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Single radial immunodiffusion (SRID)

ISSN 2398-2942

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Synonym(s): SRID

Overview

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

Uses

Alone

  • The main application of SRID to companion animal veterinary medicine is for the quantification of the major classes of serum immunoglobulin.
  • For the dog, quantification of serum immunoglobulin (Ig) G Blood biochemistry: gamma globulin , IgM and IgA are generally available.
  • Quantification of canine complement factors C3 and C4, and feline IgG, have also been documented using this method.
  • The test would predominantly be used to determine abnormally low levels of one or more serum immunoglobulins, as a screen for presumptive immunodeficiency disease.
  • The most common canine immunodeficiency is IgA deficiency which is defined by persistently subnormal levels of serum IgA, usually determined by SRID.
  • More complex immunodeficiencies, eg that which occurs in weimaraner dogs Weimaraner immunodeficiency may be characterized by multiple immunoglobulin class deficiencies.
  • Elevated levels of serum immunoglobulin may also be investigated using SRID, eg dogs with autoimmune disease or chronic infectious disease may have hypergammaglobulinemia due to elevation in serum IgG.
  • The test may be used to detect subnormal levels of serum complement components, as a prelude to more specialized testing to confirm complement deficiency.

In combination

  • Serum protein electrophoresis (SPE) Serum protein electrophoresis may be considered a screening test for abnormalities in gamma globulins.
  • Hypo- or hypergammaglobulinemia may be further investigated by quantifying the major classes of serum immunoglobulin.
  • Identification of a monoclonal gammopathy by SPE would indicate further identification of the nature of this protein by immunoelectrophoresis (IEP) or SRID.
  • Defects in complement components may be confirmed by a range of specialist functional tests of the complement pathways.

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 canine 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 canine IgG, or a standard serum pool of known immunoglobulin concentration.
  • Generally, 4 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 hour 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 canine IgG, IgM and IgA quantification is not widely available within the UK.
  • The test is performed by Langford Veterinary Diagnostics, University of Bristol.
  • Test kits for determination of canine IgG, IgM and IgA concentration are commercially available from ICN-Flow. However, these are costly, have a limited shelf life, and are sold for assay of relatively large numbers of serum samples (the kits include three plates, each designed to test 14 serum samples) per test. This would generally preclude in-practice use of these test kits.
  • Reagents for performing SRID for canine immunoglobulin determination are also marketed by the Bethyl Company. However, the use of these cannot be recommended as the nature of the standards used gives spuriously high values, particularly for IgA.

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, saliva or duodenal juice.
  • Such samples must be analyzed 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 from VetMedResource and PubMed.
  • 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 Immunopathol 64, 107-121.
  • Harley R, Gruffydd-Jones T J & Day M J (1998) Determination of salivary and serum immunoglobulin concentrations in the cat. Vet Immunol Immunopathol 65, 99-112.
  • Day M J, Power C, Oleshko J & Rose M (1997) Low serum immunoglobulin concentrations in related weimaraner dogs. JSAP 38, 311-315.
  • Schreiber M, Kantimm D, Kirchhoff D, Heimann G & Bhargava (1992) Concentrations in serum of IgG, IgM and IgA and their age-dependence in beagle dogs as determined by a newly developed ELISA. Eur J Clin Chem Clin Biochem 30, 775-778.
  • Day M J & Penhale W J (1988) Serum immunoglobulin A concentrations in normal and diseased dogs. Res Vet Sci 45, 360-363.
  • Day M J, Kay P H, Clark W T, Shaw S E, Penhale W J & Dawkins R L (1985) Complement C4 allotype association with and serum C4 concentration in an autoimmune disease of the dog. Clin Immunol Immunopathol 35, 85-91.

Other sources of information

  • Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing.

Organisation(s)

  • Clinical Immunology Laboratory, Langford Veterinary Diagnostics, Department of Clinical Veterinary Science, University of Bristol, UK (offers SRID for canine IgG, IgM and IgA).