Avidin biotin complex

Staining procedure for paraffin sections using ABC system 

Deparafftnize and rehydrate tissue sections. Rinse in distilled water for 5 min. 

When using HRP as an enzyme marker, incubate the sections for 15 min in 0.3% H2O2 in either methanol or water to quench endogenous peroxidase (see Sect. 5.2). If endogenous peroxidase activity does not present a problem, this step may be omitted. 

Incubate sections for 20 min with PBS containing 5% normal serum of species in which the secondary antibodies were raised. 

Endogenous biotin may be a cause for nonspecific background staining (see Sect. 5.4). To eliminate this unwanted staining, apply an avidin/biotin block (for instance Avidin/Biotin blocking kit from VECTASTAIN, Cat. No. SP-2001). Usually, paraffin tissue sections are free from endogenous biotin, and this step may be omitted. 

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The avidin-biotin complex, known for its extremely high affinity (Green, 1975), has been studied experimentally more extensively than most other protein-ligand systems. The adhesion forces between avidin and biotin have been measured directly by AFM experiments (Florin et al., 1994 Moy et al., 1994b Moy et al., 1994a). SMD simulations were performed on the entire tetramer of avidin with four biotins bound to investigate the microscopic detail of nnbinding of biotin from avidin (Izrailev et al., 1997). 

The simulations of the avidin-biotin complex (Izrailev et ah, 1997) showed that a major difficulty involved in studies of the binding and flexibility of... 

Izrailev et al., 1997] Izrailev, S., Stepaniants, S., Balsera, M., Oono, Y., and Schulten, K. Molecular dynamics study of unbinding of the avidin-biotin complex. Biophys. J. 72 (1997) 1568-1581... 

Iodoacetyl-LC-biotin has been used to localize the SH thiol of myosin by use of an avidin-biotin complex visualized by electron microscopy (Sutoh et al., 1984) and to determine the spatial relationship between SHj and the actin binding site on the myosin subfragment-1 surface (Yamamoto et al., 1984). 

Bayer, E.A., and Wilchek, M. (1980) The use of the avidin-biotin complex as a tool in molecular biology. Meth. Biochem. Anal. 26, 1-45. 

Wilchek, M., and Bayer, E.A. (1988) The avidin-biotin complex in bioanalydcal applications. Anal. Biochem. 171, 1-32. 

S.M. Hsu, L. Raine, and H. Fanger, A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin. Pathol 75, 734-738 (1981). 

Whereas antigen-retrieval technique serves to amplifying the immunocytochemical signal at the predetection phase, conventional methods of signal amplification, such as avidin biotin complex (ABC) and soluble enzyme-anti-enzyme immune complex techniques (peroxidase-anti-peroxidase complex and alkaline phosphatase-anti-alkaline phosphatase complex PAP and APAAP respectively), are applied in the phase of detection. For many years, the PAP and APAAP procedures represented the most sensitive and reliable and hence most popular techniques in many pathology laboratories. However, today these techniques are only rarely used, being substituted by modem more sensitive methods. 

Avidin-biotin complex (ABC) is based on the high affinity that streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) have for biotin. Biotin is a naturally occurring vitamin. One mole avidin will bind four moles biotin. ABC method affords a several-fold higher antigen detectability than those achieved in the standard indirect method. 

The procedure given here summarizes the localization of tissue antigens using a primary antibody, biotinylated secondary antibody, avidin-biotin complex, and DAB chromagen on fresh frozen brain... 

Avidin-biotin complex Prepare the ABG according to the instructions provided by the manufacturer and allow the solution to conjugate for at least 30 min at room temperature before applying to slides. Sections should incubate with the ABG for at least 30 min. Rinse sections with buffer. 

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). 

Swanson, P., Hagen, K., and Wick, M. (1987) peroxidase-antiperoxidase (ABPAP) Avidin-biotin-complex. Am. J. Clin. Pathol. 88, 162-176. 

The Avidin-Biotin Complex (ABC) Method and Other Avidin-Biotin Binding Methods ... 

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

Add the avidin-biotin complex to the slide, making sure to cover the entire specimen. Work quickly to avoid any drying. Cover the chamber and incubate for 30 min. 

Elias, J., Margiotta, M., and Gabore, D. (1989) Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin complex (ABC), and the peroxidase-labeled avidin-biotin (LAB) methods. Am. J. Clin. Pathol. 92,62. 

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. 

Avidin-biotin complex (Vector ABC Elite kit) (Vector Laboratories) ABC solution should be made fresh 10 min before use, so that complexes have time to form according to instractions of manufacturer. 

Flowever, the focus of the major part of the chapters lies on the couphng chemistry used for DNA immobilization. Successful immobihzation techniques for DNA appear to either involve a multi-site attachment of DNA (preferentially by electrochemical and/or physical adsorption) or a single-point attachment of DNA (mainly by surface activation and covalent immobihzation or (strept)avidin-biotin linkage). Immobilization methods described here comprise physical or electrochemical adsorption, cross-linking or entrapment in polymeric films, (strept)avidin-biotin complexation, a surface activation via self-assembled monolayers using thiol linker chemistry or silanization procedures, and finally covalent coupling strategies. 

Y Tanaka, S Terabe. Studies of enantioselectivities of avidin, avidin-biotin complex and streptavidin by affinity capillary electrophoresis. Chromatographia 49 489-495, 1999. 

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