Immunoassays techniques

The basic principles of immunoassay were first reported by Person and Yalow (1) and since then it has become an extremely powerful technique in the determination of a broad spectrum otcompounds. The power of the technique lies chiefly in the areas of specificity and versatility. 

Immunoassay is based on the antigen (or hapten)-antibody binding reaction which is both reversible and non-covalent  

If a label is covalently attached to the antigen in such a way that it does not block the reaction site for the antibody, the presence of the label will not significantly affect the binding reaction. Thus, in a situation in which a mixture of labelled (Ag ) and unlabelled (Ag) antigen react with antibody, competition for the antibody binding site occurs  

An alternative approach to immunoassay was described by Miles and Hales (2) and termed immunoradiometric assay (IRMA), since it initially used isotopically labelled antibodies. In this type of assay the labelled component is a specific antibody (Ab ), the process again being based on the antigen-antibody binding reaction  

This method may now become more important with the introduction of monoclonal antibodies (3) see also Chapter 1 of this volume. The major disadvantage of monoclonal antibodies, however, lies in the increased cost of the product. 

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Immunochemical metliods drat utilize radioisotopic labeling can detect tire use of anabolic sex hormones drat increase die growdi in meat animals. Stilbene [588-59-0] trenbolone [10161 -33-8] and zeranol [55331-29-8] C gH2 0, can be successfully monitored by diese immunoassay techniques... 

An enzyme immunoassay technique has been employed for measuring endosulfan and its degradation products (i.e., endosulfan diol, endosulfan sulfate, endosulfan ether, and endosulfan lactone) in water at 3 ppb (Chau and Terry 1972 Musial et al. 1976). However, this technique is not currently in use in environmental residue analysis. Further research into this technique could produce a rapid, rehable, and sensitive method for identifying contaminated areas posing a risk to human health. No additional methods for detecting endosulfan in environmental media appear to be necessary at this time. However, methods for the determination of endosulfan degradation products are needed. 

The use of immunoassays for the determination of pesticides and veterinary medicines in food animals has increased since the early 1990s. The advantages of simple analysis, quick results, and high throughput make immunoassays a powerful technique for problematic matrices commonly encountered in animal agriculture. Careful development and validation are required to obtain accurate results, however. This review has demonstrated that most immunochemical techniques have been designed for use with milk samples, but a number of applications have also been developed for liver and muscle samples. The development of immunoassay techniques for residue analysis in eggs has clearly not been pursued to the extent of other edible tissues. 

All specimens were analyzed by EMIT (Enzyme Immunoassay Technique), an enzyme method based upon the competitive bonding of an enzyme and an antibody. This method yields a positive result with concentrations of 75 ng of PCP/ml or greater (Rubenstein et al. 

The determination of theophylline in plasma can also be accomplished by various immunoassay techniques.66-67 Theophylline was also determined by a polarization fluoroimmunoassays but found to have a caffeine interference.88. In a more research oriented application, the interaction of caffeine with L-tryptophan was studied using h NMR with the results indicating that caffeine interacted with tryptophan in a 1 1 molar ratio through parallel stacking.69... 

Additionally it has been our experience that mass spectrometry as a routine detection/identification technique for bacteria is not well received by microbiologists and clinicians who prefer less expensive, less complicated approaches to bacterial typing and identification, such as methods based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). For that reason we have adapted our MS approach to serve as a means of biomarker discovery that feeds candidate proteins or leads into development as PCR targets or other immunoassay techniques. 

According to the practical equipment there are useful tools, so-called test kits, which are units that contain all the reagents and a simple instrumentation in form of plates, tubes and wells. The test kits work rapidly, are easily to handle and field-portable. Frequently, biochemical principles are applied, especially immunoassay techniques which use body-antibody reactions. 

As an alternative, extremely sensitive detection can be achieved with reporter antibody probes tagged with intensely SERS-active compounds or with enzymes that react with substrates to yield SERS-active products. These methods often involve sandwich immunoassay techniques, which increase the number of required steps but offer the advantages of excellent sensitivity and the potential for label multiplexing. For example, Nie and coworkers recently reported the simultaneous detection of two types of antigens in a... 

Pankey et al.21 described a rapid, reliable, and specific enzyme multiplied immunoassay technique (EMIT ) for amitriptyline, nortriptyline, imipramine, and desipramine in sera. To overcome crossreactivity, solid phase extraction was included in sample pretreatment. Disposable 1 mL columns packed with covalently labeled silica gel were conditioned with HPLC-grade methanol (1 mL) and then with de-ionized or distilled water (1 mL). Serum (calibrator, control, or patient sample, 500 L) was applied onto the column, eluted to waste, washed with 900 /uL of wash solution containing acetonitrile (236.1 g/L) and ion-pairing reagent in acetate buffer, pH 4.2, washed with 500 fiL of mobile phase solution containing acetonitrile (393.5 g/L) in methanolic phosphate buffer, pH 7.0,... 

With enzyme-multiplied immunoassay technique (EMIT) assays, enzyme tags are used instead of radiolabels. The antibody binding alters the enzyme characteristics,... 

The introduction of enzyme immunoassay (EIA) and similar allied immunoassay techniques in early eighties showed, in fact, a brighter path towards quantitative analysis. 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Other successful homogeneous assays have been developed such as the fluorescence polarization technique made available by Abbott Laboratories of North Chicago, USA. This competitive immunoassay technique relies upon the principle... 

Which of the following immunoassay techniques are competitive in nature ... 

M. N. Kronick and P. D. Grossman, Immunoassay techniques with fluorescent phycobiliprotein conjugates, Clin. Chem. 29, 1582-1586(1983). 

Proteins bound to the surfaces of synthetic membranes retain their antigenicity and are accessible to antibody probes. The most common membrane-based immunoassay technique is called immunoblotting or, more popularly, Western blotting. In Western blotting, proteins are transferred from an electrophoresis gel to a... 

MEGX is readily detected by HPLC and fluorescence polarization immunoassay techniques [14,21,25,40,41]. The test is simple, normally requiring a onetime blood sampling, and informative because it depends on the capacity of the hepatic enzymes to metabolize lidocaine. While the analysis of lidocaine metabolites is rapid, this method has not been adapted for continuous hepatic function monitoring, which may be possible with the radiolabeled analogues such as Tc-Sn-lidocaine iminodiacetic acid [42]. 

Enzyme Multiplied Immunoassay Technique (EMIT). This technique employs enzyme-labelled antibiotics which react analogously to the fluroimmunoassay in that a reduction of enzjrme activity is attributed to antibody binding. Higher concentrations of unlabelled drug in the sample result in less enzyme-labelled drug bound to the antibody. 

The basis for this procedure is the antigen-antibody "reaction"—i e., specific binding of an antibody to the molecule being assayed. Among the many different immunoassay techniques that have been developed—e.g., radioimmunoassay (RIA), and chemoluminescence immunoassay (CIA)—a version of the enzyme-linked immunoassay (ElA) is shown here. 

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