Classification and test methods for agar diffusion test

The soluble antigen and the antibody are diffused in the semi-solid agar. If the antigen corresponds to the antibody and the ratio is appropriate, a white precipitation line appears, which is a positive reaction. The agar diffusion test can be carried out in test tubes, in dishes, and in agar on slides. It can be divided into two-way agar diffusion test and one-way agar diffusion test.

1, two-way agar diffusion test

In the two-way agar diffusion test, the semi-solid agar is poured into a dish or a slide. After it is solidified, it is perforated on the agar plate, and the antigen and the antibody are respectively injected into the small holes to spread the two. If the antigen and the antibody correspond to each other, and the concentration and ratio are appropriate, a clear sedimentation line appears between the antigen and the antibody well after a certain period of time. Two-way agar diffusion can be used to analyze multiple antigens in solution. A pair of antigens and antibody systems can only form one sedimentation line. Different antigen-antibody systems diffuse at different speeds in agar, and different precipitation lines can be formed in agar. This method is mainly used to detect various immunoglobulins, alpha-fetoprotein, hepatitis B surface antigen and the like in serum. The disadvantage is that it takes too long and the sensitivity is not high.

2. One-way agar diffusion test

The one-way agar diffusion test is a commonly used method for quantitative detection of antigens. The appropriate amount of the antibody is mixed with the agar, cast into a plate, and after solidification, the plate is perforated, and the antigen is added to the well, and the antigen is spread to the periphery of the well, and is bound to the antibody in the agar while diffusing. After a certain period of time, a white precipitate ring was formed at a suitable ratio between the two. The diameter of the precipitation ring is proportional to the concentration of the antigen. If a standard curve is prepared with different concentrations of standard antigen in advance, the antigen content in the sample can be determined from the curve. The one-way agar diffusion test is mainly used to detect the content of various immunoglobulins in serum and various complement components in serum, and has high sensitivity.

Agar diffusion test method
Materials and reagents
1, agar powder
2, plate
3, puncher
4, saline or other buffer, can add 1 / 10,000 thiomersal or sodium azide antiseptic.
Method of operation
1. Weigh a certain amount of agar powder, add physiological saline or buffer at a ratio of about 1% (0.8% to 1.5%), and boil for 20 minutes in a water bath.
2. Pour the melted agar into the dish to a thickness of 2 mm to 3 mm. Naturally cooled.
3. According to the requirements (the aperture is the diameter of the hole, the hole distance is the distance between the centers of the two holes, including the radius of the two holes), and the hole is punched according to the template. It is also possible to punch holes directly with a combination puncher. Now generally more into a plum-shaped hole map.
4. Pick out the agar in the hole, taking care not to pick the hole edge.
5. Slowly heat the flame, so that the agar on the edge of the bottom of the hole is slightly melted to seal the bottom, so as to prevent the liquid from leaking from the bottom of the hole after the sample is added.
6. Pipette the sample into the hole with a capillary dropper, taking care not to create bubbles, to fill it up. Add less, affect the degree of reaction, add more, easy to overflow, but also affect the reaction results.
7. After the addition, cover the plate cover, turn the plate over, place it in the wet plate, and freely diffuse at 37 °C for 24h~48h.
Result determination
1. When detecting antigen or comparing antigen difference, the antiserum is placed in the center hole, and the antigen to be tested or the antigen to be compared is placed in the adjacent adjacent hole. If the precipitation band is completely fused, it is proved to be the same antigen; if the two are partially connected, it indicates that they have a common antigenic determinant; if the two precipitation lines cross each other, the antigens are completely different.
2. When used for sero-epidemiological investigation, the standard antigen is placed in the central well, and the 1, 3, and 5 wells are added with standard positive serum, and the 2, 4, and 6 wells are added with the serum to be tested. The test wells were positively combined with the precipitation bands present in the positive holes. The serum to be tested has no precipitation zone or the precipitation zone that appears is completely negative with the precipitation zone of the positive control. Although there is no sedimentation zone in the hole to be inspected, the sedimentation zone of the two positive holes is close to the hole to be inspected, and both ends are weakly positive. If only one end is bent and the other end is still straight, it is judged to be suspicious and needs to be re-examined. When re-examination, the amount of sample can be increased.
There is no sedimentation zone in the sample hole, but the precipitation zone of the positive hole on both sides becomes blurred and disappears when it is close to the sample hole. It may be that the concentration of antibody in the serum to be tested is too large, causing the precipitation band to dissolve, and the sample may be diluted and re-examined. .
3. When detecting the titer of the antiserum, the antigen is placed in the central well, and the antiserum is diluted and placed in the surrounding well, so that the highest dilution factor of the serum in the sedimentation zone is the agar expansion titer of the antiserum.

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