Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) is the most widely used technology in enzyme immunoassay. The basic method is to adsorb the known antigen or antibody on the surface of solid carrier (polystyrene micro-reaction plate), so that the enzyme-labeled antigen-antibody reaction is carried out on the solid surface, and the free components in the liquid phase are washed away by washing. Commonly used ELISA methods include double antibody sandwich method and indirect method, the former is used to detect macromolecular antigens, and the latter is used to detect specific antibodies. Since Engvall and Perlman( 197 1) first reported the establishment of enzyme-linked immunosorbent assay (ELISA), it has been rapidly developed and widely used because of its advantages of rapidity, sensitivity, simplicity and easy standardization. Although the specificity of early ELISA was not high enough, it hindered its application in practice. However, with the continuous improvement of methods and materials, especially the preparation of coated antigens by genetic engineering and the blocking ELISA test of monoclonal antibodies against antigen epitopes, the specificity of ELISA has been greatly improved. In addition, the use of highly computerized ELISA detector makes ELISA more simple, practical and standardized, making it one of the most widely used detection methods. At present, ELISA has been widely used in the diagnosis of many diseases such as bacteria and viruses. In animal quarantine, ELISA has been widely used as a standard method to diagnose porcine infectious gastroenteritis, bovine paratuberculosis, bovine infectious rhinotracheitis, porcine pseudorabies and bluetongue disease. (I) Basic principles The basic principle of ELISA is that enzyme molecules bind to antibodies or anti-antibody molecules, which will not change the immunological characteristics of antibodies or affect the biological activity of enzymes. Enzyme-labeled antibodies can specifically bind to antigens or antibodies adsorbed on solid carriers. After dropping the substrate solution, the hydrogen donor contained in the substrate can be changed from colorless reduction type to colored oxidation type under the action of enzyme, and a color reaction occurs. Therefore, whether there is a corresponding immune response can be judged by the color reaction of the substrate, and the depth of the color reaction is directly proportional to the number of corresponding antibodies or antigens in the sample. This color reaction can be quantitatively determined by ELISA detector, which combines the sensitivity of enzymatic chemical reaction with the specificity of antigen-antibody reaction, making ELISA a specific and sensitive detection method. (II) Enzymes used for labeling Enzymes used for labeling antibodies or anti-antibodies must have the following characteristics: high activity and sensitivity; Stable at room temperature; Reaction products are easy to appear; Can be commercialized. At present, horseradish peroxidase, alkaline phosphatase and glucose oxidase are widely used, among which horseradish peroxidase is the most widely used. 1. Horseradish peroxidase is widely distributed in plants, and the content of horseradish is the highest. Horseradish peroxidase (HRP) extracted from horseradish is a glycoprotein, which consists of colorless enzyme protein and dark brown iron porphyrin (sugar content: 18%), with a molecular weight of about 40,000 and about 300 amino acids. The enzyme is soluble in water and ammonium sulfate solution with saturation below 50%. The maximum absorption spectra of enzyme protein and auxiliary group are 275nm and 403nm, respectively. The purity of the enzyme is expressed by RZ: RZ = OD 403/OD 275. RZ of pure enzymes is mostly above 3.0, and the highest is 3.4. Enzyme products with RZ below 0.6 are crude enzymes, and non-enzymatic proteins account for about 75%, so they cannot be used for labeling. RZ above 2.5 can be used for marking. The substrate of HRP is hydrogen peroxide, and there are several hydrogen donors for the catalytic reaction: (1) o-phenylenediamine (OPD), the product is orange, soluble, highly sensitive, with the maximum absorption value of 490nm, which can be judged by naked eyes, easily terminated by concentrated sulfuric acid, and does not change color for several hours, so it is the most commonly used one in ELISA in China; (2) Anisodamine (OD), which is orange with a maximum absorption of 400nm and stable color; (3) 5-aminosalicylic acid (5-As): This product is dark brown, with a maximum absorption value of 449nm, partially dissolved and poor sensitivity; (4) The product of o-toluidine is blue, and its maximum absorption value is 630nm. Partially soluble, unstable, acid-resistant, but fast reaction, obvious color. 2. Alkaline phosphatase is extracted from calf intestinal mucosa and Escherichia coli and consists of several isoenzymes. There are many kinds of substrates, and nitrobenzene phosphate is commonly used, which is cheap and nontoxic. The enzymolysis product is yellow and soluble, and the maximum absorption value is 400nm. The activity of this enzyme is based on the hydrolysis of 1μg disodium phenylphosphate in the reaction system of pH 10 at 37℃ for 0 min. (3) Enzyme labeling method of antibody and determination of labeling effect 1. The enzyme conjugate with good labeling method depends on two conditions: high titer antibody and high activity enzyme. The activity and purity of antibody are very important for the preparation of labeled antibody, because the specific immune response is enhanced with the improvement of antibody activity and purity. In the process of enzyme labeling, antibody activity decreases, so antibody globulin with high purity, high titer and strong antigen affinity is needed, and affinity chromatography is best used.