Peroxidase(POD)

According to the catalytic properties of HRP, hydrogen peroxide (H2O2) is generally used as one of the HRP substrates in ELISA. In the presence of a hydrogen donor (i.e. chromogenic substrate), the reaction between HRP and H2O2 is rapid and specific. As shown in Figure 4, HRP is divalent oxidized by H2O2 to form complex I, which can then undergo a two-step continuous monovalent interaction with the hydrogen donor to reduce to its initial state. Complex II is an intermediate product with one electron that has been oxidized. When there is an excess of H2O2, the enzyme activity is inhibited due to the formation of complex III or IV (to be added later). 30% H2O2 is not stable. Due to H2O2 being both a substrate and an inhibitor of HRP, in order for ELISA to obtain satisfactory measurement results, H2O2 must be limited within a certain concentration range, with a final concentration typically between 2-6 mmol/L. However, in practical research work, this point is generally rarely paid attention to. The concentration of H2O2 used by most researchers is often 2-4 times higher than the amount required for ideal reactions. HRP adsorbed on the solid phase is more susceptible to inhibition by excess H2O2 than free HRP. If the concentration of 30% H2O2 storage solution is confirmed to be 30% by measurement, then diluting it by 10000-12000 times is often an ideal substrate. The molar extinction coefficient of H2O2 is 43.6 at a wavelength of 240nm with a light path of 10mm. Therefore, this method can be used to detect the concentration of H2O2 working solution.In solid-phase ELISA, when the temperature is above 20oC, HRP activity is often low. Adding non-ionic detergents such as polysorbate-20 or TritonX-100 to the substrate solution can delay HRP inactivation and increase the reaction temperature. However, the enzyme activity protective effect of non-ionic detergents varies depending on the hydrogen donor. If 2,2 ‘- diazobis – [3-ethylbenzothiazoline] -6-sulfonic acid (ABTS) is used as the hydrogen donor, only 20% of the enzyme activity can be protected, while when ortho anisidine (ODA) is used as the hydrogen donor, the protection of enzyme activity is increased to 90%.The reason why HRP is the most widely used labeling enzyme in ELISA so far is mainly because it is easy to extract and relatively inexpensive; On the other hand, it has stable properties, is heat-resistant, and can withstand the action of organic solvents. After coupling with antigens or antibodies, its activity is rarely lost.