Immunoassays methods listed

The role of the immunoassay, especially the radioimmunoassay (RIA), in clinical biochemistry has been the major factor in the tremendous advances made in that field since its introduction in 1959 (11). At present the RIA is the most powerful analytical tool available for quantitative detection of molecules of diverse structure and function in biological fluids of human, animal and now plant origin. The immunoassay comprises a unique combination of sensitivity and specificity as well as precision and applicability. With this assay technique, it is now possible to detect and very accurately measure compounds at endogenous physiological concentrations which frequently are in the range of 10 M or lower. In Table II the major characteristics of the immunoassay are listed. This method is versatile, specific, can be utilized for almost an unlimited number of compounds and has a high throughput potential. 

The essential criteria for a useful analytical technique are specificity, sensitivity, accuracy, precision, simplicity, rapidity, economy, wide applicability, and freedom from hazard. It is well known that radioimmunoassay (RIA) was developed in 1959 by Yalow and Berson (Yl). Since then the radioimmunoassay method has been widely used in the field of clinical chemistry. Radioimmunoassay has inherent in it the advantages listed above. However, this method always requires special facilities for use and disposal of radioisotopes and consideration must be given to the fact that the labeled substances have short half-lives. Immunoassay methods are explosively increasing in use and development as an analytic technique in basic science as well as in clinical laboratory medicine (L1-L3, VI). With these points as background, efforts have been made to develop nonisotopic immunoassay methods or alternative immunoassay methods that are based on antigen-antibody reactions but do not involve use of a radioisotope. 

Table 9 lists various congenital disorders and the immunoassay methods recommended for their mass screening. Most of the substances listed in Table 9 have been measured by radioimmunoassay, but a nonisotopic immunoassay such as enzyme immunoassay is now recommended. Sections 7.1 and 7.2 describe examples of mass screening by enzyme immunoassay. 

The list above is not intended to be comprehensive, but it will serve as a general guide to immunoassay development. We must emphasize again the difference between development and implementation. Assays which work in the laboratory generally require modification before they can be used to analyze field samples. Implementation of assays that have been fully validated for field samples may require little additional commitment by the user, other than analyst training. For the near future, there may be considerable pressure to transfer immunoassay methods to the analytical lab as soon as possible after development, and the... 

It is unnecessary to provide further detail of the apparatus since some excellent descriptions are in the literature [14-20]. These allow the construction of simple and hence flexible systems, yet thoroughly examine the theory of hght measurement so that the investigator can choose the most suitable apparatus. With the growth of interest in luminescence-based immunoassay methods, there has been a rush by many manufacturers to produce luminometers to serve this potentially lucrative market. The instruments and the addresses of the companies are listed in reference 2. It is important to point out however, that these instruments are rarely suitable for research work in the field of chemiluminescence itself. They are sensitive and where the analytical requirements are well defined, they can be very convenient. More flexibility is required in the apparatus used for investigations of the fundamental light reaction and its mechanism. 

Table 10 gives a list of enzyme immunoassays of substances related to neonatal hypothyroid screening. The methods for measuring substances in dried blood can be used for mass screening and those for tests on serum can be used for babies who are recalled. 

While no real labels meet all of these needs, the properties of some of the more recently introduced labelling systems are approaching the ideal. Radioisotopes, once the only type of label used for immunoassays, have clearly been overwhelmed by current applications of fluorescent labeling methods, enzyme labels, and even coenzyme and prosthetic group labels. A variety of alternative labels has also been investigated, including red blood cells, latex particles, viruses, metals, and free radicals. Table 6.1 shows a representative listing of labels used in modem immunoassays.1... 

As of 2004, the College of American Pathologists Interlaboratory Survey listed five manufacturers as providing first generation noncompetitive immunoassays for intact PTH. Most of these commercially available methods are on fully automated immunoassay analyzers using chemiluminescence detection including ALP with 1,2-dioxetane phosphate, acridinium ester, or electrochemiluminescence (ruthenium chelate). Signal antibody is less commonly radiolabeled with L... 

In many instrumental analysis methods the instrument response is proportional to the analyte concentration over substantial concentration ranges. The simplified calculations that result encourage analysts to take significant experimental precautions to achieve such linearity. Examples of such precautions include the control of the emission line width of a hollow-cathode lamp in atomic absorption spectrometry, and the size and positioning of the sample cell to minimize inner filter artefacts in molecular fluorescence spectrometry. However, many analytical methods (e.g. immunoassays and similar competitive binding assays) produce calibration plots that are intrinsically curved. Particularly common is the situation where the calibration plot is linear (or approximately so) at low analyte concentrations, but becomes curved at higher analyte levels. When curved calibration plots are obtained we still need answers to the questions listed in Section 5.2, but those questions will pose rather more formidable statistical problems than occur in linear calibration experiments. 

The ICMs used for pesticide analysis include immimoassays (lAs) and the use of antibodies for sample preparation (e.g., for SPE and the cleanup of samples) [153], detection in flow-injection analysis, and biosensors. The earliest ICMs to be developed for pesticides analysis were lAs. There are various t) es of lAs, but the most frequently used in this context is the enzyme-linked immunosorbent assay (ELISA) [185]. ELISA is a heterogeneous assay because the antibodies or antigens are immobilized on a solid phase. Table 18.3 lists selected ELISA methods for the determination of pesticides in water samples [186-190]. Bjamason et al. have proposed an enzyme flow immunoassay (EFIA) using a protein G column for the determination of triazine herbicides in surface and wastewaters with a linear range between 0.1 and 10 pg/L [191]. 

A pTAS must miniaturize the steps listed above (and any we may have omitted). There are two types of methods to do this. First, try to miniaturize the existing components used to accomplish these steps. Second, find new methods that accomplish the same result in a novel way appropriate to a pTAS. In the first family would be efforts to make microvalves and pumps, that move solutions around, mix them, and thereby miniaturize conventional chemistry. Homogeneous immunoassays involve efforts to find non-solution equivalents, such as controlled release of encapsulated reagents [14], or virtual separation using an evanescent wave optical signal [15], and are thus in the second category. 

Immunoassay techniques have been approved for the determination of numerous analytes commonly found in hazardous wastes. Where the EPA method numbers are given in parentheses in the following list, these include pentachlorophenol (4010) 2,4-dichlorophenoxyacetic add (4015) polychlorinated biphenyls (4020) petroleum hydrocarbons (4030) polycyclic aromatic hydrocarbons (4035) toxaphene (4040) chlordane (4041) dichlorodiphenyltrichloroethane (DDT) (4042) trinitrotoluene (TNT) explosives in soil (4050) and hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) in soil (4051). ELISAs have been reported for monitoring pentachlorophenol and BTEX (benzene toluene ethylbenzene and o-, m-, and p-xylene) in industrial effluents. 

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