Enzyme-linked immunosorbent immunoblotting

Laboratory confirmation is vital to effective treatment of HSV, especially in individuals in whom a clinical diagnosis cannot be obtained. There are several methods by which a definitive diagnosis may be acquired, and these include virologic typing, serologic diagnosis, rapid point-of-care antigen detection, enzyme-linked immunosorbent assay (ELISA), immunoblot, and DNA polymerase chain reaction.27... 

The products of gene expression (mRNA and proteins) can be measured by techniques such as the following. Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radioactive probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays (ELISAs) and western blots (immunoblots) are used to detect specific proteins. 

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa has proven its worth as an enzyme label for many years. It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed using a variety of different substrates. Its activity is easily detected by eye in, for example, immunoblots, and it can be quantified by changes in absorbance, fluorescence, or luminescence for use in enzyme-linked immunosorbent assays. 

B36. Buyon, J. P., Winchester, R. J., Slade, S. G., Arnett, F., Copel, J., eta .. Identification of mothers at risk for congenital heart block and other neonatal lupus syndromes in their children. Comparison of enzyme-linked immunosorbent assay and immunoblot for measurement of anti-SS-A/Ro and anti-SS-B/La antibodies. Arthritis Rheum. 36, 1263—1273 (1993). 

Chatterjee, U., Mondal, G., Chakraborti, P., Patra, H. K., and Chatterjee, B. P 2006. Changes in the allergenicity during different preparations of pomfret, hilsa, bhetki and mackerel hsh as illustrated by enzyme-linked immunosorbent assay and immunoblotting. Int Arch Allergy Immunol 141 1-10. 

Use of immunohistochemistry, immunoblotting, and enzyme-linked immunosorbent assay to detect the partially proteinase resistant form of the prion (PrP ) protein. 

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. 

Both polyclonal and monoclonal antibodies can be used in many of the same application such as immuno-histochemistry, immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISAs). 

Purity for a small molecule is a relatively simple concept. Normally, an HPLC method is sufficient to measure the content and impurity levels of a small molecule drug. A macromolecule, such as a protein, has a much more complex behavior. Determining protein concentration by UV absorption spectroscopy can give a measure of the total protein in the product, but it will not necessarily differentiate between active protein and inactive protein (i.e., denatured or otherwise degraded). A validated method or methods to determine the biological activity of the molecule is needed. So, whereas protein concentration is usually tested as part of the specifications, it is also normally accompanied by one or more methods that measure or correlate to biological activity. This is the bioassay. These methods can be animal-based or cell-based, protein interaction assays, binding methods such as surface plas-mon resonance or ELISA (enzyme-linked immunosorbent assay) and immunoblot methods. 

Antibodies were raised in rabbits against the individual polypeptides components of the ftc. complex that were purified by preparative SDS-polyacrylamide gel electrophoresis p]. IgG was purified from the antisera by ammonium sulfate precipitation, and immunoblotting was performed as described by Towbin et al.[5] using peroxidase-labeled goat anti-rabbit IgG as the second antibody and 4-chloro-l-naphthol for the staining reaction. Assessment of the specificity of the antisera by this procedure indicated that of the four subunits, only the 14 MDa component was non-immunogenic. Antibody titers were measured by enzyme-linked immunosorbant assay. For the iron-sulfur protein, sera obtained 11 weeks after immunization exhibited a detectable reaction at dilutions up to 10. ... 

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