Protecting Consumer Health By Developing a Multiplex Amperometric Immunosensor for the Detection of Banned Antibiotic Residues in Milk
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Introduction Chemical (eg pesticides, veterinary drugs, etc.) and bacteriological contaminants (eg. foodborne pathogens) could contaminate animal and plant derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites, dyes) are banned in foodstuffs of animal origin (eg. milk, meat, eggs, etc.) in European Union because of toxicological risks for the consumer. The European Regulation has set Minimum Required Performance Limits (MRPL) [1] or Reference Point for Action (RPA) for banned substances [2]. Food containing residues of substances at or above the MRPL or RPA are declared non-compliant and consignments are rejected from the consumer’s market. Screening methods are the first stage of food control and so are essential for food safety monitoring. Conventional screening methods are microbiological methods (eg. plate tests, tube tests), immunological methods (eg. ELISA, radioimmunoassays) or physico-chemical methods (Thin Layer Chromatography, High Performance Liquid Chromatography (HPLC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). These methods sometimes lack of sensitivity or specificity; they also could be time and money consuming. There is thus a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach. Electrochemical immunosensor An innovative electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [3]. This technique presents major advantages: low cost (eg. disposable electrodes, potentiostat), promising detection limits, portability, and possible automatisation. Our laboratory has evaluated the transferability of the method. An electrochemical immunosensor has been developed for the detection of chloramphenicol residues in milk as a proof of concept. The matrix effect (milk samples) was high and so sample preparation has to be improved to reduce matrix effects. The objective is to develop an amperometric bead-based immunosensor for the multiplex detection of banned antibiotics (eg. chloramphenicol, nitrofuran metabolites, dyes) in bovine milk. Method Antibodies (Abs) against antibiotics are grafted on the surface of magnetic beads (MBs). Milk samples and antibiotic conjugated with Horseradish peroxidase (HRP) are mixed with MBs-Abs. A competition occurs between the HRP conjugates and the antibiotic residues if present in the sample, for the binding to the antibody. The MBs are washed to remove free antibiotics and conjugates. Then, a Screen Printed Carbon Electrode (SPCE) with MBs on its surface (maintained by a magnet) is soaked into a buffer solution containing hydroquinone; when adding hydrogene peroxide (H 2 O 2 ) to the solution, an amperometric signal is produced, due to the enzymatic activity of HRP and measured. The amperometric signal is inversely proportional to the antibiotic concentration in the sample. Results and Conclusions Screening methods for the detection of veterinary drugs in food products have to be validated according to the European regulation [1] and to the European guideline for the validation of screening methods [4]. After the development and the optimization of the analytical parameters (eg. sample preparation, HRP concentration, incubation time, applied potential, etc), the methods developed for single compounds will be evaluated and validated according to the European regulations. Then the single compound methods will be merged into one multiplex method if possible. The results will be presented to the conference, discussing the advantage sand drawbacks of amperometric biosensors for the screening of antibiotic residues in food products. References 1. Commission Decision (EC) N° 2002/657 of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. 2002: Official Journal of European Communities. p. 8-36. 2. Commission Regulation (EC) N° 470/2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin 2009, The European parliement and the Council: Official Journal of the European Union p. 11-22. 3. Conzuelo F, Ruiz-Valdepeñas Montiel V, Campuzano S, Gamella M, Torrente-Rodríguez RM, Reviejo AJ, Pingarrón JM. 2014. Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors. Anal. Chim. Acta. 820:32-38. 4. CRL, Guideline for the validation of screening methods for residues of veterinary medicines (initial validation and transfer). 2010: Available from:<http://ec.europa.eu/food/food/chemicalsafety/residues/lab_analysis_en.htm>: Guideline_Validation_Screening_en.pdf. p. 1-18.Keywords:
Veterinary Drugs
Objective To understand the situation of application of antibiotics in certain hospital and solve the main problems of existence.Methods Retrospective analysis was applied to investigate the use of antibiotics in the cases,which include all kinds of type Ⅰ incision patients of surgery system.Results Totally 458 cases used antibiotics,and the usage rate of antibiotics was 91.05%.The selected antibiotics included 10 groups 27 varieties and the total application amount was 750.82 received antibiotics preoperatively,255 received antibiotics during operation and 433 received antibiotics postoperatively.In the continue medication,the longest time of antibiotic use was18d,the shortest was 1d and the average was 4.38d.119 cases received antibiotics for ≤48 h(27.5%),314 for 48h(72.5%).Conclusions Problems existed in the application of antibiotics,such as non-strict handling of indications of antibiotic use,high grade of antibiotics selected and long course.Measures should be taken to normalization the management of antibiotics and improve the rationality of application of antibiotics.
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Objective To investigate the use of antibiotics in the surgical patients.Methods The use of antibiotics in 361 cases of the surgical patients was retrospectively analyzed.Results The rate of prophylactic and therapeutic use of antibiotic was 45.15 and 54.85,respectively.42.94 patients received postoperative antibiotic prophylaxis for 4 to 21 days.Percentage of patients receiving 1,2,or 3 types of antibiotics was 61.77,37.67 and 0.56,respectively.Conclusion Guidelines on the use of antibiotics in surgical patients were not sufficient.Improper use of antibiotics included the selection and prolonged use of antibiotics,inappropriate use of multiple antibiotics and the use of antibiotics during the perioperative period.Antibiotics should be used according to the guidelines and the results of microbiological analysis.
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Most clinical isolates that exhibit a multi-drug resistant phenotype owe that resistance to over-expressed efflux pumps. Compounds that are efflux pump inhibitors (EPIs) reduce or reverse resistance to antibiotics to which the bacterial strain is initially resistant. We have evaluated non-antibiotics to reduce resistance of commonly encountered bacterial pathogens to antibiotics.The effect of non-antibiotics on the susceptibility of bacteria to antibiotics was conducted by minimum inhibition concentration determinations of the antibiotic in the absence and presence of the non-antibiotic.Non-antibiotics such as chlorpromazine, amitryptiline and trans-chlorprothixene are shown to reduce or reverse resistance of a variety of bacteria to antibiotics.The results suggest that non-antibiotics may serve as adjuncts to conventional antibiotics for the therapy of problematic antibiotic infections caused by bacteria that owe their resistance to over-expressed efflux pumps.
Efflux
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Hapten
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Virginiamycin
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Objective To offer reference for the proper use of antibiotics by exploring the features of use of antibiotics in the hospitalized patients staying in the department of nephrology. Methods The data on 1 000 discharged patients who had been hospitalized in our department were retrospectively investigated and the use of antibiotics was analyzed. Results Of 1 000 patients, 876 (87.6%) received antibiotic treatment. 639 of the 876 patients (72.9%) were received single-antibiotic therapy while 237 (27.1%) received two antibiotic agents. 69.2% of the antibiotics were used for treatment and 30.8% for prevention. 89.3% of the patients received prophylactic antibiotics for 3 days or less while 10.7% used antibiotics for more than 3 days. The rate of prophylactic use of antibiotics 0.5 to 1 hour before surgery was lower. The administration route of antibiotics was applied in 100% of the patients. Conclusions The use of antibiotic is basically appropriate in this study. The rate of prophylactic use of antibiotics is higher in the hospitalized patients staying in the department of nephrology. Further studies should be conducted on the exact timing of prophylactic use of antibiotics and whether oral antibiotics can replace intravenous antibiotics for the prevention of postoperative infection.
Nephrology
Intravenous antibiotics
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As a new type detection method for heavy metal ions, immunoassay has unique characteristic and advantage.The immunoassay technology system consists of preparation of heavy metal ions monoclonal antibodies, increase of sensitivity of antibodies and immunoassay, in which obtaining antibodies is the base.The progress in immunoassay for heavy metal ions was introduced.In addition, critical issues and direction were discussed in the article.
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Quantitative immunoassay technique is the common method of quantitative detection in clinical laboratory. Several important branches of quantitative immunoassay were formed by changing the tracer or the Antigen-antibody complex separation method, including radioimmunoassay, fluorescent immunoassay, enzyme immunoassay, chemiluminescence, colloidal gold, immuno-turbidimetric analysis and homogeneous immunoassay. Different immunoassay techniques have their own characteristics, also apply to different detecting conditions in clinic. This paper reviewed several common kinds of quantitative immunoassay technology, and discussed both their advantages and disadvantages, which provide reference for the application and development of clinical testing technology. (Chin J Lab Med, 2017, 40: 478-480)
Key words:
Immunoassay; Radioimmunoassay; Fluoroimmunoassay; Immunoenzyme techniques
Chemiluminescent immunoassay
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