Bridged avidin-biotin

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.)... 

Antibody-masking enzyme tag immunoassay Adenosine 5 -monophosphate S-Acetylmercaptosuccinic anhydride Alkaline phosphatase anti-alkaline phosphatase (enzyme-antibody) complex Alkaline phosphatase 5-Aminosalicylic acid Adenosine 5 -triphosphate Aa-Benzoyl-L-arginine ethyl ester (-f-)-Biotin bromoacetyl hydrazide (-b)-Biotin Y-aminocaproic acid A-hydroxy-succinimide ester Bis-diazotized benzidine -Galactosidase (-I- )-Biotin hydrazide (-I- )-Biotin-A-hydroxysuccinimide ester (-I-)-Biotin p-nitrophenyl ester Bridged avidin-biotin (method)... 

Narang U., Anderson G.P., King K.D., Liss H.S., Ligler F.S., Enhanced biosensor performance using an avidin-biotin bridge for antibody immobilization, Proc. SPIE. 2980 187-194,1997. 

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 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 technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

The other interesting observation of this study was that Kf for directly immobilized antibody was only 50% of the avidin-biotin bridged antibody. This suggests that random coupling reduces the number of effective binding sites relative to the oriented coupling using avidin-biotin. 

MP1950/anti-P-selectln antibody attached via PEG spacer and avidin-biotin bridge Targeting activated endothelium [93]... 

After sulfo-NHS-SS-biotin-modified molecules are allowed to interact with avidin or streptavidin probes, the formed complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated component from the avidin or streptavidin detection reagent. The use of disulfide biotinylation reagents thus provides much gentler conditions to break the complex than would be required if the avidin-biotin interaction itself were disrupted (which dissociates only at 6—8 M guanidine, pH 1.5). 

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

The construction of a trilayer via incorporation of CPMV cysteine mutants has also been reported [109], In addition, there are a few further examples where CPMV particles have been bound to surfaces using different strategies and templates. For example, CPMV histidine mutants have been immobilized on Neutr-Avidin surfaces bridged with biotin-X-NTA molecules followed by decoration of the viral particles with quantum dots [107]. CPMV cysteine mutants have been successfully immobilized on maleimido-functionalized patterned templates these... 

Using the ELISA (with avidin-biotin bridges) and ELISPOT methods a stimulating effect of selected probiotic bacteria (Bifidobacterium longum, B animalis, Lactobacillus easel, L. salivarius) on the immune system of the rat and mouse has been demonstrated (higher level of specific as well as total IgGa and IgA content) (Nagy et al., 2002). 

Numerous studies on bead bio chips were therefore based on magnetic [16,17], glass or silica [18-20], and polystyrene beads [21]. The DNA immobilization chemistry of those beads could be very different, from the classical avidin/biotin affinity reaction [17,22] (Fig. 2B) to the disulfide bridging onto thiol modified silica [19] (Fig. 5A), the thiocyanate reaction onto amino-terminated latex beads [21] (Fig. 5B), and finally the hybridization-based immobilization of poly(A)-tagged probes onto poly(T)-bearing magnetic beads [16] (Fig. 5C). 

---