Terence L. Fodey

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine.

The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 μg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.

Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry.

Ractopamine (RCT) is a member of the beta-2-agonist (beta-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The Peoples Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A. In order to monitor for the illegal use of such compounds within Europe, there is a requirement to have a robust and reliable testing scheme capable of the detection of low concentrations of RCT. In the present study an optical biosensor screening assay was developed. The developed assay was compared with a liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) confirmatory procedure. These methods were used to study the ability to detect RCT in pigs following treatment. Both testing procedures were capable of detecting low microgkg(-1) concentrations of the drug in urine and liver. Liver was found to be a less suitable sample matrix, with RCT residue levels being undetectable after 5 days withdrawal of the drug. Urine samples however still contained detectable RCT residues several weeks after withdrawal. The correlation (as measured by r(2)) between the biosensor and LC-MS/MS methods was 0.99 and 0.97 for urine and liver samples, respectively. It is concluded that testing regimes based on RCT analysis in liver are less likely to detect illegal administration of the drug than those based on urine analysis. Urine samples provide an excellent matrix for the detection of RCT residues for an extended period post withdrawal.

Improved screening method for the detection of a range of nitroimidazoles in various matrices by optical biosensor

An immunobiosensor assay was developed for the multi-residue screening of a range of nitroimidazole compounds in various species and sample types including porcine, bovine and ovine kidney, avian liver, serum and eggs and bovine milk. A polyclonal antibody which binds at least seven of the major nitroimidazoles and their metabolites was raised in a sheep after inoculation with a metronidazole protein conjugate. Sample homogenates were extracted into acetonitrile and subjected to micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 20 fortified samples has shown that the method has a detection capability (CCbeta) of less than 1 microgkg(-1) (or microgL(-1)) for dimetridazole (DMZ) in all species and matrices investigated. In addition, cross-reactivity data and the analysis of a small number of fortified samples have shown that the method will also detect a range of other major parent nitroimidazoles and their metabolites including ronidazole (RNZ), ipronidazole (IPZ), metronidazole (MNZ), hydroxymetronidazole (MNZOH), hydroxydimetridazole (DMZOH) and hydroxyipronidazole (IPZOH). The cross-reactivity profile and validation data for the detection of these nitroimidazoles are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.

A review of coccidiostats and the analysis of their residues in meat and other food

Coccidiostats are used in the control of protozoan infections in different food producing animals. They are most widely used as feed additives in intensively reared species such as pigs and poultry to maintain animal health and in some cases enhance feed conversion. However, a number of these drugs are used in the control of infections in beef and lamb production. Coccidiostat residues have been frequently reported in meat and eggs in a number of countries since the late 1990s. This has prompted increased research and surveillance of coccidiostat residues in food. This paper reviews the various coccidiostat agents used in animal production, including their chemical properties, mode of action and activity. Legislation concerning coccidiostats, limits for residues in food, monitoring and occurrence of residues in food is discussed. Methods for residue determination in food, including screening and physicochemical methods are discussed in depth. The paper concludes with a synopsis of the current state of coccidiostat residue analysis and future perspectives.

Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered. These foods include bread and other bakery products, crisps, chips, breakfast cereals, and coffee. To date, the diminutive size of acrylamide (71.08 Da) has prevented the development of screening immunoassays for this chemical. In this study, a polyclonal antibody capable of binding the carcinogen was produced by the synthesis of an immunogen comprising acrylamide derivatised with 3-mercaptobenzoic acid (3-MBA), and its conjugation to the carrier protein bovine thyroglobulin. Antiserum from the immunised rabbit was harvested and fully characterised. It displayed no binding affinity for acrylamide or 3-MBA but had a high affinity for 3-MBA-derivitised acrylamide. The antisera produced was utilised in the development of an ELISA based detection system for acrylamide. Spiked water samples were assayed for acrylamide content using a previously published extraction method validated for coffee, crispbread, potato, milk chocolate and potato crisp matrices. Extracted acrylamide was then subjected to a rapid 1-h derivatisation with 3-MBA, pre-analysis. The ELISA was shown to have a high specificity for acrylamide, with a limit of detection in water samples of 65.7 microgkg(-1), i.e. potentially suitable for acrylamide detection in a wide range of food commodities. Future development of this assay will increase sensitivity further. This is the first report of an immunoassay capable of detecting the carcinogen, as its small size has necessitated current analytical detection via expensive, slower, physico-chemical techniques such as Gas or Liquid Chromatography coupled to Mass Spectrometry.

Rapid Surface Plasmon Resonance Immunoassay for the Determination of Deoxynivalenol in Wheat, Wheat Products, and Maize-Based Baby Food

A rapid screening assay (9 min/sample) has been developed and validated for the detection of deoxynivalenol in durum wheat, wheat products, and maize-based baby foods using an SPR biosensor. Through a single laboratory validation, the limits of detection (LOD) for wheat, wheat-based breakfast cereal, and maize-based baby food were 57, 9, and 6 μg/kg, respectively. Intra-assay and interassay precisions were calculated for each matrix at the maximum and half-maximum European Union regulatory limits and expressed as the coefficient of variation (CV). All CVs fell below 10% with the exception of the between-run CV for breakfast cereal. Recoveries at the concentrations tested ranged from 92 to 115% for all matrices. Action limits of 161, 348, and 1378 μg/kg were calculated for baby food, wheat-based breakfast cereal, and wheat, respectively, and the linear range of the assay was determined as 250-2000 μg/kg.

The production and characterisation of dinitrocarbanilide antibodies raised using antigen mimics.

Polyclonal antibodies were produced to detect the coccidiostat nicarbazin. Due to structural constraints of the active component of nicarbazin, dinitrocarbanilide (DNC), three different compounds that shared a common substructure with DNC were used as antigen mimics. The compounds (N-succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide (SAN), L-glutamic acid gamma-(p-nitroanilide) (GAN) and p-nitrosuccinanilic acid (NSA)) were conjugated to a carrier protein and used in the immunisation of rabbits. Five different polyclonal sera were produced and consequently characterised. The antibodies exhibited an IC(50) range of 2.3-7.6 ng/ml using a competitive ELISA procedure. Serum from one rabbit, R555, exhibited an IC(50) of 2.9 ng/ml for DNC and cross-reactivity studies showed that this serum was specific for DNC and did not cross-react with other coccidiostats such as halofuginone, toltrazuril or ronidazole.

Rapid screening for monensin residues in poultry plasma by a dry reagent dissociation enhanced lanthanide fluoroimmunoassay

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for rapid analysis of unextracted poultry plasma samples has been developed based on a novel immunoassay format: one-step all-in-one dry reagent time resolved fluorimetry. All assay specific components were pre-dried onto microtitration plate wells. Only addition of the serum sample diluted in assay buffer was required to perform analysis. Results were available one hour after sample addition. The limit of detection (mean +/- 3s) of the assay calculated from the analysis of 23 known negative samples was 14.2 ng ml-1. Intra- and inter-assay RSD were determined as 15.2 and 7.4%, respectively, using a plasma sample fortified with 50 mg ml-1 monensin. Eight broiler chickens were fed monensin at a dose rate of 120 mg kg-1 feed for one week, blood sampled then slaughtered without drug withdrawal. Plasma monensin concentrations, as determined by the fluoroimmunoassay ranged from 101-297 ng ml-1. This compared with monensin liver concentrations, determined by LC-MS, which ranged from 13-41 ng g-1. The fluoroimmunoassay described is extremely user friendly, gives particularly rapid results and is suitable for the detection and quantification of plasma monensin residues. Data from medicated poultry suggest that analysis of plasma may be useful in predicting the extent of monensin liver residues.

Comparison of porcine urine and bile as matrices to screen for the residues of two sulfonamides using a semi-automated enzyme immunoassay.

Porcine urine enzyme immunoassays for sulfamethazine and sulfadiazine have previously been employed as screening tests to predict the concentrations of the drugs in the corresponding tissues (kidneys). If a urine was found positive (> 800 ng ml-1) the corresponding kidney was then analysed by an enzyme immunoassay and, if found positive, a confirmatory analysis by HPLC was performed. Urine was chosen as the screening matrix since sulfonamides are mainly eliminated through this body fluid. However, after obtaining a number of false positive predictions, an investigation was carried out to assess the possibility of using an alternative body fluid which would act as a superior indicator of the presence of sulfonamides in porcine kidney. An initial study indicated that serum, plasma- and bile could all be used as screening matrices. From these, bile was chosen as the preferred sample matrix and an extensive study followed to compare the efficiencies of sulfonamide positive bile and urine at predicting sulphonamide positive kidneys. Bile was found to be 17 times more efficient than urine at predicting a sulfamethazine positive kidney and 11 times more efficient at predicting a sulfadiazine positive kidney. With this enhanced performance of the initial screening test, the need for the costly and time consuming kidney enzyme immunoassay, prior to HPLC analysis, was eliminated.

Screening for the coccidiostats halofuginone and nicarbazin in egg and chicken muscle: development of an ELISA

Nicarbazin and halofuginone have been widely used as coccidiostats for the prevention and treatment of coccidiosis in poultry. It has been shown that accidental cross-contamination of feed can lead to residues of these compounds in eggs and/or muscle. This paper describes a direct competitive assay for detecting halofuginone and nicarbazin, developed as qualitative screening assay. In an optimized competitive ELISA, antibodies showed 50% binding inhibition at approximately 0.08 ng ml−1 for halofuginone and 2.5 ng ml−1 for dinitrocarbanilide (marker residue for nicarbazin). Extraction from the matrix was carried out with acetonitrile followed by a wash with hexane. The assays detection capability (CCβ) for halofuginone was <0.5 µg kg−1 in egg and <1 µg kg−1 in muscle. For dinitrocarbanilide, the CCβ was estimated at <3 µg kg−1 in egg and <10 µg kg−1 in chicken muscle.