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Layman Report . Realization 5 2 Realization 2.1 Immunoassays Immunoassays are chemical tests used to detect or quantify a specific substance, the analyte, in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. The purpose of an immunoassay is to measure an analyte. Common uses include measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury. Radioimmunoassay (RIA) is a method employing radioactive isotopes to label either the antigen or antibody. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. Enzyme immunoassay (EIA) as developed as an alternative to radioimmunoassay (RIA). These methods use an enzyme to label either the antibody or antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods is the enzyme-linked immunosorbent assay (ELISA). For a long time immunoassays belong to the routine methods in biochemistry and medical diagnosis. However in the last 10-15 years their importance in different areas like the environmental analysis and food inspection increased intensely. They are used for the determination of environmental contaminates (aromatic hydrocarbons, pesticides or explosives in soil or water), food additive and pharmaceuticals. For the determination of substances with small sizes of molecules, immunoassays compete with chromatographic methods that are mainly applied today. Immunoassays have several advantages compared to other analytical methods: they are fast and inexpensive and the analysis does not require long-winded clean-up quo data GmbH 10.11.2005
Layman Report . Realization 6 steps for the samples. Thereby a high number of samples can be analysed in a short time and the needed sample volume is low. Additionally this method is suited for an automation. 2.1.1 ELISA The first step of an ELISA is coating a microtiter plate with antibodies that can bind to the analyte. For this purpose a small amount (e.g. 5 µL) of the antibody serum is diluted (e.g. 1:50.000) in a special buffer solution. This buffer solution corresponds to the characteristics (pH value, ionic strength) of the serum fluid in blood from where the antibodies were being isolated. From this solution a defined volume (200 µL) is being pipetted in the wells of the microtiter plate. The plate is shaken over a defined time and the antibodies adsorb on the surface. Afterwards excrescent antibodies are edulcorated by a special washing buffer. Now the sample that contains the analyte (i.e. the substance to be determined) is added. The next step consists of a short waiting time (pre incubation). While this time, the molecules of the analyte can bind to the antibodies that adhere to the surface. Following the so-called enzyme tracer is added. An enzymer tracer is an analyte molecule that is bound to an enzyme that catalyses a specific reaction, at the end the product of this reaction can be measured. The enzyme tracer is also diluted with a specific buffer solution. During a second incubation time, the enzyme tracer competes with the analyte over the binding sites of the antibody due to the structural similarity. The unbound analyte and enzyme tracer are being washed out afterwards, while a part of the added enzyme tracer is remaining bound on the surface. The amount of the bound enzyme tracer can be made observable by adding a special substrate solution. This solution contains two substances and their reaction is catalysed by the bound enzyme. Thereby the colourless input product becomes a coloured reaction product that is optically ascertainable. In the last step the interruption of the catalysed reaction is caused by adding sulphuric acid. Again a change of colour is taking place. Since enzyme tracer and analyte competed over the available binding sites, from the intensity of the colouration the amount of the bound analyte can be concluded. Since only the enzyme tracer possesses the property to catalyse, an intensive colouration indicates a big amount of bound tracer, but small amount of bound analyte while a faint colouration indicates a small amount of bound tracer and a big amount of bound quo data GmbH 10.11.2005
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