Enzyme-linked immunoassay is an analysis and detection technique that is commonly used in in vitro diagnostic technology and has been applied for a long time. It uses simple equipment and instruments, can be operated in various experimental environments, and has relatively high sensitivity. It has many advantages . In addition to the well-known TMB in ELISA, the substrates used in enzyme-linked immunoassays also include new Trinder’s reagents.
Chromogen substrate new Trinder’s reagent
The new Trinder’s reagent is based on the traditional Trinder’s reagent through functional group substitution optimization and improvement, with better stability and sensitivity. There are many types of it. At present, Desheng has developed and sold 9 types, including TOOS, TOPS, MAOS, ADPS and so on. The oxidation products of different Trinder’s reagents have different maximum absorption wavelengths and molar absorption coefficients, which are suitable for the detection of different biochemical indicators.
Trinder’s reagent detection principle
Trinder's reagent belongs to the oxidation-coupled chromogen substrate pair of HRP, and 4-aminoanti-anti (4-AAP) is adjacent-coupled oxidation color development (2-methyl-2 benzothiazolinone hydrazone MBTH can also be used, but application less). 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, which is greater than 540nm, and the detection wavelength is in the ultraviolet region with little interference.
Chromogen substrate TMB
TMB stands for tetramethylbenzidine, which is the enzyme-linked immunoassay substrate used in the ELISA kit. It belongs to a kind of redox chromogen substrate, and its oxidation product diphenoquinone has the largest extinction coefficient at the wavelength of 450nm (absorbed light is blue light). When the amount of enzyme HRP is small, and the hydrogen peroxide solution and TMB are excessive, blue cationic radicals are formed. Lowering the pH can convert the blue cationic 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.
Compared with TMB, the new Trinder’s reagent has a higher cost and is not as widely applicable as TMB. However, their absorption wavelength is higher, and MAOS reagent reaches 630nm. In addition to the above-mentioned chromogen substrates, Desheng can also provide various enzyme preparations required in the detection.