Alkaline phosphatase assay techniques

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,... 

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. 

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. 

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]. 

The sandwich technique can be improved even further if the second antibody is attached to an enzyme, such as alkaline phosphatase. The enzyme rapidly converts an added colorless substrate into a colored product, or a nonfluorescent substrate into a highly fluorescent product. These changes can be quantitated if the degree of change in color or fluorescence is proportional to the amount of hormone present in the patient sample. Less than a nanogram (10 g) of a protein can be measured by such an enzyme-linked immunosorbent assay (ELISA). 

In chemiluminescent-based assay systems, Mark et al. (2008) found that non-covalent immobilization of alkaline phosphatase (ALP) on the electrospun nylon 6 nanofiber membrane, nsing a mnltistacked LbL technique with the cationic polymer Sapphire 11, resulted in the highest enzyme loading when compared with other covalent immobilization methods based on glutaraldehyde cross-linking. 

Ultrasensitive enzymic radioimmunoassay (USERIA). The combination of RIA and ELISA has on occasion been used to create an assay for a given Ag (e. g. X) that is more sensitive by up to three orders of magnitude than either of these techniques used by themselves. For this assay three different Ab are required (i) an Ab to X (i.e. anti-X,), which is bonded to a solid support, (ii)a second Ab to X which binds to a different site on the X molecule (i.e. anti-Xj), and (iii) an alkaline phosphatase-labeled Ab that binds to the Fc region of anti-X2 (i.e. anti-[anti-X2]-alkaline phosphatase), along with pH]adenosine-S -monophosphate, a substrate of alka-... 

Various enzyme conjugates have been used in blotting assays (e.g., alkaline phosphatase, glucose oxidase). Correspondingly, there are numerous substrates that can be used. It is advisable to consult a textbook on histochemical techniques to identify substrates and protocols that give rise to insoluble products. Interestingly, one should note the recent introduction of chemiluminescent substrates for HRP (ECL, Amer-sham) which afford greater sensitivity and are amenable to quantification. 

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