Immunological Detection Techniques

Measured Quantity Layers in Well Advantages Disadvantages 

Antigen concentration —Enzyme-conjugated anti-antigen antibody 2 (constant) —Antigen (vanable) —Anti-antigen antibody 1 (constant) The assay requires two monoclonal or one affinity-purified antibody. One of the antibodies must be conjugated with enzyme. 

Anti-antigen antibody (screen tor hybridoma supernatants determine serum titer) —Enzyme-conjugated anti-IgG antibody (constant) —Anti-antigen antibody (variable) —Antigen (constant) Enzyme-conjugated anti-IgG antibody is available in retail.  

Antigen concentration —Enzyme-conjugated anti-antigen antibody (constant) -i- antigen (variable) —antigen (constant) Little work specific. The anti-antigen antibody must be enzyme-conjugated. Polyclonal antibodies must be affinity-purified first. 

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Luminol-based chemiluminescence methods have also been employed for detection of analytes in flowing stream analytical techniques such capillary electrophoresis (282), flow injection analyses, and hplc (267). AppHcations of the enhanced luminol methodology to replace radioassay methods have been developed for a number of immunological labeling techniques (121,283). 

Western blotting has become an important, modern technique for analysis and characterization of proteins. The procedure consists of, first, the electrophoretic transfer (blotting) of proteins from polyacrylamide gels to synthetic membranes. The transferred blots are then probed using immunological detection methods to identify proteins of specific structure and/or function. In this experiment, bovine serum will be fractionated by SDS-PAGE and the proteins blotted onto a nitrocellulose membrane. Serum glycoproteins will be identified by their specific interaction with the lectin concanavalin A. 

Recent developments show that dry chemistry has also been advancing in the sector of immunological detection methods. The homogeneous immunoassay technique facilitated access to the immunological detection method. The substrate-bound fluoro-immunoassay (SLFIA) serves as an example for the determination of theophylline concentration. However, the radial partition immunoassay paved the way for determining the concentration of numerous drugs and hormones. 

Immunological detection of ciguatoxins and related polyethers has received particular attention compared to other marine toxins. The initial RIA and enzyme immunoassay employing a polyclonal sheep anti-ciguatoxin antibody revealed cross-reactivities between ciguatoxins and other polyether toxins, suggesting the need for monoclonal antibodies. The availability of monoclonal antibodies allowed for the development of stick enzyme immrmoassay methods and solid-phase immunobead techniques (known as the paddle test), which successfully recognized toxins attached to correction fluid-coated bamboo sticks or paddles previously exposed to toxic fish tissues. 

EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

Biopolymers are employed in many immunological techniques, including the analysis of food, clinical samples, pesticides, and in other areas of analytical chemistry. Immunoassays (qv) are specific, sensitive, relatively easy to perform, and usually inexpensive. For repetitive analyses, immunoassays compare very favorably with many conventional methods in terms of both sensitivity and limits of detection. 

Fig. 3. Immunological reactions, where Ag is antigen and Ab is antibody, for detection in electrophoresis (a) Ouchtedony technique (b) single-radial diffusion (c) rocket immunoelectrophoresis and (d) crossed immunoelectrophoresis.

Protein tyrosine residues constitute key targets for peroxynitrite-mediated nitrations. Attack of various free radicals (ONOO-, N02 ) upon tyrosine generates 3-nitrotyrosine, which can be measured immunologically or by GC/MS or HPLC techniques. The detection of 3-nitrotyrosine was considered a biomarker of peroxynitrite action in vivo. Similarly, attack of HOC1 and HOBr on tyrosine generates chlorotyro-sine and bromotyrosine, respectively, both of which are measured most accurately by GC-MS. 

The rapid development and sensitivity of the mass spectrometric methods can be foreseen and in the near future the labeling can be more frequently eliminated. The identification of the cross-linked peptide can be detected first with immunological methods and then the digested and cleaved fragments with specific tandem MS techniques. The different photophores hold discrete MS fingerprints, which allow fast recognition of the modified sites. 

A most important technique which has been developed as an extension of the isotope dilution principle is that of radioimmunoassay (RIA). Analyses by this method employ substoichiometric amounts of specific binding immuno-chemical reagents for the determination of a wide range of materials (immunogens) which can be made to produce immunological responses in animals such as sheep or rabbits. It is possible to combine the specificity of an immunochemical reaction with the extreme sensitivity of radiotracer detection. Analytical methods based upon these principles have achieved wide applicability in the determination of organic compounds at trace levels. 

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