Radial immunodiffusion

Radial immunodiffusion (RID) or Mancini method, Mancini immunodiffusion or single radial immunodiffusion assay, is an immunodiffusion technique used in immunology to determine the quantity or concentration of an antigen in a sample.



A solution containing antibody is added to a heated medium such as agar or agarose dissolved in buffered normal saline. The molten medium is then poured onto a microscope slide or into an open container, such as a Petri dish, and allowed to cool and form a gel.[1][2][3] A solution containing the antigen is then placed in a well that is punched into the gel.[2][3][4] The slide or container is then covered or closed to prevent evaporation.[2][3][4]

The antigen diffuses radially into the medium, forming a circle of precipitin that marks the boundary between the antibody and the antigen.[1] The diameter of the circle increases with time as the antigen diffuses into the medium, reacts with the antibody, and forms insoluble precipitin complexes.[1][2][5] The antigen is quantitated by measuring the diameter of the precipitin circle and comparing it with the diameters of precipitin circles formed by known quantities or concentrations of the antigen.[1][2][3][6]

Antigen-antibody complexes are small and soluble when in antigen excess. Therefore, precipitation near the center of the circle is usually less dense than it is near the circle's outer edge, where antigen is less concentrated.[1][2]

Expansion of the circle reaches an end point and stops when free antigen is depleted and when antigen and antibody reach equivalence.[1][2][5] However, the clarity and density of the circle's outer edge may continue to increase after the circle stops expanding.[1]


For most antigens, the area and the square of the diameter of the circle at the circle's end point are directly proportional to the quantity of antigen and are inversely proportional to the concentration of antibody.[1][2][5][6] Therefore, a graph that compares the quantities or concentrations of antigen in the original samples with the areas or the squares of the diameters of the precipitin circles on linear scales will usually be a straight line when all circles have reached their end points (equivalence method).[1][3][5][6]

Circles that small quantities of antigen create reach their end points before circles that large quantities create.[1][2][5] Therefore, if areas or diameters of circles are measured while some, but not all, circles have stopped expanding, such a graph will be straight in the portion that contains the smaller quantities or concentrations of antigen and will be curved in the portion that contains the larger quantities or concentrations.[1][5]

While circles are still expanding, a graph that compares the quantities or concentrations of the antigen on a logarithmic scale with the diameters or areas of the circles on a linear scale may be a straight line (kinetic method).[1][2][4][5][6][7] However, circles of the precipitate are smaller and less distinct during expansion than they are after expansion has ended.[1][5] Further, temperature affects the rate of expansion, but does not affect the size of a circle at its end point.[1] In addition, the range of circle diameters for the same quantities or concentrations of antigen is smaller while some circles are enlarging than they are after all circles have reached their end points.[1][5]

The quantity and concentration of insoluble antigen-antibody complexes at the outer edge of the circle increase with time.[1] The clarity and density of the circle's outer edge therefore also increase with time.[1] As a result, measurements of the sizes of circles and graphs produced from these measurements are often more accurate after circles have stopped expanding than they are when circles are still enlarging.[1] For those reasons, it is often more desirable to take measurements after all circles have reached their end points than it is to take measurements while some or all circles are still enlarging.[1]

Measurements of large circles are more accurate than are those of small circles.[1] It is therefore often desirable to adjust the concentration of antibody and the quantity of antigen to assure that precipitin rings will be large.[1]

Radial immunodiffusion techniques

  • Measuring circles while all are expanding (kinetic method)[4]
  • Measuring circles after all reach their end points (equivalence method)[2][3]

Further reading

  • Mancini, Giuliana (1992-06-29). "This Week's Citation Classic: Refining the Angelotron" (PDF). Current Contents. 35 (26): 9. Retrieved 2017-08-07. Archived 2017-08-07 at the Wayback Machine
  • Ritzmann, Stephan E. (July 1978). "Radial Immunodiffusion Revisited. Part 1" (PDF). Laboratory Medicine. 9 (7): 23–33. doi:10.1093/labmed/9.7.23. Archived from the original (PDF) on 2017-10-29. Retrieved 2017-10-29.
  • Ritzmann, Stephan E. (August 1978). "Radial Immunodiffusion Revisited. Part 2. Application and Interpretation of RID Assays". Laboratory Medicine. 9 (8): 27–40. doi:10.1093/labmed/9.8.27.


  1. Berne, Bernard H (1974). "Differing methodology and equations used in quantitating immunoglobulins by radial immunodiffusion — a comparative evaluation of reported and commercial techniques" (PDF). Clinical Chemistry. U.S.A. 20 (1): 61–69. PMID 4203461. Archived from the original (PDF) on 2015-11-15. Retrieved 2015-11-15.
  2. Davis, Neil C.; Ho, Monto (1976). Rose, Noel; Friedman, Herman (eds.). Radial Immunodiffusion. Manual of Clinical Immunology: Chapter 2: Quantitation of Immunoglobulins. Washington, D.C.: American Society for Microbiology. pp. 5–8. ISBN 0-914826-09-3. LCCN 76017595. OCLC 1036571523. Retrieved 2019-06-14 via Internet Archive.
  3. Stanley, Jacqueline (2002). Laboratory Technique 12-1: Radial Immunodiffusion Test. Essentials of Immunology & Serology: Section II: Laboratory Techniques: Chapter 12: Precipitation. Albany, New York: Delmar Division of Thomson Learning. pp. 172–174. ISBN 978-0914826255. LCCN 2002280630. OCLC 50204319. Retrieved 2017-05-15 via Google Books. Archived 2014-11-29 at the Wayback Machine
  4. LSUMC/MIP Dental Microbiology Lab (2002). "Radial Immunodiffusion". Department of Microbiology, Immunology & Parasitology. New Orleans, Louisiana: Louisiana State University School of Medicine. Archived from the original on 2004-08-04. Retrieved 2015-11-14.
  5. (1) Mancini, G; Carbonara, AO; Heremans, JF (1965). "Immunochemical quantitation of antigens by single radial immunodiffusion". Immunochemistry. Oxford, England. 2 (3): 235–254. doi:10.1016/0019-2791(65)90004-2. PMID 4956917.
    (2) Mancini, G; Vaerman, JP; Carbonara, AO; Heremans, JF (December 1964). Peeters, Hubert (ed.). A single–radial–diffusion method for the immunological quantitation of proteins. Protides of the Biological Fluids: Proceedings of the 11th Colloquium. Amsterdam, The Netherlands: Elsevier. pp. 370–373. OCLC 54121897. Archived from the original on 2019-12-09 via Google Books.
  6. Parvez, Zaheer (1984). Radial Immunodiffusion (RID). Immunoassays in Coagulation Testing: Chapter 4: Review of Immunologic Techniques. New York: Springer-Verlag. pp. 21–22. doi:10.1007/978-1-4615-7225-1_5. ISBN 9781461572251. LCCN 83020115. OCLC 851823206.
  7. Fahey, John L; McKelvey, Eugene M (1965). "Quantitative determination of serum immunoglobulins in antibody–agar plates" (PDF). Journal of Immunology. U.S.A. 94 (1): 84–90. PMID 14253527. Retrieved 2010-05-12.
  • Bhattacharjee, Suman (2013-11-29). "Radial immunodiffusion" (video). Shomu's Biology. Retrieved 2016-06-27 via YouTube. Introductory video on radial immunodiffusion theory and technique (10:21 minutes).
  • Shaikh, Sajid (2015-09-24). "Radial immununodiffusion" (video). Retrieved 2017-05-13 via YouTube. Introductory video demonstrating radial immunodiffusion technique (3:43 minutes).
  • "Radial Immunodiffusion". Edvotek, Inc. 2017. Archived from the original (photograph) on 2017-08-07. Retrieved 2017-08-07. Photograph of precipitin circles in a Petri dish during radial immunodiffusion.
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