Immunoglobulins

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.

Measurement

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 identifying immunodeficiencies. 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, calves 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 (see separate page on FTPI)

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 bound to latex beads are added to the sample and form a precipitate with the immunoglobulins in serum. As immune-complexes form, more light will pass through and will be measured as a change in absorbance, which is proportional to the concentration of IgG 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.

Other methods

Point-of-care ELISA, latex agglutination and lateral flow assay 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. All are based on immunologic measurement of IgG.

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).

Test interpretation

Increased immunoglobulin concentration

The most common cause of increased immunoglobulins is antigenic stimulation. Neoplasia of plasma cells or B cells can result in paraneoplastic production of abnormal immunoglobulins, which helps us diagnose these diseases. The increases in immunoglobulins can frequently be inferred from finding rouleaux formation in blood smears (rare in ruminants and excessive in horses and cats, which can normally display a mild degree of rouleaux formation in red blood cells). However, fibrinogen (a beta-globulin) can be and is responsible for rouleaux formation in acute inflammatory conditions. Serum electrophoresis is useful for distinguishing between causes of hyperglobulinemia. 

• Antigenic stimulation: This usually occurs as part of an acquired immune response to antigenic stimulation, such as chronic liver disease, cancer, inflammation, or infection. Increases in all immunoglobulins may be seen in these conditions, with increases in IgA being typical of inflammation involving mucosal surfaces (e.g. lungs, gastrointestinal tract) and IgM increases reflecting a more acute reaction. 
• Neoplasia of plasma cells or B cells: Neoplasia of immunoglobulin-producing cells can result in a monoclonal or biclonal gammopathy. Plasma cell neoplasms include extramedullary plasmacytoma and multiple myeloma, which secrete IgG or IgA (rarely IgM), whereas B cell neoplasms can also produce a monoclonal gammopathy, typically involving IgM or IgG. Chronic lymphocytic leukemia and B cell lymphoma (mature cell neoplasms) can result in a monoclonal IgG gammopathy, which we have seen more frequently in horses than other species (Tallmadge et al 2015). IgM-secreting B cell neoplasms are part of the syndrome of Waldenstrom’s macroglobulinemia, which is identified more in humans than veterinary species. Immunofixation and agarose gel electrophoresis have become the go-to method for identifying the involved class of immunoglobulins in neoplastic gammopathies (Moore and Avery 2019, Moore et al 2021 part 1 and 2).

Decreased immunoglobulin concentrations

This can be physiologic in a young animal, but we typically think of immunodeficiency syndromes with low globulins. Immunodeficiencies can be inherited or acquired.

• Inherited hypogammaglobulinemia: A variety of inherited immunodeficient syndromes have been reported. Although some involve cell-mediated immunity (e.g. PSCID), they often have concurrent gamma globulin deficiencies due to impaired helper T cell function.
  • Primary severe combined immunodeficiency: This has been reported in Bassett hounds, Cardigan Welsh Corgis, Dachshunds and Arabians (full and crosses). It is characterized by a lymphopenia, decreased IgM in a presuckle foal, absent IgM and IgA post-suckling. IgM, IgG and IgA are all low after 3 months of age as maternally-derived antibodies are degraded. Animals have thymic and lymph node atrophy and die at a young age (usually when maternal antibodies disappear) of opportunistic infections, e.g. Pneumocystis carinii, adenovirus, cryptosporidiosis.
  • Agammaglobulinemia: This has been reported in foals. They have no B cells and lack Igs by 3 months of age. T cell function is normal as are lymphocyte counts. They die of repeated infections, with a poor response to therapy, by 12-18 months of age.
  • IgM deficiency: Selective IgM deficiency has been reported in horses (Arabians, Paso Fino, quarterhorses and thoroughbreds) and Dobermans. Horses usually die of fatal pneumonia, arthritis and enteritis. Dogs usually have no clinical signs as long as IgG and IgA levels are normal. Animals may have normal globulin concentrations.
  • IgA deficiency: This has been reported in various dog breeds, including Sharpeis, Beagles, Airedale terriers, and German Shepherd Dogs. They suffer from recurrent infections involving the urinary tract, respiratory tract, and skin. Animals may have normal globulin concentrations.
  • Transient hypogammaglobulinemia: This has been reported in Arabian horses and dogs. They have a delayed onset of post-natal immunoglobulin synthesis and are susceptible to adenoviral and bacterial infections.
• Acquired immunodeficiencies: These are, by far, more common than inherited immunodeficiencies.
  • Failure of transfer of passive immunity (FTPI): Animals are dependent upon ingestion of colostrum for passive immunity as immunoglobulins do not cross the placenta as they do in human beings. FTPI results when neonates fail to suckle or if dams leak colostrum pre-parturition. For diagnosis of FPTI, determination of IgG is recommended within 24 to 48 hours of birth. Point-of-care assays are measurement of IgG in foals and crias are available, however different kits are of variable accuracy. The gold standard is now the immunoturbidometric assay used on chemistry analyzers. All of these assays are antibody-based, i.e. rely on species specific anti-IgG antibodies for detection of IgG in whole blood (point-of-care assays), serum or plasma.
  • Infectious diseases
    • Viruses: Feline leukemia virus and feline immunodeficiency virus are known causes for acquired immunodeficiencies in cats. Canine distemper virus causes immunodeficiency in dogs. Bovine viral diarrhea causes immunodeficiency in cattle and Aleutian mink disease virus (a parvovirus) causes immunosuppression in ferrets.
    • Parasites: Toxoplasmosis and Theileria cause immunodeficiency. Generalized infection with Demodex canis is often found in immunodeficient dogs, however it may be a result of immunodeficiency and not its cause. Eperythrozoon wenyonii infection in cattle is associated with reduced humoral immunity.
    • Johne’s disease: Causes decreased T cell function.
  • Neoplasia: Lymphoma in cattle and horses is associated with immunosuppression. Low IgM levels can be seen in horses with lymphoma, however IgM is not considered a good biomarker of the disease due to poor sensitivity (28-50% depending on the cut-off) and specificity (35-88%) (Perkins et al 2003). Multiple myeloma in small animals and horses may be associated with markedly decreased immunoglobulin concentrations of the classes that are not forming the paraprotein (e.g. in an IgA monoclonal gammopathy, IgM and IgG concentrations may be decreased).
  • Idiopathic: Idiopathic immunodeficiency has been reported in young llamas with failure to gain weight, ill-thrift and recurrent infections. Many of these llamas have concurrent Eperythrozoon infections.