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ABSTRACT: Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with considerably less fluid volume into a measuring chamber. Once there SEMs are held in a monolayer by a magnet. Light-emitting diodes (LEDs) are focused on the chamber to illuminate the SEMs - instead of lasers and they are imaged by a charge-coupled device (CCD) detector, offering a more compact sized, transportable and affordable system. The feasibility of utilising this system to develop a 3-plex SEMs-based imaging immunoassay (IMIA) for the screening of persistent organic pollutants (POPs) was studied. Moreover the performance characteristics of 3-plex IMIA were critically compared with the conventional 3-plex flow cytometric immunoassay (FCIA). Both SEM technologies have potential for the multiplex analysis of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) in buffer and fish extract with insignificant differences in assay sensitivities. Furthermore, we developed a faster and simpler, modified QuEChERS-like generic POPs extraction from tilapia fillet using sodium hydrogen carbonate as one of the salt additives and dispersive solid-phase extraction (dSPE) as a clean-up. Finally, a preliminary in-house validation using 40 different blank and spiked tilapia fillet samples was performed in both systems and the results obtained were critically compared. The lower-cost imaging SEMs-based system performed similarly to the original flow cytometer and, in combination with the new quicker QuEChERS-like extraction, it has high potential for future rapid screening of POPs in several other sample matrices such as other fish species, vegetable refined oils and environmental samples.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 04/2013;
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ABSTRACT: A generic high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of known and unknown recombinant human sex hormone-binding globulin (rhSHBG)-binding designer steroids in dietary supplements. For screening, a semi-automated competitive inhibition binding assay was combined with fast ultrahigh-performance-LC-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS). 17β-Testosterone-D3 was used as the stable isotope label of which the binding to rhSHBG-coated paramagnetic microbeads was inhibited by any other binding (designer) steroid. The assay was performed in a 96-well plate and combined with the fast LC-MS, 96 measurements could be performed within 4 h. The concentration-dependent inhibition of the label by steroids in buffer and dietary supplements was demonstrated. Following an adjusted bioaffinity isolation procedure, suspect extracts were injected into a chip-UPLC(NanoTile)-Q-time-of-flight-MS system for full-scan accurate mass identification. Next to known steroids, 1-testosterone was identified in three of the supplements studied and the designer steroid tetrahydrogestrinone was identified in a spiked supplement. The generic steroid-binding assay can be used for high-throughput screening of androgens, estrogens, and gestagens in dietary supplements to fight doping. When combined with chip-UPLC-MS, it is a powerful tool for early warning of unknown emerging rhSHBG bioactive designer steroids in dietary supplements.
Analytical Chemistry 03/2013; · 5.86 Impact Factor
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ABSTRACT: Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively.
Analytical and Bioanalytical Chemistry 08/2012; 404(5):1361-73. · 3.78 Impact Factor
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ABSTRACT: For the analysis of thyroid transporter ligands, a triple bioaffinity mass spectrometry (BioMS) concept was developed, with the aim at three different analytical objectives: rapid screening of any ligand, confirmation of known ligands in accordance with legislative requirements, and identification of emerging yet unknown ligands. These three purposes share the same biorecognition element, recombinant thyroid transport protein transthyretin (rTTR), and dedicated modes of liquid chromatography-mass spectrometry (LC-MS). For screening, a rapid and radiolabel-free competitive inhibition MS binding assay was developed with fast ultrahigh performance-liquid chromatography-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS) as the readout system. It uses the nonradioactive stable isotopic thyroid hormone (13)C(6)-L-thyroxine as the label of which the binding to rTTR is inhibited by any ligand such as thyroid drugs and thyroid endocrine disrupting chemicals (EDCs). To this end, rTTR is either used in solution or immobilized on paramagnetic microbeads. The concentration-dependent inhibition of the label by the natural thyroid hormone l-thyroxine (T4), as a model analyte, is demonstrated in water at part-per-trillion and in urine at part-per-billion level. For confirmation of identity of known ligands, rTTR was used for bioaffinity purification for confirmation of naturally present free T4 in urine. As a demonstrator for identification of unknown ligands, the same rTTR was used again but in combination with nano-UPLC-quadrupole time-of-flight-MS (nLC-Q-TOF-MS) and urine samples spiked with the model "unknown" EDCs triclosan and tetrabromobisphenol-A. This study highlights the potential of BioMS using one affinity system, both for rapid screening and for confirmation and identification of known and unknown emerging thyroid EDCs.
Analytical Chemistry 06/2012; 84(15):6488-93. · 5.86 Impact Factor
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ABSTRACT: Persistent organic pollutants (POPs) are environmental and food-related contaminants of global public health concern and known to be carcinogenic and endocrine disruptors. Their monitoring is essential, and an easy-to-use, rapid, and affordable multianalyte screening method with simplified sample preparation can be a valuable tool prior to instrumental analysis. For this purpose, a flow cytometric immunoassay (FCIA), based on a spectrally encoded microbeads technology, was developed for the multiplex detection of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (BDEs) in buffer and fish extracts. The sensitivities of the assays in the three-plex FCIA format were similar to the individual FCIAs for the marker compounds benzo[a]pyrene (BaP), 3,3',4,4'-tetrachlorobiphenyl (PCB77), and 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in buffer with IC(50) values of 0.4, 20, and 2 μg L(-1), respectively. Apart from the three markers, we could detect at least 14 other POPs. Extracts of fish with different fat content, prepared with a simplified extraction and cleanup procedure, had an insignificant influence on the overall three-plex FCIA performance, with the exception of some impact on the PAHs detection. The performance of the three-plex FCIA, in combination with the simple extraction procedure, is adequate for regulatory control in accordance with the required limits.
Analytical Chemistry 10/2011; 83(22):8696-702. · 5.86 Impact Factor
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ABSTRACT: A prototype imaging surface plasmon resonance-based multiplex microimmunoassay for mycotoxins is described. A microarray of mycotoxin-protein conjugates was fabricated using a continuous flow microspotter device. A competitive inhibition immunoassay format was developed for the simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN), using a single sensor chip. Initial in-house validation showed limits of detection of 21 and 17 ng/mL for DON and 16 and 10 ng/mL for ZEN in extracts, which corresponds to 84 and 68 μg/kg for DON and 64 and 40 μg/kg for ZEN in maize and wheat samples, respectively. Finally, the results were critically compared with data obtained from liquid chromatography-mass spectrometry confirmatory analysis method and found to be in good agreement. The described multiplex immunoassay for the rapid screening of several mycotoxins meets European Union regulatory limits and represents a robust platform for mycotoxin analysis in food and feed samples.
Analytical and Bioanalytical Chemistry 07/2011; 400(9):3005-11. · 3.78 Impact Factor
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ABSTRACT: Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography-quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants.
Analytical and Bioanalytical Chemistry 07/2011; 400(9):3085-96. · 3.78 Impact Factor
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ABSTRACT: A multi-mycotoxin immunoassay-using the MultiAnalyte Profiling (xMAP) technology-is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, zearalenone and T-2-toxin in an inhibition immunoassay format. Sets of six mycotoxin-protein conjugates and six specific monoclonal antibodies were selected, and we observed good sensitivities and no cross-interactions between the assays in buffer. However, detrimental effects of the feed extract on the sensitivities and in some cases on the slopes of the curves were observed and different sample materials showed different effects. Therefore, for quantitative analysis, this assay depends on calibration curves in blank matrix extracts or on the use of a suitable multi-mycotoxin cleanup. To test if the method was suitable for the qualitative detection at EU guidance levels, we fortified rapeseed meal, a feed ingredient, with the six mycotoxins, and all extracts showed inhibited responses in comparison with the non-fortified sample extract. Contaminated FAPAS reference feed samples assigned for a single mycotoxin showed strong inhibitions in the corresponding assays but also often in other assays of the multiplex. In most cases, the presence of these other mycotoxins was confirmed by instrumental analysis. The multiplex immunoassay can be easily extended with other mycotoxins of interest, but finding a suitable multi-mycotoxin cleanup will improve its applicability.
Mycotoxin Research 02/2011; 27(1):63-72.
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Anke K Trilling,
Hans de Ronde,
Linda Noteboom,
Adèle van Houwelingen,
Margriet Roelse,
Saurabh K Srivastava, Willem Haasnoot,
Maarten A Jongsma,
Arend Kolk,
Han Zuilhof,
Jules Beekwilder
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ABSTRACT: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.
Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4 × 10(-10) M.
A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.
PLoS ONE 01/2011; 6(10):e26754. · 4.09 Impact Factor
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ABSTRACT: Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC(50) of 0.3 microg L(-1) using a monoclonal antibody (Mab22F12) against BaP, similar to the IC(50) of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are under development.
Analytica chimica acta 07/2010; 672(1-2):9-14. · 4.31 Impact Factor
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ABSTRACT: The European Union has banned the use of recombinant bovine somatotropins (rbST, growth hormones) to increase milk yield in dairy cattle. As direct detection of rbST in serum is problematic, methods based on the detection of changes in multiple rbST-dependent biomarkers have high potential for monitoring rbST abuse. In this study immunoassays were developed for total insulin-like growth factor 1 (IGF-1) in cow sera. Ultimately aiming at combination with other rbST-dependent biomarker assays two multiplex formats were studied and compared critically, a multi-channel surface plasmon resonance (SPR)-based biosensor and flow cytometry combined with color encoded microbeads. Moreover, a new dedicated sample pretreatment was developed for the dissociation of complexed IGF-1 in serum, while keeping other biomarkers in solution. Compared to the SPR biosensor immunoassay, the flow cytometric immunoassay (FCIA) was more sensitive, less antibody-consuming and less vulnerable to necessary but interfering reagents from the sample treatment. In an initial in-house validation study the developed FCIA showed to be fast, specific, robust, and a high repeatability and reliability, and generated realistic IGF-1 values for bovine serum, without compromising the potential for simultaneous detection of other biomarkers. Due to the xMAP technology, in which 100 different bead sets can be measured simultaneously, the total IGF-1 assay can easily be extended with other immunoassays for candidate biomarkers. Preliminary results about a FCIA for IGF-1 multiplexing with insulin-like growth factor binding protein 2 (IGFBP2) are presented which strongly supported both the FCIA multiplex format as well as the generic nature of the developed sample pretreatment.
The Analyst 05/2010; 135(5):1147-52. · 4.23 Impact Factor
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ABSTRACT: Monitoring of antimicrobial drug residues in foods relies greatly on the availability of adequate analytical techniques. Currently, there is a need for a high-throughput screening method with a broad-spectrum detection range. This paper describes the development of a microarray biosensor, based on an imaging surface plasmon resonance (iSPR) platform, for quantitative and simultaneous immunodetection of different antibiotic residues in milk. Model compounds from four major antibiotic families: aminoglycosides (Neomycin, Gentamicin, Kanamycin, and Streptomycin), sulfonamides (Sulfamethazine), fenicols (Chloramphenicol), and fluoroquinolones (Enrofloxacin) were detected using a single sensor chip. By multiplexing seven immunoassays in a competitive format, we were able to measure all the target compounds at parts per billion (ppb) levels in buffer and in 10x-diluted milk. The assays for Neomycin, Kanamycin, Streptomycin, Enrofloxacin, and Sulfamethazine were sensitive enough for milk control at maximum residue levels as established in the European Union. The overall performance of the biosensor was determined to be comparable to that of conventional four-channel surface plasmon resonance (SPR)-based biosensors, in terms of assay sensitivity and robustness. Combining the advantages of a SPR sensor and a microarray, utilization of the biosensor described here offers a promising alternative to the existing methods and is highly relevant for multianalyte food profiling.
Analytical Chemistry 09/2009; 81(18):7743-9. · 5.86 Impact Factor
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ABSTRACT: The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct biosensor immunoassay, based on a highly specific monoclonal antibody, was developed to detect the presence of hazelnut proteins in olive oils. The sample preparation was easy (extraction with buffer); the assay time was fast (4.5 min only) and the limit of detection was low (0.08 microg/g of hazelnut proteins in olive oil). Recoveries obtained with an olive oil mixed with different amounts of a hazelnut protein containing hazelnut oil varied between 93% and 109%.
Analytical and Bioanalytical Chemistry 04/2009; 395(1):119-26. · 3.78 Impact Factor
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ABSTRACT: Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two inhibition assays using the main thyroid hormone transport proteins, T4 binding globulin (TBG) and transthyretin (TTR), in combination with a T4-coated biosensor chip were optimized and automated for screening chemical libraries. The transport protein-based biosensor assays were rapid, high throughput and bioeffect-related. A library of 62 chemicals including the natural hormones, polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals was tested with the two assays. We discovered ten new active compounds with moderate to high affinity for TBG with the TBG assay. Strikingly, the most potent binding was observed with hydroxylated metabolites of the brominated diphenyl ethers (BDEs) BDE 47, BDE 49 and BDE 99, that are commonly found in human plasma. The TTR assay confirmed the activity of previously identified hydroxylated metabolites of PCBs and PBDEs, halogenated BPA and genistein. These results show that the hydroxylated metabolites of the ubiquitous PBDEs not only target the T4 transport at the TTR level, but also, and to a great extent, at the TBG level where most of the T4 in humans is circulating. The optimized SPR biosensor-based transport protein assay is a suitable method for high throughput screening of large libraries for potential thyroid hormone disrupting compounds.
Toxicology and Applied Pharmacology 10/2008; 232(1):150-60. · 4.45 Impact Factor
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ABSTRACT: Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this automated FCI, a previously developed biotinylated multi-sulfonamide mutant antibody (M.3.4) was applied in combination with fluorescent beads, directly coated with a sulfathiazole derivative, and streptavidin-phycoerythrin (SAPE) for the detection. With this FCI, at least 11 different sulfonamides could be detected (more than 50% inhibition at the 100 ng mL(-1) level) and, after an incubation of 1h, measurements were rapid (10s per sample). For the application with raw milk, a 96-well microplate-based filtration step was included into the protocol to remove disturbing milk fat particles. Because of differences in sensitivity towards different sulfonamides, the FCI was considered and validated as a qualitative screening assay. For sulfadoxine, the most applied sulfonamide in Dutch dairy cattle, the detection capability (CCbeta) was <50 microg L(-1) and this level seems feasible for five other sulfonamides. For sulfadiazine, the CCbeta was <200 microg L(-1) and this level seems feasible for four other sulfonamides. A major advantage of the applied xMAP-technology, with its 100 different color-coded bead sets, is the possibility to develop multiplex immunoassays for the simultaneous detection of several antibiotics.
Analytica chimica acta 07/2008; 620(1-2):142-9. · 4.31 Impact Factor
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ABSTRACT: An automated fluorescent microsphere-based flow cytometric triplex immunoassay, using the Luminex 100 flow analyzer with MultiAnalyte Profiling (xMAP) technology, was developed for the simultaneous detection of proteins from three vegetable sources as potential fraudulent adulterants in milk powder. In the final triplex inhibition immunoassay, soluble wheat proteins (SWP) and proteins from soy and pea were coupled to three different microsphere sets. A mixture of these microsphere sets was transferred to a microtiter plate well together with the sample and a mixture of three affinity-purified polyclonal antibodies raised against the proteins and labeled with a fluorophore (Alexa 532). After incubation for 1.5 h at room temperature in the dark, the fluorescence intensities on the microspheres were directly measured (no wash procedure) in the Luminex during 10 s per well (100 microspheres per set). The sensitivities of the three assays for plant protein extracts were determined as 0.5-0.6 microg/mL at 50% inhibition. For the detection of the vegetable proteins in milk powder, the samples were dissolved in buffer (0.1 g in 10 mL) and further diluted (20 times) to create a 50% inhibition at approximately 0.5% of the vegetable proteins in the total protein content of milk powder. With the help of calibration standards, prepared under conditions comparable to those for sample materials, the triplex immunoassay proved to be quantitative above 0.1%, although concentrations in high-heated milk powders were underestimated. Due to the xMAP technology, in which 100 different microsphere sets can be distinguished, this triplex immunoassay can easily be extended to detect other possible adulterants.
Journal of Agricultural and Food Chemistry 06/2007; 55(10):3771-7. · 2.82 Impact Factor
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ABSTRACT: Flumequine (Flu) is one of the fluoroquinolones most frequently applied for the treatment of broilers in The Netherlands. For the detection of residues of Flu in blood serum of broilers, a biosensor immunoassay (BIA) was developed which was fast (7.5 min per sample) and specific (no cross-reactivity with other (fluoro)quinolones). This inhibition assay was based on a rabbit polyclonal anti-Flu serum and a CM5 biosensor chip coated with Flu which could be detected in the range of 15-800 ng mL(-1). For the detection of Flu in muscle, an easy extraction procedure in buffer was selected and the measuring range was from 24 to 4000 ng g(-1). Average recoveries of 66 till 75% were found with muscle samples spiked at 0.5, 1 and 2 times the maximum residue limit (MRL in muscle = 400 ng g(-1)) and the decision limit (CCalpha) and the detection capability (CCbeta) were determined as 500 and 600 ng g(-1), respectively. Incurred muscle samples were analysed by the BIA and by LC-MS/MS and a good correlation was found (R2 = 0.998). Serum and muscle samples from with Flu treated broilers were analysed and the concentrations found in serum were always higher than those found in muscle (average serum/muscle ratio was 3.5) and this proved the applicability of the BIA in serum as predictor of the Flu concentration in muscle.
Analytica chimica acta 04/2007; 586(1-2):312-8. · 4.31 Impact Factor
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ABSTRACT: Fluoroquinolones (FQs) are synthetic antibiotics of broad-spectrum antibacterial activity widely used to treat infections in farmed fish, turkeys, pigs, calves and poultry. Monitoring these substances residues is therefore regulated by law. For the detection of FQs, we studied the feasibility of coupling the simultaneous screening of several FQs, using a dual surface plasmon resonance (SPR) biosensor immunoassay (BIA), in parallel, with an analytical chemical methodology for their identification. Six FQs were simultaneously screened at or below their maximum residue level (MRL) in chicken muscle using a multi-FQ BIA for norfloxacin, ciprofloxacin, enrofloxacin, difloxacin and sarafloxacin, and a specific BIA for flumequine. The two BIAs were serially coupled in a multi-channel SPR biosensor featuring a dual BIA in a competitive inhibition format. The samples non-compliant during the screening with the dual BIA were further concentrated and fractionated with gradient liquid chromatography (LC). The effluent was splitted toward two 96-well fraction collectors resulting in two identical 96-well plates. One was re-screened with the dual BIA to identify the immunoactive fractions and direct the identification efforts toward the relevant fractions in the second well-plate with high resolution LC-electrospray time-of-flight mass spectrometry (ESI-TOFMS). The system not only allows the possibility to screen and identify known FQs, but also to discover unknown chemicals of similar structure which show activity in the dual BIA.
Analytica chimica acta 04/2007; 586(1-2):259-68. · 4.31 Impact Factor
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ABSTRACT: The use of growth hormones (recombinant somatotropins (rSTs)) is approved in several countries, e.g. the USA, Brazil and Australia to enhance growth or lactating performances of livestock. Their use in the EU is banned, however, due to the widespread application, the illegal use within the EU cannot be excluded. To screen for rSTs in injection preparations, a biosensor immunoassay (BIA) using surface plasmon resonance (SPR) technology was developed. Compared to existing analysis methods for rSTs, like radio immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), this technique provides a rapid (7 min) alternative. A direct BIA was compared to an indirect (inhibition) BIA and the performances of several antibodies against (r)STs were compared in the indirect BIA. In the final inhibition assay, using rabbit anti-bovine rST, extracts from several injection preparations were shown to contain bovine rST (rbST). The limit of detection for rbST in the assay is 0.008 microg mL(-1) which is far below the expected concentrations in injection preparations. Although the cross-reactivities for STs of other species were low, screening of injection preparations for porcine, equine and human ST was feasible through the analysis of less diluted extracts. Tryptic digestion followed by nano-electrospray liquid chromatography-ion trap tandem mass spectrometry (nano-LC-MS/MS) was used to identify STs.
Analytica chimica acta 04/2007; 586(1-2):239-45. · 4.31 Impact Factor
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ABSTRACT: Novel surface plasmon resonance-based biosensor assays for the bioeffect-related screening of chemicals with thyroid-disrupting activity are described. Two thyroid transport proteins (TPs), thyroxine binding globulin (TBG) and recombinant transthyretin (rTTR), were applied in an inhibition assay format in a Biacore 3000 using CM5 biosensor chips coated with l-thyroxine (T4), the main hormone of the thyroid system. Assay conditions were optimized for the natural thyroid hormones, and known thyroid disruptors and structurally related compounds were selected as model compounds to be tested in both assays for their relative potency (RP) compared to T4. The chosen compounds were halogenated phenols, halogenated bisphenols, bisphenol A, 3,5-dichlorobiphenyl, and its hydroxylated metabolite 4-hydroxy-3,5-dichlorobiphenyl (4-OH PCB 14). The TBG-based assay was highly specific for T4, and the rTTR-based assay was sensitive toward several compounds, the highest sensitivity (RP = 4.4) being obtained with 4-OH PCB 14, followed by tetrabromobisphenol A (RP = 1.5) and tetrachlorobisphenol A (RP = 0.75). For the bioeffect-related screening of known and identification of possible new thyroid disruptors, the TPs-based biosensor assays were more sensitive (IC(50) of 13.7 +/- 1.3 and 8.6 +/- 0.7 nM for the rTTR and the TBG-based assay, respectively), easier to perform, and faster alternatives (10 min/sample) than the currently used methods such as radioligand binding assays and immunoprecipitation-HPLC.
Analytical Chemistry 03/2006; 78(4):1107-14. · 5.86 Impact Factor
