Immunoprecipitation specific antigen

This assay method (RIPA) is used primarily in research. It is too technically demanding for routine use in clinical laboratories. HIV is cultured in radio-labeled cells, or viral proteins are directly labeled with a radioisotope. The virus is disrupted and then exposed to the test specimen. Specific antigen-antibody complexes are concentrated and isolated by immunoprecipitation. After exten-... 

The basic principle behind the ChIP method is relatively simple It is based on the selective enrichment of a chromatin fraction containing a specific antigen (e.g. transcription factors, DNA binding proteins, modified histones, etc.) by an immunoprecipitation step. Specific (important, see below ) antibodies that recognize a protein of interest or the modified form of a protein can be used to determine the relative abundance of it within DNA regions. 

Isotyping of monoclonal antibodies 20 Competitive radioimmunoassays 21 Immunoprecipitation of specific antigen 22... 

Immunohistochemistry is a powerful method for identification of proteins in cells and tissues, but control procedures are necessary. The specificity of the results depends on two independent criteria the specificity of the antibody and of the method used (Burry 2000). The antibody specificity is best determined by immu-noblot and/or immunoprecipitation. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG (must be the same species as primary antibody at the same concentration), and a positive control, testing the primary antibody with tissues known to contain the antigen. 

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. It involves the interaction between an antigen and its specific antibody. Antigen-antibody interactions may produce a network of many antigen molecules cross-linked by antibody molecules, which result in insolubilization and precipitation of the complex (Williams 2000). 

Immunoprecipitation followed by SDS-PAGE and immunoblotting, is routinely used in a variety of applications to determine the molecular weights of protein antigens, to study protein-protein interactions, to determine specific enzymatic activity, to monitor protein post-translational modifications and to determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to... 

Fig. 6.2. Prominent parasite antigens and host components in Echinococcus granulosus sheep cyst fluid (SCF). Labelled fluid was immunoprecipitated with lane 1, normal rabbit serum lane 2, rabbit anti-SCF lane 3, preabsorbed rabbit anti-SCF lane 4, rabbit anti-sheep whole serum. Antigens were reduced prior to electrophoresis. The major parasite antigens are shown arrowed at Mr 38000, 20000, 16000 and 12000. D.F., diffusion front. (Reprinted with permission from Molecular and Biochemical Parasitology, 25, Shepherd, J. C. McManus, D. P., Specific and cross-reactive antigens of Echinococcus granulosus hydatid cyst fluid, 1987, Pergamon Journals Ltd.)...

This example shows that immunochemical methods are not neccessarily highly specific. Cross reactions with immunoprecipitable material or solubilization of antigen complexes can falsify the assay to a considerable extent. Currently more sophisticated techniques bearing a higher specifity than the older methods are employed They are used in many laboratories for routine purposes The most reliable... 

---