Antibodies immunochemical detection

Irth, H., Oosterkamp, A. J., Van der Welle, W., Tjaden, U. R., and Van der Greef, J., On-line immunochemical detection in liquid chromatography using fluorescein-labelled antibodies. J. Chro-matogr. 633, 65-72 (1993). 

Immunochemical detection is possible in one step, if the detecting antibody carries the signal-forming principle. But also cascades of steps are used to identify the first-bound antibody and by it the antigen immobilized by blotting. Antibodies form these cascades, i.e., if the first bound antibody is from species A, e.g., rabbit, a second antibody from other species, e.g., goat, directed against the primary, is used. 

Nakamura, M., Kindaichi, K., and Kagayama, M. 1991. Immunohistochemical and immunochemical detection of a similar epitope in enamel proteins and monocyte macrophage protein recognized by mouse monoclonal antibody MOMA-2. Arch. Oral Biol. 36 619-622. 

Figure 11.8. Detection of NOS-dependent S-nitrosylation in vivo, a Immunochemical detection procedure. A primary antibody directed against nitrosothiol groups was used in combination with an enzyme-linked secondary antibody. The dye released by the enzyme is insoluble and thus will precipitate close to the site of formation, b S-nitrosylationinaorticrings. Samplesweie treated with Phenylephrine (PE), acetylcholine (Ach), and the inhibitor N-methylarginine (L-NAME) as indicated. Brown stain deposits indicate S-nitrosylation. The traces above the histology panels illustrate the contraction and relaxation responses evoked by the dmgs.

Immunochemical technology is rapidly advancing in many areas development of field-portable formats, specific antibody generation, detection systems, quality control and quality assurance measures, and new applications. It is not a panacea but should be used when deemed to provide the most appropriate analysis. 

Immunochemical methods are rapidly gaining acceptance as analytical techniques for pesticide residue analysis. Unlike most quantitative methods for measuring pesticides, they are simple, rapid, precise, cost effective, and adaptable to laboratory or field situations. The technique centers around the development of an antibody for the pesticide or environmental contaminant of interest. The work hinges on the synthesis of a hapten which contains the functional groups necessary for recognition by the antibody. Once this aspect is complete, immunochemical detection methods may take many forms. The enzyme-linked immunosorbent assay (ELISA) is one form that has been found useful in residue applications. This technique will be illustrated by examples from this laboratory, particularly molinate, a thiocarbamate herbicide used in rice culture. Immunoassay development will be traced from hapten synthesis to validation and field testing of the final assay. 

In the cytoblot assay, double immunochemical detection was applied the primary antibody detects, for example, phosphorylated proteins while a second antibody linked to horseradish peroxidase provides a chemiluminescence readout (Stockwell, Haggarty, and Schreiber, 1999). The applicability of the cytoblot assay was demonstrated by natural product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the antiproliferative effect of rapamycin. In another application, a similar setup was used to identify the phosphorylation pattern of multiple signaling proteins in response to known kinase inhibitors (Chen et al., 2005). 

Kato Y, Wu X, Naito M, Nomura H, Kitamoto N, Osawa T. Immunochemical detection of protein dityrosine in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody. Biochem Biophys Res Commun 2000 275 11—15. 

The most widely used immunological approaches are those based on enzyme-conjugated antibodies, offered as kits that allow immunochemical detection of allergens and toxic components in foods. These products are popular in that they offer ease of automation, the possibility of analyzing a large number of samples in a single approach, besides relying on safe chemicals and simple protocols. Either direct or competitive enzyme-linked immunosorbent assay... 

Specific Antibodies for Immunochemical Detection of Wood-Derived Hemicelluloses... 

General Strategy Immunochemical Detection Using Anti-Polysialic Acid and Anti-KDN Antibodies... 

Since antibody probes are extremely sensitive to variations, even subtle ones, in protein conformation, they are potent but rather delicate tools for the study of protein folding or unfolding. For the first time, Anfinsen s group developed a quantitative immunochemical approach to study the refolding of staphylococcal nuclease (Sachs et aL, 1972a,b,c, 1974 Eastlake et ai, 1974 Furie et aL, 1974, 1975). From that, different methods using specific antibodies for detection and characterization of intermediates in protein... 

Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

Va.ria.tions in Methods. The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be cmde antisemm, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is mn, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

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