Multiplex biosensor immunoassays for antibiotics in the food chain

The use of antibiotics in food-producing animals may result in unwanted residues in food products. The main objective of the present research was to study the development and application of fast and automated multiplex surface plasmon resonance (SPR)-based biosensor immunoassays (BIAs), based on multi-component antibodies and/or combined immunoassays in serially connected flow channels, for the detection of selected antibiotics in the food chain. The scientific challenges to deal with were: the development of multi-sulfonamide monoclonal antibodies (Mabs) against the generic structure of sulfonamides and the evaluation of mutated recombinant antibodies (Rabs) derived thereof, finding of the best BIA format with aminoglycosides as model compounds and solving foreseen matrix and combined immunoassay interferences, and to study the use of antibiotic concentrations in blood serum as predictors for concentrations in edible tissue. Broiler’s blood serum, easy to collect in slaughterhouses, was chosen for the detection of sulfonamides and quinolones which are frequently used in poultry. With a Mab raised against sulfamethazine (21C7), the BIA could detect at least eight sulfonamides in ten times diluted broiler serum with limits of detection (LODs) far below the desired detection limit. Other less performing Mabs were developed against the generic part of sulfonamides. The best Mab-producing hybridoma cell-line (27G3) was used by the University of Turku to develop better performing mutated Rabs and the mutant-based BIA in broiler serum was found to be the most sensitive towards most of the sulfonamides. The assay was fast (5 min per sample), robust (>1000 runs per chip) and the sample preparation was easy (dilution in buffer only). The Rab-based multi-sulfonamide immunoassay was applied to analyze serum samples from broilers treated with sulfamethoxazole and sulfadiazine and the concentrations found were higher than the concentrations found in tissue by LC-MS/MS. This, and the good correlation with tissue concentrations, made this assay suitable to predict levels in edible tissue. A similar result was obtained with the specific BIA for flumequine. Unique direct BIAs for the detection of aminoglycosides in milk were developed with Mab-coated chips. However, the inhibition assays with aminoglycosides on the chips were found to be more robust. For the simultaneous detection of five aminoglycosides in milk, the sensor chip surfaces in the four serially connected flow channels were covered with four aminoglycosides. In combination with a mixture of four specific antibodies, gentamicin, neomycin, kanamycin and (dihydro) streptomycin could be detected in milk far below the maximum residue limits (MRLs) and within 7 min. In conclusion, serum and milk are suitable sample materials for the biosensor detection of antibiotics in the food chain. Such assays are fast, robust, automated, easy to handle, and require simple sample preparations (dilutions in antibody-containing buffer). In principle, such assays can be combined with assays for the detection of anti-pathogens, which broadens the application area in a food safety control system. However, the four-channel biosensor systems are too limited and the antibodies too specific for the simultaneous detection of more antibiotics. More extended multiplex systems (e.g. imaging SPR sensors or multiplex flow cytometry-based systems) need to be explored in which the knowledge obtained in the present research will likely be of great value

Imaging surface plasmon resonance for multiplex microassay sensing of mycotoxins

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 sample

Development of a multiplex flow cytometric microsphere immunoassay for mycotoxins and evaluation of its application in feed

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

Effect analysis of transient scenarios for successful water management strategies

Recent scenario studies on water management focus on one or two projection years and the effects on the water system and functions. The future is however more complex and dynamic. Therefore, we analyse transient scenarios in order to evaluate the performance of water management strategies. Current available simulation tools are not suitable for this purpose. Therefore, we have developed and used a tool to simulate 50-100 year long time series and that is good and fast enough to simulate the effects of these scenarios and strategies on the water system and the interaction with the human system. We present the first step by means of a case study

Literatuuronderzoek naar het voorkomen en het bepalen van Zearalenon en Zeranol

Dit verslag heeft tot doel een literatuuroverzicht te geven omtrent het voorkomen en bepalen van Zearalenon en Zeranol in landbouwprodukten. Zearalenon is gevonden in de meeste delen van de wereld waar mais of andere granen groeien. Varkens zijn het meest gevoelig voor estrogene effecten t.g.v. het toedienen van Zearalenon. Volgens de literatuurgegevens moet het mogelijk zijn om Zearalenon te bepalen in graanprodukten tot een niveau van 2 ug/kg . Het bepalen van Zeranol in vlees en urine zou mogelijk moeten zijn tot een niveau van 1 ug/kg

Het ontwikkelen van een snelle methode voor het bepalen van aflatoxine MI in melk

Doel is het ontwikkelen van een snelle methode voor het bepalen van aflatoxine M1 in melk met als detectiegrens 0,005 µg/l

Het ontwikkelen van een snelle methode voor het bepalen van aflatoxine B1 in veevoeders

De tolerantie van aflatoxine B1 in veevoeder moet omlaag

Literatuuronderzoek omtrent polycyclische aromaten

Dit verslag heeft tot doel een literatuuroverzicht te geven omtrent het voorkomen van polycyclische aromaten en methoden voor het aantonen en bepalen van deze in relevante produkten. Polycyclische aromaten (PCA) zijn algemeen verspreid in het milieu en kunnen carcinogeen, syncarcinogeen of cocarcinogeen zijn. Zij ontstaan door de onvolledige verbranding van organisch materiaal en kunnen door bepaalde microorganismen worden gesynthetiseerd. Voedingsmiddelen kunnen door luchtstof of door "roken" met PCA verontreinigd worden. De door luchtstof verontreinigde grootbladerige groentesoorten, zoals groenekool bevatten in het algemeen grotere hoeveelheden aan PCA dan b.v . gerookte vis. In vergelijking met lucht en water bevat slib, uit zuiveringsinstallaties, enorm grote hoeveelheden aan PCA (mg/kg niveau). In Duitsland geldt voor gerookte vleesprodukten een tolerantie van 1 ug Benzo(a)pyreen per kg vlees en uit onderzoek is gebleken dat deze grens vele malen werd overschreden. Reversed phase vloeistofchromatografie met fluorescentie- en U.V. detectie is de meest geschikte combinatie voor het bepalen van PCA. Voor de bevestiging kunnen dunnelaagchromatografie of capillaire gaschromatografie met een FID of een massaspectrometer gebruikt worden

Het gehalte aan ochratoxine A in varkens- en pluimveevoeders

Schimmels welke ochratoxine A produceren komen regelmatig voor op landbouwprodukten. In landen als onder andere: Zweden, Denemarken en Canada werd ochratoxine A aangetroffen in landbouwprodukten en tevens in varkens- en kippenieren. Om een inzicht te verkrijgen in hoeverre het Nederlandse veevoeder (mengvoeders) besmet is met ochratoxine A werden 42 veevoeders onderzocht