Detection using avidin-biotin

Bayer, E.A., Ben-Hur, H., and Wilchek, M. (1987a) Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology. Anal. Biochem. 161, 123-131. 

The first aptasensor reported was particularly based on optical detection [66]. The 58-mer RNA aptamer selective to L-adenosine was immobilized onto the core of multimode fiber using avidin-biotin method. The detection was based on competitive binding of FITC-labeled L-adenosine with unlabeled analyte. This sensor also allowed to study the kinetics of binding and determine equilibrium constants. 

The choice of blocking buffer is sometimes critical for sensitive detection. Milk based blocking solutions are not recommended for use avidin-biotin system because milk contains biotin, which may directly cause competition with biotinylated antibody." Bovine Serum Albumin was not selected in this system in order to avoid the cross-reactivity. Therefore, gelatin was chosen as the blocking agent. 

Biotin labelled antibodies are effectively detected using the biotin binding proteins avidin and streptavidin. Streptavidin is usually preferred as the secondary reagent because avidin is ycosylated and has a high pi and these properties can give rise to non-specific binding in some situations. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

Psoralen-PEOj-Biotin has been used to label double-stranded DNA for detection using (strept)avidin reagents (Henriksen et al., 1991 Wygrecka et al., 2007). The psoralen photoreactive group provides better insertion yields than typical phenyl azide-based systems, such as the standard photobiotin probe discussed previously in this section. 

Biotinylated oligosaccharides are convenient probes of carbohydrate interactions, because the biotin label can be captured or detected using an avidin or streptavidin derivative. For instance, immobilized streptavidin can be used to purify glycoconjugates that have been labeled... 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

A common application for (strept)avidin-biotin chemistry is in immunoassays. The specificity of antibody molecules provides the targeting capability to recognize and bind particular antigen molecules. If there are biotin labels on the antibody, it creates multiple sites for the binding of (strept)avidin. If (strept)avidin is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive antigen detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through its multiple biotinylation sites is the key to dramatic increases in assay sensitivity over that obtained through the use of antibodies directly labeled with a detectable tag. 

Similar techniques can be used to devise (strept)avidin-biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind... 

Other fluorescent probes also may be used to label (strept)avidin molecules for detection of biotinylated targeting molecules. Chapter 9 reviews many additional fluorescent labels, such as quantum dots, lanthanide chelates, and cyanine dye derivatives, all of which may be used in similar protocols to create detection conjugates for (strept)avidin-biotin-based assays. 

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