Radial immunodiffusion (RID) or Mancini method is also known as Single radial immunodiffusion. An immunodiffusion technique, used in immunology to determine the quantity or concentration of an antigen in a sample.
1. RADIAL IMMUNO DIFFUSION
Submitted to Submitted by
Dr.R.Saranya J. Joyce Angel
Assistant Prof Of Microbiology I M.Sc. Applied Microbiology
Sacred Heart College Sacred Heart College
Tirupattur Tirupattur
2. IMMUNODIFFUSION 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.
3. 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 long incubation time.
4. 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 in 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 known concentration of
antigen are placed in separate wells, while control and “unknown”
samples are placed in other remaining wells.
5. A single or series of standards containing known concentration
of antigen are placed in separate wells, while control and
“unknown” samples are placed in other 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 hour the diameter of circular precipitates
formed around the wells then they are measured.
6. By measuring the diameter precipitin rings of the formed by known
“standard antigen concentrations” a standard curve can be
constructed.
The diameter of precipitin rings are directly proportional to antigen
concentration.
(source: fishersci.com)
The precipitin ring is formed around the well of samples.
The diameter of the precipitin ring is measured and noted.
7. The antigen diffusing radially in the gel that contain 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 indicate
specific antigen-antibody interaction.
Absence of precipitin ring suggest absence of reaction.
The greater the amount of antigen in the well, the farther the ring
will form from the well.
8. 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
9. Limitations
Long reaction time (18-48 hours)
It has also been proposed that the results of Mancini’s test is
influenced by the presence bound metal cations in the test samples
(protein).
Single diffusion method of precipitation is considered relatively
wasteful than other methods.
The test has been recently replaced by more sensitive and
automated methods, such as nephlometry and enzyme-linked
immunosorbent assays.
10. Reference
Stephan E. Ritzmann; Radial Immunodiffusion Revisited. Part 1, Laboratory
Medicine, Volume 9, Issue 7, 1 July 1978, Pages 23–34.
https://www.slideshare.net/prashanthkumarguddeti/antigen-antibody-
interactions-63139960
Lydyard, P.M., Whelan,A.,& Fanger,M.W. (2005).Immunology (2 ed.).London:
BIOS Scientific Publishers.
Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India:
Elsevier India.
