Avidin, biotin binding

Fig. 21. (Top) Schematic and chemical structure of the biosensor molecule used by Spence et al. (Bottom) Detection of biotin-avidin binding with xenon NMR. The HP Xe NMR spectra of the functionalized xenon biosensor alone and in the presence of protein. These spectra show a new resonance and marked broadening in response to the increasing amounts of avidin. (Courtesy of Connie Chung and the Pines group, adapted from ref. 314 with permission. Copyright 2004, American Chemical Society.)...

Many biotinylated succinimide esters are now available. Most of these variations alter the size of the spacer arm between the succinimide coupling group and the biotin. The additional spacers could facilitate avidin binding and, thus, may be critical for some applications (9). 

This method, either standard or elite (increased molar ratio), remains the forefront of much of the immunocytochemistry being performed today, and will be the main subject of this chapter. There are new methods, though, that are being used with increased frequency, such as the labeled-avidin binding method, sometimes called the streptavidin-binding method, and a newer catalyzed amplification method that uses avidin, biotin, peroxidase, and a biotinyl tyramide to achieve even more sensitivity. These methods will be discussed at the end of this chapter. 

Some tissue samples, especially fresh or frozen ones, can have intrinsic biotinbinding capabilities that must first be blocked before using an ABC type of detection. The simple Blocking Kit from Vector can be used to add avidin to the specimen to bind biotin, followed by biotin to bind avidin. Incubations of 15 min with brief PBS rinses should suffice. 

High-quality reagents needed for biotin-avidin immunostaining are all available commercially. Kits are also available commercially for biotinylating antibodies (see Chapter 7). The major consideration in biotinylating antibodies is the use of biotin with a carbon spacer arm at least 1 nm long, since the binding site of biotin on avidin and probably streptavidin is in a deep depression (4). 

The basic methods to quantify biotin in food are bioassays, avidin-binding assays or fluorescent derivative assays. Although the avidin-binding assay of biotin and its metabolites after the separation by HPLC is considered as one of the best cnrrently available methods [587], it is still not largely diffnsed. 

In the avidin-biotin (ABC) method a complex of avidin and biotinylated tracer containing the free avidin binding sites is applied to the biotinylated antibody. As a high... 

Sections (5 xm thick) of formalin-fixed and paraffin-embedded tissues are placed on slides, dried overnight at 37°C, deparaffinized with xylene, and rehydrated with descending concentrations of ethanol. They are treated with 3% hydrogen peroxide in methanol for 5-10 min and then rinsed with PBS. The slides are immersed in 10 mM citrate buffer (pH 6.0), and heated for 2 min at 100% power, followed by 8 min at 80% power. After being rinsed in PBS, the sections are incubated in an appropriate primary antibody. They are rinsed in PBS and then treated for 4-8 min with egg white avidin solution (2.5 g of egg white avidin in 0.1 mM PBS) (Ventana Medical Systems). Avidin binds to endogenous biotin. 

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