Avidin bridging molecule

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 avidin/biotin system is rapidly becoming an important tool for EIA since (i) avidin has an exceptionally high affinity for (+ )-biotin (lO M" ) (ii) (-l-)-biotin is very easily coupled to antibodies and to many enzymes often without any loss of activity and, (iii) avidin is very stable and has several binding sites so that it may be used as a bridging molecule between two biotinylated molecules. The use of the avidin/biotin system produces superior detectabilities and low background levels. 

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. 

The only disadvantage to the use of NHS-biotin or sulfo-NHS-biotin is the lack of a long spacer group off the valeric acid side chain. Since the binding site for biotin on avidin and streptavidin is somewhat below the surface of the proteins, some biotinylated molecules may not interact as efficiently with (strept)avidin as when longer cross-bridges are used (Green et al., 1971 Bonnard et al., 1984). 

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

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... 

Fig. 17.3. Anti-enzyme antibodies linked by a bridge (anti-Ig) to the primary antibody can immobilize enzyme molecules (at ), which can be revealed subsequently. This can be repeated by several layers (double bridge, etc.) until the avidity of the antibodies used becomes the limiting factor. Instead of anti-enzyme, avidin can be used if enzyme and primary antibodies are biotinylated. The last step (addition of enzyme) is not illustrated. In the PAP-method, POase-anti-POase complexes are preformed.

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