Antibodies enzyme selection

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... 

Carrier protein Macromolecule to which a hapten is conjugated, thereby enabling the hapten to stimulate the immune response. catELISA Similar to an ELISA, except that the assay detects catalysis as opposed to simple binding between hapten and antibody. The substrate for a reaction is bound to the surface of the microtitre plate, and putative catalytic antibodies are applied. Any product molecules formed are then detected by the addition of anti-product antibodies, usually in the form of a polyclonal mixture raised in rabbits. The ELISA is then completed in the usual way, with an anti-rabbit second antibody conjugated to an enzyme, and the formation of coloured product upon addition of the substrate for this enzyme. The intensity of this colour is then indicative of the amount of product formed, and thus catalytic antibodies are selected directly. 

In general, the strengths of the ELISA are its selectivity and specificity, whereas its weaknesses are related to precision of measurement. Each assay varies — depending on the antibody, enzyme, enzyme conjugate, and measurement, as well as on assay format. Each should be validated so that its unique performance characteristics are known. 

Murry, J. (2000). Monoclonal antibody treatment of solid tumors a coming of age. Semin. Oncol. 27(6), 64-70. Senter, P. Springer, C. (2001). Selective activation of anticancer prodrugs by monoclonal antibody-enzyme conjugates. Adv. Drug Deliv. Rev. 53(3), 247-264. 

The ability to select the relative size of the antibody—enzyme complex is important, depending on the assay application. Low-molecular-weight conjugates may be more optimal for immunohistochemical staining or blotting techniques where penetration... 

Antibody inhibition, which involves an evaluation of the effects of inhibitory antibodies against selected CYP enzymes on the metabolism of a drug candidate by human liver microsomes. Because of the ability of antibodies to inhibit selectively and noncompetitively, this method alone can potentially establish which human CYP enzyme is responsible for metabolizing a drug candidate. Unfortunately, the utility of this method is limited by the availability of specific inhibitory antibodies. 

Chemical and antibody inhibition represent the second and third approaches to reaction phenotyping. They typically involve an evaluation of the effects of known CYP enzyme inhibitors or inhibitory antibodies against selected CYP enzymes on the metabolism of a drug candidate by pooled human liver micro-somes. As in the case of correlation analysis, chemical and antibody inhibition experiments must be conducted with pharmacologically relevant concentrations of the drug candidate in order to obtain clinically relevant results. 

During the evolution of a natural enzyme, selection is not solely dependent on rate improvement. Therefore, there is no requirement for enzymes to be kinetically perfect, and it should be possible to develop catalytic antibodies that are faster than their natural counterparts. The designed substrate 52 has a rate of 1.4 s-1 with 84G3-catalyzed retro-aldol reaction (Zhong et al., 1999). Its kinetic parameters hold the current world record for antibody catalysis (KM = 4.2 /ulM, = 2 X... 

Isothiocyanate-terminated polyethylene glycol derivatives, (II) and (III), were prepared by Smith [2] and Acharya [3], respectively, and were effective in selectively reacting with biomolecules including antibodies, enzymes, and proteins. 

Sensitive, selective detection of biochemically active compounds can be achieved by employing antigen-antibody, enzyme-substrate, and other receptor-protein pairs, several of which have been utilized in the development of piezoelectric immunoassay devices. The potential analytical uses of these materials has been reviewed, particularly with respect to the development of biochemical sensors [221-224], The receptor protein (e.g., enzyme, antibody) can be immobilized directly on the sensor surface, or it can be suspended in a suitable film or membrane. An example of the sensitivity and response range that can be... 

This method of selecting catalytic sites significantly depends on spontaneous processes, in contrast to the development of artificial enzymes and catalytic antibodies. The selection process is based on self-assembly, selforganization and self-optimization. Therefore, this selection approach bears the characteristics of supramolecular chemistry. A similar concept is used in natural evolution processes, resulting in the complicated life forms we see around us today. Therefore, it is clear that we can design the self-organizational processes used in supramolecular chemistry to proceed according to the concepts followed by this natural evolutionary process. 

The chemical interest of these trace amount proteins stems from the fact that they can be used as catalysts or enzyme analogs for almost any chemical reaction (Tramontano et al., 1986 Pollack et al., 1986 Lemer et al., 1991). The fundamental difference between antibodies and enzymes does not relate so much to the protein structures as to the structure and lifetime of the substrates. Antibodies selectively bind molecules in their ground state, whereas enzymes selectively produce and then bind more strongly to short-lived transition states. Antibody-antigen complexes tend toward precipitation, whereas enzyme-transition state complexes react to enzyme-product complexes, which immediately dissociate. In both cases, however, the same noncovalent bonds are used. 

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