Immuno-diffusion Technique
Immuno-diffusion is a technique used for the detection or measurement of antibodies and antigens by their precipitation, which involves diffusion through a substance such as agar or gel agarose.
Single Radial Immunodiffusion
Radial immunodiffusion (RID) or Mancini method is also known as Mancini immunodiffusion or single radial immunodiffusion assay.
It is an immunodiffusion technique, used in immunology to determine the quantity/concentration of an antigen in a sample.
This technique is very simple and cost-effective; however, it may require a long incubation time.
Principle
• Radial immuno-diffusion is a type of precipitation reaction used in immunology to determine the quantity of an antigen by measuring the diameters of circles of precipitin complexes.
• Specific antigens to the impregnated antibodies diffuse through the agar in all directions from the well and react with the antibody present, forming visible precipitate or a precipitin ring.
Procedure
• Incorporate antibodies (antiserum) specific for the antigen of interest into molten agarose. Then pour the mixture into Petri dish.
• Carefully, circular wells are cut and removed from the plates.
• Ab in gel, ag in well.
• A single or series of standards containing a known concentration of antigen are placed in separate wells, while control and “unknown” samples are placed in the remaining wells.
• As the antigen diffuses radially, a ring of precipitate will form in the area of optimal antigen – antibody concentration.
• When its concentration becomes equivalent to that of the antibody fixed in the gel a ring of ag-ab precipitate(precipitin) is formed. (zone of equivalence).
• After 24 to 48 hours, the diameter of circular precipitates formed around the wells is then they are measured.
• By measuring the diameter of the precipitin rings formed by known “standard antigen concentrations” a standard curve can be constructed.
• The diameter of precipitin rings is directly proportional to antigen concentration.
• The precipitin ring is formed around the well of the samples.
• The diameter of the precipitin ring is measured and noted.
• The antigen diffuses radially in the gel that contains specific antibody.
• To plot the standard curve, square the diameter value & plot antigen concentration on the x-axis & the diameter squared on the y-axis.
• Plot the point & draw the line. Calculate the value of the unknown antigen concentration from this graph.
Interpretation
• The presence of a precipitin ring around the antigen wells indicates specific antigen-antibody interaction.
• Absence of a precipitin ring suggests absence of reaction.
• The greater the amount of antigen in the well, the farther the ring will form from the well.
Applications
• Immuno-diffusion techniques are mostly used in immunology to determine the quantity or concentration of an antigen in a sample.
• Estimation of the immunoglobulin classes in sera.
• Estimation of IgG, IgM antibodies in sera to influenza viruses. To determine relative concentrations of antibodies in serum.
• Estimate serum transferrin and alpha-fero protein.
• To compare properties of two different antigens.
• To determine the relative purity of an antigen preparation
• For disease diagnosis
• Serological surveys
Limitations
• Long reaction time (18-48 hours)
• It has also been proposed that the results of Mancini’s test is influenced by the presence of bound metal cations in the test samples (protein).
• The single diffusion method of precipitation is considered relatively wasteful than other methods.
• The test has recently been replaced by a more sensitive and automated methods, such as nephelometry and enzyme-linked immunosorbent assays.
Reference
Stephan E. Ritzmann; Radial Immunodiffusion Revisited. Part 1, Laboratory Medicine, Volume 9, Issue 7, 1 July 1978, Pages 23-34.
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Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India:
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