Radioimmuno-convection electrophoresis assay

Summary

When an antibody (or antigen) labeled with a radioactive element is precipitated with the corresponding antigen (or antibody), the precipitation line is confirmed by autoradiography. It is suitable for (1) rapid diagnosis of diseases (2) semi-quantitative determination of antigens (3) analysis of the relative concentration of antibodies.

Operation method

radioimmunoassay convection electrophoresis

Principle

When an antibody (or antigen) labeled with a radioactive element is precipitated with the corresponding antigen (or antibody), the precipitation line is confirmed by autoradiography. The so-called autoradiography is the process by which rays emitted by the labeled isotope in the complex during the metamorphosis process act on the silver halide crystals of the light-sensitive material to produce a latent image, which is then visualized by developing the image. In this way, precipitation lines invisible to the naked eye can be detected, thus increasing the sensitivity of the reaction, and this method can be applied to all reactions in gels.

Materials and Instruments

Radioisotope-labeled antigens Antibodies
Developing solution Fixing solution
X-ray film Panchromatic film Electrophoresis apparatus Electrophoresis tanks

Move

I. Materials
1. Radioisotope-labeled antigens or antibodies
2. X-ray film or full-color film
3. developing and fixing solutions
4. Others as for convection immunoelectrophoresis
Operation Methods
1. Prepare the gel plate according to convection immunoelectrophoresis and punch the holes.
2. Add antigen to the negative end of the wells and antibody to the positive end. When adding the antibody sample, quickly add the labeled antigen (2,000~3,000 cpm) and mix it, do not add the labeled antigen after the antigen has been absorbed, so as to avoid the formation of false positives due to the lagging surface of the labeled antigen.
3. The electrophoresis current is 4 mA for a 2.6 cmx7.5 cm plate.
4. After electrophoresis, the plate was immersed in 3% HCl and rinsed for 8 h. The solution was changed twice.
5. Rinse with running water overnight.
6. Dry at room temperature or by blower.
7. Exposure In the darkroom, the film is cut to the same size as the plate, and then the drug side is densely connected to the gel side, and then a glass plate is pressed onto the film, wrapped tightly in black cloth, and tied tightly with leather straps. the exposure time is related to the isotope used and the shelf life. 181I, 5 h to 10 h in one week; 24 h in 1 to 2 weeks; 48 h in 2 to 3 weeks. 125I, 10 h in 2 months.
8. Processing method for panchromatic films, water washing, developing at 20°C for 10 min, water washing, fixing, water washing, cool drying, and observing the results.
Results Determination
If a clear black image is formed between the antigen and antibody holes, it will be judged as positive.

Caveat

1. When the specimen is lipemia, old blood specimen or due to α2-macroglobulin can cause false positives, it is close to the anode in the shape of an arc. In order to exclude this false positive can be identified by reference electrophoresis, i.e., the spacing between the positive control holes and the holes to be examined is 0.2cm, and the antiserum holes are played in the middle of the two holes at a distance of 0.4cm, and the antiserum holes are added with the samples, and then electrophoresed, and the two precipitates are positive when they coincide with the line, and negative when they intersect with each other, and the other conditions are the same as the test above.

2. The pH value, ionic strength, voltage and electrophoresis time of electrophoresis buffer have influence on the test results and should be controlled.

3. The electrophoresis time should be extended appropriately with the increase of the distance between the holes. When the distance between two holes is 1cm, the electrophoresis time is 1.5--2h; when the distance between holes is 0.6cm, the electrophoresis time is 1h.

4. The ratio of antigen to antibody concentration should be appropriate, which can be adjusted by diluting the antigen to avoid false negatives.

Common Problems

1. The result is judged as positive if a clear black image is formed between the antigen-antibody wells.

2. The method is simple, quick and 8-16 times more sensitive than the biphasic immunodiffusion method. 3.

3. high specificity and high affinity diagnostic antibodies must be used, otherwise the results are difficult to interpret.

4. Agar with high electroosmotic effect must be used as support medium.

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