Antigen preparation method

Antigen preparation method

Many substances in nature can be used as antigens, but the antigens used as diagnostic reagents must be monospecific, that is, purified antigens, so the required antigens must be extracted from complex components. Here are some main preparations method.

Preparation of granular antigen

Granular antigens mainly refer to cell antigens or bacterial antigens. The most commonly used cell antigen is sheep red blood cells used for preparing hemolysin. Its preparation method is relatively simple, using fresh sheep red blood cells, washed with sterile saline three times, and finally prepared into a cell suspension with a concentration of 106 cells / mL. Bacterial antigens are mostly prepared from liquid or solid cultures after bacterial collection and post-treatment. Some eggs and cell membrane components can be made into particulate antigens. Granular antigens are in the form of emulsions, mostly using intravenous immunization, and rarely using adjuvants for intradermal injection.

Soluble antigens mainly include proteins, glycoproteins, lipoproteins, bacterial toxins, enzymes, complement, etc. However, the composition of these proteins is relatively complex and requires purification before immunization. The usual purification process is carried out as follows.

(1) Preparation of tissue and cell crude antigens Fresh or ultra-low temperature sample tissues are cleaned, cut into small pieces and then crushed. Commonly used crushing methods include high-speed tissue masher and grinding methods. After the slurry is centrifuged, the supernatant contains the material that can extract soluble antigen-crude antigen.

(2) Preparation of soluble antigens of tissue cells Cells that can be used to prepare antigens include normal cells, pathological cells or passage cells. When preparing the antigen, the cells need to be broken up to release the antigen. The preparation methods include repeated freezing and thawing method, alternating hot and cold method, ultrasonic breaking method, autolysis method, lysozyme treatment method, etc.

(3) Ultracentrifugation and gradient density centrifugation method Ultracentrifugation is an effective means to separate subcellular parts and protein macromolecules. It is further divided into differential centrifugation and gradient centrifugation. The method of centrifugal purification of antigen is based on the specific gravity characteristics of the antigen. It is currently only suitable for a small number of large-molecule antigens, such as IgM, C19, and thyroid protein. Isolation and Purification.

(4) Selective precipitation method Selective precipitation is the use of various precipitants or changes in certain conditions to promote the precipitation of antigen components, so as to achieve the purpose of purification, mainly including salt precipitation precipitation method, organic solvent precipitation method, water-soluble non-ionic polymerization Material precipitation method, etc.

(5) Affinity chromatography, gel filtration and ion exchange chromatography A variety of chromatographic methods can be used to purify a certain protein from complex components.

(6) Identification of purified antigen Common methods include polyacrylamide gel electrophoresis, crystallization, immunoelectrophoresis, and immunodiffusion. It is common to use several methods in combination to determine reliable results.