Analytical techniques - immunoassay

Immunoassay as an analytical technique was introduced by Rosalind Yalow and Solomon Berson in 1960 with their use of anti-insulin antibodies to measure the concentration of the hormone in plasma. This advance, for which Rosalind Yalow was awarded the Nobel prize, was probably the most important single advance in biological measurement of the following two decades. Examples of the use of immunoassay may now be found in almost all areas of analytical biochemistry. 

Despite many novel developments in immunoassay design the principles are confined to two broad approaches those that rely on the competition between antigens labelled with a molecule which may be readily observed (for example, a radioisotope) and unlabelled antigens for a limited number of antibody binding sites and those in which the antibody is available in excess and for which there is no competition for binding sites. 

The basis for this technique lies in the competition between the test antigen and a labelled antigen for the available binding sites on a fixed amount of antibody. While the binding sites are traditionally associated with an antibody, any source of specific reversible binding sites may be used to create an assay in this format. Examples of such are specific transport proteins such as thyroxine-binding globulin and certain cellular receptors such as opiate or benzodiazepine receptors. Under these circumstances the equilibrium mixture may be represented thus  

This approach to immunoassay is characterized by the fact that the antibody is present in excess and is generally also labelled. Because the labelled antibody is in excess there is no requirement for the setting up of an equilibrium since all of the test antigen may be sequestered by the excess of antibody. 

In immunometric assays, unlike competitive systems, the amount of labelled antibody bound is directly proportional to the amount of unlabelled antigen present rather than inversely proportional. 

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Materials characterization techniques, ie, atomic and molecular identification and analysis, are discussed in articles the tides of which, for the most part, are descriptive of the analytical method. For example, both infrared (ir) and near infrared analysis (nira) are described in Infrared and raman SPECTROSCOPY. Nudear magnetic resonance (nmr) and electron spin resonance (esr) are discussed in Magnetic spin resonance. Ultraviolet (uv) and visible (vis), absorption and emission, as well as Raman spectroscopy, circular dichroism (cd), etc are discussed in Spectroscopy (see also Chemh.itmtnescence Electro-analytical technique Immunoassay ZvIass spectrometry Microscopy Microwave technology. Plasma technology and X-ray technology). 

Immunoassays. Immunoassays (qv) maybe simply defined as analytical techniques that use antibodies or antibody-related reagents for selective deterrnination of sample components (94). These make up some of the most powerflil and widespread techniques used in clinical chemistry. The main advantages of immunoassays are high selectivity, low limits of detection, and adaptibiUty for use in detecting most compounds of clinical interest. Because of their high selectivity, immunoassays can often be used even for complex samples such as urine or blood, with Httle or no sample preparation. 

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. 

The advantages of immunoassay technology relative to other analytical techniques have been discussed in several reviews, and include the following ... 

In the past, general chapters and reviews have been published, related to the characteristics of CL as analytical technique [7-9], mainly in the liquid phase [10-14], and its use as detection mode in flowing streams and immunoassay [15-17]. Two extensive reviews reported on the specific application of CL reactions according to the nature of the analyte (inorganic species, enzymes and nucleotides, acids and amines, carbohydrates, steroids, polycyclic aromatic compounds, and drugs) and covering the literature from 1983 to 1991 [18] and from 1991 to mid-1995 [19]. 

The properties of the antibody used in an immunoassay will in large measure define its usefulness as an analytical technique. Both polyclonal and monoclonal antibodies have been used in immunoassays. 

There are many analytical techniques available that measure total petroleum hydrocarbon concentrations in the environment, but no single method is satisfactory for measurement of the entire range of petroleum-derived hydrocarbons. In addition, and because the techniques vary in the manner in which hydrocarbons are extracted and detected, each method may be applicable to the measurement of different subsets of the petroleum-derived hydrocarbons present in a sample. The four most commonly used total petroleum hydrocarbon analytical methods include (1) gas chromatography (GC), (2) infrared spectrometry (IR), (3) gravimetric analysis, and (4) immunoassay (Table 7.1) (Miller, 2000, and references cited therein). 

In addition to being necessary for all forms of life, biopolymers, especially enzymes (proteins), have found commercial applications in various analytical techniques (see Automated instrumentation, clinical chemistry Automated instrumentation, hemtatology Biopolymers, analytical techniques Biosensors Immunoassay) in synthetic processes (see Enzyme applications, industrial Enzyme applications in organic synthesis) and in prescribed therapies (see Enzyme applications, THERAPEUTICS IMMUNOTHERAPEUTIC AGENTS Vitamins). Other naturally occurring biopolymers having significant commercial importance are the cellulose (qv) derivatives, eg, cotton (qv) and wood (qv), which are complex polysaccharides. 

Hormones - [BIOSENSORS] (Vol 4) - [PROTEINS] (Vol 20) -analysis of [BIOPOLYMERS - ANALYTICAL TECHNIQUES] (Vol 4) -brain oligopeptides [HORMONES - BRAIN OLIGOPEPTIDES] (Vol 13) -detection of [BIOPOLYMERS - ANALYTICALTECHNIQUES] (Vol 4) -effect of growth regulators [GROWTHREGULATIONS - ANIMAL] (Vol 12) -immunoassay for [IMMUNOASSAY] (Vol 14) -mineral nutrients effect on [MINERAL NUTRIENTS] (Vol 16)... 

Radiopaque -use m immunoassay [BIOPOLYMERS - ANALYTICAL TECHNIQUES] (Vol 4) - [PROSTHETIC AND BIOMEDICALDEVICES] (Vol 20)... 

JM Van Emon, JN Seiber, BD Hammock. Immunoassay techniques for pesticide analysis. In J Sherma, ed. Advanced Analytical Techniques. New York Academic Press, 1989, pp 217-263. 

Prior to application of immunoassays for detection of antigens, it is important to do a cost/benefit analysis to establish whether it adds value as compared to other analytical techniques. The value of immunoassay depends on its (1) performance versus other analytical techniques, (2) ease of use, (3) type of environment i.e., on site or in the field, (4) cost, and (5) return on investment (ROI) (48, 79, 80)... 

Food products are prone to accidental or deliberate abuse anywhere in the food processing/storage/distribution/consumption cycle, starting from raw materials to finished products. Faced with this problem a food analyst needs rapid, sensitive, reliable and cost effective techniques. Immunoassays are ideally suited for this type of tasks and their role in food diagnostics is expanding due to the numerous analytes like adulterants/additives, allergens and contaminants/toxins that it can detect. For more detailed information on applications of immunoassays one can refer to some of the recent review articles and books (11-13, 38, 44). 

The availability of MIP microparticles through this synthetic method has also stimulated the development of analytical techniques that make use of them as sensing elements. Apart from competitive radioassays [30] and immunoassays [32], which were already performed with ground bulk polymers, the small, regular size of the beads prepared by dispersion/precipitation polymerisation enables their use in CEC [45, 46], scintillation proximity assays [35], fluorescent polarisation assays [47], and chemiluminescence imaging [48]. 

As with any analytical technique, generation of a reproducible standard curve with minimal error is critical. An assay calibration consists of several steps during which the value of the primary standard is transferred to the calibrators used in the final assay [22]. Immunoassay optimization is usually difficult due to protein heterogeneity and matrix effects and these factors, heterogeneity and matrix effects, will also affect MIP based assays [22]. 

PCBs can be conveniently determined by most of the common analytical techniques which include GC-ECD, GC-HECD, GC-FID, GC/MS, HPLC, NMR, and enzyme immunoassay. Among these, GC-ECD and GC/MS are by far the most widely used techniques for the determination of PCBs in the environmental samples at a very low level of detection. While the former can detect the PCBs at subnanogram range, the mass selective detector (GC/MS) identifies the components relatively at a higher detection range, 10 to 50 times higher than the ECD detection level. GC/MS, however, is the best confirmatory method to positively confirm the presence of PCBs, especially in heavily contaminated samples. Aqueous and nonaqueous samples must be extracted into a suitable solvent prior to their analysis. 

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