Components of the immune system called antibodies are found in the liquid portion of blood and help protect the body from harm. Antibodies can be used also outside the body in a laboratory-based assay to help diagnose disease caused by malfunctions of the immune system or by infections.

This virtual laboratory will demonstrate how such a test, termed an enzyme-linked immunosorbent assay (ELISA), is carried out and some of the key experimental problems that may be encountered. Students will learn about the assay procedure and the equipment and materials that are needed. By completing this exercise, students will gain a better understanding of experimental design, key concepts in immunological reactions, and interpretation of data.

Concepts Covered

  • The basis of humoral immunity
  • The foundation for ELISA
  • Potential errors in conducting an ELISA
  • Sensitivity and specificity of a diagnostic test

    Potential Experimental Problems

    ELISA is used in many laboratories to determine whether a particular antibody is present in a patient's blood sample. Although the procedure is routine and straightforward, it involves a number of variables, such as reagent selection, temperature, volume measurement, and time, which if not adjusted correctly can affect subsequent steps and the test outcome. This virtual laboratory has been developed so that when a mistake is made, you will not get the correct answer. The program keeps track of errors made throughout the experiment and generates a report at the end.

    Limitations of the Test

    This general test has some important limitations.

    First, a positive result correctly confirming the presence of antibody does not necessarily mean the patient is sick. The body can continue to produce antibodies even though the person may have had the disease earlier and recovered.

    Second, people may be poor producers of antibody or may have some interfering substance in their blood. The amount of antibody, consequently, may be too low to measure accurately or may go undetected. This result is termed a false negative.

    Third, a positive result may occur if an unrelated antibody reacts with the antigen nonspecifically. Unlike a true-positive result where the specific antibody is detected, however, this positive reaction is false. Testing many patients and running tests more than once helps lab workers distinguish a true from a false result. To avoid simple experimental mistakes leading to incorrect results, scientists conduct tests using duplicate (or, sometimes, more than two) samples.

    Begin with the Background describing the experiment.