TOPICAL assay methods

This text is devoted solely to peptidase enzymes, although the information can be extrapolated to othersystems. The text covers a wide range of topics, including assay methods, mechanisms of action, inhibition, and technological applications of peptidases, in a relatively straightforward manner. 

The assay methods for these enzymes have not received the critical attention they deserve and there is in the literature a vast number of papers (close to 2000 on topics related to catalase and almost 1000 on peroxidase) in which the results are obscured by faulty and erroneous assay methods, or by a lack of adequate data on the mechanism of enzyme action. It is the purpose of this paper to appraise various assay methods critically in the light of the detailed knowledge of the chemistry and mechanism of action of the hydroperoxidases now available. 

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

The discussion above was concerned with the effects of solution conditions on enzyme activity, hence reaction velocity. Equally important for the purpose of assay design is the influence of specific solution conditions on the detection method being used. This latter topic is beyond the scope of the present text. Nevertheless, this is an important issue for screening scientists whose job is often to balance the needs of biochemical rigor and assay practicality in development of an HTS assay. An... 

Many methods are available for determining food antioxidant capacity, which is an important topic in food and nutrition research. However, there is a great need to standardize these methods because the frequent lack of an actual substrate in the procedure, the system composition, and the method of inducing oxidation could limit their accuracy. In fact, antioxidant activities in complex systems cannot be evaluated satisfactorily using a single test, and several test procedures may be required. The search for more specific assays that can be more directly related to oxidative deterioration of foods and biological systems should be the objective of future investigations. 

What follows is not an exhaustive or up-to-the-minute survey of the methods available for protein quantitation, but a practical guide to selecting the appropriate assay for each stage of drug development. A case study further illustrates the application of the standard protein methods to the drug development process. The reader is referred to reviews on the topic for further details.12... 

The great advantage of chromatography for lipophilicity determination is its ability to estimate log P or log D with a relative accuracy for non-pure samples, with a limited quantity of compound. Furthermore, these methods are relatively easily to automate and they only require an HPLC or UPLC apparatus. Many approaches have been described and this topic was recently reviewed [5, 27]. That is why the focus of the following section is on strategies developed to increase the throughput and the assay dynamic range. 

He was employed at the Mint for forty years, first as assayer, then as verifier, and finally as Director of Assays. His residence was at the Mint also, and it was there that he died in 1890. According to Tis-sandier, his life, always calm and methodical, was entirely consecrated to the science that he loved with passion and to his family that he cherished no less (34). He must have been a man of broad interests, for he published papers on such varied topics as water analysis, the... 

As a number of excellent articles have been published that review various aspects of the biological roles of L-ascorbic acid,8 the biosynthesis of L-ascorbic acid in plants and animals,7-11 and radicals derived from L-ascorbic acid,12 these topics will not be treated. Likewise, the methods by which L-ascorbic acid is assayed13 and the uses of L-ascorbic acid and related molecules in a wide variety of assays and oxidation-reduction systems will not be discussed. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Aerosols used for inhalation therapy are generally packaged in containers with metered values. The standard procedure is to discharge the entire contents of the container for assay. For betamethasone dipropionate and betamethasone valerate topical aerosols, the contents are discharged into a volumetric flask and the propellants carefully boiled off. Precautions should be taken, as many of these propellants are flammable. The residue is diluted to volume with isopropanol-acetic acid (1000 1) and filtered [50]. Another approach is to discharge the contents into ethanol or dilute acid. An alternative is to immerse the canister in liquid nitrogen for 20 min, open the canister, evaporate the liquid contents, and dissolve the residue in dichloromethane. A unit spray sampling apparatus for pressurized metered inhalers has been described [51]. The components in an aerosol product that can be the cause of assay variance have been studied [52]. A method to quantify the volatile components of aerosol products has been developed [53]. 

The choice of the assay to be performed depends on the context in which it will be used, the origin of the target cells, and the nature of the tested compounds. Among different assays, the parameters that vary are cell culture method, exposure time and concentrations, recovery time after exposure, and the method used to quantify the pharmaceutical effect. This topic is briefly covered in this chapter and is discussed in detail by Freshney (2005). 

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