Enzyme Linked Immunosorbent Assay ELISA Enzyme Linked Immunosorbent

Enzyme Linked Immunosorbent Assay (ELISA)

• • • Enzyme Linked Immunosorbent Assay (ELISA) Term Was Coined By Engvall and Pearlmann in 1971 Similar To RIA, Except No Radiolabel Can Be Used To Detect Both Antibody and Antigen Very Sensitive, pg/m. L Relies on Monoclonal Abs • ELISA may be run in a qualitative or quantitative format.

• ELISA results are reported as a number using a spectrophotometer, spectrofluorometer, or other optical device. This test is determining the "cut-off" point between a positive and negative result. • Unknowns that generate a signal that is stronger than the known sample are called "positive"; those that generate weaker signal are called "negative.

• Most commonly, ELISAs are performed in 96 well (or 384 -well) usually polystyrene microtiter plates, which will passively bind antibodies and proteins

Advantages of ELISA Less costly and safest. Easy visualization of results with high level of accuracy. Specific and highly sensitive assay that can detect protein at the picomolar to nanomolar range. Easily automated for performance of large numbers of tests. Require minimal reagents. Qualitative detection or Quantitative measurement of either antigen or antibody. Wells can be coated with antigens or antibodies. Can be done by personnel with only minimal training.

Applications of ELISA Analysis of hormones, vitamins, metabolites, and diagnostic markers. Therapeutic drug monitoring. Diagnostic procedures for detecting infection.

Requirements for ELISA test 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. Micro-titer plates: 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.

Enzyme labels • Enzyme labels are used to detect the binding of antigenantibody complex. • It should have high specific reactivity. • It should be easily coupled to antigen-antibody complex and must stable. • Enzymes used in labelling should not be normally present in the patient samples. • Examples of enzyme labels are Horse radish peroxidase, Alkaline phosphatase, and Glucose oxidase.

Stages in ELISA 1. The adsorption of either antigen or antibody to the micro-titer plate. 2. The addition of the test sample and subsequent reagents. 3. The incubation of reactants (formation of antigen-antibody complex). 4. The separation of bound and free reactants by washing. 5. The binding of enzyme to the target antigen or antibody. 6. The addition of substrate (production of a visible signal). 7. The visual or spectrophotometric reading of the assay.

Types of ELISA assay – Direct ELISA – Indirect – Sandwich – Competitive