How to Do an Elisa

An enzyme-linked immunosorbent assay (ELISA) is a technique for detecting a known antibody in a sample. The ELISA test has applications in immunology, where it's used as a diagnostic tool in medicine, and it's also used as a quality-control measure in other industries. The ELISA requires at least one antibody that is specific to the antigen being tested for and some means of making the antigen detectable.

Difficulty: Moderate

Instructions

1. Prepare the sample. This procedure varies widely according to the specific ELISA test. For example, in an ELISA for HIV, the blood serum is diluted 400 times before applying it to a plate that contains HIV antigens.

2. Wash the plate to remove all the other components of the blood serum. Apply a secondary antibody that binds to the HIV antibodies to the plate and wash the plate again. This secondary antibody is chemically linked to an enzyme which the plate now contains in proportion to the amount of secondary antibody.

3. Apply a substrate to make the enzyme detectable. This typically causes the enzyme to change color or fluorescence. The results of an ELISA must be reported as a number so the color change must be quantified in some way.

4. Establish the threshold for a positive result. This is the most difficult part of the ELISA because the result can cover a wide range. An minimum value must therefore be selected to identify a positive result.

5. Perform an ELISA on a control sample with a known concentration of the analyte and set the signal that this sample produces as the standard. Any test sample that produces a stronger signal than the control sample is a positive result.

References

• University of Arizona

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