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At least 721 records · Page 40Linked to original sources
PubMed2006

A highly sensitive immunoassay resistant to autoantibody interference for detection of the diabetes-associated autoantigen glutamic acid decarboxylase 65 in blood and other biological samples.

BACKGROUND: Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells. GAD65 may also be released by transplanted islets undergoing immunological rejection. To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary. Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies. METHODS: A novel, magnetic bead-based assay was developed based on GAD65 capture by a monoclonal antibody directed to the only region of the protein known not to be significantly targeted by autoantibodies. A subsequent denaturation step allows sensitive immunodetection to proceed using anti-GAD65 polyclonal antibodies that would otherwise potentially be blocked by bound autoantibodies. RESULTS: The GAD65 assay worked equally well with serum and plasma as with a solution of bovine serum albumin (BSA). The limit of blank was 31 pg/mL and did not differ significantly in the BSA solution (27 pg/mL). Mean recovery of GAD65 from the plasma of control subjects and GAD65 autoantibody-positive and -negative subjects with type 1 diabetes was 101 +/- 4.6%, 88 +/- 7.8%, and 99 +/- 7.0% (+/- SEM), respectively. The assay was used to quantify both recombinant GAD65 and the GAD65 content of human and rodent islets and other tissue extracts that were added to human plasma samples. CONCLUSIONS: A sensitive, autoantibody-resistant GAD65 assay has been developed that is compatible with detection in serum and plasma and therefore will likely also work with a variety of other biologic fluids. This assay may enable the use of circulating GAD65 as a biomarker of islet damage or transplant rejection and will facilitate in vivo studies of the pathogenesis of anti-GAD65 autoreactivity.

Autoantibodies↗
PubMed1996

Analysis of gamma-(cholesteryloxy)butyric acid in biologic samples by derivatization with 5-(bromomethyl)fluorescein followed by high-performance liquid chromatography with laser-induced fluorescence detection.

This report describes the first application of 5-(bromo-methyl)fluorescein (5-BMF) for the quantitation of a pharmaceutically relevant carboxyl-containing analyte in a biological matrix. An analytical method for quantitation of gamma-(cholesteryloxy)butyric acid (CBA), a relatively new antitumor agent, in different tissues of Sprague-Dawley rats was developed. 5-BMF was employed to form a stable and spectrally well-characterized conjugate of CBA. The derivatization yield was maximized by optimizing several reaction variables. The conjugate was separated by high-performance liquid chromatography and quantitated by a laboratory-constructed argon ion laser fluorometer. The detection limits for CBA were 4.6 x 10(-9) and 6.34 x 10(-11) M by conventional and laser-induced fluorescence (LIF), respectively. A derivatization limit of detection of 1.85 x 10(-9) M was achieved by LIF for the conjugate The analytical method was useful for quantitation of CBA in various tissues in the picogram per milliliter range.

Animals↗
PubMed1984

Determination of morphine in biological samples by gas chromatography-mass spectrometry. Evidence for persistent tissue binding in rats twenty-two days post-withdrawal.

Combined gas chromatography-mass spectrometry with capillary and packed column gas chromatography and a deuterium-labelled internal standard was used to determine morphine in biological specimens from rats 22 days after abrupt withdrawal. Morphine was extracted from urine and body organs at pH 9 and the pentafluoropropionyl derivatives were made for analysis by gas chromatography-mass spectrometry. The stationary phase was OV-17 and the mass spectrometer was focused on m/z 414 for morphine and m/z 417 for the internal standard, [NC2H3]morphine. With fused-silica capillary columns, the sensitivity of the assay was increased about ten-fold over packed columns. Urinary excretion of total morphine (free + conjugated) was 22 ng/h (range 11-51 ng/h, n = 8) at 22 days post-withdrawal. Free morphine was mainly detected in the lung (1.8-6.5 ng/g, n = 7), kidney (1.5-4.0 ng/g, n = 7) and liver (1.8-4.6 ng/g, n = 4). Traces of morphine were also detected in brain of some rats. Treatment with the opiate antagonist naltrexone, 10 mg/kg on four consecutive days before death, failed to change the urinary excretion pattern or the concentration of free morphine in body organs. The biological significance of the residual morphine, if any, remains to be determined.

Animals↗
PubMed2005

Amino acid analysis by micellar electrokinetic chromatography with laser-induced fluorescence detection: application to nanolitre-volume biological samples from Arabidopsis thaliana and Myzus persicae.

Amino acids were derivatised with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence. The optimised MEKC background electrolyte conditions were: 40 mM sodium cholate, 5 mM beta-cyclodextrin in 20 mM aqueous borate buffer, pH 9.1, with 7% v/v acetonitrile. Using these conditions, 19 amino acids were separated within 17 min. The limits of detection were in the range of 7.6-42.2 pmol/mL and limits of quantitation from 0.05-0.14 nmol/mL. The method was systematically validated for injection volume error, migration time variation, calibration linearity, accuracy, precision, and recovery. Nanolitre volume samples of phloem sap of individual sieve element cells from the plant Arabidopsis thaliana and honeydew from the aphid Myzus persicae were directly analysed with this method. Quantitative amino acid concentrations in these two biological matrices were profiled for the first time. This method is particularly important because it allows the complete profile of the amino acids obtained from individual phloem elements, allowing cell to cell and plant to plant variation to be quantified, which to date has not been possible with Arabidopsis thaliana.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Microwave treatment of biological samples for methylmercury determination by high performance liquid chromatography-cold vapour atomic fluorescence spectrometry.

A simple and rapid microwave-assisted alkaline digestion procedure was developed in combination with high performance liquid chromatography-ultraviolet post-column oxidation-cold vapour atomic fluorescence spectrometric detection for methylmercury determination in biological tissues. Since the stability of methylmercury in methanolic potassium hydroxide solution under microwave irradiation was verified, the microwave-assisted extraction procedure was optimized in terms of quantitative recovery of methylmercury and minimum time required. The alkaline extracts were subjected to clean-up steps with dichloromethane and hydrochloric acid in order to reduce matrix interferences in methylmercury determination. The effects of matrix interferences were checked by comparison of the slopes corresponding to calibration and standard addition curves. The accuracy of the method was evaluated by the analysis of two biological certified reference materials, NRC TORT-2 and BCR 463. The results obtained by the proposed method were in good agreement with the certified values of methylmercury concentration in both materials. The detection limit was 10 microg kg(-1) and the relative standard deviation was < 8% for methylmercury concentrations ranging from 0.15 to 3.0 mg kg(-1).

Animals↗
PubMed1999

The IAEA worldwide intercomparison exercises (1990-1997): determination of trace elements in marine sediments and biological samples.

Four major worldwide intercomparison exercises for the determination of trace elements in various environmental matrices were completed by the IAEA Marine Environment Laboratory since 1990: SD-M-2/TM, deep sea marine sediment; IAEA-350, tuna fish homogenate; IAEA-356, contaminated coastal sediment and IAEA-140, sea plant (Fucus sp.). These intercomparison exercises aim at enabling individual laboratories to monitor their performance. The results of these exercises allowed us to make an overall evaluation of the quality of data provided for environmental assessment and to identify the trends of analytical performance in the determination of trace elements over the years. The number of participants in each exercise varied between 68 and 130, and permits statistical evaluation of the performance for a number of elements. For each intercomparison exercise, the performance of the participant laboratories was assessed by comparing reported results with established reference values calculating 'Z-scores'. The results show that for each sample matrix, the values reported by some laboratories were far from satisfactory in the earlier exercises, in particular for Cd, Cr and Pb. Nevertheless, over time, a general improvement of performance can clearly be seen for all elements. Moreover, there was a noticeable increase in the number of laboratories with good performance in the two most recent exercises, observed both for biological and for sediment matrices. However, the determination of trace elements such as Cd, Cr, Pb and Hg in low level environmental samples still remains a major challenge to the analysts. For this reason and in order to assess the current performance of laboratories for low environmental levels of contaminants, the future intercomparison exercises will concentrate on low level sediment and fish samples.

Animals↗
PubMed1982

A new improvement of the sensitivity and specificity of radioimmunoassay for thyroliberin. Application to biological samples.

A new approach to increase the sensitivity and the specificity of a radioimmunoassay for thyroliberin (TRH) is described. The obtention of the anti-TRH antiserum used in this study has been previously reported (8): it exhibits a higher affinity for mono and di-iodo TRH derivatives. The method consists in iodinating the standards and the biological extracts with a large excess of reagents (NaI + chloramine T) under definite conditions of pH and concentrations. Mono iodo [[I125]-His2]-TRH purified by TLC was used as a tracer. This method both increases the sensitivity by 5 folds and improves the specificity by excluding or highly reducing the cross-reactivity obtained with closely related peptides. It has been successfully applied to mouse hypothalamic tissue extracts. It presents the advantage to measure low amounts of TRH in mouse tissue samples such as posterior pituitary and anterior pituitary.

Animals↗
PubMed1988

Improved assay for bismuth in biological samples by atomic absorption spectrophotometry with hydride generation.

This simple, rapid, sensitive, reliable, and economical assay for bismuth in plasma, erythrocytes, and urine is based on atomic absorption spectrophotometry with hydride generation. Acid digestion eliminates the problem of foaming, which hitherto has complicated such assay of bismuth in plasma and erythrocytes. The detection limit of the assay has been improved to 0.1 micrograms/L, as compared with a previously documented limit of 2.5 micrograms/L. Average recovery exceeded 95% in all biological fluids. Economy of use derives from elimination of need for electrodeless discharge lamps and atomic absorption grade borohydride. Determination of basal concentrations of bismuth in clinical samples of body fluids gave reference intervals of 0.1-3.5 micrograms/L for plasma, 0.3-4.6 micrograms/L for urine.

Adult↗
PubMed1987

Methods for determination of lipid peroxidation in biological samples.

Interest in the pathological consequences of lipid peroxidation has led to the development of a number of analytical approaches to the quantitation of products. However, the various analytical methodologies employed often do not measure the same chemical classes of products, and apparent discrepencies have been observed, particularly in studies of lipid peroxidation in biological systems. This review provides a brief discussion of some of the strengths and weakness of methods currently used for the determination of lipid peroxidation in biological tissues.

Animals↗
PubMed2001

Determination of grayanotoxins in biological samples by LC-MS/MS.

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

Animals↗
PubMed1997

Gas chromatographic method using electron-capture detection for the determination of musk xylene in human blood samples. Biological monitoring of the general population.

Musk xylene (2,4,6-trinitro-1,3-dimethyl-5-tert.-butylbenzene, MX), a synthetic musk often used in different fragrances and soaps to substitute the natural musk, is a potential contaminant of humans. In this publication, a specific and sensitive detection method for the determination of musk xylene in human blood samples is described. The clean-up of the blood samples includes an extraction step followed by a solid-phase adsorption to separate MX from other plasma components. Separation and detection was carried out by capillary gas chromatography and an electron capture detector (GC-ECD). The results were verified using qualitative capillary gas chromatography and a mass selective detector with electron impact ionisation (GC-EI-MS). epsilon-Hexachlorocyclohexane (epsilon-HCH) is used as internal standard. The reliability of the GC-ECD method has been proved. The relative standard deviations of the within-series imprecision were 12.7% for samples with a concentration of 0.5 microg/l and 2.1% for samples with a concentration of 5.0 microg/l, whereas the relative standard deviations for the between-day imprecision were 14.9% (0.5 microg/l samples) and 3.4% (5.0 microg/l samples). The losses during sample treatment were between 10.1% and 17.8%. No interfering peaks were observed. The absolute detection limit was 0.1 microg/l plasma. A total of 72 human blood samples were analysed to determine the MX concentrations within the general population. In 66 of the 72 human blood samples, the MX concentrations ranged from 0.10 to 1.12 microg/l plasma for the described method. In six samples no MX was detected. The median concentration was 0.24+/-0.23 microg MX/l plasma. The 95 percentile was 0.79 microg/l. No correlation could be found between MX concentrations and smoking habit, broca index, age, sex as well as fish consumption habits. Nevertheless, the results demonstrate the exposure of the general population to MX.

Chromatography, Gas↗
PubMedDate not supplied

[Isolation of riboflavin-binding apoprotein from chicken egg protein and its use for determining riboflavin in biological samples].

A riboflavine-binding protein has been isolated from egg white and its properties have been characterized. The apoprotein-employing techniques for riboflavine detection in foods, urine, and blood serum are highly competitive with conventional labor-consuming procedures and with the high expensive HPLC method in sensitivity and selectivity, and can be recommended for riboflavine detection in biological objects.

Animals↗
PubMed1977

XAD-2 resin drug extraction methods for biologic samples.

The XAD-2 resin extraction procedure is rapidly becoming an important tool for both the clinical and forensic toxicology laboratory. Although different investigators use different elution solvents and techniques, the basic procedure is to adsorb drugs onto the XAD-2 resin and then elute these drugs with an appropriate organic solvent. A number of parameters have been shown to be involved in obtaining good results. These include pH of sample, flow rate, type and volume of eluting solvent. These parameters can be adjusted so that maximum overall drug recoveries are obtained. Since XAD-2 extraction of urine by Fujimoto and Wang [2] was introduced, the procedure has been extended to all types of biologic specimens. Various elution techniques have been reported [12, 13], namely, the differential elution of drugs from the resin into acid and neutral, and basic drug fractions. Overall, the techniques vary from rapid simple procedures for drugs of abuse in urine to more elaborate methods of extraction of autopsy material. The extraction procedure itself is flexible enough so as to suit the needs of almost any toxicology laboratory.

Bile↗
PubMed2002

Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization.

We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.

Aminobutyrates↗
PubMed1992

Quantitative determination of natriuretic peptides in human biological samples with a bioassay using cultured cells.

Up to now, members of the natriuretic peptide family have usually been determined by radioimmunoassays using antibodies more or less specific for the distinct peptides so far identified. However, natriuretic peptides differing significantly in their amino acid sequence from the one against which the antibody has been raised cannot be determined by this means, and still unknown natriuretic peptides cannot be detected. We therefore developed a new bioassay system sensitive to all members of the natriuretic peptide family by taking advantage of the biological activity of these hormones, the activation of the guanylate cyclase/cyclic GMP system. In this assay, cultured cells are incubated with the natriuretic peptides, and the amount of cyclic GMP produced by the cells is determined by radioimmunoassay. From the relative stimulation of the cellular cyclic GMP production, the concentration of natriuretic peptides in the sample is determined after calibration with synthetic atrial natriuretic peptide. For a qualitative identification of the various peptides, the bioassay is combined with a reversed-phase HPLC step. Using cultured bovine aortic endothelial or bovine kidney epithelial cells for the bioassay, we achieved detection limits of 1 fmol or 50 fmol, respectively, for human atrial as well as brain natriuretic peptide. Intra-assay coefficients of variation of 4.3% (aortic endothelial cells, at 0.65 nmol/l peptide) and 5.8% (kidney epithelial cells, at 6.5 nmol/l peptide) were obtained. The total content of natriuretic peptides as well as the amounts of the individual natriuretic peptides following HPLC separation were determined in extracts of human atria obtained at aortocoronary bypass operations.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta↗
PubMed1992

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.

Acrylamides↗
PubMed1983

Simultaneous analysis of 1-beta-D-arabinofuranosylcytosine, 1-beta-D-arabinofuranosyluracil and sodium salicylate in biological samples by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic column switching system is described for the rapid and complete separation of 1-beta-D-arabinofuranosylcytosine (Ara-C), 1-beta-D-arabinofuranosyluracil (Ara-U) and sodium salicylate using an internal standard of sodium cefmetazole. The system is highly selective and separates these compounds from interfering compounds commonly in biological matrices. The system was tested by following the pharmacokinetics of Ara-C after rectal administration in the presence of sodium salicylate which is an aid to drug absorption. The chromatographic system is also suitable for monitoring levels of Ara-C and its metabolite Ara-U after intravenous administration of Ara-C.

Animals↗
PubMed1989

[Biometrico-statistical evaluation of a biological sample].

The total animal population percentage composition, found during period May-August 1979 on tunny-fishing coco-fibres nets in Camogli (Genoa), has been valued in relation to the depth. In particular, connections between population and presence of some fish species draughted has been studied, considering ecological, reproductive and trophic habits of fishes, in order to value relations between the influence these populations exert on fishing and biological characteristics of the quantitatively more meaningful fish species.

Animals↗