General enzyme-linked immunosorbent

Benzodiazepines are an important group of drugs with tranquilizing properties. Available immunochemical methods include radioimmunoassays (164, 165), a radioreceptor assay (166), and nonseparation immunoassays such as the widely used enzyme-monitored immunotest (EMIT) and fluorescent polarization immunoassays (167, 168). Such assays generally require sophisticated apparatus and dedicated laboratories. However, a relatively simple enzyme-linked immunosorbent assay was recently described for screening benzodiazepines in urine (169). 

Particle Concentration Fluorescence Immunoassay. The PCFIA is a solid-phase immunoassay in which proteins are attached to polystyrene particles by adsorption or covalent coupling for the solid phase and fluorescent-labeled reagents are utilized for product detection.22 The general principles of the assay are similar to those of the enzyme-linked immunosorbent assay (ELISA), which has been reviewed extensively elsewhere.23 PCFIAs are performed in specially designed 96-well format Fluoricon assay plates utilizing an automated Screen Machine (Idexx Corporation, Research Product Division, Portland, ME). 

One of the most important requirements for successful protein purification is the availability of an accurate, rapid, and quantitative assay that is specific for the protein of interest. If the protein has no known enzymatic activity, the amount of the protein present after each purification step must be analyzed by some general method such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, or an enzyme-linked immunosorbent assay (ELISA). If... 

Serum samples are analyzed for antibody titer. When the titer is sufficiently high, the animal is bled. Serum samples are generally screened for the presence of the antibody by enzyme-linked immunosorbent assay (ELISA). ELISA methods are discussed later in this chapter. 

Urine sampling may be conducted on a spot basis (e.g. end-of-shift or morning void), or as a complete 24 h sample. This latter sample, while more easily interpretable, is often difficult to obtain from workers. Sample collection is relatively simple and noninvasive, although issues of privacy and confidentiality need to be addressed. Laboratory analysis is generally complex, and therefore expensive. New techniques, such as enzyme-linked immunosorbent assays (ELISAs) show promise as simple and cost-effective analytical methods. Such methods, while useful to indicate exposure, are not suitable for quantifying exposure. 

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

Antibodies can be used for a variety of applications in the molecular characterization of receptors and receptor-hgand interactions. Antibodies can be used for the detection of receptors in tissue shces. Western blot experiments [48], or ELISAs (enzyme-linked immunosorbent assays) [49]. They can also be used in competition experiments to map the binding epitope of a hgand [50]. Even though the use of antibodies is routine, fhere is no general protocol for fheir generation. 

Ricin can be detected in the blood or other bodly fluids of exposed animals using competitive radioimmunoassays or enzyme-linked immunosorbent assays (ELISA). These methods generally do not distinguish between active ricin molecules versus partially degraded or otherwise inactivated toxin. The postexposure time limit for accurate antibody-based detection of ricin in biological samples varies and depends on the route of exposure and the absorbed dose. In the laboratory, ELISA detects ricin in oro-nasal swabs of NHP exposed to ricin aerosol up to 24 h after exposure (Franz and Jaax, 1997). Likewise, ELISA detects ricin in selected tissues of laboratory rats up to 48 h after an i.m. challenge (Leith et al., 1988). 

Enzyme-linked immunosorbent assays (ELISAs) have long been used to quantify cytokines and other proteins that are secreted from cells. ELISAs enable the user to assay multiple samples, to obtain reproducible quantitative results, and to design studies with quantifiable endpoints. Intracellular activities intimately involved in apoptosis, such as control of mitochondrial permeability to holocytochrome c and activation of a specific caspase, are quantified by the ELISAs described in this chapter. The cytochrome c ELISA is generally used to quantify the activities of the proteins belonging to the Bcl-2 family. The active caspase ELISA quantifies a specific active caspase among a background of latent caspase and other active caspases. 

For the determination of binding properties of biotinylated proteins and conjugates, use flexible 96-wells microplates (Nunc). The general design of the method is similar to classical enzyme-linked immunosorbent assay (ELISA) and RIA methods. Use the described procedure for immobilization of streptavidin, antigen, biotinylated or nonmodified antibody, or catalase. 

Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. Both polyclonal and monoclonal antibodies have their advantages, and or disadvantages, but in general the production of monoclonal antibodies is more time consuming and requires tissue culture facilities and skills. The use of either monoclonal or polyclonal antibodies in some of the applications may require that the antibody is in a purified form. They can be purified by a variety of methods described in the next few chapters. The availability of commercially available kits primarily designed for the purification of IgG and IgM classes of antibodies derived from all common animal species should also be mentioned. 

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