Alkaline phosphatase-antialkaline

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. 

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. 

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). 

In addition to peroxidase-mediated immunologic assays, there are also alkaline phosphatase-antialkaline phosphatase (APAAP) reagents available (11). The general overall principle governing these immunologic assays remains the same. 

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. 

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