Bidirectional agar and unidirectional agar diffusion test
Soluble antigens and antibodies are used to diffuse in semi-solid agar. If the antigens correspond to antibodies and the proportion is appropriate, white precipitate lines will appear, which is a positive reaction. Agar diffusion tests can be performed in test tubes, in flat dishes, and in agar on slides. It can be divided into two categories: two-way agar diffusion test and one-way agar diffusion test.
Agar diffusion test classification
1. Two-way agar diffusion test: It is to pour semi-solid agar into a flat dish or on a slide. After it solidifies, punch holes in the agar plate, and inject the antigen and antibody into the small holes respectively, so that the two can diffuse each other. If the antigen and antibody correspond to each other, and the concentration and ratio are appropriate, after a certain period of time, a clearly visible precipitation line appears between the antigen and antibody pores. Two-way agar diffusion method can be used to analyze a variety of antigens in solution. A pair of antigen and antibody systems can only form a precipitation line. Different antigen and antibody systems diffuse at different speeds in the agar, and can form different precipitation lines in the agar. This method is mainly used to detect various immunoglobulins,alpha-fetoproteinHepatitis B surface antigen, etc. The disadvantage is that it takes too long and the sensitivity is not high.
2. One-way agar diffusion test: It is a commonly used method for quantitative detection of antigens. Mix an appropriate amount of antibody with agar, pour it into a plate, and after solidification, punch holes in the plate. The antigen is added to the hole, and the antigen will spread around the hole. While diffusion, it binds to the antibody in the agar. After a certain period of time, a white precipitation ring is formed at the appropriate ratio of the two. The diameter of the precipitation ring is proportional to the concentration of the antigen. If a standard curve is made with different concentrations of standard antigens in advance, the content of the antigen in the specimen can be obtained from the curve. One-way agar diffusion test is mainly used to detect the content of various immunoglobulins in the specimen and various complement components in the serum, with high sensitivity.
Second, agar immunodiffusion test method
Materials and reagents
1 agar powder
2 hole punch
3 flat dishes
4. Normal saline or other buffers can be added with one ten thousandth of thiomersal or sodium azide as preservative.
How to operate:
1. Weigh a certain amount of agar powder, add normal saline or buffer at a ratio of about 1% (0.8% to 1.5%), and boil and melt in a water bath for 20 minutes.
2. Pour the melted agar into a flat dish to make the thickness 2mm~ 3mm. Cool naturally.
3. Punch according to the requirements (aperture is the diameter of the hole, hole spacing is the distance between the centers of the two holes, including the radius of the two holes). It can also be punched directly with a combination punch. Now it is generally punched into a plum blossom-shaped hole diagram.
4. Pick out the agar in the hole and be careful not to break the edge of the hole.
5. Heat slowly on the flame to melt the agar on the bottom edge of the hole a little, and seal the bottom to avoid liquid leakage from the bottom of the hole after adding the sample.
6. Use a capillary dropper to absorb the sample and add it to the hole. Be careful not to produce bubbles, and fill it up as the degree. If you add less, it will affect the degree of reaction. If you add more, it will be easy to overflow and also affect the reaction result.
7. After adding, cover the flat plate, turn the flat plate over, place it in a wet plate, and diffuse freely at 37 ° C for 24h to 48h.
III. Result judgment
1. When used to detect antigens or compare antigen differences, place the antiserum in the center hole, and place the antigen to be tested or the antigen to be compared in the surrounding adjacent holes. If the precipitation band is completely fused, it is proved to be the same antigen; if the two are partially connected, it indicates that the two have a common epitope; if the two precipitation lines cross each other, it indicates that the two antigens are completely different.
2. When used for serum epidemiological investigation, the standard antigen is placed in the center hole, and the standard positive serum is added to the surrounding 1, 3, and 5 holes, and the serum to be tested is added to the 2, 4, and 6 holes respectively. Those who are completely fused with the precipitation band appearing in the hole to be tested and the positive hole are judged positive. Those who have no precipitation band in the serum to be tested or who have completely crossed the precipitation band appearing with the positive control are judged negative. Although no precipitation band appears in the hole to be tested, when the precipitation band of the two positive holes is close to the hole to be tested, those who are bent at both ends are judged weakly positive. If only one end is bent and the other end is still straight, it is judged as suspicious and needs to be re-examined. During re-examination, the amount of samples can be increased.
There is no precipitation band in the sample hole, but the precipitation band of the positive hole on both sides becomes blurred and disappears when approaching the sample hole. It may be that the antibody concentration in the serum to be tested is too large, resulting in the dissolution of the precipitation band. The sample can be diluted and re-examined.
3. When used to detect the titer of the antiserum, the antigen is placed in the central hole, and the antiserum is diluted in multiple ratio and then the surrounding hole is placed. The highest dilution multiple of the serum with the precipitation zone is the titer of the antiserum.
IV. Precautions: Irregular precipitation lines may be caused by overflow of sample, irregular pore shape, cracking edge, leakage at the bottom of the hole, no level during incubation, agar drying during diffusion, protein denaturation at high temperature, or bacterial contamination caused by no preservatives.
