Avidin—biotin assay

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... 

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... 

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 (Bugawanefrz/., 1990 Lloyd etal., 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). 

Biotinylated enzymes can be used as detection reagents in avidin—biotin assay procedures. Particularly, in the bridged avidin—biotin (BRAB) approach or the ABC technique (Chapter 13, Section 2), a biotin-labeled enzyme is used as the signaling agent... 

FIGURE 3.1.2 The principle of antigen determination by the immune methods with avidin-biotin signal amplification, (a) Labeled avidin-biotin assay (LAB assay) (b) Bridged avidin-biotin assay (BRAB assay) and (c) Avidin-biotin complex method (ABC assay). (From jQdrychowski, L., Pol. J. FoodNutr. Sci., 12/53(SI 2), 32, 2003b. With permission.)... 

Agiamamioti K, Triantis T, Papadopoulos K, Scorilas A (2006) 10-(2-Biotinyloxyethyl)-9-acridone a novel fluorescent label for (strept)avidin-biotin based assays. J Photoch Photobio A 181 126-131... 

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.

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). 

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

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. 

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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