Use of Avidin—Biotin in Assay Systems

A common application for 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 molecule, it creates multiple sites for the binding of avidin or streptavidin. If avidin or streptavidin 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 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 avidin—biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind their complementary DNA target through the use of avidin-labeled complexes (Bugawaneffl/., 1990 Lloyd era/., 1990). Direct detection of hybridized probes can be accomplished, in a manner similar to that for LAB, by incubating with an avidin-enzyme conjugate followed by substrate development. BRAB-like and ABC-like assays also can be utilized to further enhance a DNA probe signal (Chapter 17, Section 2.3). 

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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.

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

In a recent example, Kim et al. showed how the combination of several types of nanoparticles could also be wisely used for the design of signaling protocols. They designed a FRET-based inhibition assay to determine the avidin concentration in solution with AuNPs and QDs. The ensemble involves the use of streptavidin-conjugated QDs that interact with biotin-AuNPs through well-known streptavidin-biotin chemistry. This system was not luminescent due to FRET interaction between QDs and AuNPs. Addition of avidin to this ensemble caused the luminescence to increase gradually because the AuNPs were displaced from the streptavidin-functionalized QDs as a consequence of avidin-biotin interactions (Fig. 8). 

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). 

It is often important to determine the extent of biotin modification after a biotinylation reaction is complete. Measuring biotin incorporation into macromolecules can aid in optimizing a particular (strept)avidin-biotin assay system. It also can be used to assure reproducibility in... 

The highly specific interaction of avidin with the small vitamin biotin can be a useful tool in designing assay, detection, and targeting systems for biological analytes (see... 

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