Immunodiffusion is a diagnostic test. Antigens and antibodies diffuse and react with each other in an agar medium to form a precipitate. It is used to detect and quantify antibody/antigen content. There are two common ways of diffusion. One way is that one of the antibodies/antigens remains fixed while the other reagent moves. The other way is that the antigen and antibody move towards each other.
Compared to other available methods, immunodiffusion assays have the advantages of being inexpensive, reliable, and reproducible. We briefly describe the single immunodiffusion technique procedure, which can provide you with possible reagent/solution and specific assay services. It can be used in immunology to determine antigen content, identify immunoglobulin classes, evaluate antibodies, etc.
|Stages||Solutions and Reagents|
|Agar Preparation||Antibody solution, ionic agar or saline agar, PBS|
|Standard Preparation||Buffer, standard antigen solution|
In the immunodiffusion method, the antibodies are uniformly distributed in an agar gel and the sample containing the target antigen is placed in wells within the gel for diffusion. The antigen reacts with the antibody to form a precipitation loop. 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 good cutter to cut the wells in a gentle manner, then remove excess agarose.
Dilute the standards at different concentrations according to the instructions for the standard reference protein product. The serial dilution method can be used.
Number the wells at the bottom of the plate. Load different concentrations of standards using the same pipette tip, adding them sequentially from low to high concentrations. Use a new pipette to add the sample to be tested in the wells. Place the agar plate in the incubator and incubate for 24-48 h.
Carefully hold up the plate and let the light shine. An opaque precipitate ring around each well can be observed. Measure the diameter of the precipitate ring, plot and fit a standard curve, and calculate the concentration of the unknown antigen from this graph.
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.
Immunodiffusion is a technique widely used for antigen quantification. Precipitation in the gel is thought to provide more specific and sensitive results than other available methods. If you would like to detect antigen-antibody complexes and determine the relative amount of antigen/antibody, please consider and contact us for immunodiffusion assay services. Or if you would like to learn about various other immunoassay techniques, you can also contact us.
For research use only. Not intended for any clinical use.