The Use of Immunochemical Methods

Few examples of immunochemical methods for cytostatic agents have been reported (Table 8). Within the context of work performed by Aherne et al. [33], on the use of immunochemical methods in the analysis of microcontaminants in water samples, was reported the use of a RIA for the detection of methotrexate with a LOD of 6.25 ng mL-1. With the exception of a hospital effluent (concentration of 1 ng mL 1 of methotrexate was found), all samples (river and potable water) were negative. 

The Use of Immunochemical Methods in Studies on Proteins Pierre Grabar... 

The use of immunochemical methods to study homogeneity and purity, and to elucidate polysaccharide structure, has been discussed.271 The following discussion will deal with the application of immunochemical methods to polyglycoses. 

Structures.—Mechanisms of the activation and inhibition of enzymes by antibodies and the use of immunochemical methods for studying the structure of the active site, quaternary structure, and conformational changes in enzymes during the interaction with substrates and other ligands have been discussed. ... 

Several common methods for the detection of these compounds in environmental media have been proposed such as microbiological assay and conventional chromatographic methods. However, only one example of the application of immunochemical methods to the analysis of environmental samples has been reported [84]. In this case, erythromycin could be measured at concentrations higher than 10 pg L 1 using the Charm II6600/7600 assay in water samples proximal to livestock farms. 

When the results obtained lie above the MRL, both qualification and quantification are becoming very important. Immunochemical tests that are often used for surveillance of registered drugs are prone to cross-reactions that may influence the result. It is therefore important to confirm and quantify the results of immunochemical methods with an independent physicochemical method. 

Five, genetically distinct, /8-D-fructofuranosidases have been described for strains of Saccharomyces hybrids. The behavior and kinetics of each enzyme are very similar.362,451 W. L. Smith and Ballou have purified the mannan-protein /3-D-fructofiiranosidases of three strains of Saccharomyces cerevisiae whose cell walls have differences in mannan structure.452 By use of immunochemical methods, they found that the structure of each /3-D-fructofuranosidase mannan is similar to that of the cell wall of the corresponding strain only. Mutations affecting the structure of the one also produced similar changes in the other. 

Despite all of these obstacles, the development of immunochemical methods has continued over the last two decades, and in many cases they have proved to be fast, cost-effective, easy to use and fit for purpose technologies, that can make an important contribution in the field of environmental analysis. This chapter provides a brief description of the basics and principles of immunoassay, together with a selection of examples of environmental applications, especially with respect to the European Union Water Framework Directive (WFD). 

These studies emphasize the potentiality of the immunochemical approach in the study of conformational equilibria, in which one state is very little populated precluding the use of physicochemical methods. By immuno-chemistry as well as by hydrogen exchange, it is possible to reach the equilibrium constant in the absence of denaturant (see Chapter 6). 

The concept of immunoassay was first described in 1945 when Landsteiner suggested that antibodies could bind selectively to small molecules (haptens) when they were conjugated to a larger carrier molecule. This hapten-specific concept was explored by Yalow and Berson in the late 1950s, and resulted in an immunoassay that was applied to insulin monitoring in humans. This pioneering work set the stage for the rapid advancement of immunochemical methods for clinical use. 

Several attempts have been made to set up immunochemical techniques for dioxin analysis (reviewed in [230,238,239]). Frequently the detectability and selectivity accomplished have not been considered appropriate for the direct analysis of environmental samples. We should notice that due to the poor solubility of PCDDs and PCDFs in water, the levels of these contaminants in aqueous samples is very low. For this reason analysts usually prefer the use of chromatographic and spectrometric methods that perform using organic solvents. However, the speed and high sample throughput that can be accomplished with the immunochemical methods have prompted several research groups and companies to establish immunochemical methods. 

Considering all aspects, sex hormones, antibacterials, and antineoplastic agents were identified by Christensen as the three most relevant groups of chemicals concerning their potential human risk as a consequence of drug exposure via the environment [10]. Immunochemical methods for hormones and antibiotics have already been discussed above. In this section we will describe methods based on the use of antibodies for the analysis of analgesics, NSAIDs, and cytostatic agents. 

A PFIA, commercialized by Abbot, is one of the most common immunochemical methods used in clinical laboratories to analyze salicylic acid (SA) in serum. The assay also recognizes gentisic acid (GA) but is insensitive to salicylamide, salicyluric acid, and conjugates of S A and of its metabolites [187]. 

Recent trends in pesticide analysis in food aims for reduced sample pretreatments or simplified methodologies (as QuEChERS approaches), the use of online purification processes, the use of new adsorbents (such as molecular imprinted polymers (MIPs) and nanomaterials) for the extraction and clean-up processes, and focused on the development of large multiresidue methods, most of them based on LC-MS/ MS. In spite of the relevant role of LC-MS/MS, GC-MS-based methods still play an important role in pesticide analysis in food. Despite the development achieved in the immunochemical approaches, the need for multi-residue methods has supported the development and use of instrumental techniques. 

Immunochemical approaches are cheaper, readily adaptable, rapid, portable, and reduce the need for expensive analytical equipment. They can also be used to simultaneously assay a large number of samples over a short period of time. One of the major factors that still hmits the use of this technique in the detection of a wider range of PPCPs in the environment is the lack of suitable antibodies sensitive to most PPCPs that occur in the environment. Furthermore, immunoassay accuracy can be susceptible to cross reactions and other effects from the matrix, giving false positives in some instances (Huang and Sedlak, 2001). Thus, it is recommended that immunoassay analytical results be validated with GC- or LC-based methods. 

Level II methods are those that are not unequivocal but are used to determine the concentration of an analyte at the level of interest, and to provide some structural information. For example, these methods may employ molecular, functional-group, or immunochemical properties as the basis of the analytical scheme. Hence, these methods are often reliable enough to be used as reference methods. Level II methods commonly separate the determinative from the identification procedures, and may also be used to corroborate the presence of a compound or class of compounds. Tims, a combination of two level II methods may provide attributes suitable for a level I method. The majority of analytical methods presently available and used by regulatory control agencies are level II methods. 

Although both polyclonal and monoclonal antibodies have been effectively used in immunochemical assays, only the latter can provide the high specificity required in some applications. Antibody specificity, on the other hand, is both a major advantage and disadvantage for immunochemical methods. It allows for highly selective detection of analytes but at the same time may complicate the development of multiresidue methods. Moreover, production of monoclonal antibodies requires special expertise and it is much more expensive than polyclonal antibodies. Thus, in cases where a range of analytes similar in molecular structure are required to be determined, a polyclonal may be more suitable than a monoclonal antibody. 

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