Heterophile Antigens: Definition, Examples, and Diagnostic Tests

A child recovers from a simple sore throat, and few weeks later develops a damaged heart valve. The bacteria are long gone. What harms the heart is the child's own antibodies, made against the throat infection, which happen to also fit a protein in heart muscle. The immune system cannot tell the two apart.

This accidental resemblance between unrelated molecules is the idea behind heterophile antigens, and the same principle that makes them dangerous in disease also makes them useful in the laboratory, where one antigen can be used to detect antibodies raised against a completely different one.

Common antigens (or antigens of similar nature) that are possessed by a variety of phylogenetically unrelated species are known as heterophile antigens.

Antibodies induced by these antigens cross-react with related heterophile antigens. Such antibodies are called heterophile antibodies: an antibody produced against an antigen of one species can react with the antigen of another.

The term heterophile antibody applies to antibodies having the capacity to react with certain antigens, which are quite different from, and phylogenetically unrelated to the one which evoked antibody response.

Heterophile antigens are involved in the pathogenesis of certain diseases, such as infectious mononucleosis, rheumatic fever, glomerulonephritis, etc.

The M protein of Group A beta-hemolytic streptococci (Streptococcus pyogenes) and proteins of the human heart are heterophilic. In rheumatic fever, antibodies raised against the streptococcal M protein cross-react with cardiac myosin and related muscle proteins, and this misdirected attack damages heart tissue, including the valves. This is the classic example of molecular mimicry, where the immune response to a pathogen accidentally targets a look-alike self-protein.

Figure: Heterophile antigen

Forssman antigen is another example of heterophile antigen. It is a lipid carbohydrate complex (glycolipid) widely distributed in man, animals, birds, plants and bacteria. Forssman antigen was named after the Swedish pathologist John F. Forssman who reported, ‘antibodies produced in rabbits against organ homogenates from guinea pigs reacted with sheep erythrocytes, causing hemolysis’.

Molecular mimicry: one mechanism, two consequences

Heterophile antigens matter because of a single underlying fact: two molecules from completely unrelated sources can share a similar enough shape that one antibody binds both. This accidental resemblance is called molecular mimicry, and it cuts two ways.

In disease, it is harmful. When the body makes antibodies against a pathogen and those antibodies also fit a self-protein, the immune response turns on the body's own tissue. Rheumatic fever is the textbook case: anti-streptococcal antibodies attack cardiac myosin. Some forms of glomerulonephritis follow the same pattern, with cross-reacting antibodies deposited in the kidney.

In the laboratory, the same cross-reactivity is useful. If a patient's antibody, raised against antigen A, also reacts with an unrelated antigen B, then B can be used as a cheap and convenient stand-in to detect that antibody. This is exactly how the classic heterophile tests work: they use an easily available antigen to detect antibodies that were actually provoked by something else. The specific tests are covered in the next section.

So the same property, cross-reactivity, is a cause of disease in the body and a diagnostic tool on the bench. Keeping that dual role in mind organizes the whole topic.

Diagnostic Applications of Heterophilic Test

Heterophilic antigens can be used in various serologic tests. Antibody against one antigen can be detected in the patient’s serum by employing a different antigen which is heterophile (cross-reactive) to the first antigen.

• Weil-Felix reaction: Used for the diagnosis of typhus fever.  Antibodies produced against Rickettsial antigens are detected by using cross-reacting Proteus antigen.
• Paul-Bunnell test: The classic heterophile test for infectious mononucleosis, caused by the Epstein-Barr virus. Patients with mononucleosis produce heterophile antibodies that cross-react with sheep red blood cells, so agglutination of sheep erythrocytes by the patient's serum supports the diagnosis. The rapid slide version of this principle is the Monospot test, which is covered in detail in a separate article.
• Cold agglutinin test: Patient suffering from primary atypical pneumonia caused by Mycoplasma pneumoniae produces antibodies that cross-reacts with human O blood group RBC. Agglutination of human O group erythrocytes at 4‎°C by the sera of suspected patient sample confirms the diagnosis.  Alternatively in Streptococcus MG test, such antibodies are detected using Streptococcus MG antigens.

How to remember

Heterophile = "other-loving." "Hetero" (different) plus "phile" (loving): an antigen that reacts with antibodies from a different, unrelated species. The name itself is the definition.

One mechanism, two faces: cross-reactivity harms the body (rheumatic fever, glomerulonephritis) and helps the lab (Paul-Bunnell, Weil-Felix, cold agglutinins). If you remember the mechanism is shared, you never have to memorize the two lists separately.

The three classic tests, by the stand-in antigen they use:

• Paul-Bunnell → sheep red cells → infectious mononucleosis (EBV)
• Weil-Felix → Proteus bacteria → typhus (rickettsiae)
• Cold agglutinin → human O red cells at 4°C → atypical pneumonia (Mycoplasma)

Don't confuse the two "cardio" antigens: rheumatic fever is cardiac myosin (heterophile mimicry). Cardiolipin is the syphilis test antigen. Different word, different disease.

Key exam facts in one table

Where students get confused

"Heterophile antibodies are made against the disease organism." Not exactly, and this is the key subtlety. In the diagnostic tests, the antibody is provoked by one thing (the Epstein-Barr virus in mononucleosis) but is detected using an unrelated antigen (sheep red cells). The antibody is not anti-sheep and not always anti-EBV in a targeted sense; it is a cross-reacting antibody.

"Rheumatic fever damages the heart because of cardiolipin." No. The rheumatic fever target is cardiac myosin. Cardiolipin is the antigen used in syphilis serology. This is one of the most common mix-ups in immunology.

"Forssman antigen is a protein." No. It is a glycolipid, a lipid-carbohydrate complex, found across many unrelated species.

"The Paul-Bunnell test uses EBV antigen." No. It uses sheep red cells. The whole point of a heterophile test is that it detects the antibody without using the antigen that actually caused it.

"Cross-reactivity is always a laboratory artifact." No. The same cross-reactivity that enables these tests also causes real disease when antibodies against a pathogen attack a similar-looking self-protein.

References

1. Cunningham MW. Molecular mimicry, autoimmunity, and infection: the cross-reactive antigens of group A streptococci and their sequelae. Microbiol Spectr. 2019;7(4). https://doi.org/10.1128/microbiolspec.gpp3-0045-2018
2. Punt J, Stranford SA, Jones PP, Owen JA. Kuby Immunology. 8th ed. W.H. Freeman; 2019.
3. Votava M, Bartosová D, Krchnáková A, et al. Diagnostic importance of heterophile antibodies and immunoglobulins against Epstein-Barr virus capsid antigen in children. Acta Virol. 1996;40(2):99–101.