Saturday, April 17, 2010

ELISA :Enzyme linked immunosorbent assay

ELISA :Enzyme linked immunosorbent assay, is a technique where an antibody or and antigen is identified accurately this test is also known as ELISA, enzyme immunoassay or EIA, in ELISA , a combination of immunological and biochemical principles are employed.Radio Immunoassay was the only method available for detection of an antgen or antibody before ELISA was invented.

Principle of ELISA technique:

In ELISA technique , an unknown amount of antigen is immobilized or affixed over a surface of a polystyrene microtiter plate, and a specific antibody is washed over this surface where the unknown amount of antigen is immobilized or affixed, so that a immunological reaction occurs and they form a antigen-antibody complex . The antibody which is being washed over the fixed antigen is linked to an enzyme, for the purpose triggering biochemical reaction which can be made visible or measured as a signal of completion of a antigen antibody reaction . In fluorescence ELISA the reaction triggered by enzyme produces a fluorescence when light of suitable wavelength is passed on the sample , the magnitude fluorescence be measured .

In ELISA one of example of enzyme is peroxidase which reacts with substrate tetra 3’3,5’5 methylebenzidine which in produces a colour as a indicator for presence of a antigens or antibody.
ELISA requires at least one antibody which is specific for the particular antigen.

Procedure :
1.The sample containing unknown an unknown amount of antigen is first immobilized on a solid support of polystyrene microtiter plate this involves phenomenon known as adsorption , if the antigen is poorly adsorbed , then it is first linked to an antibody which has a good affinity for the surface to get adsorbed , this is also known as sandwich" ELISA

2.After the sample containing antigen is immobilized then the detection antibody is added over it which forms a complex with the immobilized antigen.This detection antibody is linked to an enzyme covalently or through bioconjugation,

3.After each step the plate is washed carefully with a solution of mild surfactant to remove unbound nonspecific antigens and proteins . In the final wash step the plate is added with substrate which is acted upon by the tagged or linked enzyme and produces a visible or detectable signal, which can be measured to quantify the amount of antigen present in the sample.

ELISA techniques also make use of a indicating immunological or biochemical polymerase chain reaction (real time PCR) , fluorogenic, electrochemiluminescent for the purpose of ease of detection and measure.
Newer ELISA techniques also make use of non enzyme-linked antibodies instead of this they make use of a non-enzymatic reporter.

ELISA techniques are used as quality control tool in manufacturing of biopharmaceutical and in various pharmaceuticals manufacturing. It is also used for detection of HIV antigen.

No comments: