Production of polyclonal antibodies directed to recombinant methionyl bovine somatotropin.

The administration of recombinant methionyl bovine somatotropin (rMbST) to dairy cows to increase milk yield remains a common practice in many countries including the USA, Brazil, Mexico, South Africa and Korea, whereas it has been forbidden within the European Union (EU) since 1999. A rapid screening immunoanalytical method capable of the unequivocal determination of rMbST in milk would be highly desirable in order to effectively monitor compliance with the EU-wide ban for home-made or imported dairy products. For decades, the production of specific antibodies for this recombinant isoform of bovine somatotropin (bST) has remained elusive, due to the high degree of sequence homology between both counterparts (e.g. methionine for rMbST in substitution of alanine in bST at the N-terminus). In this study, we compared several immunizing strategies for the production of specific polyclonal antibodies (pAbs), based on the use of the full-length recombinant protein, an rMbST N-terminus peptide fragment and a multiple antigen peptide (MAP) which consists of an oligomeric branching lysine core attached to the first two N-terminus amino acids of rMbST, methionine and phenylalanine (MF-MAP). The immunization with KLH-conjugated MF-MAP led to the production of the pAb with the highest rMbST/bST recognition ratio amongst the generated battery of antibodies. The pAb exhibited a specific binding ability to rMbST in a competitive antigen-coated ELISA format, which avidity was further improved after purification by rMbST N-terminus peptide-based affinity chromatography. These results suggest that immunodiscrimination between structurally related proteins can be achieved using immuno-enhanced immunogens such as MAPs.

Rapid multi-residue and multi-class qualitative screening for veterinary drugs in foods of animal origin by UHPLC-MS/MS.

Multi-class UHPLC-MS/MS was developed for the analysis of more than 160 regulated or banned compounds of various classes: anthelmintics including benzimidazoles, avermectins and others; antibiotics including amphenicols, beta-lactams, macrolides, pyrimidines, quinolones, sulphonamides and tetracyclines; beta-agonists; corticosteroids; ionophores; nitroimidazoles; non-steroidal anti-inflammatory agents; steroids; and tranquillisers. Samples were extracted with acetonitrile, without any additional purification step, and analysed by using UHPLC-MS/MS. Validation was done in accordance with the guidelines laid down by European Commission Decision 2002/657/EC for qualitative screening methods. This simple method proved applicable to routine screening for residues in egg, honey, milk and muscle samples at half the maximum concentration permitted by the European Union for each drug. In most cases, the target value was set at 5 µg kg(-1) for unauthorised compounds.

Development of ELISAs for detecting domoic acid, okadaic acid, and saxitoxin and their applicability for the detection of marine toxins in samples collected in Belgium.

Okadaic acid, a diarrhetic shellfish poison, domoic acid, an amnesic shellfish poison, and saxitoxin, a paralytic shellfish poison, are three of the best-known marine biotoxins. The mouse bioassay is the method most widely used to detect many of these toxins in shellfish samples, but animal welfare concerns have prompted researchers to seek alternative methods of detection. In this study, three direct competitive enzyme-linked immunosorbent assays (ELISAs), each based on antibodies raised in rabbits against a conjugate of the analyte of interest, were developed for marine biotoxin detection in mussel, oyster, and scallop. One assay was for okadaic acid, one for saxitoxin, and one for domoic acid usually detected and quantified by high-performance liquid chromatography-ultraviolet light (HPLC-UV). All three compounds and a number of related toxins were extracted quickly and simply from the shellfish matrices with a 9 : 1 mixture of ethanol and water before analysis. The detection capabilities (CCbeta values) of the developed ELISAs were 150 microg kg(-1) for okadaic acid, 50 microg kg(-1) for domoic acid, and 5 microg kg(-1) or less for saxitoxin. The assays proved satisfactory when used over a 4-month period for the analysis of 110 real samples collected in Belgium.

Multi-residue method for detecting coccidiostats at carry-over level in feed by HPLC-MS/MS.

A multi-residue HPLC-ESI-MS/MS method has been developed for the simultaneous extraction, detection and confirmation of the 11 coccidiostats referenced by Regulation 2009/8/EC (lasalocid sodium, narasin, salinomycin sodium, monensin sodium, semduramicin sodium, maduramicin ammonium alpha, robenidine hydrochloride, decoquinate, halofuginone hydrobromide, nicarbazin, and diclazuril) in feedstuffs at carry-over level. The sensitivity of the method allows quantification and confirmation for all coccidiostats below target concentration. The method was in-house validated and meets all criteria of European legislation (2002/657/EC). The precision of the method was determined under repeatability and within-laboratory reproducibility conditions; RSD(r) and RSD(R) were below the maximum permitted values for every tested concentration. The specificity was checked by analysing representative blank samples and blank samples fortified with potentially interfering substances (benzimidazoles, corticosteroides, triphenylmethane dyes, quinolones, nitrofurans, nitroimidazoles, phenicols); no interference were found. Concerning quantification, a quadratic regression model was fitted to every calibration curve with a regression coefficient r2 above 0.99 on each data set. Finally, the expanded uncertainty U was calculated with data obtained within the laboratory while applying the method during validation and in routine tests.

Validation of an optical surface plasmon resonance biosensor assay for screening (fluoro)quinolones in egg, fish and poultry.

A surface plasmon resonance biosensor immunoassay has been developed for multi-residue determination of 13 (fluoro)quinolone antibiotics in poultry meat, eggs and fish. The following performance characteristics were determined according to the guidelines laid down for screening assay validation in European Decision 2002/657/EC: detection capability, specificity/selectivity, decision limit, repeatability, ruggedness and stability. The detection capability estimated for norfloxacin, the reference fluoroquinolone, was below 0.5, 1 and 1.5 ng g⁻¹ for poultry meat, egg and fish, respectively. The screening assay proved specific and showed satisfactory sensitivity below the MRL levels even though flumequine and oxolinic acid had lower cross-reactivities. A wide range of non-MRL substances were also detected at concentrations below 10 ng g⁻¹. Repeatability was good with both intra- and inter-assay coefficients of variation 56%; ruggedness was also demonstrated.

Development of an optical surface plasmon resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry meat.

The aim of this study was to develop an optical biosensor inhibition immunoassay, based on the surface plasmon resonance (SPR) principle, for use as a screening test for 13 (fluoro)quinolones, including flumequine, used as veterinary drugs in food-producing animals. For this, we immobilised various quinolone derivatives on the sensor chip and tested binding of a range of different antibodies (polyclonal and one engineered antibody) in the presence and absence of free (fluoro)quinolones. The main challenge was to detect flumequine in an assay giving good results for the other compounds. One antigen-antibody combination proved satisfactory: polyclonal antibodies raised against a dual immunogen and, on the sensor chip, a fluoroquinolone derivative. It was the first time that this concept of the bi-active antibody was described in the literature. The assay, optimised for detection in three matrices (poultry muscle, fish, and egg), was tested on incurred samples prepared by liquid extraction followed by two washing steps. This rapid, simple method proved adequate for detecting at least 13 (fluoro)quinolones at concentrations below established maximum residue levels (MRLs). The reference molecule norfloxacin could be detected in the range of 0.1-10 microg kg(-1) in extracts of egg and poultry meat and in the range of 0.1-100 microg kg(-1) in extracts of fish. The determined midpoints of these calibration curves were about 1, 1.5 and 3 microg kg(-1) in poultry meat, egg and fish, respectively.

Sanitary control in bovine embryo transfer. How far should we go? A review.

Embryo transfer is a globally executed technique which, when properly done, has both economic and sanitary advantages. International guidelines are available to prevent infection of the embryo with pathogens, both originating from the donor animals as from the environment. This manuscript describes the bacteria, viruses, protozoa, fungi and prions that are of major concern in the context of embryo transfer in cattle. In addition, the actual scientific knowledge on these pathogens is evaluated in terms of the current international and national guidelines and legislation.

Efficient and sensitive detection of residues of nine coccidiostats in egg and muscle by liquid chromatography-electrospray tandem mass spectrometry.

We present a method based on electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) for determining in muscle and eggs the following nine coccidiostats: halofuginone, diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, monensin, lasalocid, narasin, salinomycin, and maduramicin. Dinitrocarbanilide-d8, nigericin, and diclazuril-bis were used as internal standards. The method uses extraction in acetonitrile followed by a clean-up on an SiOH solid-phase extraction column. High-performance liquid chromatography (HPLC) separation was performed on a Purospher C(18) column (125 mm x 3 mm i.d.) protected by a guard column, the mobile phase being a water-acetonitrile gradient (each gradient component containing 0.1% formic acid) at a flow rate of 1 ml min(-1). For unequivocal identification of each analyte, two ions were detected and chosen for multiple reaction monitoring (MRM). Validation was carried out on spiked muscle and egg samples. The method described meets all the criteria of Decision 2002/657/EC and is easy to use in routine analysis. Validation results are presented with the measured CCalpha and CCbeta values. This whole method allows extraction and analysis of up to 24 samples per day.