Shi-Yuan Sheu

Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan.

An ELISA was developed for routine examination for extensive monitoring and screening programs for the residues of salbutamol in swine serum, animal feed, meat, and meat-related products destined for human consumption in Taiwan. Objectives of the study were to investigate the use of a new immunoassay for the detection of salbutamol residues in swine meat and animal feed samples, and to compare with a commercial kit in field test screens. A fast, simple and reliable sample preparation method for the determination of salbutamol was established. Field trials with 222 swine meat and 120 animal feed samples that were taken from local meat markets, auction markets and feed mills. The application and the results of two ELISA kits (a homemade and a commercial kit) for the screening of salbutamol were presented. Adopting 2microg kg(-1) salbutamol as a cut-off value for swine meat, the commercial beta-agonist ELISA had a sensitivity of 85.3% and a specificity of 95.2% versus GC-MS at a cut-off of 2microg kg(-1). The homemade salbutamol ELISA had a sensitivity of 100% and a specificity of 90.9% and gave no false-negative rate results. Furthermore, adopting 20microg kg(-1) salbutamol as a cut-off value for animal feed, both the commercial and homemade ELISA showed 100% sensitivity and 100% specificity of the assays. In conclusion, a sensitive, specific salbutamol polyclonal antibody-based ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.

Development and fast screening of salbutamol residues in swine serum by an enzyme-linked immunosorbent assay in Taiwan.

The analysis of salbutamol in swine serum is the more practical basis for large scale surveillance programs in Taiwan. Objectives of the study were to develop a new assay and to compare with a commercially available kit in field test screens. A simple and reliable enzyme-linked immunosorbent assay (ELISA) to monitor the presence of beta-agonist, salbutamol, in 1,358 field samples of swine serum that were collected from local meat markets was described. The method proved to be suitable and sensitive for the detection of beta-agonist residues caused by growth promoting dosage. The limit of detection of the developed ELISA directly performed on diluted serum was 0.25 ng/mL. The application and the results of two ELISA kits (homemade and commercially available) for the screening of salbutamol were presented. For further confirmation, all samples that showed to be ELISA positive for salbutamol residues were analyzed by GC-MS. Adopting 1 ng/mL salbutamol as a cutoff value, the commercial beta-agonist ELISA had a sensitivity of 89.2% and a specificity of 86.7% versus GC-MS at a cutoff of 1 ng/mL. The homemade salbutamol ELISA had a sensitivity of 81.1% and a specificity of 98.6% and gave a low proportion of false-positive rate results. The reliability of the developed kit in terms of the percentage of false-positive rate results is evaluated. In conclusion, a sensitive, specific salbutamol ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.

Development and residue screening of the furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ), in cultured fish by an enzyme-linked immunosorbent assay.

A sensitive and specific polyclonal enzyme-linked immunosorbent assay (ELISA) for the determination of tissue-bound metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedures allow for the detection of protein-bound AOZ in the form of a 2-nitrophenyl derivative (2-NP-AOZ) in the sample supernatant or extract after acid hydrolysis and derivatization with o-nitrobenzaldehyde. The polyclonal rabbit antibodies were produced with the immunogen hapten, 2-NP-HXA-AOZ, and the 50% inhibition values (IC(50)) of 0.14 microg kg(-1) of AOZ was achieved with the most sensitive antibody A0505. The mean lower detection limit of the ELISA method is about 0.025 microg kg(-1). According to the test preparation record, the detection limit is 0.1 microg kg(-1), which is well below the minimum required performance limits (MRPLs) for tissue-bound residues of AOZ at 1 microg kg(-1) in the European Communities. In the present study, we investigated the use of homemade ELISA, a new immunoassay, to monitor the presence of the furazolidone marker residue in 370 samples of cultured fish. Adopting 0.3 microg kg(-1) AOZ as a cutoff value, the ELISA has a sensitivity of 100% and a specificity of 98.5% versus high-performance liquid chromatography-mass spectrometry (HPLC-MS) at a cutoff of 0.3 microg kg(-1) and gives no false-negative rate results. From the practical point of view, the homemade kit could be advantageously used for the screening of large groups of animal-edible tissue samples and the kit employed has good reliability even in routine application for the control of the illegal use of the drug.

Porous gelatin/tricalcium phosphate/genipin composites containing lumbrokinase for bone repair

Bone cell activities are very important in bone remodeling. This study investigates the effects of lumbrokinase on bone cell activities in cultures. Moreover, a biodegradable composite (GGT) containing genipin-crosslinked gelatin and β-tricalcium phosphate was prepared to carry lumbrokinase (GGTLK). Rat calvarial bone defects were filled with GGT and GGTLK composites. Bone healing was monitored in vivo by bioluminescence imaging and micro-CT. Lumbrokinase was found to have a dose-dependent effect on bone cell activities. Low concentrations (<1μg/ml) of lumbrokinase increased the viability, total alkaline phosphatase activity and mobility of osteoblasts, the number of total calcified nodules and the expression of osteopontin and osteocalcin; however, they considerably reduced the total tartrate-resistant acid phosphatase activity of osteoclasts. IVIS images revealed a stronger fluorescent signal in GGTLK-treated animals than in GGT-treated animals. Micro-CT analysis revealed that GGTLK induced more new bone formation than did GGT. These observations suggest that lumbrokinase released from GGTLK composite can enhance bone tissue regeneration.

Evaluation of Xanthine Oxidase Inhibitory Potential and In vivo Hypouricemic Activity of Dimocarpus longan Lour. Extracts

Background: Longan is a fruit tree known to contain many phenolic components, which are capable of protecting people from oxidative damage through an anti-inflammatory mechanism. It may be also worthwhile to study the effect on lowering uric acid activity. Materials and Methods: This study investigates the lowering of uric acid using longan extracts, including flowers, pericarps, seeds, leaves, and twigs, on potassium-oxonate-induced hyperuricemia mice and its inhibitory actions against xanthine oxidase (XO) activities. Results: The findings revealed that ethyl acetate fraction of longan extracts exhibited strong XO-inhibitory activity, and the flower extracts (IC50 = 115.8 μg/mL) revealed more potent XO-inhibitory activity to those of pericarps (118.9 μg/mL), twigs (125.3 μg/mL), seeds (262.5 μg/mL), and leaves (331.1 μg/mL) in vitro. In addition, different dosages of longan extract (50–100 mg/kg) were administered to hyperuricemic mice. The lowering effect of longan extracts on uric acid at 75 mg/kg markedly reduced plasma uric acid levels in decreasing order: Flowers (80%) > seeds (72%) > pericarps (64%) > twigs (59%) > leaves (41%), compared with allopurinol (89%). Finally, 10 isolated phytochemicals from longan flowers were then examined in vitro. The results indicated that proanthocyanidin A2 and acetonylgeraniin A significantly inhibited XO activity in vitro. This is the first report providing new insights into the urate-reducing effect of phenolic dimer and hydrolyzable tannin, which can be developed to potential hypouricemic agents. SUMMARY Longan flower extracts possess more potent XO-.inhibitory activity than pericarps, twigs, seeds, and leaves in vitro The lowering effect of longan flowers and seeds extracts markedly reduced plasma uric acid levels as compared to allopurinol in vivo The extract proanthocyanidin A2 and acetonylgeraniin A were demonstrated potent XO inhibitory activity in vitro. Abbreviations used: PO: Potassium-oxonate, XO: xanthine oxidase, HE: n-hexane, EA: ethyl acetate, i.p.: intraperitoneal, PBS: phosphate-buffered saline, AP: allopurinol, PUA: plasma uric acid.

ESTABLISHMENT OF A COMPETITIVE ELISA FOR DETECTION OF FLORFENICOL ANTIBIOTIC IN FOOD OF ANIMAL ORIGIN

Florfenicol (FF) is a synthetic antibiotic with a broad antibacterial spectrum and the high therapeutic effectiveness that has been developed specifically for veterinary use. Obviously, FF adulterated in animal supplies is one of essential global concerns. A competitive ELISA for the detection of florfenicol in food of animal origin (swine, chicken, and fish) is described. Influence of immunoconjugate structure on the assay sensitivity and specificity was investigated. The new ELISA showed much lower than the MRPLs for FF at 100–3,000 mg kg−1 in the European Communities and the sensitivity of our ELISA method was superior to that described in other reports. According to the test preparation record, the limit of detection of the developed ELISA performed on meat species was 0.3 µg kg−1 (IC50 value 1.9 µg kg−1). The method developed permits FF concentrations to be determined in the range 0.3–24.3 µg kg−1. A low cross-reactivity with florfenicol amine (FFA), thiamphenicol (TAP), and chloramphenicol (CAP) was displayed (16.2%, 9.5%, and 9.4%, respectively). Recovery in different food samples (swine, chicken, and fish) averages between 87–115%. The method can be applied for inspection of animal supplies for trace florfenicol residues.

Screening Procedures for Chloramphenicol Residue Determination in Serum, Milk, and Feedstuff Using Immunochromatographic Assay

The technology developed from this study is based on an immunochromatographic procedure that utilizes antigen-antibody properties using membrane carriers with immobilized immunoreactants and provides rapid and out-of-laboratory techniques. An evaluation of a colloidal gold-based immunochromatographic assay for chloramphenicol (CAP) residue detection in serum, milk and feed is described. Polyclonal antibodies against CAP were produced using CAP-IPA-MAA-ABA-bovine serum albumin (CAP-ed-BSA) conjugate as the immunogen, which exhibited no cross-reactivities with applied competitors in the studied concentration range. The test strip assay could be completed within 7 min, with a visual detection limit of 0.3 μg kg−1 CAP in serum and milk and 0.5 μg kg−1 CAP in feed. The established strip assay performed similarly as a commercial ELISA assay: the CAP test strip revealed a sensitivity of 90% (89–92%) and a specificity of 94% (91–98%), which showed appreciable accuracy and precision. The described strip is rapid, simple and cost-effective as well as sensitive and specific enough for reliable and accurate on-site screening.

Detection of Residues by Use of Solid-Phase Extraction and Reversed Phase High-Performance Liquid Chromatography after Oral Administration of Amoxicillin in Bass Muscle

An experimental trial was performed to establish an adequate depletion period of amoxicillin in bass. The amount of amoxicillin was applied by oral administration at the dose of 20 and 40 mg/kg body weight, and was given at a daily dose for five consecutive days. Amoxicillin levels were determined using a solid-phase extraction and high-performance liquid chromatography (HPLC) method. The drug was eluted from Mightysil RP-18 GP 250-4.6 mm φ5 m column with a mobile phase consisting of 0.005 M KH2PO4 buffer with pH 6.8-MeOH (90:10, v/v), at a flow rate of 1 mL/min. The effluent was monitored using a UV detector set at 210 nm. Each analysis required no longer than 15 mm. The validated concentration ranges of this method were 0.01 μg/mL to 10 μg/mL for working standard solutions, and 0.04 to 10 μg/g for muscle. Mean absolute recoveries were 78%. The limits of detection (LCD) and limits of quantitation (LOQ) for bass muscle were 0.04 and 0.1 quantitation g/g, respectively. After 5 consecutive oral doses of amoxicillin, residues in 6-h-postdosing (T(subscript max)) bass were at the maximum 0.84 and 1.18 μg/g (C(subscript max)) toward 20 and 40 mg/kg b.w. dosage, residue levels after a 72-h depletion decreased to below the methods limit of quantitation after last oral treatment. At the dosage (40 mg/kg b.w.), we suggested that the recommended withdraw period was 5 days.

The effect of platelet-rich fibrin on autologous osteochondral transplantation: An in vivo porcine model

BACKGROUND This work aimed to evaluate the efficacy of cartilage transplantation to the medial femoral condyle±platelet-rich fibrin (PRF) augmentation in a porcine model. The hypothesis of the study was that PRF may act as a bioactive cell scaffold to fill defects and enhance cartilage regeneration. METHODS Thirty-two knees of 16 miniature pigs were randomly assigned to four groups. The critical-size osteochondral defects (8x5mm) in femoral condyle of both knees were treated with one of the following: group 1－untreated controls; group 2－cartilage fragments alone; group 3－PRF alone; group 4－PRFT+cartilage fragments. After completion of the surgical implantation, the periosteal patch harvested from the proximal tibia was sutured onto the cartilage of the medial condyle to cover the implanted defects. Animals were sacrificed at six months after treatment. The regenerated cartilages were assessed by gross inspection and histological examination. RESULTS The best results were obtained with the repair tissue being hyaline-like cartilage (group 4). The grading score of histological evaluation demonstrated that group 4 had better matrix, cell distribution and cartilage mineralization than group 2 and group 3. PRF showed a positive effect on the cartilage repair; the procedure was more effective when PRF was combined with autologous chondrocytes. CONCLUSIONS This approach may provide a successfully employed technique to target cartilage defects in vivo. Larger groups and longer periods of study may provide more definitive and meaningful support for using this therapeutic approach as a new way of cartilage regeneration.

ESTIMATION OF THE WITHDRAWAL PERIOD FOR AMOXICILLIN IN POMPANO (TRACHINOTUS BLOCHII) SERUM AND MUSCLE

Amoxicillin is one of the most popular antibiotics used in aquaculture. To ensure the residue of amoxicillin in the serum and the muscle of pompano (Trachinotus blochii) is below the established tolerance levels; we used reversed phase high-performance liquid chromatography (HPLC) to measure the amoxicillin in pompano after oral administration. The pharmacokinetics of amoxicillin in the serum and the muscle of pompano were established by single oral administration or administrations for five consecutive days at the dose of 40 mg/kg. After different depletion periods, amoxicillin residue was analyzed in both the serum and the muscle fillets by HPLC. The verified concentrations of this method were 0.01 to 10 μg/mL; the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.04 and 0.1 μg/g. The concentration of amoxicillin in the serum of pompano reached the maximum threshold of 7.36 μg/g after 0.5 h depletion in the experiment with a single dose of amoxicillin (40 mg/kg of body weight). In addition, the maximal concentration (1.96 μg/g) of amoxicillin was detected in the muscle 2 h after depletion. Furthermore, the residue levels had decreased below the limit of detection after 48 h and 72 h in the serum and the muscle. Here we also suggested that 5 days of withdrawal were required after the multiple dosing of amoxicillin.