Avidin peroxidase complex

Bronckers, A.J.J., Gay, S., Finkelman, R.D., and Butler, W.T. (1987) Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs Comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement./. Histochem. Cytochem. 35, 825-830. 

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. 

A wide spectrum of hepatic lesions has been reported in AIDS (H4), but it is not known whether the changes are related to the presence of HIV-1. Therefore, sections from livers of autopsied patients with AIDS were examined for the presence of HIV-1 antigen p 24 (core) and gp 41 (envelope) by the avidin-biotin-peroxidase complex methods using monoclonal antibodies. The most common histologic abnormalities were steatosis, portal inflammation, Kupffer cell hyperplasia, and focal hepatocellular and bile duct damage. Immunoreactivity for HIV-1 antigens was demonstrated in 80% of cases. 

Hsu, S., Raine, L., and Eanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577. 

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

Fig. 1. Diagram illustrating the molecular interactions of the ABC procedure. The primary antibody against the antigen of interest is linked to the avidin-biotinylated peroxidase complex via a biotinylated secondary antibody raised against immunoglobulin of the animal species used to generated the primary antibody (A, immunoglobulin , biotin A, avidin , peroxidase).

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

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. 

The following procedure is based on the Vectastain ABC kit from Vector (suppliers appendix). It uses an avidin-biotin complex to attach horseradish peroxidase (HRP) or alkaline phosphatase (AP) to the biotinylated secondary antibody. Avidin-biotin systems are capable of extremely high sensitivity because multiple reporter enzymes are bound to each secondary antibody. In addition, the detergent Tween 20 is a popular alternative to protein blocking agents when using nitrocellulose or PVDF membranes. 

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... 

The sections are incubated for 72 hr at 4°C with primary antiserum in PBS containing 2% normal serum and 0.25% gum arabic (Sigma). The sections are then incubated overnight at 4°C with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), diluted 1 200. They are next incubated overnight at4°C with avidin-biotinylated peroxidase complex (ABC) diluted 1 100 in PBS containing 0.25% gum arabic. Note that the peroxidase-antiperoxidase procedure can also be used. 

Cattoretti G, Berti E, Schiro R, D Amato L, Valeggio C, Rilke F. Improved avidin-biotin-peroxidase complex (ABC) staining. Histochem J 1988 20(2) 75-80. 

Biotin-conjugated rabbit anti-mouse 3. Avidin - biotin complex 4. Peroxidase conjugated biotin... 

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). 

---