Biotin- strept avidin interaction

FIGURE 5.5 Schematic representations of the two immunosensor formats (a) immunosensor based on the biotin treptavidin interaction and (b) immunosensor based on rabbit IgG-modified SPCEs. (Reprinted from [27] widi permission from Elsevier.) 

Electrochemical Sensors, Biosensors and Their Biomedical Applications 

---

Predominant protein blockers include bovine serum albumin (BSA), nonfat dry milk (NFDM), casein, and fish gelatin. NFDM, used at 0.1 0.5%, is inexpensive but preparations vary in quality. Some NFDM preparations contain histones that interfere with anti-DNA determinations or inhibitors of the biotin (strept)avidin interaction such as biotin itself. Casein is a chief component of NFDM and is often used alone as a blocking agent. 

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

One of the strongest noncovalent interactions between a small molecule and a protein is found in the biotin-(strept)avidin association. This strong supramolecular interaction (K 10 M ) was first utilized by Wilson and White-sides in 1978 to prepare an asymmetric Rh(I) catalyst for the hydrogenation of olefins. They demonstrated that the biotinylated Rh(f)(ndb) diphosphine complex (3a in Figure 10.9) at 0.2 mol% loading catalyzed the quantitative hydrogenation of Af-acetamidoacrylic acid, affording the (S)-form of iV-acetamidoalanine with 41% ee, while the product was obtained in racemic form in the absence of avidin (Scheme 10.12) [42]. 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

The only disadvantage to the use of NHS-biotin or sulfo-NHS-biotin is the lack of a long spacer group off the valeric acid side chain. Since the binding site for biotin on avidin and streptavidin is somewhat below the surface of the proteins, some biotinylated molecules may not interact as efficiently with (strept)avidin as when longer cross-bridges are used (Green et al., 1971 Bonnard et al., 1984). 

Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents.

The following sections discuss the concept and use of the (strept)avidin-biotin interaction in bioconjugate techniques. Preparation of biotinylated molecules and (strept)avidin conjugates also are reviewed with suggested protocols. For a discussion of the major biotinylation reagents, see Chapter 11 and Chapter 18, Section 3. 

Use of (Strept)avidin—Biotin Interactions in Assay Systems... 

Since a biotinylated molecule potentially is able to interact with (strept)avidin at its biotin binding sites just as strongly as biotin in solution, the degree of biotinylation may be determined using the HABA method as well. Comparison of the response of a biotinylated protein, for example, with a standard curve of various biotin concentrations allows calculation of the molar ratio of biotin incorporation. 

---