Instructions for using peroxidase that catalyzes Trinder’s reagent
In enzyme-linked immunoassay, the substrate Trinder's reagent for detection requires enzyme catalysis. Enzyme refers to peroxidase POD or HRP. Whether in kits or experimental detection, POD enzyme preparations are critical. Therefore, if you want to do a better test, you need to use peroxidase, and you need to understand its physical and chemical properties.
Trinder’s reagent and peroxidase for enzyme immunoassay
The relationship between peroxidase and pH buffer:
First of all, the catalytic activity of all enzymes requires the pH of the reaction system. Of course, peroxidase POD is no exception. It is necessary to configure a suitable pH buffer to maintain the pH of the reaction system. However, peroxidase generally contains a variety of isoenzymes, so the optimum pH range is relatively wide. HRP is widely used and is a kind of POD, which refers to horseradish peroxidase extracted from plants.
Under the acidic state, the heme peroxidase is partially separated from the protein, and the enzyme protein changes from the natural state to the reversible state, with reduced activity and low thermal stability; in the neutral and alkaline state, the enzyme is in the natural state, and the protein structure Contains α-helical structure, which is stable, and the structure is destroyed after acidification, resulting in β-structure.
The relationship between peroxidase and temperature:
In addition to the pH requirements, the POD reaction also requires temperature. There is a big difference in the optimum temperature of peroxidase from different sources, generally 35-60℃. For example, the optimum temperature for the POD of potato is 55℃, while the POD of cauliflower is only 35-40℃.
The thermal deactivation characteristics of POD are biphasic and reversible. It contains parts with different heat-resistant properties. The heat-labile parts are quickly deactivated during heat treatment, while the heat-resistant parts are slowly deactivated at the same temperature. Different sources of POD also have different heat resistance. Generally, the higher the POD enzyme activity of plants, the higher the heat resistance. Low moisture content also increases the heat resistance of POD. Lowering the pH and increasing the concentration of sodium chloride will also increase the rate of enzyme inactivation.
It can be seen from the above that in the detection process of Trinder’s reagent, the appropriate temperature and pH need to be controlled to ensure the catalytic activity of peroxidase and to ensure the accuracy and sensitivity of the detection. The use of biological buffers is also essential. Desheng provides Trinder’s reagents, buffers, enzyme preparations and a series of reagents for in vitro diagnosis.
In chemiluminescence analysis, the luminescence intensity of acridine ester is influenced by various factors, such as reaction medium, temperature, time, and excitation light source energy. To achieve good detection results, it is necessary to comprehensively consider and optimize these factors. Meanwhile, attention should be paid to controlling and standardizing experimental conditions to ensure accurate and reliable results. Thoroughly studying these influencing factors will help promote the development of chemiluminescence analysis methods.