Rocket immunoelectrophoresis (RIE)

Rocket immunoelectrophoresis

Rocket immunoelectrophoresis (RIE) is a quantitative detection technology that combines unidirectional immunodiffusion with electrophoresis, which is essentially an accelerated unidirectional diffusion test. During the experiment, the antibody was mixed in agar, the antigen in the sample well was placed on the negative terminal, the antibody did not move during electrophoresis, and the antigen moved toward the positive electrode. As the amount of antigen gradually decreased, the base area of ​​antigen swimming became narrower and narrower The precipitation line formed by the antigen-antibody molecule complex gradually narrows, forming a precipitation peak of insoluble complex shaped like a rocket. When the antibody concentration in agar is fixed, the height of the peak is positively correlated with the amount of antigen. Therefore, a known standard antigen is used as a control, the antigen concentration is the abscissa, and the height of the peak is the ordinate. A standard curve is drawn. The concentration of the sample to be tested can be based on The height of the precipitation peak is calculated from the standard curve. If a fixed concentration of antigen is added to the agar, the antibody content can be detected (called reverse rocket electrophoresis).

Rocket immunoelectrophoresis

①②③④ is the standard antigen; ⑤⑥ is the specimen

There are many factors that affect rocket electrophoresis, so the following points should be noted during operation:

1. The agar used should be non-electroosmotic or very small, otherwise the shape of the rocket is irregular.

2. Pay attention to the determination of the end point time of electrophoresis. If the top of the rocket electrophoresis is unclear cloud or round, it means that the end point has not been reached.

3. When the number of specimens to be tested is large, the electrophoresis plate should be bridged on the electrophoresis tank and the power is turned on (the current should be small) before loading.

4. When quantifying IgG, due to the same properties of antigen and antibody, the rocket peak is spindle-shaped due to electroosmosis. In order to correct this phenomenon, formaldehyde can be combined with the amino group on IgG (formylation) to change the originally charged amphiphilic IgG In order to carry only negative charges, the electrophoresis speed is accelerated, the electroosmosis effect is offset, and a rocket peak extending toward the positive electrode appears.

Rocket electrophoresis can only measure the content of Peng / ml as an antigen quantification. If it is below this level, it is difficult to form a visible precipitation peak. Adding a small amount of 125I-labeled standard antigen for co-electrophoresis can form an invisible rocket peak in the agar containing antibody. After washing and drying, it can be developed with X-ray film, and radiographic imaging can occur. This is the current immunoself-imaging technology. The concentration of antigen can be calculated based on the degree of autoradiographic rocket peak reduction (competition method). Immune white imaging technique can measure the antigen concentration in ng / ml.

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