High detection assay

Luminescence has been used in conjunction with flow cells to detect electro-generated intennediates downstream of the electrode. The teclmique lends itself especially to the investigation of photoelectrochemical processes, since it can yield mfonnation about excited states of reactive species and their lifetimes. It has become an attractive detection method for various organic and inorganic compounds, and highly sensitive assays for several clinically important analytes such as oxalate, NADH, amino acids and various aliphatic and cyclic amines have been developed. It has also found use in microelectrode fundamental studies in low-dielectric-constant organic solvents. 

Gonzalez JE, Negulescu PA (1998) Intracellular detection assays for high-throughput screening. Curr Opin Biotech-no 624-631... 

In general, CE is simple, rapid, and low cost because it needs neither laborious treatment of the samples nor long times of analysis. However, its high detection limit is a major limitation of CE. CE is often poorly reproducible. Enzymatic assay is more suitable for quantifying one organic acid in honey samples because it is specific, precise, and accurate. GC is more suitable for analyzing volatile or semivolatile chemicals. HPLC is versatile and reproducible. However, common HPLC detectors such as UV-VIS are not very sensitive for organic aliphatic acids. 

Rotshteyn Y, Weingarten B. 1996. A highly sensitive assay for the simultaneous determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma by high-performance liquid chromatography with electrochemical and fluorescence detection. Ther Drug Monit 18(2) 179-188. 

Key words High-throughput assay. Two-dimensional electrophoresis. Automation, System, Simultaneous detection. Protein... 

Only a few works report a simple combination of conventional sample cleanup procedures with UV detection. Assay of CLO (OXA was internal standard) based on a protein precipitation using MeCN in acidic solution (pH 3.6) and LLE with chloroform was reported for milk samples. The organic layer was evaporated to dryness, and the residue was reconstituted in the mobile phase and injected into the chromatographic system with UV detection. Relatively clean chromatograms and high recoveries (75-84%) were obtained using this assay (55). 

A succinylated casein derivative that has nearly all its amines blocked can be used as a substrate in protease assays (Hatakeyama etal., 1992). As the casein is degraded by a protease, free amines are created from a-chain cleavage and release of a-amino groups. The creation of amines can be monitored by an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS Section 4.3). The procedure forms the basis for a highly sensitive assay for protease activity. 

J. E. Gonzalez and P. A. Negulescu, Intracellular detection assays for high-through-put screening, Curr. Opin. Biotechnol. 1998, 9, 624- 631. 

A high-throughput assay for bacterial RNA polymerase has been successfully developed and validated using a 96-well, automated format [70], The reaction mixture contained a DNA template, nucleotide substrates (NTPs), supplemented with a-33P-labeled CTP in Tris-acetate buffer (pH 6.8). The polymerase reaction was carried out at 34°C for 40 min (providing linear kinetics). The effect of dimethylsulfoxide (DMSO), the usual solvent for test compounds used in a screen, was taken into consideration. The radiolabeled RNA transcripts were allowed to bind diethyl aminoethyl (DEAE) beads, which were then separated via filtration, and radioactivity associated with the wells was quantitated to measure the RNA polymerase activity. The standard deviation of the measured activity was typically < 15% of the average. Use of this assay to screen for RNA polymerase inhibitors from chemical libraries and natural products led to the identification of DNA intercalators (known to inhibit RNA polymerase activity), rifampicin (a known inhibitors of RNA polymerase), and several derivatives of rifampicin from Actinomycetes extracts. Therefore this assay can be reliably utilized to detect novel inhibitors of bacterial RNA polymerase. 

In summary, BOCILLIN FL and fluorescein-labeled penicillins should be used for routine detection of PBPs, and 3H-penicillins are better suited for kinetic characterizations of PBPs due to their low energy level. For the development of high-throughput assays for PBPs, BOCILLIN FL and 3H-penicillins are clearly superior to other labeled penicillins. 

Analogous to the majority of dihydropyridines, nifedipine s, nitrendipine s, and nimodipine s chemical structures - a 2- or 3-nitrophenyl substituent in the 4-position combined with the dihydropyridine diester structure - results in a high response in electron capture detection (ECD), thus allowing high detection sensitivity and sufficient assay specificity towards endogenous compounds, metabolites or common co-medications (Muck et al. 1994). 

The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia colt proteins, and retroviral contamination.2 The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for E. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa-... 

Shum, D. et al. 2008. A high density assay format for the detection of novel cytotoxic agents in large chemical libraries. J. Enz. Inhib. Med. Chem. 23, 931-945. 

Picric acid and picramic acid have been identified as possible metabolites of tetryl exposure (Zambrano and Mandovano 1956). Picramic acid has been detected in rabbits fed 32.3-40.0 mg/kg/day for up to 30 days. The concentrations of picramic acid in the urine of these animals were estimated to be between 0.05 and 0.33 mg/L however, the colorimetric assay used to detect the metabolite was only semiquantitative. Picric acid could not be detected in the treated animals, possibly because of the high detection limit of the colorimetric assay used (1 mg/L). Sulfoconjugates were also detected in the urine and were found to increase with duration of exposure. These would only be useful biomarkers of exposure if they could be identified and quantitated. No other biomarkers to identify or quantify exposure to tetryl were located. No studies have been designed to identify interactions between tetryl (or its metabolites) and target molecules or cells within exposed humans or animals. 

---