Antiperoxidase antibody

Fig. 1. Diagram illustrating the molecular interactions of the PAP procedure. The PAP complex is comprised of horseradish peroxidase bound to an antiperoxidase antibody generated in the same animal species as the primary antibody which recognized the tissue antigen of interest. The primary antibody and the PAP complex are linked via a secondary antibody generated in a second animal species against immunoglobulin of the primary animal species (A, immunoglobulin , peroxidase enzyme).

FIGURE 1.6 Enzyme bridge method. A second antibody is used to link (bridge) the primary antibody to an antiperoxidase antibody, which, in turn binds to free peroxidase. Boxed asterisk represents antigen determinant on primary and secondary antibodies. Px, peroxidase label. From Taylor CR, Cote RJ. Immunomicroscopy A Diagnostic Tool for the Surgical Pathologist. 2nd ed. Philadelphia WB Saunders 1994 12. 

TgAb Antithyroglobulin antibody TPOAb Antiperoxidase antibody TSBAb TSH receptor inhibiting antibodies TSH Thyrotropin... 

Hashimoto s thyroiditis). The prevalence of antiperoxidase antibody (TPOAb) in asymptomatic pregnant women ranges between 6% and 19.6% (Stagnaro-Green et ai, 1990 Glinoer et ai, 1991), while it rises to 50% in women with type 1 diabetes (Jovanovic-Peterson and Peterson, 1988). 

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). 

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. 

The sections are incubated in a primary antibody (diluted appropriately) for 72 hr at 4°C in a sealed humid chamber the incubation is carried out by applying droplets of the antibody to the sections. After being rinsed in the buffer, the sections are incubated for 90min in secondary antiserum diluted 1 50 with PBX (0.3% Triton X-100, 0.01% sodium azide and 0.1 M phosphate buffer) and then treated for 1 hr under agitation in peroxidase-antiperoxidase (PAP), diluted 1 100 with PBX, in a sealed humid chamber in both cases. 

An immunohistochemical examination of PSA using polyclonal antibodies by the peroxidase antiperoxidase (PAP) method and by the technique of biotin-streptavidin-alkaline phosphatase has been successfully carried out (Zaviacic et al., 1994). Immunoelectron microscopy in conjunction with the protein A-gold complex can also be used for localizing PSA in human prostate (Sinha et al., 1987). The procedure for immunofluorescence localization of PSA is given below. 

A variety of techniques (Fig. 1), which use a variable combination of antibody steps, enzyme labels, and chromogens, are employed in immunohistochemistry. The most common immunohistochemical methods are the PAP (peroxidase-antiperoxidase), ABC (avidin-biotin) peroxidase or alkaline phosphatase, and the APAAP (alkaline phosphatase-anti-alkaline phosophatase) techniques however,... 

Noncovalent binding of HRP to antibody, also known as unlabeled antibody binding, is described in great detail by Sternberger (1). Instead of the use of bifunctional reagents, IgG class antibodies to HRP are used to form a soluble semicyclic immune complex consisting of two antibody and three enzyme molecules. The molecular weight of the peroxidase antiperoxidase, PAP complex is 400 - 430 kDa. 

Antibodies have been raised to HRP and used to construct peroxidase-antiperoxidase layers in assays to increase signals and thus sensitivities. This is particularly useful where high background signals are expeaed, because the signal-to-noise ratio can be increased many-fold (27). 

Depending on the crossreactivity of the antibody used, the sensitivity of the immuno-slot-blot assay may be increased by using sandwich-techniques for signal amplification, as used in immunohistochemistry, e.g., alkaline phosphatase-antialkaline phosphatase or peroxidase-antiperoxidase (seethisvol., Chapter 10). 

Fig. 12. Diagram showing sequence for detecting viral antibody by the peroxidase-antiperoxidase (PAP) method of Stemberger et al. This method uses a bridging antibody (anti-primate antibody) between the human and simian antibody. Artificial (chemical) linkage of enzyme with antibody is replaced by an antigen-antibody reaction.

Sequential localization of two antigens in the same specimen by peroxidase-antiperoxidase (PAP) complexes without intermittent antibody removal... 

Sternberger LA, Sternberger NH (1986) The unlabeled antibody method comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification. J Histochem Cytochem 34 599-605... 

The ABC detection system has been shown to be more sensitive than most other detection system (5, 6), primarily because of the large size of the preformed ABC complexes, which result in amplification of the signals. Alternative detection systems for immunohis-tochemical analysis include the peroxidase-antiperoxidase (PAP) (1) and the alkaline phosphatase-antialkaline phosphatase(APAAP) systems (7) (see Chapter 24). More recently, newer commercially available polymer-based detection systems have been developed that combine the secondary antibody and detection enzyme into a single reagent. Polymer-based detection systems overcome problems that may occur with biotin-link based systems, have excellent sensitivity, and can be applied to frozen section immunohistochemistry. Polymer-based detection systems are discussed in other chapters of the manual. (XREF). 

Petrali P., Hinton M., Monarty C., and Sternberger A (1974) The unlabeled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxi-dase-antiperoxidase complex. /. Htstochem Cytochem 11, 782-801 Pickel V M, Joh T H, Field P M, Becker C G, and Reis D. J (1975a) Cellular localization of tyrosine hydroxylase by immunohisto-chemistry / Histochem Cytochem 13, 1-12 Pickel V, M., Joh T H, and Reis D J (1975b) Ultrastructural localization of tyrosine hydroxylase in noradrenergic neurons of brain Proc Natl. Acad. Set. USA 71, 659-663... 

Fig. 3. Immunoblot of poly(ADP-ribose) synthetase recognized by monoclonal antibodies. Purified poly(ADP-ribose) synAetase and human placenta extract were resolved by SDS-polyacrylamide gel electrophoresis on a 7.5% acrylamide gel, and transferred to nitrocellulose membranes. A, gel stained with Ooomassie blue B-D, strips incubated with monoclonal antibodies G6, D4, and X3, respectively. Bound antibodies were detected by the peroxidase-antiperoxidase technique as describe (4).

Fig. 2. Increased class two antigen expression on endothelia 3 days after diabetes induction. Black spots mark small vessels in the periphery and especially the ductular region of an islet arrow. Staining with monoclonal antibody peroxidase-antiperoxidase magnification x 450...

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