Avidin-biotin reagents

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 B3.4.1 Serial dilution of primary antibody directed against the 97-kDa catalytic subunit of the plant plasma membrane ATPase. The blot was developed with HRP-coupled avidin-biotin reagents according to the Alternate Protocol and visualized with 4-chloro-1-naphthol (4CN). Note how background improves with dilution...

Fig. 7.3 Avidin-biotin reagents. Avidin is a protein from either egg whites or from bacteria and is not found in tissues or cells. Biotin, a very small molecule, is also known as vitamin B7. Four biotin molecules can attach to one avidin, with an affinity that is extremely high making the binding almost permanent...

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

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. 

Notes. When using biotin-labeled secondary antibodies instead of enzyme-labeled antibodies, you have first to detect biotin with enzyme-labeled (strept) avidin and proceed further with the Substrate Step (9). Do not add normal serum, non-fat dried milk, culture media or other potential sources of biotin to (strept)avidin-containing reagents. This may result in reduced sensitivity. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate. 

Avidin-biotin complex horseradish peroxidase reagent (Vectastain ABC HRP Kit, Vector Laboratories, Inc., Burlingame, CA). 

Fig. 3. Examples of standard curve for biotin assay with avidin-HABA reagent.

While membrane is being incubated with secondary antibody, prepare avidin-biotin-HRP or -AP complex. Mix 2 drops Vectastain reagent A and 2 drops reagent B into 10 ml TTBS (nitrocellulose or PVDF) or TBS (nylon). Incubate 30 min at room temperature, then further dilute to 50 ml with TTBS or TBS. 

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). 

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