Avidin-biotin complex methods

Avidin-biotin complex methods Avidin acts as 

Avidin and biotin can be used in different EIH systems. In the direct method, either biotinylated antibody is reacted with avidin- 

The high detectability of these techniques may be due to the presence of multiple biotin molecules on the proteins. Non-specific staining can be considerable at high concentrations of ABC which may be related to the fact that biotin is an important coenzyme for transcarbamylation and may be present in some tissues to bind ABC. Pretreatment with avidin and biotin, respectively, prior to the serological detection, could remedy this problem in some cases. 

Incubation conditions are as in the other techniques, i.e. 1 h at room temperature in a humid chamber. Primary antibodies (dilution of serum 200-4000 times) are first complexed to the antigen in the tissue. After washings, biotin-labeled anti-Ig (dilution 1 200) is added, followed by incubation with ABC (8 pg/ml avidin and 2 pg/ml biotin-POase). 

Detection of different epitopes in the same preparation Multiple staining methods to distinguish different epitopes offer important advantages. In particular, the availability of monoclonal antibodies is likely to result in the use of this method on an increasing scale. 

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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). 

FIGURE 3.1.2 The principle of antigen determination by the immune methods with avidin-biotin signal amplification, (a) Labeled avidin-biotin assay (LAB assay) (b) Bridged avidin-biotin assay (BRAB assay) and (c) Avidin-biotin complex method (ABC assay). (From jQdrychowski, L., Pol. J. FoodNutr. Sci., 12/53(SI 2), 32, 2003b. With permission.)... 

Fung K, Messing A, Ixe VM-Y,Trojanowski JQ. A novel modification of the avidin-biotin-complex method for immuniohistochemical studies of transgenic mice with murine monoclonal antibodies../ Ilistochem. Cytochem 1992 40 1319-28. 

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

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. 

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. 

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

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. 

In the avidin-biotin (ABC) method a complex of avidin and biotinylated tracer containing the free avidin binding sites is applied to the biotinylated antibody. As a high... 

The avidin-biotin complex (ABC) method and the streptavidin-biotin (SAB) method are more sensitive than the peroxidase-antiperoxidase (PAP) method for histochemical techniques. The strong noncovalent attraction between biotin and avidin or streptavidin is exploited in many histochemical, immunohistochemical, and in situ hybridization... 

Avidin-biotin complex (ABC)—the most sensitive of all avidin-biotin methods (Figure 3.1.2)... 

Streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) both possess four binding sites for biotin. The biotin molecule is easily conjugated to antibodies and enzymes. In the avidin-biotin complex (ABC) method secondary antibodies are conjugated to biotin and function as links between tissue-bound primary antibodies and an avidin-biotin-peroxidase complex (5). 

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