How Does This Work?

The interaction of antigen and antibody outside the body--in the laboratory--can be used to determine if a patient has an infectious or an autoimmune disease. The test measures whether a specific antibody associated with an illness can be found in a patient's blood. A positive result indicates that the antibody is there and implies that the person has encountered a particular disease.

This exercise begins with removing red and white blood cells, which can interfere with the test, from a patient's blood sample. The watery fluid that remains is called serum. A portion of serum possibly containing the antibody is allowed to react with the target antigen. A correct match causes the antigen and antibody to bind together.

Detection becomes possible when a second antibody is added. This antibody is prepared from the serum of an animal injected previously with human antibody; the human antibody in this case serves as an antigen and the animal thus produces an antibody against the human antibody. Once isolated, the second antibody can be chemically linked to a system that can produce a detectable signal.

In ELISAs, the antigen antibody complex is exposed to the second antibody, which binds to the antibody portion of the complex (against which it was formed), creating a sandwich-type structure (Figure 1). The signaling system consists of an enzyme attached to the second antibody. When the appropriate chemical is added, the enzyme converts it to a colored substance that can be measured.

This test quantifies how much enzyme is present by the amount of color produced. The more enzyme present, the more secondary antibody must be attached. The amount of secondary antibody present is determined by the amount of target, or first antibody, available. Finally, because the first antibody binds to antigen, the more antigen that is accessible, the more first antibody will be retained. The measure of color, therefore, reflects the amount of antigen initially present.

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