| More and more people concerned food safety with the development of economy, because it's related to the health and life security of everybody and it's the key problem that been concerned by Chinese government all the time. Since dairy products have been one of the most important foods in daily consumption, close and extensive concerns about the safety of dairy products are continuously rising. Antibiotic residues are the most harmful contaminants in dairy products besides the illegal additives like melamine. The traditional technologies aren't capable to detect multiple antibiotic residues rapidly, sensitively and high-throughput. To resolve this problem, a novel high-throughput suspension array technology has been established for detection of multiple antibiotic residues. The aim of the study is to implement quantitative detection of the multiple residues rapidly, accurately and efficiently.Five kinds of antibiotics, including chloramphenicol, tetracycline, gentamicin, streptomycin, and tylosin, which are commonly used for the medication of cows, are regarded as the targets. The experiment was based on the indirect competitive method. The goal that up to 5 kinds of antibiotic residues can be detected at the same time has been achieved by competition between the specific antibodies and the multiple antigens of antibiotic residues which labeled on the fluorescent beads in liquid phase. In addition, the sensitivity and stability of suspension array technology was improved by optimization and screening the experimental conditions, and the detecting cost reduced at the same time. A novel milk samples treatment that appropriate for suspension array had been explored and applied. The operation of this technology is simple, treatment is rapid, and hardly has any effect on detection sensitivity, stability etc. The veracity of suspension array was further certified by compared with traditional detection technology. The suspension array detection is simple, rapid, easy to operate and reliable in the experiment.In this paper, the followings have been included:1. The coupling of different antibiotic antigen on fluorescent bead Glutaraldehyde method and carbodiimide method were employed for the synthesis of the protein conjugates of some targets. The optimum coupling concentration of antigens were selected based on the report fluorescence, which obtained by different coupling concentration of antigens. EDC and NHS were employed to couple the different antibiotic antigen on fluorescent bead 2. Conditions optimization of Luminex assay. Multi reaction conditions, such as concentration of electrolyte of reaction buffer, reaction buffers, detection models (employing of biotin-secondary antibody complex), incubation time, reagents addition method, the dose of antibody, the dilution proportion of SA-PE and the dose of antibody in simultaneous detection, are selected and optimized.3. Simultaneous detection for multiple antibiotic residuals, and the detection sensitivity, specificity and the methodological comparison. By means of optimizing the condition and exploring a variety of the tested target concentration gradient, the standard curves of the five single channel suspension array have been polted. Based on this situation and the optimization of the dose of antibody in simultaneous detection, the standard curves of the five multiple channel suspension array have also been polted. The Logistic regression are employed to fitting the standard curves, and the curves shows good relationship, coefficient of determination R2 were higher than 0.9945 the detection sensitivity has obvious advantage compare with the technology used by national standard. Specific recognition was carried on and it proved excellent recognition between various paired antigen-antibody on the beads. And in the aspect of specificity for structurally similar and non-similar drugs, it showed no significant cross-reaction and interference, except for two kinds of tetracyclines with the monoclonal antibody of tetracycline. Traditional indirect competitive method and liquid chromatogram method have been employed for the detection of multiple antibiotic residues in comparison with suspension array. The sensitivities of suspension for the detection of the targets are higher than the traditional methods. Furthermore, simply operation, rapid (the whole detection takes in 2 hours, including the treatment of milk samples) and high-throughput have the advantage over indirect competitive method and liquid chromatogram method.4. Pre-treatment method of milk samples for suspension array detection After the comparison of different treatment of milk samples, a novel treatment of milk samples for Luminex suspension array has been established. The treatment based on diethyl ether is established. The operation is simple, the process is fast and effective, and the detection sensitivity and the reaction stability have been greatly improved. It also provide clues for the further development of food treatment. The whole process is simple and time-saving, only completed in 20 min. Spike recovery experiments and the testing of actual samples has also shown that this method is an effective sample treatment, and has little impact on targets and immunoreaction. It meets completely the needs of sample preparation for Luminex suspension array.5. The detection of milk samples by Luminex suspension array technology The treated milk samples were detected by suspension array, and the results were calculated by standard curve. By comparing with results of ELISA obtained from the same sample, the precision of suspension array was higher and the errors was more less.The detection range of the five antibiotics by suspension array is floolwed: tylosin 0.026~400 ng/mL,gentamicin 0.4~25.6 ng/mL,tetracycline 0.05~781.25 ng/mL,streptomycin 0.08~1250 ng/mL,chloramphenicol 0.5~7812.5 ng/mL. Luminex suspension array are mostly applied to the detection of protein and nucleic acid molecules at home and abroad, the detection of different types of antibiotics in milk hasn't been systemically reported. This study has established the high-throughput detection method for multiple antibiotic residues in milk by Luminex suspension array, and the milk actual samples are analyzed and evaluated. This method is sensitive, rapid and stable. It provides a novel method for multiple antibiotic residues in milk and also provides the foundation of research and development of detection kits. |
PDF Full Text Request