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.

Here are some articles which will be useful for you in further understanding of aspects of sterile dosage form manufacturing and regulatory affairs and good manufacturing practice in pharmaceutical industry

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Requirements of documents for validation of sterilisation process

How to investigate OOS out of specification results

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Aspects of validation of manufacturing process in sterile pharmaceuticals

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