Immunoglobulins are produced by B lymphocytes and plasma cells. They are considered delayed response proteins as they take 1-3 weeks to increase in response to inflammation or antigenic stimulation. Immunoglobulins are composed of two heavy chains of the same class (eg. IgG, IgM or IgA) and two light chains (either κ or λ). An increase in the concentration of immunoglobulins is referred to as a gammopathy. If this increase consists of many different immunoglobulin clones, this indicates a polyclonal gammopathy, which is typical of infectious or inflammatory conditions. The finding of an abundance of one clone of immunoglobulin indicates a monoclonal gammopathy and is highly suggestive of a neoplastic proliferation of B lymphocytes or plasma cells.
The immunoglobulin class that is most commonly measured is IgG, but IgM and IgA can also be measured. The two main indications for measuring immunoglobulins are to evaluate for failure of transfer of passive immunity (FTPI or FPT) in large animal neonates, and (less commonly) as a supporting test to evaluate monoclonal gammopathies. The classic method for measuring IgG, IgM, and IgA is radial immunodiffusion (RID). A newer and more accurate method for measurement of IgG in foals and crias is also available, called a turbidometric immunoassay (TIA).
Radial immunodiffusion (RID)
Radial immunodiffusion provides more accurate concentrations of serum immunoglobulins. Antisera to specific immunoglobulins (anti-IgG, anti-IgM, or anti-IgA) are incorporated into agarose plates. Patient samples are dispensed into wells cut into the agarose. The sample (containing the antigen) diffuses into the gel. When antibody-antigen complexes form, a precipitation ring can be visualized in the agarose. The width of the ring is compared to that produced by a standard curve obtained from a solution of known immunoglobulin concentration and is proportional to the concentration of that immunoglobulin class in the serum.
Indications for radial immunodiffusion testing are:
- To differentiate between inflammatory polyclonal gammopathies and neoplastic monoclonal gammopathies (used primarily in dogs, cats and horses for this purpose).
- To identify failure of transfer of passive immunity (FTPI) in neonatal foals, calves and crias (more to follow on this topic under causes of hypoglobulinemia).
Turbidometric immunoassay (TIA)
This method uses an automated chemistry analyzer to quantify immunoglobulins, so is more accurate with a quicker turnaround time than the RID method. The intensity of a light beam passing through serum is measured. Anti-IgG antibodies are added to the sample and form a precipitate with the immunoglobulins in serum. A light beam is again passed through the reaction tube, and the light intensity that passes straight through the tube changes in proportion to the concentration of immunoglobulin in the sample.
The TIA method is only available for certain species (currently horses, cattle, and camelids), and only for IgG class. So if you need to measure IgA or IgM, or need to measure immunoglobulins in other species, then you will have to use the RID. The TIA is mostly used for detection of failure of transfer of passive immunity.
Point of care ELISA and latex agglutination tests are available for measurement of serum IgG in foals. They are rapid and easy to use, but are less sensitive to decreased IgG than RID or TIA.
Other crude tests to estimate immunoglobulin concentrations are available but reliability is lower and varies by species. These include measurement of total protein by refractometry or biuret, globulin measurement, and methods dependent on protein precipitation (ZnSO4 turbidity, Na2SO3 precipitation, and glutaraldehyde coagulation test).