Purification antigen

Pritchard, D.I., Williams, D.J., Behnke, J.M. and Lee, T.D.G. (1983) The role of IgGl hypergammaglobinaemia in immunity to the gastrointestinal nematode Nematospiroides duhius. The immunochemical purification, antigen specificity, in vivo anti-parasite effect of IgGl from immune serum. Immunology 49, 353-365. 

SDS gel electrophoresis is often the last step in antigen purification. Antigens in gels can be processed in the following ways (I prefer C). 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

The basic process technology in vaccine production consists of fermentation for the production of antigen, purification of antigen, and formulation of the final vaccine. In bacterial fermentation, technology is weU estabHshed. For viral vaccines, ceU culture is the standard procedure. Different variations of ceU line and process system are in use. For most of the Hve viral vaccine and other subunit vaccines, production is by direct infection of a ceU substrate with the vims. 

Fractionation. The process by which components are extracted firm bacterial eells or from the medium in whieh the baeteria are grown and obtained in a purified form. The polysaccharide antigens of Neisseria meningitidis are separated from the bacterial cells by treatment with hexadecyltrimethylammonium bromide and those of Streptococcus pneumoniae with ethanol. The purity of an extracted material may be improved by resolubilization in a suitable solvent and precipitation. After purification, a component may be dried to a powder, stored indefinitely and, as required, incorporated into a vaccine in precisely weighed amounts at the blending stage. 

Purification of biopharmaceuticals often involves the removal of materials with physical characteristics very similar to the desired product, such as failure sequences from DNA synthesis or misfolded proteins from bacterial fermentations. The contaminants, however, may have biological characteristics very different from the desired product, including different antigenicities, bioactivities, and specificities. There are even systems in which the... 

Narum, D. L., Welling, G. W., and Thomas, A. W., Ion-exchange-immuno-affinity purification of a recombinant baculovirus Plasmodium falciparum apical membrane antigen, PF83/AMA-1, /. Chromatogr. A, 657, 357, 1993. 

Chen, Z., Prestigiacomo, A., Stamey, T. Purification and characterization of prostate-specific antigen (PSA) complexed to alpha-1 antichymotrypsin Potential reference material for international standardization of PSA immunoassays. Clin. Chem. 41, 1273-1282 (1995). 

Sensabaugh, G., Blake, E. Seminal plasma protein p30 Simplified purification and evidence for identity with prostate specific antigen. J. Urol. 144, 1523-1526 (1990). 

Sugane, K and Oshima, T. (1983) Purification and characterization of excretory and secretory antigen of Toxocara canis larvae. Immunology 50, 113-120. 

Smith, T.S., Munn, E.A., Graham, M., Tavemor, A.S. and Greenwood, C.A. (1993) Purification and evaluation of the integral membrane protein HI 1 as a protective antigen against Haemonchus contortus. InternationalJournalfor Parasitology 23, 271-280. 

Proteins such as antibodies, enzymes, hormones and vaccine antigens can be used to prevent, diagnose and treat a range of diseases. Such molecules are therefore of paramount importance in health and medicine. Historically, many of these proteins have been isolated from human or animal sources. However, the low quantities present in such source material coupled with safety risks and high purification costs have limited the availability of protein therapeutics and vaccines for many types of disease. 

Several plant vims coat proteins, including those of TMV, CPMV, AlMV and Tomato bushy stunt vims (TBSV), have been used to produce and deliver antigenic determinants from a variety of viral and bacterial pathogens. These data have been summarized in numerous publications and several reviews [12,13]. The ease of virus purification coupled with enhanced peptide immunogenicity when fused to carrier molecules makes this approach very attractive for vaccine development. 

Kurzinger, K., Springer, T. A. (1982). Purification and structural characterization of LFA-1, a lymphocyte function-associated antigen, and Mac-1, a related macrophage differentiation antigen associated with the type three complement receptor. J. Biol. Chem. 257,12412-18. 

Apart from reduced yield, down-stream processing can cause minor or even bigger modifications in the structure of the biomolecule. Often, these modifications do not affect the activity of the product, but may change its antigenicity. Along with virus safety, the reduction of such risks is a main objective in the down-stream processing of such biomolecules. Chromatographic purification,... 

If the purpose of gel electrophoresis is to identify low-abundance proteins (e.g., low-copy-number proteins in a cell extract or contaminants in a purification scheme), then a high protein load (0.1 to 1 mg/ml) and a high-sensitivity stain such as silver or fluorescence should be used. When the intention is to obtain enough protein for use as an antigen or for sequence analysis, then a high protein load should be applied to the gel and the proteins visualized with a staining procedure that does not fix the proteins in the gel, e.g., colloidal CBB G-250 (Subsection 8.2.8.1). Furthermore, for purposes of quantitative comparisons, stains with broad linear ranges of detection response should be used. 

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