Equis ISSN 2398-2977

Single radial immunodiffusion (SRID)

Contributor(s): Alastair Foote, Vetstream Ltd

Overview

  • Single radial immunodiffusion (SRID) is an agar gel precipitation test used to detect and quantify the binding of antigen and antibody.

Sampling

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Tests

Methodologies

  • An agarose gel is prepared, into which is incorporated an antiserum specific for the molecule to be detected by the radial diffusion assay.
  • In the present context, these would be antisera specific for either equine IgG   IgG  , IgM or IgA.
  • Antisera specific for the heavy chain (Fc portion) of these molecules are required.
  • Wells are cut into the gel, and into these are loaded a predetermined volume of test serum or standard control.
  • The standards consist of either purified immunoglobulin of the type to be assayed, eg purified equine IgG, or a standard serum pool of known immunoglobulin concentration.
  • Generally, four standards are used to generate a standard curve, which is needed to determine the immunoglobulin concentration of a test sample. These comprise the standard of maximal immunoglobulin concentration, and three further doubling dilutions of this standard, ie undiluted standard, ½ dilution, ¼ dilution and 1/8 dilution.
  • When the wells in the agarose gel have been loaded, the plate is incubated in a humid chamber for a 24 h period. During that time the immunoglobulin contained within the serum diffuses from the test well into the surrounding agarose gel. Here, it will interact with the antiserum and a zone of precipitation will form around the well.
  • The larger the diameter of this precipitin ring, the greater the concentration of immunoglobulin in the original sample.
  • Following the period of incubation, the diameters of the precipitin rings obtained for each standard and test serum sample are measured.
  • A standard curve is constructed (line of best fit) on a semi-logarithmic scale, and the immunoglobulin concentrations within the test samples can be calculated.

Availability

  • SRID for equine IgG, IgM and IgA quantification is not widely available within the UK and is mainly used in research establishments.

Validity

Sensitivity

  • The sensitivity of radial diffusion for immunoglobulin quantification is to 0.1 mg/ml.
  • Samples with less than this amount will be reported as having <0.1 mg/ml. This degree of sensitivity means that the radial diffusion test is not suitable for measuring immunoglobulin levels in relatively dilute samples such as cerebrospinal fluid, bronchoalveolar lavage fluid, tears, salive or duodenal content.
  • Such samples must be analyuzed by the relatively more sensitive enzyme linked immunosorbent assay (ELISA)   Enzyme linked immunosorbent assay (ELISA)  , but this test is only performed on a research basis.

Specificity

  • The specificity of the assay is determined by the specificity of the antisera incorporated into the agarose gel.
  • These must be specific for immunoglobulin heavy chain (Fc portion), as any reactivity with light chain will negate the specificity of the test.

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references fromPubMed.
  • de Camargo M M, Kuribayashi J S, Bombardieri C R & Hoge A (2009)Normal distribution of immunoglobulin isotypes in adult horses.  Vet J182(2), 359-361PubMed.
  • Barton M H, Hurley D, Norton N, Heusner G, Costa L, Jones S, Byars D & Watanabe K (2006)Serum lactoferrin and immunoglobulin G concentrations in healthy or ill neonatal foals and healthy adult horses. J Vet Intern Med20(6), 1457-1462PubMed.
  • Pellegrini-Masini A, Bentz A I, Johns I C, Parsons C S, Beech J, Whitlock R H & Flaminio M J (2005) Common variable immunodeficiency in three horses with presumptive bacterial meningitis.  JAVMA227(1), 114-122, 187PubMed.
  • McFarlane D, Sellon D C & Gibbs S A (2001)Age-related quantitative alterations in lymphocyte subsets and immunoglobulin isotypes in healthy horses.  Am J Vet Res62(9), 1413-1417PubMed.
  • German A J, Hall E J & Day M J (1998)Measurement of IgG, IgM and IgA concentrations in canine serum, saliva, tears and bile. Vet Immunol Immunopathol64(2), 107-121PubMed.


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