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.

1. Selection of working concentration

In immunoenzyme technology, the first factor to be determined is the working concentration of the enzyme marker. Because small changes in the concentration of enzyme markers can cause large fluctuations in the test results. In addition, because the concentration is too high, non-specific reactions can be increased, while the concentration is too low can affect the sensitivity of the measurement. Therefore, the working concentration must be accurately titrated before the formal test.

The titration method of enzyme-labeled antibody is: physical adsorption of the antigen (or antibody) on the solid phase carrier, and then a series of diluted enzyme-labeled antibody (or anti-Ig antibody) and the antigen (or antibody) adsorbed on the carrier The reaction determines the titer of the enzyme-labeled antibody based on the degree of color reaction between the enzyme and the substrate, or the working concentration.

The steps are: first dilute the antigen (or antibody) with 0.05M PH9.6 coating buffer to about 10μg / ml, add 0.1ml to the polystyrene plate well, 4 ℃ **, and wash the buffer the next day Wash 3 times. The enzyme-labeled antibody was diluted with 1% BSA-PBS solution to 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600 ... (depending on the antibody titer), and added to the reaction wells respectively , Two wells per dilution, 0.1ml per well, washed after incubating at 37 ° C for 1 hour. Then add substrate solution, 0.1ml per well, 37 ℃ for 10-30 minutes. The reaction was stopped with 2M H2SO4 0.05ml.

As a result, the OD value of each well was mainly read by ELISA colorimeter. The titration curve is drawn with OD as the vertical coordinate and the concentration of the conjugate as the horizontal coordinate. According to the curve, the dilution of the enzyme-labeled antibody when the OD value is about 1.0 and the slope of the curve is maximum is the working concentration of the marker.

See the ELISA section for reagents and equipment for the test.

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It should be noted that this method is a direct ELISA method, and the measured working concentration may differ from the optimal concentration in practical application by several titers. This requires the establishment of an ELISA experimental system, so the actual working concentration should be further determined (square array method can be used) to achieve the most suitable experimental conditions.

2. Enzyme preparation and its substrate

Any enzyme that is non-toxic and can exhibit a colored chemical reaction can be used as a label in principle. However, the enzyme used as a labeled antibody should meet the following requirements: (1) convenient source and easy purification; (2) high specific activity and stable properties; (3) enzyme activity and amount can be 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.

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 (German Reinheit Zahl). 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.

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:

HRP

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 sensitivity4 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.

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