Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

Biosensor immunoassay of ivermectin in bovine milk

A rapid and sensitive biosensor immunoassay was developed for determination of ivermectin residues in bovine milk. A detection limit of 16.2 ng/mL was achieved. A Biacore optical biosensor based on surface plasmon resonance was used, and a range of extraction techniques was investigated. In the final assay procedure, ivermectin was extracted with acetonitrile followed by C-8 solid-phase extraction cleanup. It was proven experimentally that 2 methods of milk storage, freezing or addition of mercury-containing compounds as preservatives, could be used without considerable change in detected concentrations (samples were fortified with ivermectin after storage). The average values for milk samples spiked at 100 and 50 ng/mL concentrations were 102.6 and 51.5 ng/mL, respectively. Extraction and analysis of 20 milk samples were performed within a single working day.

DETECTION OF IRRADIATED CHICKEN MEAT BY ANALYSIS OF LIPID EXTRACTS FOR 2-SUBSTITUTED CYCLOBUTANONES USING AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY

The means to detect the irradiation of food has been investigated for many years. In recent times radiolytic products, termed 2-alkylcyclobutanones (2-CBs), have been identified as excellent markers of irradiation in lipid-containing foods. An ELISA test was developed, which was capable of detecting a number of these compounds in irradiated chicken meat. A polyclonal antiserum was raised to a 2-CB containing a terminal carboxyl group conjugated to a carrier protein. This antiserum was highly specific for cyclobutanones containing C-10 and C-12 side chains. During assay validation the limit of detection of the assay was calculated to be 0.064 pg of 2-CB per gram of fat, within- and between-assay variations ranged from 6.7 to 18%. During experimental studies, chicken meat irradiated at doses ranging from 2.5 to 10 kGy were assayed and correctly identified as being treated. Quantitative comparisons between the ELISA and CC-MS revealed a good correlation (r(2) = 0.913) between the two methodologies in concentrations of 2-CB detected in irradiated samples.

Reduction of sample matrix effects - The analysis of benzimidazole residues in serum by immunobiosensor

Regulatory authorities, the food industry and the consumer demand reliable determination of chemical contaminants present in foods. A relatively new analytical technique that addresses this need is an immunobiosensor based on surface plasmon resonance (SPR) measurements. Although a range of tests have been developed to measure residues in milk, meat, animal bile and honey, a considerable problem has been encountered with both serum and plasma samples. The high degree of non-specific binding of some sample components can lead to loss of assay robustness, increased rates of false positives and general loss of assay sensitivity. In this paper we describe a straightforward precipitation technique to remove interfering substances from serum samples to be analysed for veterinary anthelmintics by SPR. This technique enabled development of an assay to detect a wide range of benzimidazole residues in serum samples by immunobiosensor. The limit of quantification was below 5 ng/ml and coefficients of variation were about 2%.

Determination of carazolol residues in porcine tissue by radioreceptor assay

A radioreceptor assay was developed for the determination of the p-blocker carazolol in porcine muscle and kidney. The method involves a simple alkaline extraction procedure using diethyl ether followed by a competitive assay between carazolol residues and [H-3]-dihydroalprenolol ([H-3]-DHA) using solubilised beta2-adrenoceptors isolated from a transfected cell line. The limit of detection (LOD) was determined using 20 reference blank samples of pig kidney and pig muscle. LODs for muscle (0.93 mug kg(-1)) and kidney (1.47 mug kg(-1)) were well below their respective European community maximum residue limits, (MRLs 5 and 25 mug kg(-1), respectively). The assay was used to investigate if carazolol residues persisted in pig tissues for up to 30 h post-intramuscular injection at the recommended dose rate (10 mug carazolol/kg body weight). The highest mean +/- S.D. concentrations were detected at 1 h post-injection in kidney (10.84 +/- 1.3 mug kg(-1)) and muscle (3.59 +/- 0.2 mug kg(-1)) which were less than the respective MRLs. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of carazolol residues in pig meat. (C) 2002 Elsevier Science B.V. All rights reserved.

Detection of streptomycin residues in whole milk using an optical immunobiosensor

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples (similar to3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).

Detection of unwanted residues of ivermectin in bovine milk by dissociation-enhanced lanthanide fluoroimmunoassay

Avermectins are frequently used to control parasitic infestations in many animal species. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. An immunoassay screening test for the detection acid quantification of ivermectin residues in bovine milk has been developed. After an extensive extraction procedure, milk samples were applied to a competitive dissociation-enhanced lanthanide fluoroimmunoassay using a monoclonal antibody against an ivermectin-transferrin conjugate, The monoclonal antibody, raised in Balb C mice, showed cross-reactivity with eprinomectin (92%), abamectin (82%) and doramectin (16%). The limit of detection of the assay (mean + 3 SD), calculated from the analysis of 17 known negative samples, was calculated as 4.6 ng/mL. Intra- and inter-assay RSDs were determined as 11.6% and 15.8%, respectively, using a negative bovine milk sample fortified with 25 ng/mL ivermectin. Six Friesian milking cows were treated with ivermectin, three with a pour-on formulation of the drug and three with an injectable solution at the manufacturer's recommended dose rate. An initial mean peak in ivermectin residue concentration was detected at day 4 (mean level = 47.5 ng/mL) and day 5 post-treatment (mean level = 26.4 ng/mL) with the injectable form and pour-on treatment, respectively. A second peak in residue concentration was observed using the DELFIA(R) procedure 28 days post-treatment in both treatment groups (23.1 ng/mL injectable and 51.9 ng/mL pour-on). These second peaks were not confirmed by HPLC and must at this Lime be considered to be false-positive results. By day 35 after treatment the mean ivermectin residue concentration of both groups fell below the limit of detection of the assay. Copyright (C) 2000 John Wiley & Sons, Ltd.

Detection of monensin residues in poultry liver using an enzyme immunoassay

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for the detection and quantification of monensin residues in liver has been developed, Samples (3 g) were extracted with acetonitrile-water and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised against a monensin-transferrin conjugate, The limit of detection (mean + 3s) calculated from the analysis of 12 known negative samples was 2.91 ng g(-1). Intra- and inter-assay RSD were determined as 8.5 and 10.6%, respectively, using a liver sample fortified with 20 ng g(-1) monensin, A pharmacokinetic study in which 70 six week old broilers were fed monensin at a rate of 120 mg kg(-1) in their feed for 14 d resulted in mean monensin liver residues of 102 ng g(-1). However these had fallen below the limit of detection of the assay within the 3 d withdrawal period recommended by the manufacturer.

Detection of ivermectin residues in bovine liver using an enzyme immunoassay

Ivermectin, a member of the avermectin group, is frequently used to control parasites in many food producing animal species. A method for the detection and quantification of ivermectin residues in bovine liver has been developed. Liver samples (4 g) were extracted with acetonitrile and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised in rabbits against an ivermectin-transferrin conjugate, The limit of detection of the assay (mean +/- 3s) calculated from the analysis of 24 known negative samples was 1.6 ng g(-1), Intra- and inter-assay RSDs were determined as 8.8 and 14.6%, respectively, using a negative bovine liver sample fortified with 100 ng g(-1) of ivermectin. Four Friesian steers were treated with a pour-on application of ivermectin at a dose rate of 0.5 mg kg(-1) body mass then withdrawn and killed at 7, 14, 21 and 28 d, Livers mere removed and ivermectin residue concentrations determined using the proposed immunoassay procedure, Seven days post-treatment the ivermectin liver concentration was determined as 52.7 ng g(-1), decreasing to 4.1 ng(-1) at 28 d, All immunoassay results were confirmed using high-performance liquid chromatography (HPLC), The immunoassay and HPLC results for invermectin ranged from 1 to 58 ng g(-1) and were in close correlation (r = 0.99).

A simple competitive enzyme immunoassay for the detection of the trypanocidal drug isometamidium

A new competitive enzyme immunoassay technique has been developed for the determination of concentrations of the trypanocidal drug isometamidium chloride (Samorin) in bovine serum. The method has been shown to be highly repeatable and reproducible, and it has several advantages over previous immunoassay techniques for the drug. There are fewer incubation steps overall; microtitre plates may be of coated in batches and stored frozen for future use; and the competition incubation is overnight and is followed only by a brief colour development stage of 10 min. Coefficients of variation (CVs) of duplicate samples were similar to 5%, and mean response variances of untreated cattle (n = 57) were small (CV, 10%). Partitioning of variance showed 77% of this variability to be intrinsic to the samples, and the remaining 23% was due to the procedure. The limit of detection was approximately 0.5 ng ml(-1), which was considered to be satisfactory for the intended use of the method. The drug could be detected in serum of treated cattle for up to 10 weeks following treatment, and determinations showed a high level of reproducibility.

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

Mass limits for the progenitor star of supernova 2001du and other Type II-P supernovae

The supernova SN 2001du was discovered in the galaxy NGC 1365 at a distance of 19 +/- 2 Mpc, and is a core-collapse event of Type II-P. Images of this galaxy, of moderate depth, have been taken with the Hubble Space Telescope approximately 6.6 yr before discovery and include the supernova position on the WFPC2 field of view. We have observed the supernova with the WFPC2 to allow accurate differential astrometry of SN 2001du on the pre-explosion frames. As a core-collapse event it is expected that the progenitor was a massive, luminous star. There is a marginal detection (3sigma) of a source close to the supernova position on the pre-discovery V -band frame, but it is not precisely coincident and we do not believe it to be a robust detection of a point source. We conclude that there is no stellar progenitor at the supernova position and derive sensitivity limits of the pre-discovery images that provide an upper mass limit for the progenitor star. We estimate that the progenitor had a mass of less than 15 M-circle dot . We revisit two other nearby Type II-P supernovae that have high-quality pre-explosion images, and refine the upper mass limits for the progenitor stars. Using a new distance determination for SN 1999gi from the expanding photosphere method, we revise the upper mass limit to 12 M-circle dot . We present new HST images of the site of SN 1999em, which validate the use of lower spatial resolution ground-based images in the progenitor studies and use a new Cepheid distance to the galaxy to measure an upper mass limit of 15 M-circle dot for that progenitor. Finally we compile all the direct information available for the progenitors of eight nearby core-collapse supernovae and compare their mass estimates. These are compared with the latest stellar evolutionary models of pre-supernova evolution which have attempted to relate metallicity and mass to the supernovae type. Although this is statistically limited at present, reasonable agreement is already found for the lower-mass events (generally the II-P), but some discrepancies appear at higher masses.

Controlling the divergence of high harmonics from solid targets: a route toward coherent harmonic focusing

Harmonic generation from relativistically oscillating plasma surfaces formed during the interaction of high contrast lasers with solid-density targets has been shown to be an efficient source of extreme ultraviolet (XUV) and X-ray radiation. Recent work has demonstrated that the exceptional coherence properties of the driving laser can be mirrored in the emitted radiation, permitting diffraction limited performance and attosecond phase locking of the harmonic radiation. These unique properties may allow the coherent harmonic focusing (CHF) of high harmonics generated from solid density targets to intensities on the order of the Schwinger limit of 10(29) W cm(-2) with laser systems available in the near future [Phys. Rev. Lett. 93, 115002 (2004)] and thus pave the way for unique experiments exploring the nonlinear properties of vacuum on ultra-fast timescales. In this paper we investigate experimentally as well as numerically the prospect of focusing high harmonics under realistic experimental conditions and demonstrate, using particle in cell (PIC) simulations, that precise control of the wavefronts and thus the focusability of the generated harmonics is possible with pre-shaped targets.

Transport Properties Investigation of Aqueous Protic Ionic Liquid Solutions through Conductivity, Viscosity, and NMR Self-Diffusion Measurements

We present a study on the transport properties through conductivity (s), viscosity (?), and self-diffusion coefficient (D) measurements of two pure protic ionic liquids—pyrrolidinium hydrogen sulfate, [Pyrr][HSO4], and pyrrolidinium trifluoroacetate, [Pyrr][CF3COO]—and their mixtures with water over the whole composition range at 298.15 K and atmospheric pressure. Based on these experimental results, transport mobilities of ions have been then investigated in each case through the Stokes–Einstein equation. From this, the proton conduction in these PILs follows a combination of Grotthuss and vehicle-type mechanisms, which depends also on the water composition in solution. In each case, the displacement of the NMR peak attributed to the labile proton on the pyrrolidinium cation with the PILs concentration in aqueous solution indicates that this proton is located between the cation and the anion for a water weight fraction lower than 8%. In other words, for such compositions, it appears that this labile proton is not solvated by water molecules. However, for higher water content, the labile protons are in solution as H3O+. This water weight fraction appears to be the solvation limit of the H+ ions by water molecules in these two PILs solutions. However, [Pyrr][HSO4] and [Pyrr][CF3COO] PILs present opposed comportment in aqueous solution. In the case of [Pyrr][CF3COO], ?, s, D, and the attractive potential, Epot, between ions indicate clearly that the diffusion of each ion is similar. In other words, these ions are tightly bound together as ion pairs, reflecting in fact the importance of the hydrophobicity of the trifluoroacetate anion, whereas, in the case of the [Pyrr][HSO4], the strong H-bond between the HSO4– anion and water promotes a drastic change in the viscosity of the aqueous solution, as well as on the conductivity which is up to 187 mS·cm–1 for water weight fraction close to 60% at 298 K.