Immunolabeling technology is to mark some substances that are both easy to measure and highly sensitive to specific antigen or antibody molecules, and to enhance the amplification effect of these markers to show the nature and content of the antigen or antibody in the reaction system. Commonly used markers include fluorescein, enzymes, and radionuclides. The immunodetection technology for labeling with these three markers is called the three major immunolabeling technologies. Currently, the immunomarkers used include chemiluminescent substances, ferritin, and colloidal gold. 0 x + W + F9 Q) {/ h4 S1 V Horseradish peroxidase (horse radish peroxidase, HRP, EC.1.11.1.7) is the most thoroughly studied peroxidase in plants. As early as 1930s, some people started to separate this from horseradish Enzyme, and later prepared crystals. In this experiment, horseradish was used as the raw material. After water extraction, ammonium sulfate and acetone were fractionated, purified by zinc ion, dialyzed to remove salt, and freeze-dried to obtain high-purity horseradish peroxidase. Car Organizers,Seat Back Organizers,Trunk Organizers,Car Seat Organizer,Backseat Car Organizer Ningbo Yinzhou Hengxi Winbate Household Product Manufacturer , https://www.winbatehousehold.com
1. Principle Horseradish Peroxidase (HRP)
Horseradish peroxidase is a protein containing heme, HRP is widely distributed in the plant kingdom. It is a glycoprotein composed of colorless enzyme protein and brown iron porphyrin, with a sugar content of 18%. HRP is composed of multiple isozymes, with Mr around 40,000 and an isoelectric point of 7.2. Soluble in water, solubility is 5% (W / V), the solution is brownish red and transparent. The optimal pH catalyzed by the enzyme is slightly different due to different hydrogen donors, but most of them are around pH5. The enzyme is soluble in water and ammonium sulfate solution below 58%. HRP is soluble in ammonium sulfate solutions below 0.58 saturation, but insoluble above 0.62 saturation. The enzyme is optimal for pH 7.0 (for guaiacol as a hydrogen donor). At room temperature, HRP is stable within a few weeks, heated to 63 ° C, and stabilized within 15 min. The redox potential is very low, at pH 6.08, E 0 = -0.2070V, and at pH 7.71, E′0 = -0.2787V. The maximum absorption spectra of HRP prosthetic groups and enzyme proteins are 403nm and 275nm, respectively, and the purity of the enzyme is generally expressed by the ratio OD403nm / OD275nm RZ (German Reinheit Zahl). *? # p. n2 ~ ​​1 w. X3 b
Enzyme markers include enzyme-labeled antigens, enzyme-labeled antibodies and enzyme-labeled SPA. The quality of enzyme markers is directly related to the success of immunoenzyme technology, so it is called a key reagent. The most commonly used enzyme labels are enzyme-labeled antibodies, which are formed by linking enzymes and specific antibodies through appropriate methods. The quality of enzyme-labeled antibodies mainly depends on the enzymes and antibodies with good purity, strong activity and high affinity, followed by a good preparation method. At present, high-quality enzymes (such as horseradish peroxidase, HRP for short) are already available in China. High-quality antibodies can be obtained by extraction and purification. In the preparation method, the method with high yield, which does not affect the activity of the conjugate and does not mix interfering substances, and is easy to operate is suitable. % O + R (k8 y, u & R!}
Enzyme preparations and their substrates can be used as labels in principle for enzymes that are non-toxic and can exhibit colored chemical reactions. However, the enzyme used as a labeled antibody should meet the following requirements: 3 D / Y2 U # ~ S d (r * a "Z X8 g
(1) Convenient sources and easy purification;
(2) High specific activity and stable properties; 9 z & g $ X- l $ D) C9 r # U7 a0 C
(3) Enzyme activity and quantity energy + I w / ^ & O9]
Determined by a simple method. The enzymes currently commonly used in immunoenzyme technology are horseradish peroxidase (HRP) and alkaline phosphatase (AP), followed by glucose oxidase, β-galactosidase, lysozyme and malate dehydrogenase Wait. 8]-v7 V! ~ （C （E-} "W $ A: e" I "s
Horseradish Peroxidase (HRP) is the most commonly used because of its high specific activity, stability, small molecular weight, and easy preparation of pure enzymes. HRP is widely distributed in the plant kingdom, and is high in horseradish. It is a glycoprotein composed of colorless enzyme protein and brown iron porphyrin, with a sugar content of 18%. HRP is composed of multiple isozymes with a molecular weight of 40,000 and an isoelectric point of PH3-9. The optimal pH catalyzed by the enzyme is slightly different due to different hydrogen donors, but most of them are around PH5. The enzyme is soluble in water and ammonium sulfate solution below 58% saturation. The maximum absorption spectra of the prosthetic group of HRP and the enzyme protein are 403nm and 275nm, respectively, and the purity of the enzyme is generally expressed by the ratio OD403nm / OD275nm RZ (Reinheit Zahl in German). High-purity enzyme RZ value should be around 3.0 (up to 3.4). The smaller the RZ value, the more non-enzymatic protein. It is worth noting that purity does not indicate enzyme activity. For example, when the enzyme is denatured, the RZ value can remain unchanged. * k4 | 7 U # q # b% ^-t "\
The HRP catalytic reaction requires substrates hydrogen peroxide (H2O2) and hydrogen donor (DH2). Most of the hydrogen donors are colorless vat dyes, and colored oxidative dyes (D) can be generated through the reaction. The process of the enzymatic reaction is as follows:
HRP2 ~ 2 y (w9 E + M
DH2 ＋ H2O2──── → D ＋ 2H2O
There are many types of hydrogen donors, and the characteristics of the products formed are different. For example, the reaction product of DAB (3.3-diaminobenzidine) is an insoluble precipitate and has an electron density, so it is suitable for immunoenzyme staining or electron microscopic observation. 5AS (5-aminosalicylic acid) was used for ELISA in the early days, but its solubility is not large enough, and the blank hole is not easy to control to colorless, and it is rarely used now. The characteristic of OT (o-tolidine) is that it can produce bright blue-green products with high sensitivity, but it is greatly affected by temperature in the reaction, and because the products are unstable, it needs to be measured in a short time. The currently widely used and satisfactory hydrogen donors are: OPD (o-phenylenediamine) and TMB (tetramethylbenzidine). The product formed by the former is deep orange or brown, and the product of the latter is blue-green. Both have good solubility. The color is stable in dark places. The blank can be nearly colorless. The latter is reported to be higher than the former in sensitivity. 4 More than times. In addition, there is another kind of hydrogen donor called ABTS [2, 2-side-nitro-bis (3-ethylbenzothipyrroline-6sulfonic acid)], the reaction product is blue-green, and the sensitivity and stability are both it is good. Especially in terms of carcinogenic potential, both ABTS and TMB are worthy hydrogen donors.
Since H2O2, the substrate of HRP, is itself an enzyme inhibitor, the amount of H2O2 used in the enzymatic reaction cannot be excessive. It should be controlled to reach a peak after a short period of time after reaction (indicating that H2O2 has been exhausted). In this way, even if the time is extended, the color of the reaction product will not be increased. + c0 `# W2} $ k * k & p
There are many methods for cross-linking enzymes and antibodies, and different methods can be used depending on the structure of the enzyme. For the preparation of HRP conjugates, glutaraldehyde two-step method and sodium periodate method can be used. The periodate oxidation method is commonly used, and this method is only suitable for enzymes with a high sugar content. Sodium periodate oxidizes the polysaccharides on the surface of HRP molecules to aldehyde groups, which form Schiff bases with the amino groups on the antibody molecules to bind. The latter can be further reduced with NaBH4 (or ethanolamine) to generate stable enzyme-labeled antibodies.
2. Experimental procedure-W a $ D （e2 i0 b
Preparation of HRP