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ELISA

What the assay tells you

The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding. Depending on what variation you use, it will detect antigen (hormones, enzymes, microbial antigens, illicit drugs) or antibody (anti-HIV in the screening test for HIV infection) in body fluids or tissue culture supernatents.

What you need to do the assay

• Purified antigen (if you want to detect or quantify antibody).
• Purified antibody (if you want to detect or quantify antigen).
• Standard solutions (positive and negative controls).
• Sample to be tested.
• Microtiter dishes: plastic trays with small wells in which the assay is done.
• Wash fluid (buffer).
• Enzyme-labeled antibody and enzyme substrate.
• ELISA reader (spectrophotometer) for quantitative measurements.

How the assay is done

To detect antibody (indirect ELISA):

• Coat the microtiter plate with purified antigen by letting an antigen solution sit in the wells for 30-60 minutes. Wash away unbound antigen with buffer and cover any sites that might nonspecifically bind antibody with unrelated protein (such as solution of powdered milk), again washing away unbound protein.
• Add serum sample to be tested for specific antibody to plate and allow specific antibody to bind to the antigen. Wash off unbound antibody.
• Add anti-Ig that will bind to Fc region of specific antibody (for example, anti-human gamma chain that will bind human IgG). The Fc region of the anti-Ig is covalently linked with enzyme. Wash off unbound antibody-enzyme complex.
• Add chromogenic substrate: colorless substrate that the enzyme will convert to a colored product. Incubate until color develops; measure color in a spectrophotometer. The more color that is detected, the more specific antibody is present in the unknown sample.
• Negative controls include
  • omit the antigen
  • omit the test antiserum or substitute with an antibody that will not bind the antigen.
• Positive control substitutes known positive serum for unknown serum.

To detect antigen (sandwich ELISA):

• Coat the microtiter plate with purified antibody to the antigen. Wash away unbound antibody and cover any sites that might nonspecifically bind with unrelated protein.
• Add sample to be tested for antigen to plate and allow antigen to bind antibody. Wash off unbound antigen.
• Add enzyme-labeled specific antibody to a different epitope of the antigen to make a "sandwich"; wash away unbound antibody.
• Add chromogenic substrate for enzyme that will be converted to a colored product.
• Negative control omits unknown antigen; positive control uses known antigen.

How to interpret the results

The amount of colored product is proportional to the amount of enzyme-linked antibody that binds, which is directly related to the amount of antibody that was present to bind antigen or antigen that was present to bind antibody. If known amounts of antigen or antibody are added, a standard curve can be constructed which will allow the amount of unknown antigen or antibody to be determined.

Additional comments

The ELISA is probably the most commonly used immunological assay because of its versatility, sensitivity (ability to detect small amounts of antigen or antibody), specificity (ability to discriminate between closely related but antigenically different molecules), and ease of automation. Although some of the substrates are carcinogenic, they are generally considered safer than radioisotopes used in RIA (radioimmunoassay). By binding the initial layer to plastic, washing away unbound reagents becomes rapid and requires no expensive equipment - the plate can be flodded with buffer and shaken over a beaker to remove the wash fluid and unbound antigen or antibody. The process can be easily automated for performance of large numbers of tests. False positives can result from impure reagents: for example, tissue culture-grown HIV used in the HIV ELISA can react with antibodies to tissue-culture grown influenza present in someone who has just had an influenza vaccination.

Useful web sites

Biology project interactive ELISA tutorial: http://www.biology.arizona.edu/immunology/activities/elisa/main.html

Interactive virtual ELISA assay: http://www.hhmi.org/biointeractive/immunology/vlab.html

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