A sensitive continuous-flow bioluminescent system for determining ethanol in serum and saliva

A continuous-flow system, based on NAD(P)H:FMN oxidoreductase and bacterial luciferase co-immobilized on a nylon coil placed in front of a photomultiplier, and alcohol dehydrogenase separately immobilized on a second nylon coil, is described for the assay of ethanol in serum and saliva. The flow is air-segmented and 5–50-μl samples are required. The sample throughput is 25–30 h^?1 with no carryover. The detection limit is 1 μmol l^?1 ethanol (50 pmol injected) and the response is linear between 50 and 2500 pmol of ethanol. The relative standard deviation is 3–10% for both within-day and between-day assays, and serum and saliva can be analyzed directly. The immobilized enzymes are satisfactorily stable and up to 900 samples can be analyzed with one enzyme reactor.     

A rapid and sensitive high-performance liquid chromatography assay for rofecoxib in human serum

Rofecoxib is a selective cyclooxygenase (COX)-2 inhibitor that is approved for the treatment of acute pain and osteoarthritis in adults. A sensitive and rapid high-performance liquid chromatographic (HPLC) method of determining rofecoxib in human serum is described. Alkalinized plasma samples are extracted into an organic solvent containing an internal standard and evaporated under nitrogen. The dried sample residues are reconstituted with mobile phase and analyzed by HPLC. The method uses 100 microL of the sample and is linear from 20 to 2000 ng/mL of rofecoxib. Precision and accuracy studies are performed. Stability of the drug in serum over four weeks is documented. This new method is simple, sensitive, precise, and accurate. Its use will translate into faster laboratory turnaround time, and the small sample volume required (100 microL) makes this assay suitable for pediatric patients. This assay will expedite pharmacokinetic studies and the therapeutic drug monitoring of rofecoxib and possibly other COX-2 inhibitors.     

An optimized luciferase bioluminescent assay for coenzyme A

A new bioluminescent method for coenzyme A (CoA) quantification is described. It is based on the enzymatic conversion of dehydroluciferyl-adenylate (L-AMP) into dehydroluciferyl-coenzyme A (L-CoA) by firefly luciferase (E.C. 1.13.12.7) (LUC), which causes a flash of light that can be measured in a luminometer. The method was subjected to optimization using experimental design methodologies to obtain optimum values for the concentrations of L-AMP ([L-AMP]), luciferase ([LUC]), ATP ([ATP]) and luciferin ([LH₂]). This method has a linear response over the range of 0.25–4 μM of CoA, with a limit of detection (LOD) of 0.24 μM and a limit of quantification (LOQ) of 0.80 μM. The assay has a relative standard deviation of about 7%. By coupling this optimized procedure to bioluminescent detection, a sensible and robust method can be obtained for the analysis of CoA.     

Development of a highly sensitive enzyme-linked immunosorbent assay for bisphenol A in serum

4,4'-Isopropylidenediphenol, bisphenol A (BPA), was derivatized to BPA-carboxymethylether (BPA-CME), BPA-carboxypropylether (BPA-CPE) and BPA-carboxybutylether (BPA-CBE), and then linked to bovine serum albumin (BSA). The BPA-BSA conjugates were injected into female New Zealand White rabbits, which then generated six kinds of polyclonal antibodies. In addition, BPA and bisphenol B (BPB)-enzyme conjugates were derivatized to BPA-CME, BPA-CPE, BPA-CBE, BPA-carboxyphenylether (CPhE) and BPB-CPE, and then linked to horseradish peroxidase (HRP). An enzyme-linked immunosorbent assay (ELISA) was developed and the specificity of the antibodies was confirmed by comparison with pre-immune serum and by competitive assays using different dilutions of BPA standards. Although anti-BPA antibodies cross-reacted with BPB by more than 13.6% at all dilutions used, cross-reaction with phthalates and phenols occurred only less than 0.1%. The combination with the highest sensitivity was obtained using anti-BPA-CME-BSA antibody and BPA-CPhE-HRP conjugate. ELISA successfully detected BPA in human serum at concentrations as low as 0.3 ng mL(-1), and over a measurable range of 0.3-100 ng mL(-1). Recovery tests were carried out by adding BPA to three kinds of human serum, and ranged from 89.7 to 97.3%, from 85.4 to 94.9% and from 81.9 to 97.4%, respectively. The correlation between the results from ELISA and gas chromatography-mass spectrometry (GC-MS) for BPA in spiked serum was r2 = 0.990, indicating that the proposed method is a potential tool for screening a large number of human serum samples.     

A simple and sensitive solid-phase radioimmunoassay for the assay of human TSH antibody

A simple and sensitive radioimmunoassay procedure is described for the screening and detection of specific antibodies in hybridoma cell lines. The specific procedure was developed to screen for antibodies against human thyrotropin (hTSH), but the procedure is applicable to screening for any desired antibodies. The immunoglobulin G(IgG) fraction of goat anti-mouse IgG is used to coat wells of microtiter plates. Anti-hTSH antibodies are measured by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated hTSH. Unlabeled hTSH may also be detected by its ability to inhibit binding of 125I-hTSH to the coated wells. This assay technique meets the demands of simplicity, sensitivity, reproducibility, and rapidity as a screening assay of hybridoma cell lines capable of secreting anti h-TSH.     

A mushroom (Agaricus bisporus) tissue homogenate based alcohol oxidase electrode for alcohol determination in serum

To construct homogenized tissue based biosensors by using plant tissue materials is a relatively new development in the biosensor technology. In this study, a homogenized mushroom (Agaricus bisporus) tissue based electrode in which alcohol oxidase activity was developed by immobilizing with gelatin and cross-linking agent glutaraldehyde at dissolved oxygen probe for determination of ethyl alcohol. The electrode response depends linearly on ethyl alcohol concentration between 0.2 and 20 mM. In the optimization studies, phosphate buffer (pH 7.5, 50 mM) and 35 degrees C were obtained as the optimum conditions. By using the electrode prepared we have made approximately 60 measurements in 10-h period, and response time of the electrode is 2 min for each measurement.     

A sensitive and specific high performance liquid chromatographic assay for imidazole dipeptides and 3-methylhistidine in human muscle biopsies, serum and urine.

A sensitive and specific high performance liquid chromatographic method is described for measuring imidazole dipeptides and 3-methylhistidine in human muscle biopsies, serum and urine. Muscle extract, serum or urine was reacted with o-phthaldialdehyde and the derivatives were separated by reversed phase chromatography with column switching and fluorescence detection.     

A sensitive radiochemical assay for Coenzyme A

Summary A simple and sensitive assay for Coenzyme A (CoA) is described. The method is based on coupling the enzymatic reactions of acetyl-CoA synthetase and acetyl-CoA carboxylase. CoA is converted into acetyl-CoA with acetyl-CoA synthetase in the presence of excess ATP and acetate. Acetyl-CoA is subsequently converted into malonyl-CoA with acetyl-CoA carboxylase in the presence of excess ATP and KH¹⁴CO₃. The formation of labelled acid-stable material (i.e. malonyl-CoA) is determined. Under conditions of the assay CoA is quantitatively converted into malonyl-CoA. This procedure permits the detection of as little as 15 pmoles of CoA in biological samples. Elimination of acetyl-CoA synthetase from the reaction mixture allows for the determination of acetyl-CoA.

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Empfindlicher radiochemischer Assay für Coenzym A

Zusammenfassung Ein einfacher und empfindlicher Assay für Coenzym A (CoA) wird beschrieben, der auf einer Kopplung der enzymatischen Reaktionen von Acetyl-CoA-synthetase und Acetyl-CoA-carboxylase beruht. CoA wird mit Hilfe von Acetyl-CoA-synthetase in Gegenwart von überschüssigem ATP und Acetat zu Acetyl-CoA umgesetzt. Dieses wird anschließend mit Acetyl-CoA-carboxylase in Gegenwart von überschüssigem ATP und KH¹⁴CO₃ zu Malonyl-CoA umgewandelt. Die Bildung von markierter säurestabiler Substanz (d.h. Malonyl-CoA) wird bestimmt. Unter den Versuchsbedingungen ist die Umsetzung von CoA zu Malonyl-CoA quantitativ. Noch 15 pMol CoA können in biologischem Material erfaßt werden. Durch Entfernung von Acetyl-CoA-synthetase aus dem Reaktionsgemisch kann auch Acetyl-CoA bestimmt werden.

     

GLC assay of khellin in human serum and urine

Summary A sensitive, specific and rapid GLC assay for khellin in serum and urine has been developed using flame ionization The method involves the addition of hyoscine as an internal standard, extraction with chloroform and the use of a 3% OV-17 on Gas Chromosorb column. The suitability of this procedure for serum sample analysis from rats after oral administration of the drug is reported.     

A sensitive spectrofluorimetric determination of human serum albumin with chrome azurol S

A simple and sensitive method for assay of human serum albumin (5–80 μg) is based on binding of chrome azurol S by the albumin and determination of the bound dye spectrofluorimetrically. The method is sufficiently sensitive for application to spinal fluid (20–100 μl).     

A sensitive fluorimetric assay for cationic surfactants

Summary A new fluorimetric assay for cationic surfactants is based on their capability of quenching the fluorescence of 8-octadecyloxypyrene-1,3,6-trisulfonate (PTS-18). It is specific for cationic surfactants which can be determined in the 40–400 g concentration range. The method is considered to be advantageous over former methods in that it only requires addition of the sample solution to the fluorophore solution, followed by measurement of fluorescence intensity of the probe. This is in contrast to existing methods where the detergent/dye ion pair has to be extracted before measurement.     

Bioluminescence and exogenous compounds: physico-chemical basis for bioluminescent assay

Bioluminescent systems are convenient objects to study mechanisms of influence of exogenous molecules on living organisms. Classification of physical and physico-chemical mechanisms of the effects of luminous bacteria Photobacterium leiognathi on bioluminescent reactions is suggested. Five mechanisms are discussed: (1) change of electron-excited states' population and energy transfer, (2) change of efficiency of S-T conversion in the presence of external heavy atom, (3) change of rates of coupled reactions, (4) interactions with enzymes and variation of enzymatic activity, (5) nonspecific effects of electron acceptors. Effects of various groups of chemical compounds are discussed according to the classification suggested. The compounds are: a series of fluorescent dyes, organic oxidizers, organic and inorganic heavy-atom containing compounds, and metallic salts. Applications of fluorescence time-resolved and steady-state techniques, as well as bioluminescence kinetics study, are discussed. The patterns of exogenous compounds' influence form a physico-chemical basis for bioluminescent ecological assay.     

Dissociation and rate constants of some human liver alcohol dehydrogenase isoenzymes.

ADH from human liver forms binary complexes with NADH, associated with a blue shift of the peak of the fluorescence emission of NADH. The wavelength shift is the same for all isoenzymes but the accompanying intensification of the fluorescence is different. The fluorescence is further increased by the formation of the very tight ternary enzyme-NADH-isobutyramide complexes. These properties are similar to those for the horse liver ADH, as well as the molecular weight of E=40 000 per active site of the dimer molecule (EE). "Stopped-flow" determined velocity constants (ER in equilibrium E+R) were found to be in good agreement with ethanol activity constants previously determined by activity measurement, confirming the validity of the ordered ternary complex mechanism also for the human ADH. No single isoenzyme activity as high as that reported by Mourad and Woronick or Drum has been found.     

A simple and sensitive assay for the quantitative analysis of paclitaxel in human and mouse plasma and brain tumor tissue using coupled liquid chromatography and tandem mass spectrometry

The development and validation of an assay for the determination of paclitaxel in human plasma, human brain tumor tissue, mouse plasma and mouse brain tumor tissue is described. Paclitaxel was extracted from the matrices using liquid-liquid extraction with tert-butyl methyl ether, followed by chromatographic analysis using an alkaline eluent. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicate that calibration standards in human plasma can be used to quantify paclitaxel in all tested matrices. In human samples, the validated range for paclitaxel was from 0.25-1000 ng ml(-1) using 200 microl plasma aliquots and from 5 to 5000 ng g(-1) using 50 microl tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma). In mice, the ranges were 1-1000 ng ml(-1) and 5-5000 ng g(-1) using 50 microl of mouse plasma and 50 microl of tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma), respectively. The method can be applied to studies generating only small sample volumes (e.g. mouse plasma and tumor tissue), but also to studies in human plasma requiring a lower limit of quantitation. The assay was applied successfully to several studies with both human and mouse samples.