Avidin-biotin-peroxidase complexes

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. 

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. 

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. 

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). 

Working examples of practical algorithms used in the author s laboratory are presented in Chapter 12. The statistical data used to construct them were gathered over a period of several years using the following specimens that were fixed routinely in 10% neutral-buffered formalin primary antibody incubations at 4°C for 16 to 18 hours the Elite avidin-biotin-peroxidase complex method of immunodetection (Vector Laboratories, Burlingame, Calif) and the antibody reagents listed in Table 11.1. 

Avidin-Biotin-Peroxidase Complex Biotinylated Secondary Antibody Primary Antibody... 

To detect HA on the surfaces of PP films, a colorimetric enzyme-based assay was used. PP/PSS and PP/HA bilayer films (0.15 pm) were blocked using 3% BSA and then incubated in biotinylated HA binding protein (bHABP) solution. Avidin-biotin-peroxidase complex (Vectastain kit PK-4000, Vector Laboratories) and o-phenylenediamine dihydrochloride (OPD) substrate were used to create a colored product. Absorbances w e measured at 490 nm. 

In this method, samples of freshly excised tissues were fixed by boiling them in a 10% neutral formaldehyde solution for 5 min. The fixed tissues were then embedded in paraffin and cut into 3 pm thick sections. The sections on glass slides were deparaffinized and dehydrated by passage through xylene and a graded alcohol series. Then they were incubated with a primary antibody against poly(ADP-ribose). The antibody used, IgG monoclonal antibody lOH, recognized the linear structure of poly(ADP-ribose) described previously (3). Bound antibody was stained with die avidin biotin peroxidase complex or by an indirect immunofluorescence technique. 

Because lymphocytes characterize the cellular inflammatory reaction in both PBC and PSC and because evidence of autoimmune dysfunction is common to both disease conditions, we have characterized the T and B cells present in the blood as well as in the portal areas of the liver in both conditions. This was accomplished utilizing 25 biopsies from 20 patients with PBC and 20 biopsies from patients with PSC. Ten biopsies obtained from patients without evidence of liver disease were used as normal control tissues- The technique utilized was the avidin-biotin-peroxidase complex method utilizing monoclonal antibodies for total T cells (0KT3), T helper-inducer cells (0KT4) and suppressor-cytotoxic cells (OKT8)[7,8]. The specific details of these techniques, as used in our laboratories, have been described in detail elsewhere[8,9,10]. 

Other significant improvements were the development of the enzymatic antigen retrieval methods by Huang et al. [18] and the improvement of systems for secondary antibody detection with the introduction of the avidin-biotin-peroxidase complex (ABC) and the labeled streptavidin-biotin complex (LSAB) by Hsu etal. [19-22]. 

Hsu SM, Raine L (1982) Versatility of biotin-labeled lectins and avidin-biotin- peroxidase complex for localization of carbohydrate in tissue sections. J Histochem Cytochem 30 157-161... 

Hsu SM, Raine L, Fanger H (1981) The use of antiavidin antibody and avidin-biotin-peroxidase complex in immimoperoxidase technics. Am J Chn Pathol 75 816-821... 

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