Monospot test: Principle, Procedure, Uses

The monospot test, also known as the Paul-Bunnell test, is a form of the heterophile antibody test used to rapidly screen infectious mononucleosis (mono).  Epstein–Barr virus (EBV) causes the disease infectious mononucleosis. Blood, serum, or plasma is taken and is looked for the presence of heterophile antibodies.

When a person is infected with Epstein-Barr virus (EBV), the immune system makes antibodies to fight this infection which is detected in the blood using this test. Monospot testing can usually detect antibodies two to nine weeks after infection.

Paul and Bunnell were the first scientists who suggested hemagglutination of sheep RBC to detect heterophile antibodies associated with infectious mononucleosis.


Serum/Plasma: Serum or plasma may be used with the monospot test, provided an acceptable medical technique collects whole blood. Avoid using grossly hemolyzed sera. The serum should be clear and free of bacterial contamination. Specimens can be stored at 2 to 8°C for 24 hours. For prolonged storage, specimens should be frozen (-20°C).


A sample of the patient’s serum or plasma is placed on a microscope slide and mixed with guinea pig kidney antigen and preserved horse erythrocytes (these reagents can be purchased from commercial suppliers of kits). Thoroughly mix the reagents and rock the slide for a minute. Suppose heterophil antibodies are present in the serum/plasma and the blood clumps (agglutinates). This result usually indicates a mono infection. It typically is not used to diagnose mono that started more than six months earlier. The results of a monospot test are usually ready within one hour.

Heterophile-antibody test


  1. Normal (negative): The blood sample does not form clumps (no heterophil antibodies are detected).
  2. Abnormal (positive): The blood sample clumps (heterophil antibodies are detected). If the blood sample clumps, you probably have mono.

Accuracy of Monospot Test

Studies have shown that monospot test produces both false-positive and false-negative results. Results should be correlated with clinical and hematological findings.

  1. False Positive Reactions: The antibodies detected by monospot can be caused by conditions other than infectious mononucleosis. It may occur in persons with:
    • Leukemia or lymphoma
    • Hepatitis
    • Rubella
    • Systemic lupus erythematosus (SLE)
    • Toxoplasmosis
  2. False Negative Reactions
    • Children, especially those younger than two years, are more likely than adults to have a negative monospot test (25%–50%), even when they have infectious mononucleosis.
    • It takes time to develop antibodies if the test is done within the first few weeks of becoming infected with EBV (approx. 25% false-negative rate in the first week). This may lead to a false-negative result.
    • If the first test does not indicate mono but still has symptoms, the test may be repeated.

Uses of Monospot Test:

The monospot test is not recommended for general use. Monospot test may indicate that a person has a typical case of infectious mononucleosis, but does not confirm the presence of EBV infection.

References and further readings

  1. Marshall-Andon T, Heinz P. How to use … the Monospot and other heterophile antibody tests. Archives of Disease in Childhood – Education and Practice 2017;102:188-193.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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