An indirect competitive assay-based aptasensor for detection of oxytetracycline in milk.

Oxytetracycline (OTC) is a common antibacterial agent used for the control of animal diseases. OTC abuse can seriously affect human health; therefore, we developed a biosensor using single-stranded DNA (ssDNA) aptamers for the detection of OTC. The binding probe aptamers for OTC were selected by a Systematic Evolution of Ligands by the exponential enrichment (SELEX) process and identified by the enzyme-linked aptamer assay (ELAA). Among the selected 5 aptamers, aptamer OTC3 showed the strongest affinity (Kd=4.7 nM) and highest specificity for OTC compared to structurally similar antibiotics, tetracycline and chlortetracycline. OTC was detected using indirect competitive ELAA. The limit of detection and quantitation with aptamer OTC3 were 12.3 and 49.8 µg/L, respectively, in milk and showed recovery rates of more than 90% in OTC-spiked milk. This biosensor method with high sensitivity and specificity based on indirect competitive ELAA can be applied to OTC detection in food products on-site because of the simplicity of detection.

Enhancing immunoassay detection of antigens with multimeric protein Gs.

This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)(3) linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO(2)-MNPs, Fe(3)O(4)@SiO(2)) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO(2)-MNPs was a time of 60 min and a concentration of 100 µg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO(2)-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50 ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO(2)-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation.

Risk assessment of growth hormones and antimicrobial residues in meat.

Growth promoters including hormonal substances and antibiotics are used legally and illegally in food producing animals for the growth promotion of livestock animals. Hormonal substances still under debate in terms of their human health impacts are estradiol-17ß, progesterone, testosterone, zeranol, trenbolone, and melengestrol acetate (MGA) . Many of the risk assessment results of natural steroid hormones have presented negligible impacts when they are used under good veterinary practices. For synthetic hormonelike substances, ADIs and MRLs have been established for food safety along with the approval of animal treatment. Small amounts of antibiotics added to feedstuff present growth promotion effects via the prevention of infectious diseases at doses lower than therapeutic dose. The induction of antimicrobial resistant bacteria and the disruption of normal human intestinal flora are major concerns in terms of human health impact. Regulatory guidance such as ADIs and MRLs fully reflect the impact on human gastrointestinal microflora. However, before deciding on any risk management options, risk assessments of antimicrobial resistance require large-scale evidence regarding the relationship between antimicrobial use in food-producing animals and the occurrence of antimicrobial resistance in human pathogens. In this article, the risk profiles of hormonal and antibacterial growth promoters are provided based on recent toxicity and human exposure information, and recommendations for risk management to prevent human health impacts by the use of growth promoters are also presented.