Immunobiosensor analysis- of clenbuterol in bovine hair

Due to the potential speed and flexibility of surface plasmon resonance (SPR)-biosensor analysis, the technique is currently being exploited for detection of residues in food. In the present study an antibody-based inhibition assay for analysis of clenbuterol in cattle hair was developed using a Biacore 2000 instrument. Analysis of clenbuterol with and without enhancement with a secondary antibody was compared. A fast extraction method for hair, which enabled direct analysis in the biosensor with no clean up other than microscale ultrafiltration was developed. In contrast to most of the published methods in the area, no organic solvent or solid phase extraction step was needed. The biosensor method was validated by analysis of hair samples from animals treated with clenbuterol and the results were compared to results from GC-MS analysis. The correlation was good (r ²=92.7%). The detection limit of the new method was 10 ng g^?1 hair.     

Long-term detection of clenbuterol in human scalp hair by gas chromatography-high-resolution mass spectrometry

A method for the detection of clenbuterol in human scalp hair by gas chromatography-high-resolution mass spectrometry (GC-HRMS) is described. The sample preparation involved chemical digestion of the protein structure, which was achieved by incubating the hair with 1 M KOH at 70 degrees C. A single extraction step with tert.-butyl methyl ether provided approximately 90% of the analyte, which was dried and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) to yield clenbuterol N,O-bis-trimethylsilyl (TMS). Hair was collected from four pregnant women who were therapeutically treated with Spiropent (clenbuterol-HCl) and from the infant of one female patient. Hair samples were taken during the application time and two to six months after completion of clenbuterol administration. The detection limit of the method was approximately 4 ng clenbuterol/g hair when 25 mg hair material were processed and 2 ng/g for 50 mg hair samples (corresponds to 4 pg per injection). The method allows clenbuterol to be measured retrospectively for up to at least six months. The levels of clenbuterol determined in hair ranged from 2 to 236 ng/g. No clenbuterol was found in the hair of the infant, which was taken five and a half months after delivery. To improve sample preparation, an additional purification step via immuno affinity chromatography (IAC) was integrated. The IAC purified extracts showed reduced biological background interference and an improved limit of detection (0.8 ng/g).     

Determination of clenbuterol residues in bovine hair by using diphasic dialysis and gas chromatography-mass spectrometry.

A method for the determination of clenbuterol (4-amino-3,5-dichloro-alpha[(tert.-butylamino)methyl]-benzyl alcohol hydrochloride) in hair of living cows has been developed. Hair samples were digested in an alkaline medium. The diphasic dialysis technique is a semi-permeable membrane technology developed for the direct extraction of relatively low-molecular-mass analytes such as clenbuterol. In this case, we used sodium citrate buffer to homogenize the digested hair, dichloromethane was used as the extraction solvent at 37 degrees C, and stirring was applied at 150 rpm for 4 h. The analysis was carried out using gas chromatography-mass spectrometry. The calibration curve for clenbuterol in hair was linear in the range from 12.5 to 400 ng g(-1). The detection limit of clenbuterol was 5 ng g(-1) and the quantification limit was 12.5 ng g(-1), in hair. A good inter-day reproducibility was obtained (R.S.D. = 7.08%). The repeatability and intra-day reproducibility (50 ng g(-1) of hair, n = 10) show R.S.D.s of 7.1 and 9.5%, respectively.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Determination of DDT in model samples by using solid-phase extraction and indirect competitive enzyme immunoassay

Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol l^-1 and 4.2 nmol l^-1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9 mg·ml^-1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3 nmol l^-1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C₁₈ columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0-161.0%, and 81.5-111.6% at standard addition 50 μg·kg^-1, and 200 μg·kg^-1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg^-1.     

Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development

Green fluorescent protein (GFP) transgenic (GFP⁺) mice express GFP in most tissues except erythrocytes and hair. Immune responses of GFP⁺ mouse and their application to studies of lymphocyte development were investigated. Flow cytometric analyses revealed that differentiation patterns of lymphocytes from GFP⁺ mice are equivalent to those from parental C57BL/6 mice. There was no difference in mature T-cell proliferative ability in response to allogeneic stimulator cells or anti-CD3 stimulation between GFP⁺ and C57BL/6 mice. Furthermore, the anti-OVA antibody response of GFP⁺ mice was also the same as that of C57BL/6 mice. Taken together, these results show no immunological differences between GFP⁺ and C57BL/6 mice. Bone marrow transplantation and in vitro thymus reconstitution experiments were performed in an attempt to apply the GFP⁺ mice to the analysis of lymphocyte development. When bone marrow cells from GFP⁺ mice were transplanted, T and B lymphocytes containing GFP developed normally in scid recipients. Next we examined intrathymic T-cell development by hanging drop culture methods. GFP⁺ and CD4⁺8⁺ immature T-cells developed normally from bone marrow cells in the reconstituted thymus. The experimental system using hematopoietic cells from GFP⁺ mice is a powerful tool for visualizing lymphocyte development.     

Determination of clenbuterol in bovine urine using gas chromatography-mass spectrometry following clean-up on an ion-exchange resin.

A gas chromatographic-mass spectrometric method was developed for the determination of residues of clenbuterol in bovine urine. The method involves a simple cation-exchange clean-up and concentration of clenbuterol in the acidified urine, followed by ethyl acetate extraction. The analyte is determined as the di-trimethylsilyl derivative and quantitated against an internal standard of penbutolol. Using a 5-ml sample of urine, a detection limit of 0.07 ng/ml can be achieved with recoveries close to 100% for fortification levels of 0.2 and 1 ng/ml. By increasing the sample volume to 50 ml, a detection limit below 0.01 ng/ml was achievable with recovery averaging 70%. The coefficient of variation of the assay ranged from 15% at 0.01 ng/ml (50-ml sample) to 6% at 1 ng/ml (5-ml sample). It was demonstrated that the method can detect the presence of clenbuterol in bovine urine at sub-ppb (ng/ml) levels using low resolution GC-MS with electron impact (EI) ionization.     

An enzyme‐linked immunosorbent assay for the direct analysis of beta‐agonist drugs in urine and sera

A sensitive competitive solid‐phase enzyme immunoassay for the detection of tert‐butylic beta‐agonist drugs was developed. An antiserum was raised in rabbits against the diazo‐conjugate of clenbuterol and the human serum albumin. In the assay, the anticlenbuterol antibody was incubated with the salbutamol‐enzyme conjugate and unlabelled standard or samples in microtitre wells precoated with affinity‐purified antirabbit IgG. The absolute detection limit for clenbuterol and mabuterol was 0.3 ng/ml (15 pg/well) and 50% relative binding at 1.0 ng/ml (50 pg/well). The absolute detection limits for salbutamol and terbutaline were 0.9 and 1.3 ng/ml, respectively. The cross‐reactivity of iso‐propylic parent compounds was quite low (<1%). Urine and sera were tested without any extraction step. Sample blanks (96 urine and 76 sera from five different sources) showed low matrix effect, suggesting a limit of decision for bovine urine of 0.6 ng/ml clenbuterol equivalent. Mean recoveries of clenbuterol in spiked urine and serum samples ranged from 90 to 120% (mean recovery 96%). Comparative analysis by gas chromatography/mass spectrometry showed that false negative results do not occur. The assay is simple, rapid, sensitive, multiresidual, reliable and cost‐effective. The test is adaptable to high sample throughput laboratories as well as field test for rapid screening of a few samples in slaughterhouses.     

Use of an automated DNA analysis system (DARAS®) for sequence-specific recognition of Neisseria meningitidis DNA

Objectives To combine use of the polymerase chain reaction (PCR) for rapid diagnosis of meningococcal meningitis with a novel automated detection system for sequence-specific recognition of PCR products.
Methods   DNA was extracted from cerebrospinal fluid (CSF) by a quick boil-lysis method, followed by PCR with primers specific for Neisseria meningitidis . Sequence-specific recognition of N. meningitidis DNA was performed with an automated DNA analysis system (DARAS^®) and the data were compared with results following agarose gel electrophoresis or conventional microbiological culture.
Results   The DARAS^® system had a sensitive detection limit of 10² meningococci/mL with spiked samples, compared with a detection limit of 10⁴ meningococci/mL following agarose gel electrophoresis. When the system was used to examine 74 CSF samples, the 19 CSF samples positive for N. meningitidis by conventional microbiological methods were also all positive in the DARAS^® system and the 55 samples negative by DARAS^® for meningococci were also negative by conventional microbiological methods.
Conclusion The sensitivity and specificity of the DARAS^® system makes it a useful tool for the diagnosis of meningococcal meningitis. The system is user-friendly, requires minimal hands-on time and generates data in an informative numerical format.     

Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol

A surface plasmon resonance biosensor method was developed to measure zilpaterol residues in sheep urine. A CM-5 sensor chip previously reacted with ethylenediamine to produce an aminoethyl group was coupled with 4-carboxybutyl zilpaterol activated using EDC/NHS. Five polyclonal and four monoclonal antibodies were screened for their suitability to detect low levels of zilpaterol using the biosensor technology. Total binding was greater for polyclonal than monoclonal antibodies, but a less diluted antibody solution was required for polyclonal antibodies. A fixed antibody concentration and various concentrations of zilpaterol were injected to obtain a standard curve for each antibody to allow for B₀ and IC₅₀ determination. The stability of the assay was assessed by the consistency of B₀ in repeated experiments extending at least six hours. A measure of non-specific binding allowed the assessment of the specificity of the antibody-immobilized ligand interaction. The effect of varying concentrations of urine on B₀ and IC₅₀ was evaluated to assess the degree of “matrix” effect that would be present in an assay. Based on these criteria the most promising antibody (2E10, a monoclonal antibody) was selected for further evaluation. This antibody had good sensitivity with IC₅₀=4.47±0.41 ng/ml (n=11) in buffer). Both intra- and inter-assay variation studies showed excellent recovery and reproducibility for concentrations between 2 ng/ml and 8 ng/ml. A comparison of the biosensor method with a previously developed ELISA demonstrated that both methods give equivalent results (slope of the correlation plot = 1.02) with a high correlation (r²=0.91) between them.     

Quantification of κ-casein in milk by an optical immunosensor

κ-casein (κ-CN) plays an important role in the stability and coagulation properties of milk. An immunoassay to quantify κ-casein in milk, using an optical biosensor based on surface plasmon resonance (SPR) measurement, has been developed. The assay consists of a two-step sandwich strategy, with two anti-κ-casein antibodies directed against each extremity of the casein to quantify only native κ-casein. The analysis time per sample was less than ten minutes. The antibody-coated surface could be used for more than 150 determinations. The detection limit was established at 0.45 μg.ml^-1 and the intra- and inter-assay variation coefficients were 4.28% and 6.8%, respectively. The method was applied to raw milk in order to quantify intact κ-casein, with no pretreatment of the sample. It also allowed the monitoring of κ-CN concentration during milk coagulation after addition of rennet.     

Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log₁₀ 6.37 cfu ml^-1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log₁₀ 8.0 cfu ml^-1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml^-1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.     

Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals

Externally applied ATP (100 μM) induced membrane currents in type I and type II vestibular hair cells enzymatically isolated from guinea-pig semicircular canals. In whole-cell voltage-clamp and with 140 mM K⁺ in the pipette solution, ATP evoked an inwardly directed current in 58% of the cells when held at potentials below −40 mV. In the remainder, external ATP produced an outward current. After block of the K currents, an inward current activated by ATP was revealed at −50 mV. Intracellular Ca²⁺ levels were monitored using the Ca²⁺ indicator Fura-2 and were found to rise in both hair cell types in response to ATP. These results strongly suggest that ATP directly controls the entry of Ca²⁺ into crista hair cells which can then further modulate K⁺ currents.     

Dexamethasone and clenbuterol detection by enzyme immunoassay in Bovine Liver Tissue: A new multiresidue extraction procedure

A fast and simple extraction procedure was developed for simultaneous determination in bovine liver of two veterinary drugs, widely used as growth promoters in meat production: dexamethasone (a synthetic corticosteroid drug) and clenbuterol (a beta2‐adrenergic agonist drug). Liver samples were extracted by acetonitrile, without any clean‐up step. Two different ELISAs, specific for the two classes of drugs, were used to determine the residue concentration in the extracts. The intra‐ and inter‐extraction variability was determined at different concentrations: the intra‐extraction coefficients of variation (CVs) were between 2.5 and 17.7% for dexamethasone and between 0.9 and 9.8% for clenbuterol; the inter‐extraction CVs were between 2.0 and 16.8% for dexamethasone and between 0.5 and 10.8% for clenbuterol. Recovery ranged from 92 to 154% for dexamethasone and from 78 to 105% for clenbuterol. The limit of detection was 1.43 ng g^?1 and 0.43 ng g^?1, respectively. The limit of quantification for dexamethasone was 2.09 ng g^?1 and for clenbuterol was 0.72 ng g^?1. The combination of the new extraction procedure with an ELISA detection permitted the rapid semi‐quantitative determination of both dexamethasone at its maximum residue level (MRL: 2.5 ng g^?1 in liver tissue), and clenbuterol at low concentration level.     

ELISA of streptomycin in buffer and milk: Effect of reagents' structure and analysis format on assay performance

The influence of immunoreagents' structure on assay performance was investigated and a range of ELISAs for streptomycin in direct and indirect format was developed. Streptomycin was conjugated with proteins (bovine serum albumin (BSA) for immunization and ovalbumin (OVA) for immobilization on a plate) by two different methods. Streptomycin was derivatized with carboxymethoxylamine (CMO) and then coupled to a protein or the protein was activated with adipic acid dihydrazide (ADH) and then coupled with streptomycin. A conjugate with horse-radish peroxidase was synthesized using streptomycin-ADH derivative. With the indirect ELISA the most sensitive assay for polyclonal antisera against streptomycin-oxime-BSA in combination with homologous and heterologous conjugates (limit of detection 2.5 ng ml^-1) was developed, whereas for a combination 'antisera against streptomycin-ADH-BSA/heterologous conjugate' higher background level of a calibration curve was observed. Besides the level was very high (about 60%) for homologous conjugate. In a direct ELISA similar sensitivity was achieved only for antisera against streptomycin-oxime-BSA (limit of detection 3.0 ng ml^-1). Chemiluminescent detection allowed to increase the assay sensitivity by several times (limit of detection 0.5 ng ml^-1) but led to the worse reproducibility (CV 16%). A sensitive and simple direct ELISA for analysis of streptomycin in milk products without preliminary sample preparation was developed (limit of detection 3.2 ng ml^-1). In the indirect ELISA an influence of fat content of a milk product on assay performance was observed.     

Voltage-Gated Na+ Channel Activation Induces Both Action Potentials in Utricular Hair Cells and Brain-Derived Neurotrophic Factor Release in the Rat Utricle During a Restricted Period of Development

The mammalian utricular sensory receptors are commonly believed to be non-spiking cells with electrical activity limited to graded membrane potential changes. Here we provide evidence that during the first post-natal week, the sensory hair cells of the rat utricle express a tetrodotoxin (TTX)-sensitive voltage-gated Na⁺ current that displays most of the biophysical and pharmacological characteristics of neuronal Na⁺ current. Single-cell RT-PCR reveals that several α-subunit isoforms of the Na⁺ channels are co-expressed within a single hair cell, with a major expression of Nav1.2 and Nav1.6 subunits. In neonatal hair cells, 30 % of the Na⁺ channels are available for activation at the resting potential. Depolarizing current injections in the range of the transduction currents are able to trigger TTX-sensitive action potentials. We also provide evidence of a TTX-sensitive activity-dependent brain-derived neurotrophic factor (BDNF) release by early post-natal utricle explants. Developmental analysis shows that Na⁺ currents decrease dramatically from post-natal day 0 (P0) to P8 and become almost undetectable at P21. Concomitantly, depolarizing stimuli fail to induce both action potential and BDNF release at P20. The present findings reveal that vestibular hair cells express neuronal-like TTX-sensitive Na⁺ channels able to generate Na⁺-driven action potentials only during the early post-natal period of development. During the same period an activity-dependent BDNF secretion by utricular explants has been demonstrated. This could be an important mechanism involved in vestibular sensory system differentiation and synaptogenesis.     

Optical biodetection of cadmium and lead ions in water

A three layer waveguiding silicon dioxide (SiO₂)/silicon nitride (Si₃N₄)/SiO₂ structure on silicon substrate was proposed as an optically efficient biosensor for calibration of heavy metal ions in drinking water. The catalytic activities of urease and acetylcholine esterase (AchE) were inhibited by the presence of cadmium (Cd²⁺) and lead (Pb²⁺) ions. The detection limit as low as 1 ppb was achieved by employing the technique of total reflection at the interface between the Si₃N₄ core and composite polyelectrolyte self-assembled (PESA) membranes containing cyclotetrachromotropylene (CTCT) as an indicator.     

Quantitative Immunoelectrophoretic Analysis of Extract from Cow Hair and Dander

Quantitative immunoelectrophoresis used for the analysis of a diabased, centrifuged and freeze-dried extract from cow hair and dander revealed 17 antigens. Five of these were identified as serum proteins. Partial identity to antigens of serum and extract from hair and dander of goat, sheep, swine, horse, dog, cat and guinea pig, and to antigens of house dust was demonstrated. Sera from 36 patients with manifest allergy to cow hair and dander selected on the basis of case history, RAST, skin and provocation test, were examined in crossed radioimmunoelectrophoresis (CRIE); sera from five persons with high serum IgE, but without allergy to cow hair and dander, and sera from five normal individuals were controls 31/36 of the sera contained IgE with specific affinity for two of the antigens of the extract. Further, two major and six minor allergens were identified. The control sera showed no specific IgE binding. A significant positive correlation was found between RAST and CRIE for the first group of patients. The approximate molecular weights of the four major allergens obtained by means of gel chromarography were: 2.4 × 10⁴, 2 × 10⁴, 2 × 10³ dalton, respectively. Using Con-A and Con-A Sepharose in crossed immunoaffinoelectrophoresis, eight of the antigens were revealed to contain groups with affinity for Con-A.     

Sample Matrix Interference in Immunoassays for Organochlorine Residues in Plant-derived Foods and Some Strategies for Their Removal

Immunoassays for two groups of organochlorine insecticides, cyclodienes (endosulfan and heptachlor) and DDT were applied to the analysis of a diverse range of plant-derived foods. Water-miscible solvent extracts of high-moisture, low-fat foods such as cauliflower, cabbage, green and red blue grapes and tomato caused little or no interference with the assays, enabling methanol or acetonitrile extracts of the foods to be analysed directly by immunoassay, after dilution in assay buffer. Reasonable recoveries of spikes of these pesticides were obtained by direct analysis of extracts of spiked commodities, with reliable detection down to 0.025 mg kg^-1 heptachlor or endosulfan and 0.1 mg kg^-1 DDT in the commodities. Acetonitrile extracts of milk could also be analysed directly for DDT. In contrast, extracts of low moisture, non-fatty (rice) and fatty (cottonseed) food commodities interfered appreciably with the assays, reducing assay colour and detection sensitivity. Some simple cleanup methods were developed to remove interference and enable detection of spiked organochlorines in these foods. Extracts of coloured foods, such as tea, coffee and spinach caused similarly major interference in the assays, and a number of simple clean-up methods were ineffective in removing interference. However, use of an immunoaffinity chromatography method for cyclodienes enabled quantitative recoveries to be obtained in extracts of several of these foods when analysed by either ELISA or gas chromatography. Direct analysis was suited for screening purposes but immunoaffinity chromatography results were more quantitative. These results indicate that ELISAs can be applied under developing country conditions to a range of diverse foods, but that cleanup strategies need to be tailored to different types of foods.