APAAP phosphatase

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. 

In addition to peroxidase-mediated immunologic assays, there are also alkaline phosphatase-anti alkaline phosphatase (APAAP) reagents available (12). The general overall principle governing these immunologic assays remains the same. 

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 immunohistochemical analysis include the peroxidase-antiperoxidase (PAP) (1) and the alkaline phosphatase-antialkaline phosphatase (APAAP) systems (7) (see Chapter 24). These approaches are conceptually and technically similar, and will not be discussed here. 

Cordell, J. L., Falini, B., Erber, W. N., Ghosh, A. K., Abdulaziz, Z., MacDonald, S., Pulford, K. A. F., Stein, H., and Mason, D. Y. (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline-phosphatase (APAAP complexes). J. Histochem. Cytochem. 32, 219-229. 

Alternatively alkaline phosphatase-coupled secondary antibody can be used with an anti-alkaline phosphatase antibody (APAAP technique). For this method the following protocol may be used ... 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

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,... 

AP had not been used extensively in immunohistochemistry until publication of the unlabeled alkaline phosphatase-antialkaline phosphatase (APAAP) procedure (2, 3). The soluble immune complexes utilized in this procedure have molecular weights of approximately 560 kDa. The major advantage of the APAAP procedure compared to the earlier peroxidase techniques was the lack of interference posed by endogenous peroxidase activity. Because of the potential distraction of endogenous peroxidase activity, the alkaline phosphatase techniques were particularly recommended for use on blood and bone marrow smears. Endogenous alkaline phosphatase activity from bone, kidney, liver and some white cells can be inhibited by the addition of 1 mM levamisole to the substrate solution (4), although 5 mM has been found to be more effective (5). Intestinal alkaline phosphatases are not adequately inhibited by levamisole. 

Nakane, P. R. and Pierce, G. B., Jr. (1966) Enzyme-labelled antibodies. Preparation and applicauon for the localization of antigens./. Hislochem. Cytochem. 14, 929-931. StembCTger, L. A. (1979) Immunocytochemistry, 2nd Ed., John Wley, New York. Mason, D. Y. (1985) Immunocytochemical labelling of monoclonal anubodies by the APAAP immunoalkaline phosphatase technique, in Techntquestnimmunocylochermstry, Vol. 3 (Bullock, G. R. and Petrusz, P., eds.), Academic, London, pp. 25-42. 

The principles of the alkaline phosphatase-antialkaline phosphatase (APAAP) technique are the same as those described for the PAP method (Fig. 1.10), except that the PAP complex is replaced with an APAAP complex. The method has had three major applications (1) staining of tissues with high levels of endogenous peroxidase,... 

PAP, peroxidase-antiperoxidase ABC, avidin-biotin conjugate APAAP, alkaline phosphatase antialkaline phosphatase B-SA, biotin-streptavidin PCNA, proiiferating cell nuclear antigen EPOS, enhanced polymer one-step staining (Dako) CARD, catalyzed reporter deposition HRP, horseradish peroxidase. 

Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic label of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) complexes. J Histochem Cytochem. 1984 32 219. 

APAAP alkaline phosphatase anti-alkaline phosphatase... 

This reporter is obviously encountered in the APAAP technique, but it can also be conjugated to biotin and used successfully in the ABC methods. The enzyme is sourced from calf intestine, this form being resistant to the blocking effect of levamisole, which is added to the substrate buffer and blocks all other types of endogenous alkaline phosphatase activity. Several chromogens can be used, the most popular being Fast Red TR salt which yields a bright red alcohol soluble reaction product (34). 

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