A Heterophile Antibody Test: Quick Diagnosis of EBV Infectious Mononucleosis

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The Epstein Barr virus (EBV), which causes infectious mononucleosis (IM) or “mono,” is one of the most common human viruses. EBV occurs worldwide, and most people become infected with EBV sometime during their lives. In the United States, as many as 90% of adults between 35 and 40 years of age have been infected. In most developed countries, including the U.S., infection with EBV most often occurs during adolescence or young adulthood, causing infectious mononucleosis in 35% to as many as 90% of infected individuals, according to a recent study.1

While treatment of IM normally entails bed rest and adequate intake of fluids, and the disease is typically self-limiting, it can be devastating in immunocompromised patients. In addition, in rare cases, IM is associated with serious complications, including myocarditis, neurologic syndromes, compromised respiration secondary to acute lymphadenopathy, anemia, and ruptured spleen. Therefore, accurate diagnosis and prompt treatment of complications are important. The differential diagnosis of IM is also useful in ruling out other mononucleosis-like illnesses induced by cytomegalovirus, adenovirus, or Toxoplasma gondii.

The Mono Spot Test for Rapid Diagnosis

The characteristic triad of fever, pharyngitis, and lymphadenopathy lasting for one to four weeks, together with the age of the patient (most typically adolescent), suggest the clinical diagnosis of IM. Laboratory tests are usually advisable for confirmation. Findings on a complete blood count (CBC) with differential include a normal to moderately elevated white blood cell count, an increased total number of lymphocytes, and greater than 10% atypical lymphocytes. The CBC is actually more useful for ruling out other illnesses that mimic IM. Specific diagnostic tests include testing for heterophile antibodies, which are IgM antibodies that do not react with EBV antigens but recognize antigenic determinants on sheep, horse, and cattle erythrocytes. If the heterophile antibody test (now universally known as the mono spot test) is persistently negative, then tests are performed for EBV-specific antibodies, because false negative reactions to heterophile antibodies occur in 10% of adults and 50% of children.

In keeping with the rapid proliferation of point-of-care diagnostic kits and devices, several products have now been CLIA-waived by the U.S. Food and Drug Administration (FDA). By March 2013, the FDA listed 55 CLIA-waived tests for infectious mononucleosis heterophile antibodies.

  • Quidel Corporation (San Diego, CA) provides the QuickVue®+ Infectious Mononucleosis Test, which is designed to detect IgM heterophile antibodies in whole blood (CLIA-waived) and serum or plasma (CLIA-nonwaived). The test consists of the Reaction Unit, a plastic housing containing a membrane strip which provides solid support for the chromatographic immunoassay. The sample well contains an absorbent pad which provides an even flow of the sample along the membrane. The first zone of the membrane is coated with blue latex beads that are conjugated to goat antihuman IgM antibodies. The second zone of the membrane contains two immobilized agents: 1) blue latex beads to provide a preprinted blue horizontal line, and 2) bovine erythrocyte extract. The third zone of the membrane contains an agent capable of binding the antibody-blue latex to provide the vertical “Test Complete” line. For the test procedure, serum, plasma, or whole blood is added to the “Add” well, followed by addition of the Developer. As the sample/developer fluid moves by capillary action across the first zone of the membrane, it mobilizes the antibody-blue latex. The fluid continues to move with the antibody-blue latex across the membrane to the immobilized bovine erythrocyte extract (antigen) zone. If the specific IM heterophile antibodies are present in the sample, a “sandwich” of solid phase/IM antibody/antibody-blue latex is formed. A vertical line will appear, resulting in a positive sign (+) in the “Read Result” window. If the antibody is not detected, the “Read Result” window will only contain the preprinted blue horizontal line, indicating a negative (–) result. As the fluid continues to move the antibody-blue latex across the membrane, it comes in contact with the reagent in the “Test Complete” window. The 5-min test is complete when a blue line appears. The built-in controls satisfy CLIA requirements for daily QC. The 20-test kit contains positive and negative external controls and can be stored at room temperature.
  • Alere, Inc. (Waltham, MA) offers the BioStar® Acceava® Mono II test kit, which is CLIA-waived for whole blood and CLIA-nonwaived for serum and plasma. The test is a rapid chromatographic immunoassay in a dipstick format intended for the qualitative detection of IM heterophile antibody. Bovine erythrocyte extract interacts with the blood sample. If the IM heterophile antibody is present in the sample, a visible blue test line will appear to indicate a positive result; a red line indicates a negative test outcome. Two drops of whole blood from a venipuncture or fingerstick sample are mixed in a tube with buffer, and the dipstick is placed in the tube. Results are available in 5 min. There are 25 tests/kit, and no refrigeration is needed for storage.
  • Jant Pharmacal Corporation (Encino, CA) provides the Accutest® Infectious Mononucleosis Rapid Test Device, which is CLIA-waived for whole blood, but can also be used for serum and plasma. The test is a rapid visual lateral-flow chromatographic immunoassay for the detection of heterophile antibodies, and can be used to help in the diagnosis of IM in patients with clinical symptoms common to IM. The test is in a cassette format. There are 20 tests/box, and results are available in 5 min.


  1. Balfour, H.H.; Odumade, O.A. et al. Behavioral, virologic, and immunologic factors associated with acquisition and severity of primary Epstein-Barr virus infection in university students. J. Infect. Dis. 2013, 207, 80–8.

Please check out our Point of Care Devices (POC Diagnostic Devices) section to find manufacturers that sell these products