Alkaline phosphatase assay principles

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. 

Use of apoenzymes for the detection of metal ions Generally, apoenzymes of metalloenzymes can be used for the detection of the corresponding metal ion. Restoration of enzyme activity obtained in the presence of the metal ion can be correlated to its concentration. This principle has been demonstrated in the detection of copper while evaluating reconstituted catalytic activities in galactose oxidase and ascorbate oxidase and also in the detection of zinc since this ion is essential for the activity of carbonic anhydrase and alkaline phosphatase [416]. The need of stripping the metal for the preparation of the apoenz5une may demand tedious procedures and a catalytic assay with the addition of the substrate is always required for detection. 

Adherence to sound principles of the assay of serum enzymes in general (F5-F7) is required for both manual and automatic techniques. Thus, one should insure that the release of product that is being measured is a linear function of the amount of alkaline phosphatase. The reaction must be carried out at the optimal pH characteristic of the enzyme with that particular buffer and substrate. The period of hydrolysis and substrate concentration should be adjusted in such a way that an initial zero-order rate of hydrolysis is obtained. The standard curve should obey Lambert-Beer s law. Arbitrary and variable dilutions of high-titer sera are not recommended. 

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