DETECTION OF IRRADIATED CHICKEN MEAT BY ANALYSIS OF LIPID EXTRACTS FOR 2-SUBSTITUTED CYCLOBUTANONES USING AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY

The means to detect the irradiation of food has been investigated for many years. In recent times radiolytic products, termed 2-alkylcyclobutanones (2-CBs), have been identified as excellent markers of irradiation in lipid-containing foods. An ELISA test was developed, which was capable of detecting a number of these compounds in irradiated chicken meat. A polyclonal antiserum was raised to a 2-CB containing a terminal carboxyl group conjugated to a carrier protein. This antiserum was highly specific for cyclobutanones containing C-10 and C-12 side chains. During assay validation the limit of detection of the assay was calculated to be 0.064 pg of 2-CB per gram of fat, within- and between-assay variations ranged from 6.7 to 18%. During experimental studies, chicken meat irradiated at doses ranging from 2.5 to 10 kGy were assayed and correctly identified as being treated. Quantitative comparisons between the ELISA and CC-MS revealed a good correlation (r(2) = 0.913) between the two methodologies in concentrations of 2-CB detected in irradiated samples.

Detection of streptomycin residues in whole milk using an optical immunobiosensor

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples (similar to3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).

Detection of unwanted residues of ivermectin in bovine milk by dissociation-enhanced lanthanide fluoroimmunoassay

Avermectins are frequently used to control parasitic infestations in many animal species. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. An immunoassay screening test for the detection acid quantification of ivermectin residues in bovine milk has been developed. After an extensive extraction procedure, milk samples were applied to a competitive dissociation-enhanced lanthanide fluoroimmunoassay using a monoclonal antibody against an ivermectin-transferrin conjugate, The monoclonal antibody, raised in Balb C mice, showed cross-reactivity with eprinomectin (92%), abamectin (82%) and doramectin (16%). The limit of detection of the assay (mean + 3 SD), calculated from the analysis of 17 known negative samples, was calculated as 4.6 ng/mL. Intra- and inter-assay RSDs were determined as 11.6% and 15.8%, respectively, using a negative bovine milk sample fortified with 25 ng/mL ivermectin. Six Friesian milking cows were treated with ivermectin, three with a pour-on formulation of the drug and three with an injectable solution at the manufacturer's recommended dose rate. An initial mean peak in ivermectin residue concentration was detected at day 4 (mean level = 47.5 ng/mL) and day 5 post-treatment (mean level = 26.4 ng/mL) with the injectable form and pour-on treatment, respectively. A second peak in residue concentration was observed using the DELFIA(R) procedure 28 days post-treatment in both treatment groups (23.1 ng/mL injectable and 51.9 ng/mL pour-on). These second peaks were not confirmed by HPLC and must at this Lime be considered to be false-positive results. By day 35 after treatment the mean ivermectin residue concentration of both groups fell below the limit of detection of the assay. Copyright (C) 2000 John Wiley & Sons, Ltd.

A microtitre plate assay for the detection of antibiotics in porcine urine

Techniques for screening porcine samples for antimicrobial residues in the EU usually involve analysis of samples taken post slaughter, and are either time consuming or expensive. Some of the positive test results at this screening stage could be avoided by allowing the animal sufficient withdrawal time following drug treatment. A method is described that can detect the presence of five major antibiotics in porcine urine at concentrations below 1 mu g ml(-1) for each of the compounds. The test uses Bacillus subtilis, which is already widely employed in antimicrobial inhibition assays, and when combined with a colorimetric substrate, p-nitrophenyl-beta-D-glucopyranoside, can detect inhibitory substances within an assay time of four and a half hours. The method, which uses microtitre plate technology, could be developed into a convenient test kit for use at farm level to determine whether animals were still excreting antimicrobials in their urine prior to their submission for slaughter.

The development of a rapid immunobiosensor screening method for the detection of residues of sulphadiazine

A rapid imununoassay using an optical biosensor (BIAcore(TM)) for determining the presence of sulphadiazine (SDZ) residues in pig bile was developed. SDZ,cas immobilised onto the surface of a dextran-coated silicon chip and a solution containing SDZ antibody, sample and buffer was injected over the chip surface. The level of antibody binding to the chip was determined after 20 s and the surface of the chip was then regenerated over a 1-min period prior to another sample injection taking place. Standard curves were constructed to allow quantification of SDZ presence in sample. Concentrations ranging from 0 to 10.64 mu g ml(-1) SDZ were detected in bile samples taken from experimentally fed pigs and randomly selected pigs taken from a local slaughterhouse. These results were compared to the concentrations of SDZ detected by high-performance liquid chromatography: in associated tissues. When concentrations in bile exceeded 0.6 mu g ml(-1) SDZ, the corresponding edible tissue was above the maximum residue level (MRL), i.e. 0.1 mu g g(-1) in 13 out of 14 cases. Wizen the bile concentration was less than 0.6 mu ml(-1) the associated tissue concentrations never exceeded rite MRL. This experiment has indicated that biosensor analysis of bile is a highly effective method for detecting violative SDZ residues in meat.

Comparison of two polyclonal antibodies for the detection of 19-nortestosterone in bovine bile by ELISA

19-Nortestosterone (beta-NT) is banned for use as a growth promoter in food animals within the European Union. For regulatory control purposes, urine and bile samples are routinely screened by immunoassay. The aim of the present study was to compare the ability of two immunoassays, using two rabbit polyclonal antibodies raised against two different NT derivatives, to detect NT residues in bovine bile. One antiserum cross-reacted with both alpha-NT and beta-NT (alpha/beta-NT), whereas the other was specific for alpha-NT. Bile samples from 266 slaughtered cattle were deconjugated and analyzed using both antibodies, with all screening positives (>2 ng ml(-1)) confirmed by high resolution gas chromatography mass spectrometry. The alpha/beta-NT and alpha-NT antibody-based ELISAs screened 39 and 44 samples positive, respectively, with NT confirmed in 22 and 39, respectively. The alpha/beta-NT antibody-based ELISA produced a false-negative rate of 44% compared to 0% for the alpha-NT antibody-based ELISA. Supplementary investigations concluded that a matrix effect was a major cause of the marked differences in false-negative rates. This result underlines the necessity to validate immunoassays in the sample matrix.

Detection of monensin residues in poultry liver using an enzyme immunoassay

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for the detection and quantification of monensin residues in liver has been developed, Samples (3 g) were extracted with acetonitrile-water and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised against a monensin-transferrin conjugate, The limit of detection (mean + 3s) calculated from the analysis of 12 known negative samples was 2.91 ng g(-1). Intra- and inter-assay RSD were determined as 8.5 and 10.6%, respectively, using a liver sample fortified with 20 ng g(-1) monensin, A pharmacokinetic study in which 70 six week old broilers were fed monensin at a rate of 120 mg kg(-1) in their feed for 14 d resulted in mean monensin liver residues of 102 ng g(-1). However these had fallen below the limit of detection of the assay within the 3 d withdrawal period recommended by the manufacturer.

Development of a dual label time-resolved fluoroimmunoassay for the detection of beta-agonists in cattle urine

beta-Agonists are among the most widely abused drugs in veterinary medicine for the illegal promotion of farm animal growth. An array of analytical procedures has been developed to detect the residues of these compounds in many biological materials. As the number of beta-agonist formulations increases, it has become increasingly difficult to devise screening techniques capable of detecting a broad spectrum of these residues in a single test. A dual immunoassay based on time-resolved fluorescence was developed that incorporated a monoclonal antibody raised to tertiary butyl amines and a polyclonal antibody to biphenolic beta-agonists. This assay was capable of detecting residues of a range of beta-agonists present in bovine urine without the need for sample extraction. The limits of detection of the assay ranged from 1 to 8.5 ng ml(-1) depending on the cross-reactivity of individual compounds with the antibodies employed in the procedure.

Detection of ivermectin residues in bovine liver using an enzyme immunoassay

Ivermectin, a member of the avermectin group, is frequently used to control parasites in many food producing animal species. A method for the detection and quantification of ivermectin residues in bovine liver has been developed. Liver samples (4 g) were extracted with acetonitrile and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised in rabbits against an ivermectin-transferrin conjugate, The limit of detection of the assay (mean +/- 3s) calculated from the analysis of 24 known negative samples was 1.6 ng g(-1), Intra- and inter-assay RSDs were determined as 8.8 and 14.6%, respectively, using a negative bovine liver sample fortified with 100 ng g(-1) of ivermectin. Four Friesian steers were treated with a pour-on application of ivermectin at a dose rate of 0.5 mg kg(-1) body mass then withdrawn and killed at 7, 14, 21 and 28 d, Livers mere removed and ivermectin residue concentrations determined using the proposed immunoassay procedure, Seven days post-treatment the ivermectin liver concentration was determined as 52.7 ng g(-1), decreasing to 4.1 ng(-1) at 28 d, All immunoassay results were confirmed using high-performance liquid chromatography (HPLC), The immunoassay and HPLC results for invermectin ranged from 1 to 58 ng g(-1) and were in close correlation (r = 0.99).

A simple competitive enzyme immunoassay for the detection of the trypanocidal drug isometamidium

A new competitive enzyme immunoassay technique has been developed for the determination of concentrations of the trypanocidal drug isometamidium chloride (Samorin) in bovine serum. The method has been shown to be highly repeatable and reproducible, and it has several advantages over previous immunoassay techniques for the drug. There are fewer incubation steps overall; microtitre plates may be of coated in batches and stored frozen for future use; and the competition incubation is overnight and is followed only by a brief colour development stage of 10 min. Coefficients of variation (CVs) of duplicate samples were similar to 5%, and mean response variances of untreated cattle (n = 57) were small (CV, 10%). Partitioning of variance showed 77% of this variability to be intrinsic to the samples, and the remaining 23% was due to the procedure. The limit of detection was approximately 0.5 ng ml(-1), which was considered to be satisfactory for the intended use of the method. The drug could be detected in serum of treated cattle for up to 10 weeks following treatment, and determinations showed a high level of reproducibility.

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.