The use of antibodies in the ELISA test.

The idea

ELISA (enzyme-linked immunosorbent assay) uses the specificity of antibodies to detect and measure a molecule. In a direct test the antigen is immobilised on the surface of a well of a microtitre plate, a monoclonal antibody with an enzyme attached and a binding site complementary to that antigen is added and binds, the well is washed to remove unbound antibody, then a substrate is added and the enzyme converts it, producing a colour change. In an indirect test, such as the HIV test, the antigen is fixed to the well, the patient's plasma is added so any complementary antibodies bind, and a second, enzyme-linked antibody that binds to those antibodies is added before the substrate. Washing between every step is essential, because unbound antibody left behind gives a false positive. The intensity of the colour is proportional to the quantity bound, so the test is quantitative.

What earns the marks

• An antibody with a binding site complementary to the antigen binding to it, forming an antigen-antibody complex.
• A second or labelled antibody, with an enzyme attached, binding to that complex.
• Washing to remove unbound antibody, since otherwise a colour change gives a false positive.
• The enzyme acting on an added substrate to produce a colour change, with the intensity of colour proportional to the quantity present.
• Interpreting a positive result with care, for example maternal antibodies crossing the placenta producing a positive result in an uninfected baby.

Common mistakes and how to put them right

• Describing the antigen-antibody binding as an enzyme-substrate reaction.

  Put it right: Only the attached label is an enzyme.

• Omitting the washing steps, which carry marks in their own right.

• Treating a positive result as proof of current infection, when antibodies persist after infection and can be maternal.

• Confusing which molecule is fixed to the well.

  Put it right: In the HIV test the antigen is fixed and the patient supplies the antibody.

• Saying the colour only shows presence, when the depth of colour gives a quantitative result.

Exam tips

• Track each molecule through the diagram in order and name it: antigen, first antibody, second antibody with enzyme, substrate, colour change.
• In 'evaluate this test' questions use the data first, how early the disease is detected and how many positives are found, before commenting on speed, cost and access to laboratories.
• Do not confuse ELISA with lateral flow (immunochromatography) tests: ELISA is performed in wells of a microtitre plate, whereas a lateral flow test uses a test strip with a control line to show the sample has moved along the strip.