Detailed Introduction of ELISA

Enzyme-linked immunosorbent assay (hereinafter referred to as ELISA): is the most widely used technique in enzyme immunoassay technology. The basic method is to adsorb the known antigen or antibody on the surface of the solid phase carrier (polystyrene micro reaction plate), make the enzyme-labeled antigen and antibody reaction proceed on the solid phase surface, and wash the free components in the liquid phase by the washing method. except. 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 determine specific antibodies.

Introduction

Since Engvall and Perlman (1971) first reported the establishment of enzyme-linked immunosorbent assays (Enzyme-Linked ImmunosorbentAssays, ELISA), ELISA has the advantages of fast, sensitive, simple, easy to standardize, etc., so that it has been rapidly developed and widely used. Although the early ELISA hindered its application in practice due to insufficient specificity, with the continuous improvement of the method and the continuous update of materials, especially the preparation of coated antigens by genetic engineering methods, the use of a specific antigen table The blocking ELISA test with a monoclonal antibody greatly improves the specificity of ELISA, and the use of a computerized ELISA tester makes ELISA more convenient, practical and standardized, making it the most widely used One of the detection methods.

Now the ELISA method has been widely used in the diagnosis of many diseases such as bacteria and viruses. In terms of animal quarantine, ELISA has become a widely used standard method in the diagnosis of swine transmissible gastroenteritis, bovine paratuberculosis, bovine transmissible rhinotracheitis, swine pseudorabies, bluetongue disease, etc.

Fundamental

The basic principle of the ELISA method is that an enzyme molecule is covalently bound to an antibody or anti-antibody molecule. This combination will not change the immunological properties of the antibody, nor will it affect the biological activity of the enzyme. This enzyme-labeled antibody can specifically bind to the antigen or antibody adsorbed on the solid-phase carrier. After dropping the substrate solution, the substrate can change the hydrogen donor contained in it from the colorless reduced type to the colored oxidized type under the action of the enzyme, and a color reaction occurs. Therefore, the color response of the substrate can be used to determine whether there is a corresponding immune response. The depth of the color response is directly proportional to the amount of the corresponding antibody or antigen in the specimen. This color reaction can be quantitatively measured by an ELISA detector, which combines the sensitivity of the enzyme chemical reaction with the specificity of the antigen-antibody reaction, making the ELISA method a specific and sensitive detection method.

characteristic

The enzymes used to label antibodies or anti-antibodies must have the following characteristics: high activity and sensitivity; stable at room temperature; reaction products are easy to visualize; and can be produced commercially. Nowadays, horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, etc. are widely used, among which HRP is the most widely used.

Horseradish peroxidase

Peroxidase (HRP) is widely distributed in plants. Horseradish has the highest content. The horseradish peroxidase (HRP) extracted from horseradish is composed of leuco enzyme protein and dark brown iron porphyrin. A glycoprotein (18% sugar content) with a molecular weight of about 40 000, composed of about 300 amino acids, and an isoelectric point of pH 3-9. The optimal pH value for catalytic reactions varies slightly depending on the hydrogen donor , Generally around pH 5. This enzyme is soluble in water and ammonium sulfate solution below 50% saturation. The maximum absorption spectra of enzyme protein and prosthetic group are 275nm and 403nm, respectively.

The purity of the enzyme is expressed in RZ: RZ=OD403/OD275

The 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% and cannot be used for labeling. Only those with RZ above 2.5 can be used for marking. The substrate of HRP is hydrogen peroxide, and there are several kinds of hydrogen donors in the catalytic reaction: ⑴O-Phenylenediamine (OPD), the product is orange, soluble, highly sensitive, with a maximum absorption value of 490nm, which can be distinguished by visual observation , Easy to be terminated by concentrated sulfuric acid, the color can not change within a few hours, is currently the most commonly used in domestic ELISA; ⑵ dianisidine (OD), the product is orange, the maximum absorption value is 400nm, the color is more Stable; ⑶5-aminosalicylic acid (5-AS): the product is dark brown, the maximum absorption value is at 449nm, partially soluble, and the sensitivity is poor; ⑷ the o-tolidine (OT) product is blue, the maximum absorption value is 630nm, partially dissolved, unstable, and intolerant to acid, but the reaction is fast and the color is obvious.

Alkaline phosphatase

It is extracted from calf intestinal mucosa and Escherichia coli, and consists of multiple isozymes. There are many types of their substrates, the most commonly used is nitrophenyl phosphate, which is cheap and non-toxic. The enzymatic hydrolysis product is yellow, soluble, and the maximum absorption value is 400nm. The enzyme activity is based on the hydrolysis of 1 μg of phenyl disodium phosphate at 37°C for 1 minute in a pH 10 reaction system.

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