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The monospot test, a form of the heterophile antibody test, is a rapid test for infectious mononucleosis due to Epstein–Barr virus (EBV). It is an improvement on the Paul-Bunnell test. The test is sensitive for heterophile antibodies produced by the human immune system in response to EBV infection. Commercially available test kits are 70-92% sensitive and 96-100% specific. 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 the horse RBCs by heterophile antibodies in patient's serum. Heterophile means it reacts with proteins across species lines. Heterophile also can mean that it is an antibody that reacts with antigens other than the antigen that stimulated it (an antibody that crossreacts). A 20% suspension of horse red cells is used in an isotonic 3-8% sodium citrate formulation. One drop of the patient’s 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 10 times. Ten micro liters of the horse red cell suspension are then added and mixed with each drop of adsorbed 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 adsorbed with guinea-pig kidney, the test is positive. If stronger with the sera adsorbed 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.
The specificity of the test is high, virtually 100%, so a positive test is useful in confirming EBV. Rarely, however, a false positive heterophile antibody test may result from toxoplasmosis, rubella, lymphoma and leukemia.
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)
It was named after the two American Physicians, John Rodman Paul (1893 – 1971) and Walls Willard Bunnell (1902 – 1965) who devised the test in 1932. 
In the past, the most common test for diagnosing infectious mononucleosis was the heterophile antibody test, which involves testing heterophile antibodies by agglutination of guinea pig, sheep and horse red blood cells. As with the aforementioned criteria, this test is specific but not particularly sensitive (with a false-negative rate of as high as 25% in the first week, 5–10% in the second and 5% in the third). About 90% of patients have heterophile antibodies by week 3, disappearing in under a year