Immunochemical Selectivity

The binding equilibrium expressed as shown above (2.2) is actually a gross oversimplification of the situation. The heterogeneity of the binding sites and multiple valency of individual antibodies lead to formation of secondary bonds that contribute to hysteresis or ripening of the antibody-antigen complex. Its ultimate form is the polymerization of a primary complex, which happens when the antigen is also polyvalent. Formation of the polymer (precipitin reaction) renders such a reaction virtually irreversible. 

The secondary bonds, which may be formed much more slowly than the primary bonds, actually contribute more to the overall affinity. For example, the primary (Coulombic) bond between bovine serum albumin (BSA) and anti-BSA IgG is 3.3kcalM 1 whereas the secondary bond (van der Waals) is 28kJ, for a total AH = 42 kJ. Because the formation of the secondary bond is much slower, it is easier to prevent formation of the strong complex rather than to try to dissociate it. This is one reason why the competitive immunoassays yield results that correlate with the equilibrium-binding constants, but any such direct-binding assays have to rely on the measurement of the initial rate of binding. 

In order to assess the utility of the immunochemical reaction for chemical sensing, we need to examine the effects of the experimental conditions on the primary association reaction. The effect of temperature is not particularly distinct for most reactions and cannot be generalized. This is due to the fact that the relative 

In contrast with sensors, sensing systems are ideal for exploitation of immunochemical selectivity. This accounts for various highly sensitive and successful competitive immunoassays. Incorporation of the manipulative step(s) opens the door for regeneration of the antibody or even for operation under virtually reversible conditions. 

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Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

The remarkable selectivity that is inherent in the reaction of an antibody with the antigen or hapten against which it was raised is the basis for the extensive use of immunoassay for the rapid analysis of samples in clinical chemistry. Immunochemical reactions offer a means by which the applicability of potentiometric techniques can be broadened. A number of strategies for incorporating immunoassay into the methodology of potentiometry have been explored... 

An electrode in which an antibody or an antigen/hapten is incorporated in the sensing element is termed an immunoelectrode . The potential response of the immuno-electrode is based on an immunochemical reaction between the sensing element of the electrode and antibody or antigen/hapten in the sample solution. One example of such an electrode is the polymer membrane electrode shown in Fig. 7. The selective response of this electrode to specific immunoglobulins is based on the interaction between antibody in solution and an antigen-ionophore complex in the membrane ... 

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. 

K. Mizutani, T. Electronic and structural requirements for metabolic activation of butylated hydroxytoluene analogs to their quinone methides, intermediates responsible for lung toxicity in mice. Biol. Pharm. Bull. 1997, 20, 571-573. (c) McCracken, P. G. Bolton, J. L. Thatcher, G. R. J. Covalent modification of proteins and peptides by the quinone methide from 2-rm-butyl-4,6-dimethylphenol selectivity and reactivity with respect to competitive hydration. J. Org. Chem. 1997, 62, 1820-1825. (d) Reed, M. Thompson, D. C. Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di- m-butyl-4-methylphenol (BHT). Chem. Res. Toxicol. 1997, 10, 1109-1117. (e) Lewis, M. A. Yoerg, D. G. Bolton, J. L. Thompson, J. Alkylation of 2 -deoxynucleosides and DNA by quinone methides derived from 2,6-di- m-butyl-4-methylphenol. Chem. Res. Toxicol. 1996, 9, 1368-1374. 

Volume 84. Immunochemical Techniques (Part D Selected Immunoassays) Edited fey John J. Langone and Helen Van Vunakis... 

On the other hand, the most severe constraint of CL analyses is their relatively low selectivity. One major goal of CL methodologies is thus to improve selectivity, which can be accomplished in three main ways (1) by coupling the CL reaction to a previous, highly selective biochemical process such as an immunochemical and/or enzymatic reaction (2) by using a prior continuous separation technique such as liquid chromatography or capillary electrophoresis or (3) by mathematical discrimination of the combined CL signals. This last approach is discussed in Sec. 4. 

Abstract A significant number of immunochemical methods have been described for the determination of the most important emerging pollutants. The present chapter is a compilation of the information available today regarding immunochemical determination of industrial residues with a high potential risk of causing negative effects in the environment, wildlife, and public health. Homogeneous immunoassays, ELISAs, FIIAs, immunosensors, and selective immunoaffinity sample treatment methods have been reported for the analysis of an important number of these substances. The bases of these methods are briefly presented. 

In recent years many efforts have been made to develop immunochemical techniques integrating the recognition elements and the detection components, in order to obtain small devices with the ability to carry out direct, selective, and continuous measurements of one or several analytes present in the sample. In this context biosensors can fulfill these requirements. Biosensors are analytical devices consisting of a biological component (enzyme, receptor, DNA, cell, Ab, etc.) in intimate contact with a physical transducer that converts the biorecognition process into a measurable signal (electrical or optical) (see Fig. 4). In... 

Immunochemical methods have been reported for both APEs and their metabolites, especially APs. A discussion of the immunochemical methodologies reported to date, the effect of the immunizing haptens employed, and the features of these techniques were recently reviewed [169]. Unfortunately, the detectability achieved is usually far from what is necessary for direct application to environmental samples. Moreover, the selectivity for APs versus APEs is not always satisfactory. Thus, Goda et al. [ 148] developed a direct ELISA for NP with a LOD of 10 pg L 1 and a working range between 70 and 1,000 pg L, but APEs with one to ten ethoxylate units are also well recognized. 

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. 

It has been widely demonstrated that immunochemical techniques offer a good alternative to conventional methodologies in many areas due to the high sensitivity and selectivity achieved for the antibodies toward the target analytes. 

Because of the complex and polymorphic nature of the Lp(a) lipoprotein, together with the homology of the apo(a) moiety with plasminogen, a number of specific problems arise concerning the immunochemical quantification of Lp(a). These include the selection of a suitable type of immunoassay, its specificity and sensitivity, and the type of antisera used in the assay (L2). Moreover, the selection of an appropriate standard and of the units of mass to express the amount of Lp(a) require careful consideration (L4). 

Much research has gone into raising the sensitivity and selectivity of immunosensors to the desired levels. Several labels have proved to ensure a high sensitivity, yet radioisotopic labels have essentially been avoided. Non-isotopic labels for immunosensors include various enzymes, catalysts, fluorophores, electrochemically active molecules and liposomes. Labelled immunosensors are basically designed so that immunochemical complexation takes place on the surface of the sensor matrix. There are several variants of the procedure used to form an immunocomplex on the matrix. In the final step, however, the label should always be incorporated into the immunocomplex for determination, as shown in Fig. 3.27.B. 

When a more specific detection system is used instead, a rigorous sample cleanup may not be necessary. This is actually the case with most of the microbiological and immunochemical detection systems applied in residue analysis. Owing to the selectivity and sensitivity of their detection principle, homogenization with an aqueous buffer is often tire only treatment required prior to analysis. Moreover, these detection systems are usually independent of the sample size as, in many cases, a single drop of milk or tissue fluid is sufficient to carry out a successful analysis. 

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