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A microassay-based procedure for measuring low levels of toxic organophosphorus compounds through acetylcholinesterase inhibition

by Frank Green
Utilizing a microtiter plate spectrophotometric system, an assay process was developed for the next poisonous organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-[2-[bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP).
The process, based mostly on the Ellman assay technique, makes use of inhibition of eel acetylcholinesterase (0.01 unit per effectively) to hold out the willpower of inhibitor concentrations for each a typical curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of each unknowns and requirements (a minimal of six concentrations have been used per commonplace curve) have been assayed 5 occasions per pattern, with three management (uninhibited) enzyme exercise factors included for every pattern.
The time required for finishing up a single plate was approx 30 min. Sensitivity for essentially the most potent acetylcholinesterase inhibitor examined was 0.Four nM below the situations used for a typical assay. It needs to be famous, nonetheless, that no try was made to optimize the assay process for sensitivity.

A microassay for the willpower of soluble and membrane-bound glutamate decarboxylase activity–influences of cations, lipid composition, and pyridoxal 5′-phosphate on the glutamate decarboxylase binding to liposomes

A radiochemical microassay for soluble and membrane-bound glutamate decarboxylase (GAD) is described. As much as 180 samples might be decided per day with a variation coefficient of two%. The tactic detects newly synthesized gamma-amino-n-butyric acid within the picomole vary and might simply be utilized to different enzymes whose substrate and product differ by cost. In an aqueous homogenate of mind (1 + 10; w/v) about 15% of the overall GAD exercise are spun down by centrifugation (1 h, 100,000g) rising to 35% of the overall GAD exercise in options with Eight mM calcium chloride or 100 mM potassium acetate. There’s related dependence on the cation focus when GAD binds to phospholipid vesicles (liposomes) in addition to dependence on lipid focus and lipid composition. The coenzyme pyridoxal 5′-phosphate has no affect on GAD binding to liposomes.

Evaluation of the androgen receptor in remoted testicular cell sorts with a microassay that makes use of an affinity ligand

A microassay for the androgen receptor was developed to analyze the mobile distribution of receptor in freshly remoted testicular cell sorts. The microassay makes use of an androgen affinity ligand, 17 beta-dihydrotestosterone bromoacetate. Binding of this ligand by the androgen receptor is fast and irreversible, which allows the event of a extremely delicate assay. The androgen receptor microassay is accomplished inside Four h and detects receptor in as little as 0.5 micrograms mobile protein.
There was no detectable binding of the affinity label by albumin or Sertoli cell-secreted proteins, together with androgen-binding protein. Androgen receptor was present in mobile sonicates of human foreskin fibroblast, rat ventral prostate, rat kidney, and rat liver. Though the relative distribution of receptor was much like that obtained utilizing a standard equilibrium binding assay, the degrees of receptor have been considerably greater utilizing the microassay. The androgen receptor microassay was subsequently used to analyze the receptor in remoted testicular cell sorts. Androgen receptor was detected in freshly remoted peritubular myoid cells (80 fmol/micrograms DNA), Sertoli cells (88 fmol/micrograms DNA), and Leydig cells (35 fmol/micrograms DNA). No androgen receptor was detected in a combined inhabitants of germ cells.
Hormones weren’t discovered to affect androgen receptor ranges in cultured peritubular cells or Sertoli cells. Electrophoretic evaluation of androgen receptor radiolabeled with the affinity ligand demonstrates a single 52-kDa type of the receptor in peritubular cells, Sertoli cells, and Leydig cells. The scale of the androgen receptor species detected within the rat testicular cell sorts was barely smaller than the 56-kDa protein detected in a human fibroblast cell line. The present research demonstrates the utility of the microassay and affinity ligand to analyze androgen receptor biology. Information point out that androgen receptors are current in a number of testicular cell sorts and recommend that the management of testicular perform by androgens in all probability entails actions on a number of cell sorts.

Microassay for screening newborns for galactosemia with use of a fluorometric microplate reader

We describe a microassay for measuring galactose (Gal) and galactose 1-phosphate (Gal-1-P) in dried blood spots. After a coupled enzyme response involving galactose dehydrogenase (GADH, EC 1.1.1.48) and alkaline phosphatase (AP, EC 3.1.3.1) in a microplate effectively, NADH fluorescence is measured by a extremely delicate fluorometric microplate reader, able to fast measurement of fluorescence (2 min per 96 samples). Inside- and between-run CVs for measurements of Gal at 90 mg/L with Gal-1-P at 130 mg/L have been each lower than 5% (n = 8), and analytical recoveries for Gal at 90 mg/L and Gal-1-P at 130 mg/L have been 98% and 92%, respectively. 5 hundred dried blood-spot samples might be assayed inside 2 h, with full calculation of outcomes by an on-line microcomputer. This fast and dependable assay system may be very helpful for the routine screening of newborns for galactosemia.

A microassay for the willpower of iodide and its software to the measurement of the iodination of proteins and the catalytic actions of iodo compounds.

A microassay system based mostly on the impact of the catalytic Sandell-Kolthoff response of iodide on the oxidation of arsenic(III) by cerium(IV) was developed to measure iodine-containing compounds. This fast assay makes use of small portions of reagents, is appropriate to be used with a photometric microplate reader, can check many samples concurrently, and eliminates issues related to the usage of radiolabeled compounds to measure iodination. It may detect picogram portions of iodide.
We report the usage of this assay to measure the conjugation of an iodine-containing hapten (iodinated Bolton-Hunter reagent, IBHR) to ovalbumin and human serum albumin. It has confirmed to be glorious for finding out the relative molar catalytic exercise of the iodine-containing compounds IBHR, thyroxine, 4-iodophenol, and lithium 3,4-diiodosalicylate. The interference by azide on the assay was investigated.
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A high-performance liquid chromatographic microassay using a liquid-solid extraction approach for etintidine in plasma

This paper describes a brand new, fast stable extraction technique for the willpower of etintidine in plasma. The tactic employs a semiautomatic pattern preparation system. Plasma samples and the inner commonplace (cimetidine) have been utilized onto octyl-bonded silica extraction columns. The extraction columns have been then subjected to Tris buffer and water wash and have been subsequently loaded onto an automated pattern injection system. The contents of the extraction columns have been eluted on-line with a cell section of acetonitrile:methanol:0.1% ammonium hydroxide (85:10:5, by quantity) onto a silica analytical column and detected by UV absorption at 229 nm.
The chromatographic situation separates etintidine from a few of its metabolites and different endogenous elements in plasma. The detection restrict for etintidine was 0.02-0.05 microgram/ml when 0.2 ml of plasma was used. This technique has been used for the willpower of plasma etintidine ranges in people and mice after oral administration of etintidine and was discovered to be appropriate for pharmacokinetic/bioavailability research of etintidine in people and animals. The tactic can be used for the quantitative willpower of cimetidine and sure metabolites of etintidine.
Frank Green