Phosphatase assay technique

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

In the application of alkaline-phosphatase-sensitive, triggerable 1,2-dioxetanes, the nucleic-acid hybridization assay is nowadays quite popular . Such techniques include viral load assays for hepatitis B and C and for human immunodeficiency viruses (HBV,... 

The whole question of the specificity was reopened with the discovery that E. coli phosphatase, contrary to an earlier statement (114), hydrolyzed a variety of polyphosphates including metaphosphate of average chain length 8 (97). It was subsequently reported that partially purified phosphatases from several mammalian tissues had appreciable PPi-ase activity at pH 8.5 (115). This was confirmed (116) and extended to include ATPase and fluorophosphatase activities (117). Proof that the same enzyme is responsible for the monoesterase and PPi-ase activities was afforded by heat inactivation studies, cross inhibition experiments, and inhibition of PPi-ase activity by L-phenylalanine, a specific inhibitor of intestinal phosphatase. It was also found that calf intestinal phosphatase couid be phosphorylated by 32P-PP and the number of sites so labeled agreed with the number of active sites determined with a monoester substrate using a stopped-flow technique (118). It would seem that the main reason for the confusion with regard to the PPi-ase activity results from the inclusion of Mg2+ in the assay. This stimulates the monoesterase activity but almost completely inhibits PPi-ase activity (117). 

Protein phosphatase inhibition is usually detected by colorimetric methods, but the development of a biosensor requires the search of other transduction techniques. Electrochemistry has been widely used in biosensors because of the simplicity, easy to use, portability, disposability and cost-effectiveness of the devices. As protein phosphatase is not an oxidoreductase enzyme, our work has been devoted to the investigation of novel enzymatic substrates, electrochemically active only after their dephosphorylation by the protein phosphatase. Nevertheless, colorimetric assays have been used for the optimisation of several experimental parameters. 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

In this technique, an aliquot of the sample under investigation is first chromatographed. A second aliquot of sample is incubated with the enzyme under the appropriate conditions of pH and temperature. After a suitable time period, the incubated mixture is chromatographed. The disappearance of the substrate peak and/or appearance of product peaks confirms the identity and purity of the chromatographic peak. In cases where specific enzymes may not be available for a certain substrate, less specific enzymes, such as phosphatases, can be used for the identification of classes of compounds. In addition, it is possible to use coupled enzyme assays to drive a reaction to completion and thus characterize a peak in the chromatogram. 

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). 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

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. 

Diversity of Detection Techniques and Assay Formats Available for Phosphatase Screening... 

Interestingly, all these compounds are inactive against mouse lAPs, therefore, unsuitable for studies of lAP function in mouse models. The objectives of the current studies were the identification of small-molecule modulators of mouse lAP and, potentially, identification of novel selective scaffolds of human lAP. To this end, we applied and optimized the chemiluminescent assays that were successfully utilized for screening human lAP, TNAP, and FLAP and developed a novel assay for mouse lAP, analogous to the assays of other isozymes. These assays utilize CDP-star, a substrate of alkaline phosphatases specifically invented for and commonly utilized in blotting techniques [19, 20]. Development and utilization of the prototype plate-reader enzymatic assay for TNAP isozyme with CDP-star substrate was previously described in detail elsewhere [21]. 

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