Question

What does ELISA stand for? What are ELISA assays used for in labs? Give at least three examples. Briefly describe two limitations of an ELISA. Please use your own words. Which portion of the blood is used for the test? Explain. In five sentences, summarize how an ELISA assay “works.” How is the test quantified?

Answer 1

ELISA- Enzyme linked immuno sorbent assay.

This assay is used to determine the antigen-antibody interaction. It is used to measure and determine the no.of antibodies in blood as well as the specificity of antibody towards infectious agents.

Example of ELISA assay include-

1. Pregnancy test kits.

2. Diagnosis of HIV infection.

3. Detection of potential food allergens.

Limitations of ELISA-

1. Possibility of false positive or false negative results.

2. Plasma constituents may affect the enzyme activity.

3. Specificity towards one antigen at a time makes this test limited to some extent.

Serum or plasma is used as blood samples in elisa assay. Antibodies specified for a particular antigen (through exposure to antigen) are found in the serum of blood. Removal of RBCs result in plasma from which antibodies are purified for detection of antigens.

Summary : The first step begins with immobilisation of antigen to be detected on a solid support (depending upon the type of ELISA performed). After the immobilisation of antigen, antibody is added to determine the antigen-antibody relationship. The antibody itself could be covalently linked to a secondary antibody already linked with an enzyme through biconjugation or only to an enzyme. Washing between every step and is an important process for removal of unbound antibodies or unwanted proteins. Final step includes the addition of substrate which gets linked with the enzyme and produces visible signal for the detection of antigen (amount of antigen).

The signal can be detected using available instruments like flourometer, spectrophotometer or luminometer.

Quantitatively, data of ELISA assay can be interpretated via comparison with a standard curve obtained through dilution of a purified antigen. This can be utilized for the detection of accurate concentration of antigen in the sample.