Detection system avidin-biotin conjugate

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. 

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). 

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). 

The use of the avidin-biotin system enables a separation of the primary and secondary steps. Previous employment of an antibody-marker for direct detection was problematic since such conjugates are very large and subject to a variety of irrelevant interactions that interfere with the desired immunochemical binding. In contrast, the biotinylated antibody can first be incubated with the cell or tissue sample following this initial interaction, the sample can be fixed and incubated subsequendy with... 

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

Another simple detection method is the polymer-based detection system. This detection system does not depend on the avidin-biotin interaction for localization of the marker enzyme to the antigen but bases on an enzyme conjugated polymer backbone vith attached secondary (link) antibody. This method has the advantage to join both secondary antibody step and the detection complex incubation step in a single step. Consequently, it is a more rapid method and the blocking of the endogenous biotin activity is not necessary. 

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