Critical binding reagents

Immunoassays are inherently sensitive and specific. However, with continued need to develop increasingly sensitive assays to support preclinical and clinical studies, there have been ongoing efforts to enhance the capabilities of these techniques. Advances in critical binding reagents, detection systems, new assay formats and automation have resulted in improved immunoassay technology. 

Although most immunoassays have used polyclonal antibodies as the critical binding reagents, development of monoclonal antibodies by Kohler and Milstein in 1975,has resulted in their widespread use, particularly in assays for macromolecules. Their unique epitope specificity conveys advantages in double antibody immunoassays for proteins, where one monoclonal antibody may be used to capture the protein by a specific subunit or epitope, and another, directed against a... 

Immunoassays generally vary in the type of critical antibody binding reagent or the detection and reporter... 

It is critical to keep in mind that existing reagents can be used for multiple formats. Eor example, polyclonal antibodies dominate the environmental field because they generally provide greater sensitivity and specificity for small molecules at a much lower cost than do monoclonal or recombinant antibodies. With some biosensors monoclonal or engineered antibodies or recombinant binding proteins may offer advantages. 

Reactants and reagents can be conveniently loaded into the dry zeolite by adsorption. This can be accomplished by intimately mixing the solid or liquid reactant and the powdered zeolite, by absorption from the gas phase, or by diffusion in a solvent slurry containing the zeolite and dissolved reactant. The choice of solvent for the slurry method is critical. It must be volatile enough to be removable at a pressure and temperature that does not result in evacuation of the reactant or its decomposition. In addition, the reactant must have a greater affinity for the interior of the zeolite than for the slurry solvent itself. The lack of affinity for the interior of the zeolite is an acute problem for non-polar hydrocarbons that lack binding sites for the intrazeolitic cations. The use of fluorocarbons such as perfluorohexane as slurry solvents takes advantage of the fluorophobicity of many hydrocarbons and has alleviated this problem to some extent.29... 

Desmocalmin, which may be the bovine equivalent to a human protein called keratocalmin (Fairley et al., 1991), was identified in the mid-1980s as a desmosome protein that binds both to calmodulin in a Ca2+-dependent manner and to reassembled keratin filaments in vitro in the presence of Mg2+ (Tsukita and Tsukita, 1985). Unfortunately, due to the loss of critical reagents, progress on this potentially interesting regulator of IF binding has been impeded, and further work will be necessary to revisit its potential role in desmosome assembly and regulation. 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

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