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.[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 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

For most antigens, the area and the square of the diameter of the circle at the circle's endpoint are directly proportional to the 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 linear scales will usually be a straight line after all circles have reached their end points (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 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 endpoint.[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 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][8] 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]

References
  1. Berne BH (1974-01-01). "Differing methodology and equations used in quantitating immunoglobulins by radial immunodiffusion--a comparative evaluation of reported and commercial techniques" (PDF). Clinical Chemistry. Washington, D.C.: American Association for Clinical Chemistry. 20 (1): 61–69. doi:10.1093/clinchem/20.1.61. ISSN 0095-1137. LCCN 58002529. OCLC 43430009. PMID 4203461. Archived from the original (PDF) on 2017-08-08. Retrieved 2015-11-15.
  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 J (2002). "Chapter 12: Precipitation: Single Radial Immunodiffusion: Laboratory Technique 12-1: Radial Immunodiffusion Test". Essentials of Immunology & Serology. Albany, New York: Delmar Division of Thomson Learning. pp. 172–174. ISBN 978-0914826255. LCCN 2002280630. OCLC 1149023866. Retrieved 2017-05-15 – via Internet Archive.
  4. LSUMC/MIP Dental Microbiology Lab (2002). "II. Lab Work: B. Radial Immunodiffusion". Exercise 3: Antigen-Antibody I. New Orleans, Louisiana: Louisiana State University School of Medicine: Department of Microbiology, Immunology & Parasitology. Retrieved 2015-11-14. Archived 2004-08-04 at the Wayback Machine.
  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. Archived 2019-12-09 at the Wayback Machine.
  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. Retrieved 2020-07-08. Archived 2020-07-08 at the Wayback Machine.
  7. Fahey JL, McKelvey EM (1965-01-01). "Quantitative determination of serum immunoglobulins in antibody–agar plates" (PDF). Journal of Immunology. Baltimore, Maryland: American Association of Immunologists. 94 (1): 84–90. ISSN 1048-3233. LCCN sf85007036. OCLC 204767467. PMID 14253527. Retrieved 2007-07-08. Archived 2018-02-25 at the Wayback Machine.
  8. 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. 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. Archived 2021-01-10 at the Wayback Machine.

Further reading
This article is issued from Wikipedia. The text is licensed under Creative Commons - Attribution - Sharealike. Additional terms may apply for the media files.