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ELISA
The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries. In the most simple form of an ELISA, antigens from the sample to be tested are attached to a surface. Then, a matching antibody is applied over the surface so it can bind the antigen. This antibody is linked to an enzyme and then any unbound antibodies are removed. In the final step, a substance containing the enzyme's substrate is added. If there was binding, the subsequent reaction produces a detectable signal, most commonly a color change. Performing an ELISA involves at lea ...
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Eva Engvall
Eva Engvall, born 1940, is one of the scientists who invented ELISA in 1971. She is an elected fellow of the American Association for the Advancement of Science. Vita Eva Engvall earned her PhD from the University of Stockholm in 1975. Her postdoctoral work was done at the University of Helsinki and City of Hope National Medical Center in California, where she was subsequently appointed to staff. In 1979, Engvall was recruited to Sanford-Burnham Medical Research Institute in La Jolla, California (then called La Jolla Cancer Research Foundation). From 1993 to 1996, Engvall held joint appointments at Sanford-Burnham Medical Research Institute and as Chairperson of the Department of Developmental Biology at Stockholm University. Research Enzyme-linked immunosorbent assay (ELISA) uses antibodies to detect proteins and other different immunogens. Eva Engvall was one of the two Swedish scientists at Stockholm University (the other was the principal investigator Peter Perlmann) who ...
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Immunoassay
An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an " analyte" and is in many cases a protein, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes. Immunoassays come in many different formats and variations. Immunoassays may be run in multiple steps with reagents being added and washed away or separated at different points in the assay. Multi-step assays are often called separation immunoassays or heterogeneous immunoassays. Some immunoassays can be carried out simply by mixing the reagents and sample and making a p ...
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Antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and Viral disease, viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can ''tag'' a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly (for example, by blocking a part of a virus that is essential for its invasion). To allow the immune system to recognize millions of different antigens, the antigen-binding sites at both tips of the antibody come in an equally wide variety. In contrast, the remainder of the antibody is relatively constant. It only occurs ...
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Assay
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called the analyte, the measurand, or the target of the assay. The analyte can be a drug, biochemical substance, chemical element or compound, or cell in an organism or organic sample. An assay usually aims to measure an analyte's intensive property and express it in the relevant measurement unit (e.g. molarity, density, functional activity in enzyme international units, degree of effect in comparison to a standard, etc.). If the assay involves exogenous reactants (the reagents), then their quantities are kept fixed (or in excess) so that the quantity and quality of the target are the only limiting factors. The difference in the assay outcome is used to deduce the unknown quality or q ...
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Microtiter Plate
A microplate, also known as a microtiter plate (''Microtiter'' is a registered trademark in the United States, therefore it should not be used generically without attribution), microwell plate or multiwell, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories. A very common usage is in the enzyme-linked immunosorbent assay (ELISA), the basis of most modern medical diagnostic testing in humans and animals. A microplate typically has 6, 12, 24, 48, 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix. Some microplates have been manufactured with 3456 or 9600 wells, and an "array tape" product has been developed that provides a continuous strip of microplates embossed on a flexible plastic tape. Each well of a microplate typically holds somewhere between tens of nanolitres to several millilitres of liquid. They can also be used to store dry powd ...
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3,3',5,5'-Tetramethylbenzidine
3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA). TMB is a white solid that forms a pale blue-green liquid in solution with ethyl acetate. TMB is degraded by sunlight and by fluorescent lights. Enzymatic assay TMB can act as a hydrogen donor for the reduction of hydrogen peroxide Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscous than water. It is used as an oxidizer, bleaching agent, and antiseptic, usually as a dilute solution (3% ... to water by peroxidase enzymes such as horseradish peroxidase. The resulting one-electron oxidation product is a diimine-diamine complex, which causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at the wavelengths of 370 and 650 n ...
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Antigen-antibody Interaction
Antigen-antibody interaction, or antigen-antibody reaction, is a specific chemical interaction between antibodies produced by B cells of the white blood cells and antigens during immune reaction. The antigens and antibodies combine by a process called agglutination. It is the fundamental reaction in the body by which the body is protected from complex foreign molecules, such as pathogens and their chemical toxins. In the blood, the antigens are specifically and with high affinity bound by antibodies to form an antigen-antibody complex. The immune complex is then transported to cellular systems where it can be destroyed or deactivated. The first correct description of the antigen-antibody reaction was given by Richard J. Goldberg at the University of Wisconsin in 1952. It came to be known as "Goldberg's theory" (of antigen-antibody reaction). There are several types of antibodies and antigens, and each antibody is capable of binding only to a specific antigen. The specificity of ...
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Enzyme Immunoassay
An enzyme immunoassay is any of several immunoassay methods that use an enzyme bound to an antigen or antibody. These may include: * Enzyme-linked immunosorbent assay (ELISA) * Enzyme multiplied immunoassay technique (EMIT) * Fluorescent enzyme immunoassay Allergies, also known as allergic diseases, refer a number of conditions caused by the hypersensitivity of the immune system to typically harmless substances in the environment. These diseases include hay fever, food allergies, atopic dermati ...s (FEIAs) * Chemiluminescent immunoassays (CLIAs) * Radioimmunoassays (RIAs) {{set index ...
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Radioimmunoassay
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies. Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains among the least expensive methods to perform such measurements. It requires special precautions and licensing, since radioactive substances are used. In contrast, an immunoradiometric assay (IRMA) is an immunoassay that uses radiolabeled molecules but in an immediate rather than stepwise way. A radioallergosorbent test (RAST) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy. Method Classically, to perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it ...
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Ligand Binding Assays
A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor. There are numerous types of ligand binding assays, both radioactive and non-radioactive.Joseph R. Lakowicz. (1991) Topics in Fluorescence Spectroscopy: Biochemical applications. As such, ligand binding assays are a superset of radiobinding assays, which are the conceptual inverse of radioimmunoassays (RIA). Some newer types are called "mix-and-measure" assays because they do not require separation of bound from unbound ligand. Ligand binding assays are used primarily in pharmacology to p ...
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Secondary Antibody
Primary and secondary antibodies are two groups of antibodies that are classified based on whether they bind to ''antigens or proteins'' directly or target another (primary) antibody that, in turn, is bound to an ''antigen or protein''. Primary A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease and they are used for the study of absorption, distribution, metabolism, and excretion (ADME) and multi-drug resistance (MDR) of therapeutic agents. Secondary Secondary antibodies provide signal detection and amplification along with extending the utility of an antibody through conjugation to proteins. Secondary antibodies are especially efficient in immunolabeling. Secondary antibodies bind to primary antibodies, which are directly bound to the target antigen(s). In immunolabeling, the primary antibody's Fab domain binds to an antigen and exposes its Fc domain to secondary antibody. Then ...
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Biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and metabolism. Over the last decades of the 20th century, biochemistry has become successful at explaining living processes through these three disciplines. Almost all areas of the life sciences are being uncovered and developed through biochemical methodology and research.Voet (2005), p. 3. Biochemistry focuses on understanding the chemical basis which allows biological molecules to give rise to the processes that occur within living cells and between cells, Karp (2009), p. 2. in turn relating greatly to the understanding of tissues and organs, as well as organism structure and function. Miller (2012). p. 62. Biochemistry is closely related to molecular biology, which is the study of the molecular mechanisms of biological phenom ...
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