutes?
I am not familiar with the substrate you mention. Is it a peroxidase substrate and does it give rise to a coloured product?
I think there are a few of points to keep in mind.
1. The velocity of the reaction will be proportional to the (bound) enzyme
concentration, so the development time may vary considerably and there is no 'magic number'.
2. Perhaps more importantly, the response time will depend on temperature. In my opinion, a common mistake it to take the assay buffer out of the fridge just before assay and to use it without allowing it even to warm to room temperature!
Adequate temperature control is essential in all enzyme assays. If Elisa development is to be done at, say, 25 degrees celcius, the assay buffer should be preincubated (without substrates) for, say, 30 minutes prior to Elisa development. As far as possible, this temperature should be maintained for the duration of the assay. (If you take the buffer straight out of the fridge, it stands to reason the assay time may be a lot longer and vary considerably from day-to-day).
3. You need to leave the assay long enough for sufficient colour development to occur, but do not let the colour 'max out'. This may take anything form 1 min to, say 50 min. Usually, this may be judged by eye.
Rate of horseradish peroxidase (HRPO) activation depending by H2O2 concentration in medium, representing a system unity (HRPO/H2O2). After HRPO-activation step, the system it is capable, in adapted conditions of pH (6.0 ± 1.5) and temperature (40 ± 20 °C), of both one electron and two-electron oxidations, depending on the substrate employed. The oxidation of 3,5,3'5'-tetramethylbenzidine by the HRPO/H202 yields colored products, that first turn in blue, pass through a green stage, and finally become yellow. The green solution is simply a mixture of the initial blue product and final yellow product. The reaction involves transfer of two electrons. In a first step, a cation radical reaction intermediate is formed which is the result of one electron transfer. In a second step, as result of a second electron transfer, the cation radical (electron donor) is further oxidized to form the yellow coloured diimine derivative of tetramethylbenzidine (electron acceptor), component with an absorbance maximum at 450 nm. Two of the intermediate cation radicals may however combine to form a blue coloured charge-transfer complex (electron donor), component with an absorbance maximum at 655 nm, which may also be considered to consist of molecule tetramethylbenzidine and a molecule of its diimine derivative; an equilibrium state, that depends on the reaction conditions, exists between these various molecules. If the enzyme reaction is terminated by lowering of the pH < 1.0, with HCl or H2SO4, an additional increase of the absorbance at 450 nm is observed. It is shown that this additional increase is partly due to a 1.4 fold increase in the molar lineic absorbance of oxidized TMB, caused by the acidic pH, as well as a quantitative shift of the existing equilibrium between TMB, oxidized TMB and their charge-transfer complex. The total absorbance increase upon acidification of the reaction mixture depends therefore on the reaction conditions as well as the reaction coordinate, but also by auto-oxidation of the charge-transfer complex under acidic conditions or a combination of all factors above reported.
Now, according my experience, assuming that the observed peroxidase activity origins from reconstituted HRP, 50 nM H2O2 trigger the formation of about 0.1 nM active HRP after 40 min incubation time, and HRP strongly amplifies the signal by conversion of TMB into its colored oxidation products (about 200 nM in 3 min). By native HRP alone, the limit of H2O2 detection using the substrate TMB is 1 μM. In my experiment for to realize an chromogen TMB one bottle, for Alfa-Wasserman Rome, at fixed concentrations of 3,5,3’,5’-tetramethylbenzidine (250 µM) and H2O2 (75 µM), enzyme concentration was varied between 2 and 100 µg. The formation of the charge transfer complex was measured as a function of time following addition of enzyme by recording absorbance at 700 nm. In each case, the final absorbance was the same, that has been inversely proportional reached between 1 and 20 mins.
When pre-incubation is carried out in the presence of hydrogen peroxide a significant increase in peroxidase activity can be detected after 10 min starting from the moment of addition of H2O2. It should be emphasized that this increase in peroxidase activity is not an artefact caused by the rise of peroxide concentration in the assay mixture containing TMB and 0.17 mM H2O2, because, firstly, peroxidase activity increases with time.
In last artifact presence can be influenced by : a) temperature of incubation : with incubation > 40 °C increase auto-oxidation of TMB, while temperature < at 10 °C diminishes sensitivity significantly; b) time of incubation : a short incubation times of 2-5 min. do not seem to be compatible with satisfactory sensitivity even if compensatory increments are introduced in temperature, H202 concentration or TMB chromogen concentration. However, since increasing the duration of incubation also increases artifact in the background, the optimal time must reflect a suitable compromise between artifact and sensitivity. Thus, the optimal TMB procedure was found to require at least 20 min. of incubation in a medium containing the highest compatible concentrations of TMB and H202.
All protocols I've seen is 30 minutes. I add the substrate to each column with a multi-channel at 10 sec intervals and repeat the 10 sec spacing when I add stop solution.
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