Bridged avidin—biotin system

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 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. 

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). 

Fig. 18.7. Schematic representation of the immobilization of the biotinylated cholera toxin B subunit using the biotin-avidin bridging system. The hydrophilic co-polymer on the transducer surface improves the permeation of the enzymatically generated quinone.

The BRAB system is essentially the same as the LAB system except that the avidin is not conjugated to an enzyme. Here, the avidin acts as a bridge to connect the biotinylated secondary antibody and biotinylated enzyme. Since the avidin has multiple biotin binding sites, this system allows more biotinylated enzyme to be complexed with a resulting amplification of signal, thus making the system potentially more sensitive than the LAB system. 

The binding affinity between streptavidin and biotin is among the strongest noncovalent bonds known to exist (A o = 10" M) Therefore, the high affinity coupling of the biotin-avidin system has been used to immobilize different biomolecules on the surface of biomaterials and biosensors. " - It consists of using avidin as a bridge between the biotinylated surface and biotinylated... 

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