Types of chromogen substrates of enzymes in enzyme-linked immunoassay

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Enzyme-linked immunoassay is a detection technology commonly used in the field of biochemical diagnosis. Due to the specific catalysis of enzymes, the detection has high sensitivity. Among them are the application of various enzymes and their chromogen substrates. This article introduces the types of chromogen substrates of the commonly used enzyme HRP.


New Trinder’s reagent:

Trinder’s reagent is a commonly used chromogen substrate for horseradish peroxidase HRP. There are many types, including more than ten kinds of TOOS, MAOS, ADPS and so on. This kind of reagent belongs to the oxidation-coupled chromogen substrate pair of HRP, and 4-aminoantipo (4-AAP) is adjacently coupled to the oxidation color development (2-methyl-2 benzothiazolinone hydrazone MBTH can also be used, but Less applications). In the presence of H202, 4-aminoantipyrine and the new Trinder’s reagent are condensed by HRP to form a red quinoneimine substance, which has a maximum absorption at a specific wavelength (above 540nm, detection wavelength ultraviolet region, less interference).



MAOS reagent, a chromogen substrate of HRP

Reference formula: Dissolve 4-AAP at a concentration of 2 mmol/L, TOOS/ADPS at a concentration of 25 mmol/L and H202 at a concentration of 0.8 mmol/L in 0.1 mol/L Good's buffer, and measure the wavelength according to TOOS/ADPS select. When the coupled chromogen substrate is used in solid-phase ELISA, the sensitivity is generally low. Generally, the dry chemical method chromogen uses TOOS, TOPS, etc. with a larger molar absorbance. The new Trinder’s reagent is often used for the enzymatic detection of clinical biochemical indicators such as glucose, triacylglycerol, creatine kinase, liver function, and kidney function.


Redox chromogen substrate TMB:

The full name of TMB is tetramethylbenzidine. Its oxidation product, diphenoquinone, has a maximum extinction coefficient at a wavelength of 450nm. If the amount of HRP is small and the amount of hydrogen peroxide solution and TMB is excessive, blue cations will be formed. Lowering the pH can transform the blue cation radicals into yellow diphenoquinone. Sulfuric acid as a terminator can stabilize the product for 90 minutes, and 1% SDS as a terminator can keep the bright blue color unchanged within 24 hours, but has a strong fading effect.


There are many chromogen substrates for HRP. For example, OPD can be oxidized and polymerized into 2,2'-diaminoazobenzene (DAB) for color development, but it has mutagenic effects on the body; ABTS is also a highly sensitive substrate for HRP. However, there is basically no application in China. The new Trinder’s reagent and TMB are mainly used as HRP substrates. Desheng is a manufacturer of chromogen substrates, which can provide a variety of chromogen substrates and corresponding buffers and enzyme preparations.