Single radial immunodiffusion (SRID) is an agar gel precipitation test used to detect and quantify the binding of antigen and antibody.
The main application of SRID to veterinary medicine is for the quantification of the major classes of serum immunoglobin. It is also sometimes used for detection of specific antigens.
Quantification of serum immunogloblin (Ig)G, IgM and IgA can be performed, as well as complement factors, eg C3 and C4.
The test is predominantly used to determine abnormally low levels of one or more serum immunoglobulins, as a screen for presumptive immunodeficiency disease. Failure of passive transfer of immunoglobulins Foal: failure of passive transfer (IgG) at birth is the most common reason for assessing IgG levels in foals.
The test may be used to detect subnormal levels of serum complement components, although primary complement deficiencies have not yet been reported in horses.
Serum protein electrophoresis (SPE) 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 immunoglobin.
Identification of a monoclonal gammopathy by SPE would indicate further identification of the nature of this protein by immunoelectrophoresis (IEP) or SRID.
Defect in complement components may be confirmed by a range of specialist functional tests of the complement pathways.
Source of test material
The sample required is clotted blood or serum by standard venipuncture Blood: collection .
Quantity of test material
1-2 ml of clotted blood provides sufficient serum for the assay.
Samples may be stored before testing for immunoglobulin conentration.
Serum harvested from clotted blood can be stored in a refrigerator (4°C/39.2°F) and tested within a week.
Alternatively, serum may be frozen to -20°C/-4°F for longer term storage, eg frozen serum samples will be suitable for analysis for many years.
Serum or clotted blood taken for quantification of immunoglobulins may be posted to the laboratory by standard means.
Serum immunoglobulins are relatively stable and heat-resistant, and there will be minimal degradation of these molecules following storage (mailing) at room temperature for 1-2 days.
Note that for quantification of complement molecules the requirements are different. Complement is extremely heat-labile, and therefore serum must be harvested from whole clotted blood and stored to -20°C/-4°F (-70°C/-94°F is optimum, but such refrigeration is not widely available outside research institutions) within 1 h of collection.
Serum for complement analysis must be stored frozen until just before use.
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.
SRID for equine IgG, IgM and IgA quantification is not widely available within the UK and is mainly used in research establishments.
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.
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.
Normal (reference) values
Generally, an IgG level in a newborn foal of <4 g/l is considered to indicate failure of transfer and a value of 4-8 g/l is considered partial failure of transfer.
A range of factors must be taken into consideration when interpreting serum immunoglobulin levels as an indicator of immunodeficiency, particularly the age.
Young foals are likely to have some level of maternal (colostrally-derived) immunoglobulin which will be detected by the test in addition to the foal's endogenously produced immunoglobulin.
The testing laboratory should provide some interpretation of the significance of the concentration of immunoglobulin obtained.
Errors and artifacts
At least some species (canine), it is known that serum IgA concentration can undergo day-to-day and diurnal variation, so a single 'point assay' of serum IgA concentration may be misleading and repeat sampling may be advisable to confirm a low serum concentration.
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