A simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence

Degradation of heparan sulfate proteoglycan by heparanase is an important process in tissue invasion by malignant tumor cells and inflammatory cells. Several heparanase assays have been reported previously, but all of them are cumbersome and time-consuming for separating degradative products from uncleaved substrates and detecting these products. This paper describes the development of a simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence based on non-radiative energy transfer. In this assay, heparan sulfate proteoglycan labeled with biotin and europium cryptate (fluorescence donor) was used as a substrate for heparanase. Degradation of the substrate by incubation with murine melanoma cell extract was detected by measuring the time-resolved fluorescence after addition of XL665 (cross-linked allophycocyanin, fluorescence acceptor)-labeled streptavidin. As heparanase activity can be simply measured by successive addition of substrate, enzyme and detection reagent onto one assay plate, our heparanase assay allows rapid processing of large numbers of samples.     

A simple and rapid liquid chromatographic assay for evaluation of potentially counterfeit Tamiflu

A simple and rapid liquid chromatographic assay for the evaluation of potentially counterfeit oseltamivir (Tamiflu has been developed and assessed. The assay uses approximately 1mg Tamiflu powder when used for authentication and content estimate. The procedure was validated using 50 replicates analysed during five independent series with a total R.S.D. of 11.2%. The assay can also be used to monitor the exact content of oseltamivir in Tamiflu capsules. One Tamiflu capsule was transferred to a 250mL volumetric flask and 150mL water was added. The flask was placed in an ultrasonic bath at 40 degrees C for 20min to dissolve the capsule. The solution was allowed to cool to room temperature before the flask was filled up to the mark (250mL). A small aliquot was centrifuged and then directly injected into the LC-system for quantification. Oseltamivir was analysed by liquid chromatography with UV detection on a Hypersil Gold column (150mmx4.6mm) using a mobile phase containing methanol-phosphate buffer (pH 2.5; 0.1M) (50:50, v/v) at a flow rate of 1.0mL/min. The assay was implemented for the analysis of Tamiflu purchased over the Internet and at local pharmacies in Thailand and Vietnam.     

A rapid and simple toxicity assay based on growth rate inhibition of Pseudomonas fluorescens

A simple and rapid test, based on growth rate inhibition (GRI) of Pseudomonas fluorescens, is described for toxicity screening and assessment. The test involves challenging standardized suspensions [0.2 optical density (OD) at 600 nm] of exponentially growing cells with various concentrations of a toxicant at 33°C and monitoring changes in the growth rate by OD measurements after 0.5, 1.0, 1.5, and 2.0 h. The toxicity end point is measured as the effective concentration of a test sample that causes 50% inhibition of growth rate (EC₅₀) during each designated time period. Several metal salt solutions, organic compounds, and complex effluents were screened using this test and data compared with those obtained using the Microtox® assay. Results indicated that the P. fluorescens GRI test could be used as a valuable part of a battery of short-term bioassays for toxicity screening and assessment.     

A novel binding assay for phospholipase A2

We have devised a rapid and simple assay for estimating the binding of pancreatic phospholipase A2 to a bilayer lipid membrane. The binding was observed to be extremely rapid at 37 degrees and was absolutely dependent upon Ca2+. Amongst several drugs known to inhibit the catalytic activity of phospholipase only mepacrine at high concentrations (500 microM) and chlorpromazine (100 microM) were active. Treatment of the enzyme with p-bromophenacylbromide did not inhibit binding. Several alcohols potentiated binding whereas detergents tended to inhibit. Amongst several purified proteins tested, only the steroid-induced anti-phospholipase protein lipocortin prevented binding. The use of this assay in screening for antiphospholipase agents is discussed.     

Determination of barbituric acid,utilizing a rapid and simple colorimetric assay

A simple, fast and sensitive method is proposed for Tryptophan (Trp) determination in pharmaceutical formulations containing other non-electroative aminoacids, vitamins and hydroxycobalamines. Optimized conditions for differential pulse voltammetry allowed the determination of Trp with detection limit of 1.7 microM in 2-30 mM linear dynamic range (n=7; r=0.999), at a bare, non-treated carbon paste electrode. Ascorbic acid interference was evaluated and eliminated by choosing a suitable baseline for determination of peak currents for Trp.     

A rapid and simple assay for the study of thromboxane B2 synthesis by intact human platelets

Conversion of 1-¹⁴C-arachidonic acid (AA) to thromboxane B₂ (TXB₂) by human platelets was studied by using a new, simple technique. Organic solvent extraction was avoided by spotting aliquots of the reaction mixture directly on thin layer chromatography (TLC) plates. In this way it was possible to study the kinetic parameters of the formation of TXB₂. Moreover, the rapidity and simplicity of the assay should be particularly suitable when studying the possible relationships between thromboxane synthesis and aggregation function in human platelets, where a large number of determinations is required.     

A rapid assay of human thyroid peroxidase activity

Impaired synthesis or action of thyroid hormones (THs) during critically sensitive periods of development can have long term adverse effects on health. Development of rapid assays to identify chemicals that impair THs physiology is an important goal for reducing risks from chemical use. Thyroid peroxidase (TPO) is a key enzyme regulating THs synthesis in thyroid gland and a vulnerable target for chemicals that disrupt THs synthesis. To develop a human-relevant, rapid assay for TPO inhibition, we have engineered two cell lines (CHO and LentiX- 293) to express active human TPO (hTPO) enzyme and applied them in a recently-described assay using a stable fluorescent product (Amplex UltraRed). Assay performance was assessed by comparing activity of 19 reference chemicals with known strong, weak or no TPO inhibitory activity. The assay using hTPO from either cell line consistently identified the relative potency of strong to moderate inhibitors and chemicals known to be inactive. Results were less consistent for chemicals reported to be weak inhibitors of rodent TPO, possibly suggesting some species specificity. Our studies support the use of hTPO from stably transfected cell lines to substitute for animal-derived thyroid microsomes for rapid high throughput screening assays to identify and characterize TPO inhibitors.     

A simple sensitive fluorimetric assay of APS-kinase activity

Adenosine phosphosulphokinase (APS-kinase or ATP:adenylylsulphate 3'-phosphotransferase; EC 2.7.1.25) catalyses the formation of 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Its activity in various tissues was measured by transferring the sulphate from PAPS, a product of APS-kinase reaction, to 4-methylumbelliferone (4-MU) to form 4-MU-sulphate (4-MUS) using phenolsulphotransferase (PST) extracted from rat liver. Desalting with Sephadex G-25, together with the addition of EDTA effectively removed the Mg2+ ions from the rat liver extract and thereby inhibited the APS-kinase activity therein in the subsequent PST reaction. 4-MUS formed was measured indirectly by a decrease in the fluorescence of 4-MU by a continuous fluorimetric assay. Kinetic data showed that the substrate, APS, at concentrations at and above 132 microM inhibited the APS kinase reaction. Pyrophosphate (PP) also inhibited the reaction. The apparent Km for APS was 14 microM. Two apparent Km values of 0.12 mM and 1.06 mM were obtained for ATP, while that for Mg2+ was 0.09 mM.     

A rapid HPLC assay for voriconazole in human plasma

This report describes a simple, rapid and reproducible method with a calibration range of 0.2–10 μg ml⁻¹ voriconazole in human plasma which is more appropriate for routine clinical use than the authors previously published method. The method utilises protein precipitation with acetonitrile as the only sample preparation involved prior to reverse phase HPLC. No internal standard was required.     

A simple,continuous fluorometric assay for HIV protease

Novel fluorogenic substrates for human immunodeficiency viral protease have been developed based on the principle of fluorescence energy transfer. Starting from a p24/p15 cleavage site-derived hexapeptide substrate, Ac-Thr-Ile-Nle-Nle-Gln-Arg-NH₂, incorporation of 2-aminobenzoic acid in place of the acetyl group as the donor and p-NO₂-Phe at the P1′ position as acceptor gave the intramolecularly quenched fluorogenic substrate. Cleavage of the substrate by HIV protease released the fluorescent N-terminal tripeptide from its close apposition to the quenching nitrobenzyl group, resulting in enhanced fluorescence. An automated assay based on 96=well microtiter plates and a fluorometric plate reader have been developed, which allow high throughput of compounds in the search for HIV protease inhibitors.     

A simple and rapid technique for preparing histological sections of brain

A rapid technique for preparing histological sections of rodent brain is described. Sectioning and staining may be completed within two hours of sacrifice.     

Air-induced writhing: A rapid broad spectrum assay for analgesics

The injection of air into the peritoneal cavity of rats produces reliable and quantifiable behavioral responses. These responses are blocked by compounds known to have analgesic properties, including opioid agonists, opioid agonist-antagonists, cyclo-oxygenase inhibitors, and amine potentiators. However, psychotropic agents were generally inactive at reasonable doses. The ability of this assay to identify a wide spectrum of analgesic agents in rats was superior to the hot plate, warm plate, and tail immersion assays.     

A simple and rapid automated radiosynthesis of [18F]fluoroacetate

Two fully automated synthetic procedures of [¹⁸F]fluoroacetate ([¹⁸F]FAC) have been developed using a modified commercial TRACERlab FX_FN synthesizer. One was a two‐step one‐pot procedure, consisting of nucleophilic [¹⁸F]fluorination of benzyl‐2‐bromoacetate as a precursor with no‐carrier‐added [¹⁸F]fluoride, hydrolysis within the same [¹⁸F]fluorination reaction vessel, and purification with/without high‐performance liquid chromatography (HPLC). The second procedure consisted of nucleophilic [¹⁸F]fluorination, hydrolysis on the column, and purification with SEP‐PAK cartridges instead of HPLC. The radiochemical purity of [¹⁸F]FAC was >95% by the two procedures. The second procedure was a simple, rapid, and fully automated synthesis of [¹⁸F]FAC with a high and reproducible radiochemical yield exceeding 60% (decay uncorrected) within the total synthesis time less than 20 min. The new, simple, and rapid on‐column hydrolysis procedure should be adaptable to the fully automated synthesis of [¹⁸F]FAC at a commercial fluoro‐deoxyglucose synthesis module. Copyright © 2008 John Wiley & Sons, Ltd.     

A simple and rapid method for the determination of thiocyanate in serum

Studies were performed to evaluate ultrafiltration as a means for isolating thiocyanate from serum prior to its colorimetric determination as a ferric chloride complex. Recovery of thiocyanate from serum was enhanced by addition of alkali prior to preparation of the ultrafiltrate, and averaged 96.8%. The reproducibility (CV) of test was 2.3%, within-day; and 3.3%, between-day. The colorimetric reaction was linear to 100 micrograms/mL. The method was tested by determining thiocyanate concentrations in samples collected from smoking and nonsmoking individuals. Thiocyanate concentrations in patient samples, collected after IV-infusion of nitroprusside, were scattered around those of the general population, and ranged from 0.3 to 20 micrograms/mL.