RID - Radial Immunodiffusion

Overview

Immunodiffusion is a detection technique used in measuring antibodies and antigens by their precipitation involving diffusion through agar. Radial immunodiffusion is a single diffusion technique whereby a solution containing the antigen is placed into wells of gel or agar surface that have had an antibody evenly distributed throughout the gel/agar. Radial immuno-diffusion is a type of precipitation reaction based on the principle that the precipitin curve states that antigen-antibody interactions form a cross-linked precipitate when a proper ratio is present.

Required materials

• Agarose

• 10x assay buffer

• Antiserum

• Standard antigens

• Glass plate

• Gel puncher

• Template

• Reagents: Distilled water, Ethanol

Protocol

1. Dilute the 10x assay buffer with distilled water to produce 1x assay buffer.

2. Add 10 ml of 1% agarose to the assay buffer, heat slowly allowing the agarose to completely dissolve, and leave to cool until around 60°C.

3. Add antibody into agarose solution and mix well.

4. Pour the agarose solution into a dish and allow it to cool until solid.

5. Once cooled and solidified cut holes into the gel.

6. Add a small amount of sample into each well.

7. Incubate gel at room temperature for several hours to allow the sample to diffuse into the gel and form a ring of precipitation.

Results

Radial immunodiffusion is used for producing quantitative estimations of antigens. The wells cut in the agarose gel will have formed rings of precipitation; the size of these indicates the concentration of samples inside the wells.