Direct Assays

Finally, receptor stimulus can be measured through membrane assays directly monitoring G-protein activation (group IV assays). In these assays, radiolabeled GTP (in a stable form for example, GTPj/S) is present in the medium. As receptor activation takes place, the GDP previously bound to the inactive state of the G-protein is released and the radiolabeled GTP/S binds to the G-protein. This is quantified to yield a measure of the rate of GDP /GTP j/S exchange and hence receptor stimulus. 

Some biomarkers only provide a measure of exposure others also provide a measure of toxic effect. Biomarkers of the latter kind are of particular interest and importance and will be referred to as mechanistic biomarkers in the present text. Some mechanistic biomarker assays directly measure effects at the site of action as described in Section 2.4 (see Chapter 4, Table 4.2, for examples). Inhibition of acetylcholinesterase is one example. Others measure secondary effects on the operation of nerves or the endocrine system (examples given in Table 4.2 and Chapters 15 and 16). 

BENZIE i FFand STRAIN J J (1999) Ferric reducing/antioxidant power assay Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration, Meth Enzymol, 299, 15-27. 

Neomycin phosphotransferase II (NPTII) extraction from cotton leaves and cottonseed. The extraction buffer consists of 100 mM Tris, lOmM sodium borate, 5mM magnesium chloride, 0.2% ascorbate and 0.05% Tween 20 at pH 7.8. The frozen leaf sample is homogenized in cold (4 °C) buffer. An aliquot of the homogenate is transferred to a microfuge tube and centrifuged at 12 000 g for 15 min. The supernatant is diluted and assayed directly by ELISA. 

The study of active transport mechanisms has grown substantially in recent years, with transport proteins such as P-gp, BCRP, and MRP-2 among the most studied [59]. Several types of in vitro assays to assess substrates of transporters have been established these include assays directed toward intestinal and biliary efflux [60]. Assays that measure passive and active transport are also used to assess penetration of the blood-brain barrier. In addition to the assays described above, transfected cell lines that overexpress transporters present in the blood-brain barrier are also employed [61]. 

Nasmyth Have you tried adding Rsk to the APC ubiquitin ligase assays directly ... 

As pointed out previously, controlled degradation reactions are very difficult with aliphatic or alicyclic hydrocarbons, and most of the relabeling work has been concentrated on aromatic reaction products. Procedures have been extensively described by Pines and co-workers (e.g., 97, 96, also 87, 89-98, 95, 98). For the present purpose, it suffices to note that the 14C contents of the methyl side-chains and the rings in aromatic reaction products are readily estimated by oxidation of the methyl to carboxyl, followed by decarboxylation, while ethyl side-chains may be oxidatively degraded one carbon atom at a time. Radiochemical assays may be made on CO2 either directly in a gas counter, or after conversion to barium carbonate, while other solid degradation intermediates (e.g., benzoic acid or the phthalic acids) may be either assayed directly as solids or burned to CO2. Liquids are best assayed after burning to CO2. 

For the assay of uric acid, a sensor based on KMn04-octylphenyl polyglycol ether is proposed [88], Uric acid can be assayed directly in urine in the 0.10— 600- ag/mL concentration range with a detection limit of 55 ng/mL. The system is free of interferences. 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

There are five categories of protein assay colorimetric assays, direct absorbance methods, fluorescence methods, amino acid analysis, and custom quantitation methods. A brief summary of the principles, advantages, and limitations of these methods follows. 

Phenotypic resistance assays directly measure the ability of HlV-1 to replicate in a cell culture in the presence of different antiretroviral drug concentrations. This process is similar to that used to determine antibiotic resistance and is, therefore, more familiar to most clinicians. The recombinant virus, composed of a virus s reverse transcripfase and protease genes, is inserted into a standard reference strain of virus. The recombinant virus is then tested in vitro for fhe amount of drug needed to inhibit virus replication by 50%, relative to the amount of drug needed to inhibit a reference strain of virus. Phenotypic resistance testing is limited by the fact that it is conducted in vitro and not in vivo. 

Hydrogenase was assayed directly in aliquots from the cultures at an OD q °f I -5-... 

Houtman, C. J., Booij, R, Jover, E., Pascual del Rio, D., Swart, K., Velzen, M. van, Vreuls, R., Legler, J., Brouwer, A. and Lamoree, M.H. (2006). Estrogenic and dioxin-like compounds in sediment fi om Zierikzee harbour identified with CALUX assay-directed fractionation combined with one and two dimensional chromatography analyses. Chemosphere, 65, 2244-2252. 

At the time of the discovery of chlorpromazine. dopamine could not be assayed directly and its role as a neurotransmitter was unknown (Carlsson, 1987). Chlorpromazine exerts a large number of pharmacological effects, all of which could, in principle, be considered responsible for its clinical action. Thus, it was first assumed that the antipsychotic action of chlorpromazine and similar... 

Nitric oxide can be assayed directly in tissues by its electrochemical oxidation on electrode surfaces (Shibuki, 1990). The technique was successfully used by Shibuki in cerebellar slices. However, the probe was fabricated in a glass micropipet coated with a thin hydrophobic chloroneoprene membrane, which makes the technique experimentally difficult. More recently, a carbon fiber electrode... 

Sulfadiazine Swine bile and Direct assay Direct competitive enzyme-linked 32-36 67... 

Protocols for preparing six environmental sample types prior to the Ames Salmonella assay were proposed at a recent panel discussion sponsored by the U.S. Environmental Protection Agency (USEPA) and the U.S. Army. Air particles, soil-sediment, and solid waste are extracted with dichloromethane, concentrated, and solvent exchanged into dimethyl sulfoxide (DMSO). Organics in water and waste water are absorbed onto XAD columns, then eluted with hexane-acetone, solvent reduced, and exchanged into DM SO. Nonaqueous liquids are assayed directly and as concentrates before they are solvent exchanged to DMSO. If bacterial toxicity or lack of dose response is observed in the Ames assay of extracts, the extracts are fractionated prior to solvent exchange. These are interim methods and have not been subjected to policy review of the USEPA or the U.S. Army. 

Cemm will be determined by performing the described cholesterol assay directly on serum. CHDL will be determined on a separated, soluble HDL fraction of serum. Very low-density lipoproteins and low-density lipoproteins are selectively removed from serum by precipitation with magnesium-phos-photungstate reagent. 

Disulfiram [bis(diethylthiocarbamoyl)disulfide] used in the treatment of alcoholism can be assayed directly by pulse polarography in an aliquot of a solution of a ground tablet dissolved in ethanol-acetate buffer (pH 4.5) [133]. A mechanism for the electrode process was proposed involving the reaction of the disulfiram with the mercury drop to form an insoluble mercuric salt, which then underwent reduction at the electrode surface. 

This method combines the advantages of liquid chromatography with the selective and sensitive receptor assay Charm H-Q test. Milk sample previously precipitated (with Mcllvaine buffer) and preconcentrated on a C8 cartridge was fractionated using an LC system. The fractions were collected according to the retention times and peak widths, and they were assayed directly with Charm II-Q test with small modifications. This method was suited for the AMO, AMP, PenG, CLO, CEF, and CEP residues in the milk samples and after small modifications for CFD, TIC, and NAF. A simple purification scheme gave recoveries from 50% for AMO to 80-90% for other /3-lactams (95). 

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