As we all know, it is very common to use Trinder's reagent and TMBZ in ELISA kit as chromogen. So what's the difference between them?

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Firstly, the new Trinder's reagent of chromogen is similar to the traditional Trinder's reagent. Taking the sodium salt of N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline ADPS as an example, it reacts with 4-AA (4-aminoantipyridine) in the presence of hydrogen oxide or POD. The N of aniline becomes C=N double bond, and the para-amino of 4-AA binds to C=N double bond. The bonds form a quinone imide structure, which is similar to the C = O double bond of p-benzoquinone, and the absorption wavelength is in the visible region, thus producing a color reaction. So the reaction here must involve 4-AA (or MBTH).

The principle of ELISA kit-linked enzyme immunosorbent assay (ELISA) is that specific antibodies are coated on the enzyme plate, and the antigens in the sample or standard sample will bind to the coated antibodies, and the free components will be washed away. Biotinized specific antibodies and horseradish peroxidase labeled avidin were added in turn. Biotinized specific antibodies bind to antigens bound to coated antibodies and biotin binds to avidin to form immune complexes. Free components are washed away. When TMB (TMBZ) was added, TMB showed blue color catalyzed by horseradish peroxidase and turned yellow after adding termination solution. OD value was measured at 450 nm wavelength by enzyme labeling instrument. The concentration of antigen was proportional to OD value. The concentration of antigen in the sample was calculated by drawing standard curve. It does not need such as chromogen ADPS, TOOS, MAOS, etc. It needs to introduce another compound (such as 4-AA, MBTH) for color reaction, but TMBZ needs color reaction under acidic conditions (HCl), which limits its application.

The principle of the new Trinder's reagent enzymatic method (enzymatic spectrophotometry) or the ELISA kit with TMB (TMBZ) as the chromogen is similar. Mainly see different types of detection using different kits, for the same substance to be tested also have different detection methods.


DeSheng Science and Technology has been developing and producing in vitro diagnostic reagents for blood testing since 2005, and has a deep research on new Trinder's reagents for chromogenic substrates.