ELISA

Enzyme-Linked Immunosorbent Assay



ABSTRACT

A laboratory technique for detecting specific antigens or antibodies by using enzyme-labeled immunoreactants and a solid-phase binding support, such as a test tube. A number of different enzymes can be used, including carbonic anhydrase, glucose oxidase, and alkaline phosphatase. Labeling is done by covalently binding the enzyme to the test substance through an enzyme-protein coupling agent such as glutaraldehyde. Products of the reaction may be detected by fluorometry or photometry. ELISA is nearly as sensitive as radioimmunoassay and more sensitive than complement fixation, agglutination, and other techniques. It is commonly used in the diagnosis of human immunodeficiency virus infections.

Enzyme-Linked Immunosorbent Assay

Introduction

ELISA, enzyme linked immunosorbent assay, has been used very successfully to measure and monitor the presence of antibodies in biological fluids.In this variant, the immunosorbent is an immobilised antigen (rabbit immunoglobulin) relevant to the particular antibody to be measured (Leng, 2008, pp.879-84). after reacting the immobilised antigen with varying concentrations following a subsequent reaction with an enzyme-labelled anti- immunoglobulin antibody.The experimental aims to:develop your aknowledge of the of a method to evaluate the use of conjugates couples to antibodies in order to visualise antibody/antigen binding, such as utilised in modern immunoassay.

Enzyme-linked immunosorbent assay is a rapid immunochemical test that involves an enzyme. It also involves an antibody or antigen (immunologic molecules). ELISA method is used for measuring a wide variety of tests of body fluids. ELISA tests detect substances that have antigenic properties, primarily proteins rather than small molecules and ions, such as glucose and potassium. Some of these substances include hormones, bacterial antigens, and antibodies. There are variations of this test, but the most basic involves an antibody attached to a solid surface, such as human chorionic gonadotropin (hCG), the commonly measured protein that indicates pregnancy (Lequin, 2005, pp.2415-8).

A mixture of purified hCG linked to an enzyme and the test sample (blood, urine, and so on) are added to the test system. If no hCG is present in the test sample, then only the hCG and the linked enzyme bind. The more hCG present in the test sample, the less enzyme-linked hCG binds. The substance the enzyme acts on is then added, and the amount of product is measured in some way, such as by a change in color of the solution. ELISA tests are generally highly sensitive and specific, and they compare favorably with radioimmune assay (RIA) tests. They have the added advantage of not requiring the use of radioisotopes or radiation-counting apparatus.

Methodology

Coating the microtitre plate (this has been done by the technicians for you). The normal rabbit serum (NRS) containing rabbit immunoglobulin is added to wells A-H in varying concentrations. Concentrations may be 100 microlitres of 1/500 down to 100 microlitre of 1/50,000. wells down to number 8 may be coated.b) Adding anti-rabbit immunoglobulin-peroxidase conjugateafter 3 washes with phosphate buffered saline (PBS, ph 7.2), to each of the coated wells should be added 100 microlitre of 1/200 anti-rabbit immunoglobulin-peroxidase conjugate. Incubate at room temperature for approximately 30 ...