Use of amine-reactive biotin or desthiobiotin derivatives at the recommended MR typically leads to a degree of labeling (DOL) of three to eight biotins (or desthiobiotins) per intact IgG molecule, which is optimal for most detection paradigms. Degrees of labeling in this range have no adverse effects on antigen binding by the antibody or any of its physicochemical properties. Thus, it is not absolutely necessary to measure the DOL before using the biotinylated antibody. As long as each antibody molecule contains at least one biotin (or desthiobiotin), the former can be detected with reporter group-labeled biotin-binding proteins. However, multiple biotins (or desthiobiotins) on the antibody do enable more than one streptavidin or avidin to bind to it and will increase the detected signal intensity.
Degrees of antibody biotinylation beyond those discussed previously may result in increased background in the researchers' experiments. However, determining the DOL obtained at different biotinylation reagent: antibody MR is crucial for optimizing the reaction conditions that result in reproducible antibody labeling. A detailed discussion of the numerous methods available for determining the DOL is beyond the present scope. The reader is referred to refs. 9-11 for more detail. We describe here only the most popular method (12). In Subheadings 3.5.1. to 3.5.3., the terms "biotin" and "desthiobiotin" are used interchangeably.
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