Avidin-biotin conjugate

FIGURE 1.9 Avidin-biotin conjugate (ABQ method. A biotinylated secondary antibody serves to link the primary antibody to a large preformed complex of avidin, biotin, and peroxidase. Boxed asterisk represents antigen determinant on primary antibody. A, avidin B, biotin Px, peroxidase label. From Taylor CR, Cote RJ, eds. Immunomicroscopy A Diagnostic Tool for the Surgical Pathologist. 3rd ed. Philadelphia Elsevier 2005 22. 

PAP, peroxidase-antiperoxidase ABC, avidin-biotin conjugate APAAP, alkaline phosphatase antialkaline phosphatase B-SA, biotin-streptavidin PCNA, proiiferating cell nuclear antigen EPOS, enhanced polymer one-step staining (Dako) CARD, catalyzed reporter deposition HRP, horseradish peroxidase. 

PBS, phosphate-buffered saline ABC, avidin-biotin conjugate AEC, aminorthyl carbazole. 

Chung D-J, Kim K-C, Choi S-H (2011) Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection. Appl Surf Sci 257(22) 9390-9396... 

Avidin-biotin systems. Stable and highly active protein-LB/LS film composites can be accessed using avidin-biotin conjugation techniques. Thus, sensor elements displaying biotinylated GOx and phycoerythrin, and streptavidin and av-idin conjugates of phycoerj tiirin, have been bound to avidin-lipid and biotinylated lipid-streptavidin LB films, or biotinylated lipid layers have been produced [71-73]. 

As with SAMs, nonredox enzymes can also be utilized for BLM sensors. For instance, highly sensitive electrochemical and ISFET sensors for urea, nicUlin, acetylcholine, thyroxine, and triazine herbicides have been produced from phospholipid BLMs with urease, penicillinase, acetylcholinesterase, and antibodies attached electrostatically, covalently, or via avidin-biotin conjugation [130-132]. 

Reaction with avidin-labeled alkaline phosphatase (AP)-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). 

Figure 22.21 Antibodies may be conjugated to liposomes using an indirect approach incorporating a (strept)avidin-biotin system. Biotinylated liposomes may be complexed with biotinylated antibodies using (strept)avidin as a bridging molecule or may be complexed with an antibody-(strept)avidin conjugate.

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. 

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