Enzyme-linked immunosorbent assay advantages

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

TIR-based techniques that use fluorescence transduction (TIRF) have been used for the majority of the applications discussed above. These platforms, while not currently the gold standard for measurement of many target analytes, do offer advantages over current technologies, such as cell culture, chromatography, and the enzyme-linked immunosorbant assay (ELISA). Advantages include the ability to perform faster, more sensitive, multiple analyte, and real-time measurements. 

The use of immunoassays in the field of agricultural research has increased dramatically in recent years, and has become a reliable analytical tool that possesses numerous advantages over standard, chemical extraction and analytical methods. A few Of these advantages (described in several review articles (1,2)), include its greater sensitivity and specificity, the increased speed of the assay, which allows greater sample thrdugh-put, the requirement for smaller samples for extraction, and the assay s improved cost effectiveness. Enzyme-linked immunosorbent assays (ELISA) have been... 

Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. Both polyclonal and monoclonal antibodies have their advantages, and or disadvantages, but in general the production of monoclonal antibodies is more time consuming and requires tissue culture facilities and skills. The use of either monoclonal or polyclonal antibodies in some of the applications may require that the antibody is in a purified form. They can be purified by a variety of methods described in the next few chapters. The availability of commercially available kits primarily designed for the purification of IgG and IgM classes of antibodies derived from all common animal species should also be mentioned. 

Another technique, the enzyme-linked immunosorbent assay (ELISA), combines the specificity of antibodies with the sensitivity of an enzyme assay. The ELISA can be performed in a variety of combinations that involve either a specific antibody or the total cellular protein immobilized on a solid support, such as the wells of a plastic microplate. In one version of the method, the sandwich ELISA, the primary antibody is bound to the wells. When a mixture of proteins is added, the protein of interest binds to the antibody, and other proteins are washed away. A second labeled antibody, specific to a different epitope on the protein, is added, and the amoruit of signal is proportional to the amoimt of the particular protein in the sample. The method can be modified to detect specific antibodies in a mixture by using their antigen as the immobilized bait. ELlSAs also have the advantage of being able to be performed in 96-weU plates so many samples can be analyzed in one experiment. 

A further alternative is to implement these approaches with the use of redox and/or nanoparticle labels. The redox and nanoparticle labels replace the familiar enzyme labels in enzyme-linked immunosorbent assays (ELISAs) and offer the advantages of simplified protocol, wider linear dynamic range, higher stability compared to enzymes, and higher separation efficiency. 

There are more or less specific color tests for starch (iodine blue) and carrageenans (using methylene blue), and in some instances the action of enzymes is diagnostic and used in reagent kits. A relatively new approach to the assay of food gums and stabilizers, one that shows many advantages in speed and specificity, is enzyme-linked immunosorbent assay, which has a number of variants. 

Specific methods Immunochemical methods have been developed for identification and specific determination of proteins that have no catalytic activity. The method is based on the special relationship between a protein and a specific antibody that has been raised against the protein. The advantages of an immunoassay lie in its high specificity and detectability. Since the specificity is independent of the activity of the antigen protein, the method measures all enzyme molecules, inactive as well as active. The methods depend on the availability of specific antisera directed against the particular enzymes under study. The antibody is raised in an experimental animal by injection of a sample of protein antigen. Selective precipitation method and enzyme linked immunosorbent assay (ELISA) are commonly used for the specific detection and reliable measurement of a particular enzyme in a raw sample, such as a tissue extract or body fluids. 

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