Antibody production, technique electrophoresis

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

A number of immunological techniques have been used in comparative studies.8,9 The most important of these is microcomplement fixation (MC F), a quantitative technique that has played a key role in many classic studies of molecular evolution and molecular systematics. By selecting proteins with different rates of evolution, a broad range of divergences can be examined. The cost of the technique is moderate, but biochemical expertise is required and the labor involved is substantial. Protein must be purified from some or all taxa for antibody production, and, for those taxa, a sizable tissue or serum sample is needed. Antibody production itself is usually done in rabbits, so an animal care facility must be available. Like isozyme electrophoresis, the large body of immunological distance data already available ensures the continued value of this technique for certain investigations. 

Over the past 40 years, capillary electrophoresis (CE) has advanced significantly as a technique for biomolecular characterization. It has not only passed the transition from a laboratory curiosity to a mature instrumental-based method for micro-scale separation, but also emerged as an indispensable tool in the biotech and pharmaceutical industries. CE has become a method of choice in research and development (R D) for molecular characterization, and in quality control (QC) for the release of the therapeutic biomolecules.In the biopharmaceutical industry, more and more CE methods have been validated to meet International Conference on Harmonization (ICH) requirements. In this chapter, we present real industrial examples to demonstrate the role of CE in R D of pharmaceutical products. The focus in this chapter is on method development analytical control for manufacturing and release of therapeutic proteins and antibodies. 

The use of capillary electrophoresis has become an integral part of the study of biopharmaceuticals, especially for the monitoring of product identity and purity. It is a powerful technique that, in many instances, has been shown to be superior to the more conventional electrophoretic techniques and complementary to the widely used high-resolution chromatographic techniques. It is particularly well suited to the study of complex mixtures such as glycoproteins and monoclonal antibodies. 

Two-dimensional gel electrophoresis has become the method of choice for protein separations, when high resolutloh Is required. Routinely, >1000 polypeptides can be separated by this technique, with >3000 possible uhder special conditions. This paper discusses the basic methods Involved, such as gel composition and solubilization buffers, as well as recent advances, such as membrane preparatlohs, gel scanning devices and the production of antibodies from gel spots. 

The determination of the animal species contributing to the meat(s) in a meat product is important for marketing purposes. It may be necessary, in some cases, to ensure the absence of meat of a particular species, such as pork. A number of methods for meat species identification are used which are based on immunological antigen-antibody reactions, on protein isolation techniques, such as electrophoresis, or on DNA analysis. 

In many cases, for the purpose of protein identification, immunochemical techniques have been combined with separation methods and with other methodologies. A good example of a combined procedure is the identification of the protease-resistant pathological form of the prion protein, in which the products of specific proteolysis of the whole tissue are first separated by electrophoresis, and identified by specific monoclonal antibodies after transfer (blotting) to a suitable membrane. Another common application of immunoblotting techniques is the identification of the food proteins (and of peptides... 

See also. Chemiluminescence Oven/iew. Derivatiza-tion of Analytes. Electrophoresis Oven/iew. Enzymes Oven/iew Immobiiized Enzymes Enzyme-Based Eiec-trodes Enzymes in Physioiogicai Sampies Industriai Products and Processes Enzyme-Based Assays. Fluorescence Clinical and Drug Applications. Immunoassays Overview Production of Antibodies. Immunoassays, Applications Clinical Food Forensic. Immunoassays, Techniques Radioimmunoassays Enzyme Immunoassays Luminescence Immunoassays. Mass Spectrometry Polymerase Chain Reaction Products. Nucleic Acids Chromatographic and Electrophoretic Methods Electrochemical Methods. Polymerase Chain Reaction. 

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