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.

Description

Preparation

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. A solution containing the antigen is then placed in a well that is punched into the gel. The slide or container is then covered, closed or placed in a humidity box to prevent evaporation.[1][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][2] 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 endpoint 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]

Interpretation
Best-fit line plots. RID often produces similar plots when the horizontal abcissa (X-axis) contains the initial antigen concentrations, the vertical ordinate (Y-axis) contains the squares of the precipitin ring diameters, and each plot is a measurement time. The entirely curved purple plot (bottom) appears when all rings are expanding. The entirely straight red plot (top) appears when all rings have reached their endpoints and have stopped expanding. Intermediate plots are partially straight and partially curved (see [7] and [8]).

For most antigens, the area and the square of the diameter of the circle at the circle's endpoint are directly proportional to the initial quantity of antigen and are inversely proportional to the concentration of antibody.[1][2][5] 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 a best-fit line plot will usually be a straight line after all circles have reached their endpoints (equivalence method).[1][3][5][6]

Circles that small quantities of antigen create reach their endpoints before circles that large quantities create do so.[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 whose wells initially contained the smaller quantities or concentrations of antigen and will be curved in the portion whose wells contained the larger quantities or concentrations.[1][5]

While circles are still expanding, a graph that compares the initial 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][9] 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 endpoint.[1] In addition, the range of circle diameters for the same initial quantities or concentrations of antigen is smaller while some circles are enlarging than they are after all circles have reached their endpoints.[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 endpoints 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][10] It is therefore often desirable to adjust the concentration of antibody and the initial quantities of antigen to assure that precipitin rings will be large.[1]

Radial immunodiffusion techniques
Straight line on a best-fit semi-logarithmic plot. RID often produces a similar plot while all precipitin circles are expanding, as in the kinetic method (see [11] and [12]).

One can determine the antigen concentration in a sample whose concentration is unknown by finding its location on a graph that charts the diameters of precipitin circles produced by three or more reference samples with known antigen concentrations. Two techniques often produce straight lines on such graphs. The techniques produce those lines on different types of graphs.

The techniques and their graphs are:

  • Measuring circles while all are expanding (kinetic method): graph charting logarithms of initial antigen concentrations vs. diameters of precipitin circles on a best-fit semi-logarithmic plot.[4]
  • Measuring circles after all reach their end points (equivalence method): graph charting initial antigen concentrations vs. squares of diameters of precipitin circles on a best-fit line plot.[2][3]

Notes
  1. Berne (1974).
  2. Davis NC, Ho M (1976). "Chapter 2: Quantitation of Immunoglobulins: Radial Immunodiffusion". In Rose N, Friedman H (eds.). Manual of Clinical Immunology. 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 (2002)
  4. LSUMC/MIP Dental Microbiology Lab (2002).
  5. (1) Mancini G, Carbonara AO, Heremans JF (September 1965). "Immunochemical quantitation of antigens by single radial immunodiffusion". Immunochemistry. Oxford, England: Pergamon Press. 2 (3): 235–254. doi:10.1016/0019-2791(65)90004-2. ISSN 0019-2791. LCCN 64009461. OCLC 53097967. PMID 4956917. Retrieved 2020-07-08 – via ScienceDirect.
    (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, Bruges, Belgium (1963). Amsterdam, The Netherlands: Elsevier. pp. 370–373. OCLC 25285708. Retrieved 2020-07-08 – via Google Books.
  6. Parvez Z (1984). "Chapter 4: Review of Immunologic Techniques: Radial Immunodiffusion (RID)". Immunoassays in Coagulation Testing. New York: Springer-Verlag. pp. 21–22. doi:10.1007/978-1-4615-7225-1_5. ISBN 9781461572251. LCCN 83020115. OCLC 851823206. Archived from the original on 2020-07-08. Retrieved 2020-07-08..
  7. Berne (1974), p. 65, Fig. 4; p. 66, text: "In Figure 4, the squares of diameters are plotted as a linear ordinate and the concentrations as a linear abscissa ..... here, linearity appears in progressively larger portions of the curve, corresponding to cessation of growth. The plot is linear over the entire range at equivalence; ...."
  8. Stanley (2002), p. 174, Fig. 12-6.
  9. Fahey JL, McKelvey EM (1965-01-01). "Quantitative determination of serum immunoglobulins in antibody–agar plates" (PDF). Journal of Immunology. Baltimore, Maryland: Williams & Wilkins Co. 94 (1): 84–90. ISSN 0022-1767. LCCN 52052893. OCLC 802743767. PMID 14253527. Archived from the original on 2018-02-25. Retrieved 2007-07-08..
  10. Kalff, MW (March 1970). "Quantitative determination of serum immunoglobulin levels by single radial immunodiffusion". Clinical Biochemistry. Elsevier. 3 (1): 91–104. doi:10.1016/S0009-9120(70)80011-X. PMID 4110625. Archived from the original on 2021-01-10. Retrieved 2021-01-10 – via ScienceDirect. 4. The coefficient of variation of the immunoglobulin determinations in one batch of test serum repeatedly quantified in the course of ten months, was taken as the measure for the reproducibility of the method. This coefficient was 8.5% for IgG, 5.8% for IgA, and 4.4% for IgM. The immunoglobulin levels in this test serum lay in the middle range of the calibration lines. The accuracy of the method increases with the height of the calibration line..
  11. Berne (1974), p. 63, Fig. 2; p. 63, text: "In Figure 2, the concentration is plotted as a logarithmic ordinate and the mean diameter as a linear abscissa .... at first the plot is linear,".
  12. LSUMC/MIP Dental Microbiology Lab (2002):
    "6. Determine the concentration of the unknown test serum by drawing a Standard Curve - plotting the known concentrations of the reference sera versus their diffusion diameters on 2-cycle semi-log paper (see example).
    7. The plot of the reference sera data should give a STRAIGHT LINE, and the concentration of the IgG in the test serum can be accurately determined."

References

Further reading
  • Bhattacharjee S (2013-11-29). "Radial immunodiffusion" (video). Shomu's Biology. Archived from the original on 2021-12-21. Retrieved 2016-06-27 – via YouTube. Introductory video on radial immunodiffusion theory and technique (10:21 minutes).
  • Shaikh S (2015-09-24). "Radial immununodiffusion (Teaching kit)" (video). Archived from the original on 2021-12-21. Retrieved 2017-05-13 – via YouTube. Introductory video demonstrating radial immunodiffusion technique (3:43 minutes).
  • "Radial Immunodiffusion (Mancini Technique)" (video). Frank Lectures. 2017-08-08. Archived from the original on 2021-12-21. Retrieved 2020-07-31 – via YouTube. Introductory lecture/slideshow illustrating radial immunodiffusion theory and technique. (6:56 minutes)
  • "Radial Immunodiffusion". Washington, D.C.: 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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