Endogenous Alkaline Phosphatase

Alkaline phosphatase is an enzyme represented by various isoforms in many tissues such as liver, bone, intestine, placenta, some tumors and in leukocytes. Addition of 1 mM levamisole to the chromogen/substrate will inhibit endogenous alkaline phosphatase activity, with the exception of the intestinal isoform. If necessary, this can be blocked with a weak acid wash, such as 0.03 0.5 N HC1 or 1 M citric acid. 

When using high-temperature antigen retrieval, you may skip blocking alkaline phosphatase, since some endogenous enzymes, such as alkaline and acid phosphatases, in contrast to peroxidase, are destroyed by boiling for even a short time at 100°C (Cattoretti et al. 1993). 

Phosphatases are numerous and important enzymes (see also Chapt. 2). They are classified as phosphoric monoester hydrolases (phosphatases, EC 3.1.3), phosphoric diester hydrolases (phosphodiesterases, EC 3.1.4), triphosphoric monoester hydrolases (EC 3.1.5), diphosphoric monoester hydrolases (pyrophosphatases, EC 3.1.7), and phosphoric triester hydrolases (EC 3.1.8) [21] [63]. Most of these enzymes have a narrow substrate specificity restricted to endogenous compounds. However, some of these enzymes are active toward xenobiotic organophosphorus compounds, e.g., alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), aryldialkylphosphatase (para-oxonase (PON1), EC 3.1.8.1) and diisopropyl-fluorophosphatase (tabunase, somanase, EC 3.1.8.2) [64 - 70]. However, such a classification is far from definitive and will evolve with further biochemical findings. Thus, a good correlation has been found in human blood samples between somanase and sarinase activities on the one hand, and paraoxonase (PON1) type Q isozyme concentrations on the other [71]. 

Variations on the ABC technique can also be used to incorporate different enzymes that result in different chromogenic products. Alkaline phosphatase ABC is one example. The difficulty with this system is the consumption of endogenous alkaline phosphatase, which is more prevalent than peroxidase and is harder to remove. However, the alkaline phosphatase enzyme does provide more product per unit than does peroxidase and is therefore a slightly more sensitive means of detection. Endogenous alkaline phosphatase can be blocked by an incubation in 3 mMlevamisole for 15 min, but some enzyme may escape consumption. Alkaline phosphatase has many substrates, too, the most popular being BCIP/NBT, which precipitates to a dark blue (see Chapter 25). 

Destroy endogenous alkaline phosphatase by treating the slides with either 0.02 N HCl at room temperature for 8 min or 20% acetic acid for 20-30 s at 4°C for 20 s. 

When unfixed cells or frozen sections are used for ELISA, it may be necessary to block endogenous alkaline phosphatase activity, and in this case, include 0.1 mil/ levamisole in the substrate solution. 

Place the sections in the acetic acid solution for 15 min to block endogenous alkaline phosphatase activity (see Chapter 24). 

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. 

Table 3. Common endogenous enzyme blocking reagents for horseradish peroxidase and alkaline phosphatase systems.

Dual endogenous enzyme block Horseradish peroxidase and alkaline phosphatase labels... 

Figure 2. Example of endogenous alkaline phosphatase in ileum stained with Permanent Red.

Specimens rich in endogenous peroxidase activity may be processed using an alkaline phosphatase detection method instead of a peroxidase method, eliminating the background. 

Figure 1. Red blood cells showing endogenous peroxidase activity (a) before, and after blocking with three percent hydrogen peroxide, (b) Alkaline phosphatase-based detection methods.

Figure 2. Placenta showing endogenous alkaline phosphatase activity (a) before, and (b) after blocking with levamisole.

Unquenched endogenous alkaline phosphatase activity may be seen in leucocytes, kidney, liver, bone, ovary bladder, salivary glands, placenta and gastro-intestinal tissue. Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme label such as horseradish peroxidase. Intestinal alkaline phosphatase is not quenched by the addition of levamisole. Pretreat the tissue with 0.03 N HCI. 115-121... 

Indicates endogenous alkaline phosphatase activity in the tissue sections. It is present in liver, kidney, Gl tract, bone, bladder, ovary, salivary gland, placenta, leukemic, necrotic or degenerated cells. 

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