Avidin-biotin reactions

After use, the electrode surface can be renewed by a simple polishing procedure for further uses, highlighting a clear advantage of this new material with respect to surface-modified approaches such as classical biosensors and other common biological assays. Biotinylated antiatrazine antibodies can be easily immobilized on the surface of the avidin-modified transducer through the avidin-biotin reaction since antibodies can be readily linked to biotin without serious effects on their biological, chemical, or physical properties. Moreover, antiatrazine antibodies can be easily immobilized on the surface of the Protein A-modified transducer without any modification of the antibodies. 

An interesting kind of covalent fixing is the avidin-biotine reaction. It is used exclusively for biosensors. In this reaction, a small molecule is somehow enveloped by a large molecule. 

The avidin-biotin reaction can be used to link several molecular monolayers which lie on top of each other with avidin and biotin molecules arranged in alternating mode. A large variety of configurations results from different combinations of the simple reaction depicted schematically in Fig. 7.28. The stability of the avidin-biotin complex is extremely high. 

Oligonucleotides are immobilized at non-metallic surfaces most commonly by means of the avidine-biotin reaction (Chap. 7, Sect. 7.4.1). 

It is often important to determine the extent of biotin modification after a biotinylation reaction is complete. Measuring biotin incorporation into macromolecules can aid in optimizing a particular (strept)avidin-biotin assay system. It also can be used to assure reproducibility in... 

If cellular localization of the antigen-antibody complex is not required, enzyme immunolabeling can be performed on cells adherent to a microtiter plate, and the color change resulting from the enzymatic reaction can be detected as a change in absorbance with an automatic plate reader (see Chapter 28). Biotinylation of antibodies and the use of the avidin-biotin complex has further extended the versatility and sensitivity of the enzymatic techniques (see Chapters 7 and 25-27). Most recently, the principles behind these techniques have been applied in combination with in situ hybridization techniques. Using nucleic acid-antibody complexes as probes, specific DNA or RNA sequences can be localized (see Chapters 46 9). 

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. 

Fig. 12 Schematic illustration of immobilization strategy using affinity reactions via avidin-biotin coupling. Biotinylated mixed monolayers improve the availability of the surface-bound avidin or streptavidin to biotin coupled probe biomolecules...

Q Yang, X-Y Liu, M Hara, P Lundahl, J Miyake. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin-biotin binding. Anal Chem 280 94-102, 2000. 

The sections are incubated for 1 hr with the primary monoclonal antibody, mouse antihuman mast cell tryptase antibody (DAKO), diluted 1 200 with 1% BSA/PBS. They are washed for 10 min in PBS using magnetic stirring, incubated with biotinylated antimouse antibody for 15 min, and washed in PBS. This is followed by adding avidin-biotin-horse-radish peroxidase for 15 min. A Vector DAB Substrate kit is applied to develop the reaction product by using nickel-DAB (5 min developing time) according to the manufacturer s instructions. This step yields a black reaction product at sites of mast cell tryptase. 

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