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

Onderzoek naar de toepassing van seleniumsolen bij de ontwikkeling van een snelle test voor het aantonen van biologisch actieve verbindingen en milieucontaminanten op ppb-nivo

Tussen Holland biotechnology bv (Hbt) en het RIKILT is overeengekomen een gemeenschappelijk onderzoek op te zetten naar de mogelijke toepassing van seleniumsolen in eenvoudige screeningstests voor de analyse van stoffen op het (sub)ppb-nivo. Een seleniumsol, gekoppeld aan antilichamen of eiwitconjugaten, wordt gebruikt als specifiek kleurlabel. Bij de tot nu toe uitgevoerde experimenten is nortestosteron (NT) als teststof gebruikt, omdat hiervoor voldoende antiserum en NT-eiwitconjugaten , alsmede monstermateriaal en analysemethoden beschikbaar zijn om een test op te zetten en te valideren. In eerste instantie werd gekozen voor het ontwikkelen van een striptest met nitrocellulose als drager. Uit de verrichtte experimenten bleek dat met een geringe hoeveelheid NT-eiwitconjugaat op de nitrocellulosestrip (overeenkomend met 0,3 ng NT) al een kleuring werd verkregen met antilichamen gelabeld met sol . Ook is een geringe hoeveelheid antistof op de strip (4,7 pmol IgG, dat theoretisch maximaal 2,6 ng NT kan binden) voldoende om een kleuring te geven met NT-eiwitconjugaat dat aan de sol gekoppeld is. Het laagste nivo toegevoegd vrij NT, waarbij tot nu toe een (visueel) kleurverschil werd waargenomen bedroeg 60 ng/ml. Vermoedelijk heeft het antilichaam een grotere aviditeit voor het NT-eiwitconjugaat dan voor het vrije NT, hetgeen de test nadelig beïnvloed. Door een ander NT-eiwitconjugaat of zelfs een conjugaat van een ander steroid te kiezen, kan mogelijk een veel gevoeligere test worden verkregen. De tot nu verkregen resultaten zijn hoopgevend en bieden voldoende perspectief om het samenwerkingsprojekt voor langere termijn te continueren

Intestinal uptake of genistein and its glycoside in the rat using various isolated perfused gut segments

Genistein receives much attention because of its potential to prevent hormone-related cancer and cardiovascular diseases. Limited information is available on the pharmacokinetics of this compound like, for instance, their intestinal uptake by humans and systematic bioavailability. In this study, the fate of the absorption of genistein and its glycoside has been analysed in various isolated perfused gut segments of the rat. In all perfused gut segments the transport of genistein was higher compared to its glycoside. Furthermore, it appeared that the resorbate (i.e. serosal side) concentration of genistein was the highest in ileac segments, whereas the transport of genistein in the various other segments tested showed no difference between intestinal compartments. Less than 0.2 f genistin appeared in the resorbate fluid of all isolated gut segments. The main site of metabolism of genistein and its glycoside appears to be located in the jejunal compartment of the rat gut. About 38 f genistein and about 29 f genistin metabolised within 2 h of perfusion. In the ileac and colonic intestinal segments, genistein metabolised for only 10&Eth;For the first time, this study demonstrated that genistin could be metabolised by epithelial cells present in isolated colonic segments. However, the metabolites of genistin did not occur at the serosal side (the resorbate) of isolated colonic segments. We assume that there is no absorption of genistin and/or its metabolites in or through colonic tissue of the rat

Screening methods and recent developments in the detection of anticoccidials

This article presents a review of the current trends in the analysis of coccidiostats in various matrices, focusing principally on screening and rapid methods. Coccidiosis is an infectious disease having a high negative impact on the animal industry. Drugs are therefore necessary to prevent and/or to combat this disease. However, it is also of crucial importance that these veterinary drugs do not enter the human food chain. European legislation has therefore established the boundaries for the use of coccidiosats and has also addressed the unavoidable problem of cross-contamination of the feed, mainly caused by the use of the same production lines. Consequently there is a need for analytical methods and/or analytical strategies for the monitoring and control of the residues of anticoccidials, both in feed and in the resulting matrices for human consumption. In the frame of the European collaborative project CONffIDENCE, such attempts to establish the required analytical tools were made, which required beforehand a review of the state of the art in this domain. Aiming at this objective, in this review we consider themost interesting publications since 2000. In essence, both a rapid approach with mainly immunoassays and chromatographic methods were developed. To date, the obstacle to routine use of the first approach has been its inability to detect more than two compounds simultaneously, but recent developments in flow cytometry have made it possible to detect six coccidiostats at once. On the other hand, an increasingly popular approach for detecting multiple coccidiostats simultaneously is liquid chromatography coupledwith tandemmass spectrometry. There remains a need to adapt these analytical methods to legislative requirements

Comparison of multi-sulfonamide biosensor immunoassays

Three different group-specific anti-sulfonamide antibodies were compared in inhibition assay formats in an optical biosensor (BIACORE 3000) using CM5 sensor chips coated with three different sulfonamide derivatives. The antibodies used were an anti-sulfamethazine monoclonal antibody (Mab) 21C7, the sulfonamide binding protein (SBP) in the Qflex Kit Sulfonamides and a recently developed mutant antibody (M.3.4). Each of these antibodies showed interactions with all 17 sulfonamides tested and one (Mab 21C7) was sensitive for the N4-acetyl metabolites also. The limits of detection of the different sulfonamides in chicken serum varied between 7 and >1000 ng ml¿1 (Mab 21C7), 15 and 340 ng ml¿1 (Qflex) and 4 and 82 ng ml¿1 (Mutant M.3.4). The mutant M.3.4 based assay was found to be the most sensitive towards most of the sulfonamides whereas the Qflex Kit Sulfonamides detected the five sulfonamides registered for application in poultry in The Netherlands within the narrowest measurement rang

Application of a multi-sulfonamide biosensor immunoassay for the detection of sulfadiazine and sulfamethoxazole residues in broiler serum and its use as a predictor of the levels in edible tissue

A multi-sulfonamide biosensor immunoassay (BIA), based on a previously developed mutant antibody (A.3.5) in an optical biosensor (Biacore 3000), was applied to analyse the serum and plasma samples obtained from the broilers treated with sulfamethoxazole and sulfadiazine. The assay was fast (5 min per sample), the sample preparation easy (dilution in antibody containing buffer only) and an equal sensitivity for the two sulfonamides was obtained with limits of detection (LOD) in serum and plasma below 10 ng ml¿1. The concentrations found with the BIA in serum and plasma of the treated broilers were comparable and higher than the concentrations found in the tissue by LC¿MS/MS. The average serum/tissue ratios for sulfamethoxazole were 6.2 (leg meat), 2.5 (liver) and 1.3 (skin + fat) and for sulfadiazine 8.7 (leg meat), 3.1 (liver) and 2.2 (skin + fat). To predict the concentrations of the two sulfonamides below the maximum residue limit (MRL) of 100 ng g¿1 in the tissue with the highest level (skin + fat), the proposed action level of the multi-sulfonamide BIA in serum is 130 ng ml¿1. A later developed mutant antibody (M.3.4), with a better sensitivity towards more sulfonamides, was applied during a survey. Serum samples (n = 300) of broilers from 30 different flocks were found negative. Concentrations betwee

Application of a multi-sulfonamide biosensor immunoassay for the detection of sulfadiazine and sulfamethoxazole residues in broiler serum and its use as a predictor of the levels in edible tissue

A multi-sulfonamide biosensor immunoassay (BIA), based on a previously developed mutant antibody (A.3.5) in an optical biosensor (Biacore 3000), was applied to analyse the serum and plasma samples obtained from the broilers treated with sulfamethoxazole and sulfadiazine. The assay was fast (5 min per sample), the sample preparation easy (dilution in antibody containing buffer only) and an equal sensitivity for the two sulfonamides was obtained with limits of detection (LOD) in serum and plasma below 10 ng ml¿1. The concentrations found with the BIA in serum and plasma of the treated broilers were comparable and higher than the concentrations found in the tissue by LC¿MS/MS. The average serum/tissue ratios for sulfamethoxazole were 6.2 (leg meat), 2.5 (liver) and 1.3 (skin + fat) and for sulfadiazine 8.7 (leg meat), 3.1 (liver) and 2.2 (skin + fat). To predict the concentrations of the two sulfonamides below the maximum residue limit (MRL) of 100 ng g¿1 in the tissue with the highest level (skin + fat), the proposed action level of the multi-sulfonamide BIA in serum is 130 ng ml¿1. A later developed mutant antibody (M.3.4), with a better sensitivity towards more sulfonamides, was applied during a survey. Serum samples (n = 300) of broilers from 30 different flocks were found negative. Concentrations betwee