Heterophile antibody test

Heterophile antibody test

The test is sensitive for heterophile antibodies produced by the human immune system in response to EBV infection.

The monospot test, a form of the heterophile antibody test,[1] is a rapid test for infectious mononucleosis due to Epstein–Barr virus (EBV). Commercially-available test kits are 70-92% sensitive and 96-100% specific.[2] It will generally not be positive during the 4-6 week incubation period before the onset of symptoms. It will also not generally be positive after active infection has subsided, even though the virus persists in the same cells in the body for the rest of the carrier's life.



The test relies on the agglutination of horse RBCs by heterophile antibodies in patient's serum. Heterophile means it reacts with proteins across species lines. [3] A 20% suspension of horse red cells is used in an isotonic 3-8 % sodium citrate formulation. One drop of the patient’s neat serum to be tested is mixed on an opal glass slide with one drop of a particulate suspension of guinea-pig kidney stroma, and a suspension of beef red cell stroma; sera and suspensions are mixed with a wooden applicator for 10 times. Ten micro liter of the horse red cell suspension are then added and mixed with each drop of absorbed serum. The mixture is left undisturbed for one minute (not rocked or shaken). Examine for the presence or absence of red cell agglutination. If stronger with the sera absorbed with guinea-pig kidney, the test is positive. If stronger with the sera absorbed with beef red cell stroma, the test is negative. If agglutination is absent in both mixtures, the test is negative. A known 'positive' and 'negative' control serum is tested with each batch of test sera.


It is indicated as a confirmatory test when a physician suspects EBV, typically in the presence of clinical features such as fever, malaise, pharyngitis, tender lymphadenopathy (especially posterior cervical; often called "tender glands") and splenomegaly.[4]

Usefulness of the test

Because the specificity of the test is high, a positive test is useful in confirming EBV. However, the sensitivity is only moderate, so a negative test does not exclude EBV.

This lack of sensitivity is especially the case in young children, many of whom will not produce the heterophile antibody at any stage and thus have a false negative test result.

In the case of delayed or absent seroconversion, an immunofluorescence test could be used if the diagnosis is in doubt. It has the following characteristics: VCAs (Viral Capsid Antigen) of the IgM class, antibodies to EBV early antigen (anti-EA), absent antibodies to EBV nuclear antigen (anti-EBNA)


  1. ^ Basson V, Sharp AA (May 1969). "Monospot: a differential slide test for infectious mononucleosis". J. Clin. Pathol. 22 (3): 324–5. doi:10.1136/jcp.22.3.324. PMC 474075. PMID 5814738. http://jcp.bmj.com/cgi/pmidlookup?view=long&pmid=5814738. 
  2. ^ Elgh F, Linderholm M, Clinical and Diagnostic Virology 7 (1996) 17-21 http://dx.doi.org/10.1016/S0928-0197(96)00245-0
  3. ^ heterophil - Definitions from Dictionary.com
  4. ^ Davidson's Principles & Practices of Medicine 20th ed