Assay techniques

Sample preparation for the modified Fischer assay technique, a standard method to determine the Hquid yields from pyrolysis of oil shale, is necessary to achieve reproducible results. A 100-g sample of >230 fim (65 mesh) of oil shale is heated in a Fischer assay retort through a prescribed temperature range, eg, ca 25.5—500°C, for 50 min and then soaked for 20 min. The organic Hquid which is collected is the Fischer assay yield (7). The Fischer assay is not an absolute method, but a quaHtative assessment of the oil that may be produced from a given sample of oil shale (8). Retorting yields of greater than 100% of Fischer assay are possible. 

N. P. Chilcott, D. A. Phillips, M. G. Sanders, I. R. Collins, and A. Gyani. The development and application of an accurate assay technique for sulphonated polyacrylate co-polymer oilfield scale inhibitors. 2nd Annu SPE Oilfield Scale Int Symp (Aberdeen, Scotland, 1/26-1/27), 2000. 

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

The fluorescent labelling of heparin with F-D by this technique did not observably alter the biologic activity of the heparin as regards to its binding to antithrombin and catalysis of antithrombin s neutralization of activated coagulation factors. F-D labelled heparins also bound to other known heparin-binding proteins in a saturable and reversible manner, as demonstrated by the dot-blot assay technique (Figure 6). 

Saxberg BEH, Duewer DL, Booker JL, Kowalski BR (1978) Pattern recognition and blind assay techniques applied to forensic separation of whiskey. Anal Chim Acta 103 201... 

The plaque procedure also permits the isolation of pure virus strains, since if a plaque has arisen from one virus particle, all the virus particles in this plaque are probably genetically identical. Some of the particles from this plaque can be picked and inoculated into a fresh bacterial culture to establish a pure virus line. The development of the plaque assay technique was as important for the advance of virology as was Koch s development of solid media for bacteriology. 

Determine the amount of adsorbed protein on the particles by using a suitable protein assay technique, such as the bifunctional chelating agents (BCA) Protein Assay (Thermo Fisher). 

The second label also may be a fluorescent compound, but doesn t necessarily have to be. As long as the second label can absorb the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce fluorescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe, N-(4-aminobutyl)-N-ethylisoluminol, and reactive fluorescent derivatives of fluorescein, rhodamine, and the cyanine dyes (Chapter 9). For a review of these techniques, see Morrison (1992). 

Herzberg, M. (1984) Molecular genetic probe, assay technique, and a kit using this molecular genetic probe. European Patent Application, 0128018. 

Toxicity. There is only one Member State (Germany) having a test which is used to assess toxic hazards of combustion gases. The test is used mainly to evaluate non-combustible materials and is based on bio-assay techniques. The philosophies of other countries consider non-combustible materials as presenting no, or negligible toxic hazard. 

Support Function Protective Clothing for Hazardous Chemicals Operations - NFPA 1993. Quincy, MA. Technical Bulletin - Assay Techniques for Detection of Exposure To Sulfur Mustard, Cholinesterase Inhibitors, Terrorism Incident Annex to the Federal Response Plan. Washington, D.C. 1995. 

Among various methods reported, we wish to describe an assay technique with Saccharomyces carlsbergensis 4228. This yeast had been used by Atkin et al. (All) for the determination of vitamin B6. We have omitted inositol from the medium and have added an excess of pyridoxine (S19). The composition of the medium is given in Table 8. The method has an accuracy of 0.1 mpg/ml. 

The establishment of quantitative methods for the determination of vitamins in body fluids and tissues by microbiological assay techniques should stimulate the search for the significance of vitamins in disease, not only in nutritional deficiency, but in the much wider field of all metabolic disturbances. Functional vitamin deficiencies are produced by malabsorption, by inhibitors of the vitamin function through products of the body, and particularly through drugs and other toxic substances. Vitamin deficiencies may be relative deficiencies whenever an individual s metabolism is deranged so as to require enhanced quantities of a given vitamin to cure or to counteract certain symptoms as, e.g., in Darier s disease (keratosis follicularis) (P2a). 

Capillary electrochromatography-mass spectrometry (CE-MS), 4 641 Capillary electrodes, 14 27 Capillary electrophoresis (CE), 4 602-603, 631-633 6 385 9 751-752 antibody based columns with, 6 402 chiral additives, 6 77-79 applications, 4 641 basic principles, 4 606-609 detectors, 4 634-635 for DNA analysis, 4 636-637 flow profiles generated, 4 608 instrumentation, 4 633 as microfluidic assay technique,... 

Various techniques have been used for the determination of organic (and total) carbon in sediments. These include both wet and dry combustion methods which depend on the quantitative conversion of the organic (or total) carbon to carbon dioxide [30-32], In addition, an approximate assay technique reported by Bremner and Jenkinson has been used [36]. 

Cyclodiene pesticides, of which endrin and its oxidized analogs are representative, can also be estimated by receptor-assay technique. Cyclodiene pesticides exert their mode of action by altering central nervous system membrane ion transport. In work reported by Saleh et al. (1993), a labeled amino acid, GABA, that binds to the chloride channel receptor is displaced by endrin (and other similar molecules), and thus serves as an assay for these pesticides. The GABA receptor was shown to be a potentially useful biomarker for organochlorine pesticides such as lindane, toxaphene, endrin, chlordane, and others. The assay involves small quantities of blood (0.1 mL), and requires only that the plasma be separated from the... 

Enzymatic assay techniques have been developed for several additives by Merck. BIOQUANT kits are available for aspartame (intense sweetener) and nitrate (preservative). Gromes et al. (1995) applied the Bioquant kit to determination of aspartame in yoghurt, quark and confectionery. For low concentrations of aspartame a blank correction procedure was necessary. Recoveries of aspartame were in the range 93-102%. 

A new hydroxynitrile lyase (HNL) was isolated from the seed of Japanese apricot Prunus mume). It accepts benzaldehyde and a large number of unnatural substrates for the addition of HCN to produce the corresponding (7 )-cyanohydrins in excellent optical and chemical yields. A new high-performance liquid chromatography (HPLC)-based enantioselective assay technique was developed for the enzyme, which promotes the addition of KCN to benzaldehyde in a buffered solution (pH 4.0). Asymmetric synthesis of (7 )-cyanohydrins by a new HNL is described (Figure 8.4). ... 

Several things may be done if the researcher has difficulty in detecting an enzyme activity of interest in a homogenate, or elsewhere. A more sensitive assay technique may be used, if one is available. The concentration of enzyme may be increased, as the rate of product formation is directly proportional to [E]. The incubation time for enzyme with substrate can be increased, although the caveats discussed in O Section 3.3.2 must be borne in mind. The reaction volume may also be increased, while maintaining concentrations of reactants constant this approach is particularly useful if product is separated and detected by chromatography, or if a column is used to separate radiolabeled substrate from product, because the increased amount of product formed in unit time will result in enhanced signal size. 

It has already been stated that a suitable quantitative assay technique must be available to measure the reaction of interest and it is assumed that the experimenter has determined optimal reaction conditions for the enzyme of interest. All kinetic assay techniques assume that v is a variable and that [S] is known as such, preparation of substrate must be meticulous in terms of ensuring that concentrations are correct, and this in turn will rely upon factors such as good weighing and pipetting techniques with calibrated instruments capable of precise, accurate, and sufficiently sensitive measurement. 

The wet assay technique to measure dust in cotton was a modification of the method described by Thibodeaux (11). A 400-mg tuft of cotton, randomly selected from a bulk sample, was subjected to multiple ultrasonic washings in methanol. Clean methanol (200 ml) was used for each of three 5-min washings. The combined methanol washings were filtered through a 17 ym sizing screen (the screen was identical for both wet and dry assay procedures) and collected on a 0.5 ym filter. Increase in filter weight provided the measure of dust content (%) in cotton by wet assay. 

Enzyme activity measured, in vitro, in extracts of the muscle (for details of assay technique, see Chapter 3). 

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