What Are TMB Peroxidase Substrates?

TMB is a chromogenic substrate used to stain in immunohistochemistry and is also used in ELISA. This substrate is also known as the 3,3’5,5 – Tetramethylbenzidine, TMBE substrate, chromogenic ELISA substrate. It looks like a white solid that turns into a blue-green liquid using ethyl acetate. It is susceptible to sunlight and fluorescent lights. There is no proof of TMB being cancerous.


It is also a substrate for horseradish peroxidase. How this is connected with TMB is through combining horseradish peroxidase with hydrogen peroxide makes the blue color appear in TMB. The way TMB is used in ELISA experiments is like an electron donor. How this works is converting peroxides into water changing the color to blue. If we want to stop this reaction, we can change the color of TMB to yellow by using acid in it.

But, do we know what ELISA really stands for. ELISA, known as the enzyme-linked immunosorbent assay, detects proteins, antibodies, and hormones.

Usually, you’ll need serum, plasma, saliva, and urine to do an essay like this. The way it works is by having an antibody bind with a target antigen. This is one of the easiest essays to be done when performing on various samples simultaneously. Follow this link to find out more about it https://medlineplus.gov/ency/article/003332.htm.

There are 4 types of ELISA experiments. Those are direct, indirect, sandwich, and competitive. When talking about a direct ELISA, the sample has to be immobilized on a plate. After that, antibodies tend to bind with a target sample. A substrate is added, such as TMB, which is the same amount as the sample.

 What is helpful from this type of ELISA is that it is a fast protocol and can be very simple. What is not so beneficial is that it is not so specific. This is because only 1 antibody is used in the experiment.

The indirect ELISA, like the direct ELISA, puts the sample on the plate directly. Unlike the direct one where there is only 1 antibody used in it, here, 2 antibodies have been used. They have been used one after the other, and after that, a substrate is also added to it.

What is useful here is the usage of 2 antibodies instead of 1. What is not so beneficial is the possibility of having a mixed reaction because of that second antibody.

The most common type is the sandwich ELISA. This is where 2 antibodies are used at the same time to sandwich the sample. After that, just like we know from before, a substrate is added in the end with the same amount as the sample. This is why this method is the most used one. It brings the best results, but what’s not so good about it is the time it takes to be done.

The competitive ELISA is used when the sandwich one can’t be used. That is when the sample that has to be tested has small molecules. Here, like the indirect ELISA, only 1 antibody is used, making the result less specific. As we know, at the very end, a substrate is added. What is so good about this method is that it can be used to test smaller molecules. Check this page out for more information.


Like every experiment or test is done, they are times when something doesn’t go the right way. And this can be because of various reasons. When talking about ELISA, a few things can happen. Those being having no signal. This can happen because of using the wrong reagents, or the assay was not set up correctly. Another thing can be when using the wrong antibody or not using the right amount of antibodies.

Another problem that can occur has a weak signal. Because of the shortage of antibodies used in the test, the settings on the plate have been making incorrectly. Sometimes the process of changing the color takes too long. This can happen because the temperature has been set up wrongly or the reagent used is too old.

A lot of things can happen when dealing with these types of tests and experiments. A significant factor in them is the TMB peroxidase substrates which can make or break the same test. These substrates can be used for many tests regarding various diseases, so it is best to know how to use them properly. Nowadays, it has been made easier for doctors and scientists to use them the right way.

They have been made to discover diseases early, and they have already made a significant change in the medical and scientific fields. By experimenting more and doing research on them, these substrates can be used for many more things. Time will only tell.

Comments are closed.