Enzymatic assay, generally methods

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... 

One of the most important requirements for successful protein purification is the availability of an accurate, rapid, and quantitative assay that is specific for the protein of interest. If the protein has no known enzymatic activity, the amount of the protein present after each purification step must be analyzed by some general method such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, or an enzyme-linked immunosorbent assay (ELISA). If... 

Enzymatic assay methods are classified as fixed-time assays, fixed-change assays, or kinetic (initial rate) assays. Kinetic assays continuously monitor concentration as a function of time pseudo-first-order conditions generally apply up to 10% completion of the reaction to allow the initial reaction rate to be determined. If the initial substrate concentration is > 10Km, then the initial rate is directly proportional to enzyme concentration. At low initial substrate concentrations (< 0.1 Km), the initial rate will be directly proportional to initial substrate concentration (cf. Chapter 2). For enzyme quantitation, a plot of initial rate against [E] provides a linear... 

Flow-based analytical procedures are already used in many fields, e.g., environment, food and health. In tandem with the developments described above, flow analysers will also be used in new and emerging fields of application such as the "omics" (metallomics, genomics and proteomics), biotechnology (enzymatic assays, platforms for bio-sensors and flow immunoassays) and quality-control applications in general. Economic resources will also be a driver for more multi-parametric flow-based methods, particularly with spectrophotometric detection. 

In general enzymatic assays have a lower sensitivity in comparison with other methods such as RIA or EIA (requiring time-consuming cycling reactions, in order to obtain a higher sensitivity). Yet the enzymatic methods have been widely applied in clinical laboratories, since they provide good assay speed combined with low cost, and simple instruments and procedures. However these methods cannot be applied for the evaluation of BA malabsorption syndrome, in which a reduction of BA has been observed. 

In order to avoid the described problems, antibody-based assays have been developed. As mentioned above, usually a primary antibody recognizes the modification, which in turn is then quantitated. One of these approaches is based on the technology of AlphaScreen (Amplified luminescent Proximity Homogeneous Assay) [51, 52], also known as luminescent oxygen channeling immunoassay (LOCI) [53], a method that can be used to study protein-protein interactions in general. In this case the enzymatic transfer of acetyl groups to a histone peptide is determined. 

As mentioned above, the central core component of a genotyping technology is contained within the assay biochemistry. In this regard, a general distinction may be made between those methods that rely primarily upon differential hybridization stringency for their specificity, and those that derive specificity primarily from the ability to detect a product of an enzymatic reaction. A number of... 

The HPLC assay method is particularly useful when it is necessary to obtain initial rate data for a study of an enzymatic activity. Optimal assay conditions for the HPLC must be established first. Usually, the optimization process involves the determination of several variables, such as the optimal substrate concentration, pH, temperature, and enzyme concentration. It is assumed that the reader is familiar with the problems associated with assay conditions such as pH, buffer, and temperature. This chapter discusses only factors that might present problems for the HPLC assay method. For additional information, see the works cited in the General References. 

Compared to enzymatic methods, immunochemical assay methods for SODs are more reliable and reproducible, because the determinations are specific to the protein moiety and immunogenicity will not change in the presence of inhibitors or activators of SOD in the tissues (G3,I2). In some cases, both enzymatic and immunochemical assays are requisite for the assessment of tissue SOD levels. In the serum, however, the activity assay is generally too insensitive to detect SOD, and immunochemical assay is much more reliable and convenient. 

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