Radial immunodiffusion (RID) is a type of immunodiffusion technique used to determine the amount or concentration of antigen in a sample. It is based on the principle that when the appropriate ratio of antigen to antibody is present, the antigen-antibody interaction forms a visible cross-linked precipitate producing a precipitating ring. The ring diameter is related to the concentration of antigen present in the sample.
RID is a simple and rapid immunoassay technique. Creative Biolabs offers RID laboratory techniques for exploring antigen-antibody interactions and quantification. We perform experiments according to standard protocols, and create standard curves to determine the concentration of unknown antigen samples. As well as we provide troubleshooting guides for common problems to help experiments run smoothly.
Stages | Solutions and Reagents |
Agarose Plate Preparation | Phosphate buffer (PBS), antibody solution, ionic agar or saline agar |
Sample Preparation | Dilution buffer, standard antigen solution, sample to be tested |
RID is a technique widely used for quantitative antigen estimation. Antibodies of known specificity are uniformly distributed in an agar gel. A sample containing the target antigen is placed in a well within the gel. The antigen then diffuses radially from the wells and forms a precipitation loop at the point of antibody-antigen equilibrium. The antigen concentration is determined by measuring the diameter of the precipitation loop and extrapolating using a standard curve.
Prepare agar plates according to plate size. Dilute the known specific antibody with PBS, preheat and pour into dissolved agar solution, mix the antibody and agar well and pour on the plate. After solidification, place the agar plate on the template and use a well cutter to cut the wells in a gentle manner, then remove excess agarose.
Prepare the standard solution using the serial dilution method. Prepare several microtubes labeled with concentrations. Add an equal amount of buffer to each tube and perform the serial dilution operation. Mix well the existing antigen samples for the standard curve.
Number the surrounding wells at the bottom of the plate, leaving the center well unmarked. First load the standard dilution according to the number and load the sample to be tested in the center well. Cover the plate without inverting the petri dish and place it in the incubation chamber for incubation.
The precipitate rings will be visible after incubation. Lift the plate to see the opaque circles around each well. Measure the diameter of the precipitate with a ruler. Plot a standard curve through known concentrations and calculate the value of the unknown antigen.
No precipitate ring
Blurred precipitation ring
Oversized or undersized precipitation ring
Too large or too small a precipitation ring can result in inaccurate ring diameter measurements.
Multi-ring
Non-circular ring
Poor calibration curve
If you would like to become familiar with RID and how to use it to determine the concentration of a specific antigen, please read our protocol and troubleshooting or contact us for detailed information.
For research use only. Not intended for any clinical use.
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