Selectivity, immunoassay development

The development of class-selective antibodies is another approach to multi-analyte analysis. The analyst may design haptens that will generate antibodies that recognize an epitope common to several compounds, as explained above for the analysis of pyrethroids by measuring PBA. Other examples of class-selective immunoassays that have been developed are mercapturates," glucuronides, pyrethroids, organophosphate insecticides, and benzoylphenylurea insecticides." ... 

Antibodies from class (1) and (2) can be used for the development of analyte-specific and group-selective immunoassay, respectively. However, production of class (3) antibodies often occurs and they are difficult to use for quantification of analyte(s) in unknown samples. In such situation, incomplete or incorrect characterization of the antibody CR in the specific assay format might lead to erroneous quantification of the analyte. In general, only the target analyte is used for calibration and the results are often expressed in equivalents of target analyte concentration , which is a more or less accurate estimation of all cross-reactants present in the sample [33,36]. 

Although high cross-reactivity is sometimes seen as a problem in immunoassay development, antibodies with a high CR for compounds within the same chemical class can favourably be used either for post-column detection of the cross-reactants separated by HPLC [60-64] or for selective SPE in columns with immobilized antibodies (immunoaffinity SPE), followed by HPLC separation of the trapped cross-reactants (see Fig. 9.12) [65]. 

At present, the aim is to ensure that a humoral response is generated. For mucosal vaccines, IgA may be more appropriate.21516 T cell-mediated responses may also be considered, and advances in knowledge and the development of different types of vaccines and/or adjuvants suggest that cell-mediated responses may be just as or more important than the humoral response, when considering the selection of species. Adjuvants can selectively switch the T helper cell response, which may influence a protection or pathology dependent response (see Section 19.2.5). However, the current understanding and interpretation of such data are limited in terms of predicitivity, and there are also limitations in the immunoassays developed for species routinely used in nonclinical testing. This is a key area where further development is required. 

Specificity of an immunoassay is usually measured by determining the extent that compounds that are structurally similar to the test analyte react in the assay. The determination of the assay reactivity of an array of potential cross-reactant is routinely performed in immunoassay development. A panel of suspect cross-reactant, should be selected on the basis of structural similarities to the test analyte and on the expected occurrence along with the test analyte in the sample. Thus, an immunoassay specific for 17/1-estradiol should be tested for reactivity with estrone, estriol, 17a-estraiol and testosterone. Other compound testing might also be indicated. 

To understand how the Department has selected target pesticides for immunoassay development, it should be understood that the EM PM Branch monitors the environment only for residues of certain pesticides. Since we have regulatory authority only over those pesticides in current use, we do not monitor the environment for pesticides such as EOT and related chemicals, which are no longer in use in the State. Therefore, although they are important environmental contaminants, they are not included in our assay development program. 

The EMSL-LV evaluates selected immunoassay methods to ensure the EPA has appropriate and necessary methods for environmental monitoring. When a developer approaches the EMSL-LV, efforts are made to find either an EPA Regional Office or a Superfund site where the immunoassay can be implemented for use. 

Immunoassays are very versatile, and if one could select but a single method, it could be the method of choice. Fortunately we have a variety of techniques available and a good analyst should know when to apply them. Table I provides some general rules for determining how difficult an immunoassay will be. The terms used are relative and possibly other dimensions to the table could be the laboratory s experience with immunoassay and the problems faced. This table does not indicate that good assays cannot be developed for hard compounds it just Indicates that the expense, skill and time required may be greater for those compounds. For instance we have developed successful immunoassays for some lipophilic, small, unstable, volatile compounds. However, such compounds would be a poor choice to use for one s first venture into immunoassay development. 

Wu, A.H.W. (2006) A selected history and future of immunoassay development and applications in clinical chemistry. Clinica Chimica Acta, 369, 119 124. 

Immunoassays represent very selective and sensitive techniques that have found application in several areas such as clinical chemistry, bioanalysis, pharmaceutical analysis, toxicological analysis, and environmental analysis. The first immunoassays developed were radioimmunoassays, which are very sensitive however, one drawback is the need to work radiochemicals. To circumvent the drawbacks, other labels such as enzymes in combination with photometric measurement have been introduced, however, at the expense of sensitivity. With the development of fluoro-immunoassays (FIAs) an improvement in sensitivity was obtained and by introducing chemiluminescence in immunoassays, a sensitivity equivalent to radioimmunoassays was achieved. In this article, different variations and techniques of luminescence immunoassays are described. 

Mak, S.K. et al., Development of a class selective immunoassay for the type II pyrethroid insectides. Anal. Chim. Acta, 534,109,2005. 

Another SPR-based immunosensor for DDT, its metabolites, and analogues in real water samples was developed by Mauriz et al. Low limits of detection (LODs), in the sub-nanogram per liter range, were attained for DDT-selective (15 ng 1 ) and DDT group-selective immunoassays (31 ng 1 ). The SPR analysis of DDT proved to be three times more sensitive than colorimetric ELlSAs without the need of labeling combined with a much shorter time of respmise. This SPR biosensor portable platform (P-SPR) is also commercialized by the SENSIA company. 

The first application of immunologically based technology to pesticides was not reported until 1970, when Centeno and Johnson developed antibodies that selectively bound malathion. A few years later, radioimmunoassays were developed for aldrin and dieldrin and for parathion. In 1972, Engvall and Perlman introduced the use of enzymes as labels for immunoassay and launched the term enzyme-linked... 

However, if a class-selective assay is desirable (for multi-analyte assays), the handle should be located at or near a position that differentiates members of the class and exposes features common to the class. Using the pyrethroid example, an ideal immunogen should retain the phenoxybenzyl moiety and link the protein from the distal acid end (Figure 9). Using such an immunogen hapten, a class-specific immunoassay was developed that was highly cross-reactive with the type I pyrethroids permethrin, phenothrin, resmethrin and bioresmethrin. ... 

Acrylonitrile metabolites have been measured in blood and urine, but, except for measurement of thiocyanate, these methods have not been developed for routine monitoring of exposed humans. Supercritical fluid extraction/chromatography and immunoassay analysis are two areas of intense current activity from which substantial advances in the determination of acrylonitrile and its metabolites in biological samples can be anticipated. The two techniques are complementary because supercritical fluid extraction is especially promising for the removal of analytes from sample material and immunoassay is very analyte-selective and sensitive (Vanderlaan et al. 1988). 

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