Procedure for the ELISA Test

ELISA is the acronym for a forensic test procedure called enzyme-linked immunoabsorbent assay. It is based on the high specificity of antibodies for a specific antigen. Researchers use chemically-modified antibodies anchored to a sample well and color-tagged antigens to determine the presence and concentration of a wide variety of compounds, drugs and pathogens. Samples can be taken from blood, urine, saliva and other sources.

Antibodies and Antigens

• ELISA normally uses several chemically-engineered components to generate a test for a given antigen. The first step in producing an ELISA test is to develop an antibody to the compound that is being tested. The antibody is then purified, modified, and anchored or immobilized on a sample well that will be used to perform the test. The compound being tested for will have a fluorescent tag added.

  Industry uses a standard rectangular plate with "sample wells"--like tiny bowls or test tubes molded within to perform multiple experiments. Plate dimensions are 127.8mm by 85.5mm and standardized so they fit analytical instruments from a variety of manufacturers. Typical plates have between six and 96 sample wells, with newer models have hundreds or more.

The Test

• When a solution containing the compound you are testing for and the color-tagged compound are put into the sample well, they compete for binding on the immobilized antibody. The more native compound, the less color-tagged compound will be able to bind to the immobilized antibody. The sample well is rinsed of unbound compound (native and colored). The sample well is evaluated for how much color-tagged compound remains, usually with a spectrophotometer.

The Results

• Less color means a higher concentration of the native compound. Researchers will set up a "standard curve" in a series of wells to which they will add a range of known concentrations of the native compound. The ELISA test is performed on all wells and the amount of color difference across the span of standards allows researchers to compare and determine unknown concentrations in the samples they are evaluating.

  ELISA tests are used to test for a wide variety of things from HIV virus to drugs, poisons and other chemicals. They have the advantage of high-throughput for large numbers of samples and when running many samples they can be very cost-effective.

  The ELISA test can also be used to detect antibodies in a test solution. In this case the antigen (portion of the compound of interest that binds to the antibody) is immobilized on the sample well, and antibodies for the test have a color tag attached.