Paul Bunnell Test

Synonyms: Heterophil agglutination test, Paul-Bunnell-Davidsohn test; Forssman antibody test

The first serological test described for infectious mononucleosis was the heterophile antibody assay developed by Paul and Bunnell. An antigen of the Ebstein Barr virus is similar (shares epitopes) to antigens on the cells of a number of animals (but not man). Following infection, 85-90% of patients with EBV infection produce heterophile antibodies which can be detected by the Paul Bunnell test.

• Sheep red blood cells agglutinate in the presence of heterophile antibodies and this is the basis for the Paul-Bunnell test.
• Horse red cells agglutinate on exposure to heterophile antibodies and this is the basis of the Monospot test.
• Clumping (agglutination) with a titre less than 1:56 is a positive result.
• Heterophile test antibodies are sensitive and specific for EBV heterophile antibodies. Positivity increases during the first 6 weeks of the illness and so the heterophile antibody test results may be negative early in the course of EBV infectious mononucleosis. The titre does not correlate with the severity of the disease.
• Agglutinins remain in the blood for 4 to 8 weeks but may remain positive in low levels for up to one year.

• Agglutination of the sheep cells is of limited value since it is not specific and only determines the presence or absence of heterophile antibodies.
• The Davidsohn differential test is used to remove other EBV-unrelated cross-reacting antibodies and so differentiate between heterophile sheep cell agglutinins in human serum due to Forssman antigen, serum sickness, and infectious mononucleosis.
• Some of the antigens that cause agglutination of sheep erythrocytes are carried on ox (beef) erythrocytes but not on the kidney cells of the guinea pig. Therefore exposure of the test serum to both guinea pig kidney cells and ox (beef) erythrocytes causes absorption of either one or both of these antibodies. The absorbed agglutinins can be removed and the resulting supernatant fluid then tested with sheep erythrocytes.

False positive heterophile antibody tests are rare but may be associated with:

• Rubella
• Cytomegalovirus
• Toxoplasmosis
• HIV
• Herpes simplex virus infection
• Malaria
• Viral hepatitis
• Rheumatoid arthritis
• Systemic lupus erythematosus
• Leukaemia
• Lymphomas
• Burkitt's lymphoma
• Pancreatic cancer

• Occur in 10% of adults and 50% of children.
• May occur if testing is performed too early in the course of the illness.
• The heterophile test is less useful in children younger than 2 years, in whom the results are frequently negative. One study found fewer than half of children aged under four to have detectable heterophile antibodies at any time.¹
• False negative results are also more likely to occur in elderly patients.²

• If a false-positive Paul-Bunnell test result is suspected, then specific testing using an EBV-based antibodies serological test may be useful.
• There is a high (90%) agreement between the heterophile antibodies tests and the Viral Capsid Antigen-IgM ELISA but the specific antibody (VCA IgM) Elisa is more sensitive.³
• The antibody response to specific EBV serological testing consists of measuring the antibody response to surface and core EBV viral proteins. For clinical purposes, the most useful EBV-specific antibodies are the VCAs (Viral Capsid antigens) and the EBNA (EBV Nuclear Antigen). Both VCA and EBNA antibodies are usually reported as IgM or IgG antibodies.
• IgM and IgG antibodies directed against the VCA of EBV are useful in confirming the diagnosis of EBV and in differentiating recent infection from previous infection.
• EBV IgM VCA titres decrease in most patients after 3-6 months but may persist in low titre for up to 1 year.
• EBV IgG VCA antibodies rise later than the IgM VCA antibodies but remain elevated with variable titres for life. Persistent IgG does not indicate chronic infectious mononucleosis and is not relevant in the assessment of chronic fatigue syndrome.⁴ False-positive VCA antibody titer results may occur on the basis of cross-reactivity with other herpes viruses, e.g. CMV, or with unrelated organisms, e.g. Toxoplasma gondii.
• As with the heterophile test, the EBV antibody response may be falsely negative early in the course of the infection. False negativity also may occur in young children (less than 2 years old).