Antiserum potency assay by two-way immunodiffusion method

Summary

The two-way immunodiffusion method can be used to (1) diagnose disease and (2) determine the potency of antibodies.

Operation method

two-way immunodiffusion (BID)

Principle

The antigen and the corresponding antibody are added to adjacent wells in the same gel plate, so that they diffuse into each other, and when they diffuse until their concentrations reach the equivalence point (comparable concentrations) a precipitation line is formed. If the antigen-antibody concentration is appropriate, the precipitation line is in the middle of the two wells. If there is an excess of antibody, the precipitation line is close to the antigen wells. If there is an excess of antigen, the precipitation line is close to the antibody well. This technique can be used to determine the potency of an antiserum.

Materials and Instruments

Human IgG Rabbit anti-human IgG
Saline Agar Thimerosal Sodium azide
Slides Levels Perforators Lidded enamel trays Alcohol lamps Toothpicks Microsamplers Constant temperature incubators

Move

1. Melt 3 ml of saline agar in a small test tube in a water bath. 2.

2. Carefully pour the melted agar onto leveled slides, each 1 mm thick. 3.

3. after solidification of the agar, make holes according to the template, the agar in the holes can be picked out with a toothpick if it is not sucked out, the center hole is for adding antigen, and the 6 holes around it are for adding antibody, the diameter of the hole is 3 mm, the distance between the holes is 4 mm, and the slides will be baked on the back of an alcohol lamp after making holes (the temperature should not be too high, and it is suitable for not burning your hands), so that agar will adhere tightly to the slides.

4. Dilute the rabbit anti-human IgG into different concentrations of 1:2, 1:4, 1:8, 1:16, 1:32 according to the two-fold dilution method.

5. Quantify the different dilutions of antibodies into the surrounding five wells in a clockwise direction from top to right to bottom to left, and add saline to the sixth well as a control (never overflow or leak on the agar gel).

6. Carefully put the plate into a wet box and place it in a 37 ℃ incubator for 24 h of diffusion, and observe it on the next day.

7. Observe the appearance of precipitation bands and draw them down. Among the diluted antigens with precipitation, the dilution of the antigen with the largest dilution is the antibody potency.

Caveat

1. The glass plate must be carefully washed and cleaned, and placed horizontally when making the gel plate, so that the thickness of the agar plate is uniform. General gel thickness of 1 ~ 2mm is appropriate.

2. Agar should be fully melted in a boiling water bath. A preparation of more agar liquid should be divided into 20 ~ 50mL small volume. In order to avoid multiple re-melting to change the concentration.

3. The concentration of agar (sugar) can be slightly adjusted depending on the temperature: summer concentration can be increased to 1.2% ~ 1.5%, winter can be used 0.8% ~ 0.2%.

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