恩诺沙星完全抗原的制备试验

目的制备恩诺沙星(EF)的完全抗原,进而为EF单克隆抗体检测试剂盒的研制奠定基础。方法将小分子的药物EF和鸡血清白蛋白(OVA)以及牛血清白蛋白(BSA)分别采用碳二亚胺法和活化酯法两种不同的偶联方法合成完全抗原,通过紫外特征光谱和抗血清效价对完全抗原的合成效果进行分析。结果2种不同的偶联方法均使OVA和BSA与EF偶联成功。偶联方法对于完全抗原的合成效果也存在差异,从紫外吸光率和偶联物结合比判断,OVA采用活化酯法效果好,未经EDA处理的OVA、活化酯法和碳二亚胺法合成的偶联物的结合比分别为10.3、20.7、16.5;而BSA采用碳二亚胺法效果好,未经EDA处理的BSA、活化酯法和碳二亚胺法合成的偶联物的结合比分别为20.5、24.6和30.8。血清效价检测发现,以BSA-EF为完全抗原的效价为25600,以OVA-EF为完全抗原的效价为6400,这表明完全抗原的合成是成功的。结论2种不同方法合成的OVA-EF和BSA-EF完全抗原,可以作为免疫原用于制备抗体以及检测时的包被原进行ELISA检测。     

硫酸镁在恩诺沙星注射液无菌检查中的应用#br#

目的 探讨恩诺沙星注射液无菌检查的方法。方法 参照《中国兽药典》2015版第一部附录1101无菌检查方法,采用薄膜过滤法,样品过滤后使用含不同浓度硫酸镁溶液的缓冲液冲洗,随后加入含不同浓度硫酸镁的培养基,根据硫酸镁的浓度设置4组试验对恩诺沙星注射液进行无菌检查。结果 按薄膜过滤法,样品过滤后使用0.1%无菌蛋白胨水溶液(含0.1mol/L MgSO4)冲洗4次,每次100mL,冲洗后向集菌器中加入100mL培养基(含0.05mol/L MgSO4),可消除恩诺沙星注射液的抑菌活性,试验菌均可正常生长。结论 该方法可用于恩诺沙星注射液的无菌检查。     

人工诱导耐恩氟沙星金黄色葡萄球菌grlA和gyrA基因突变研究

通过恩氟沙星人工多步诱导标准金黄色葡萄球菌 2 80 0 1产生MIC值为 4～≥ 1 2 8μg/ml的耐药变异株 ,选其 3株提取染色体DNA ,对gyrA和grlA基因进行PCR扩增 ,产物与pMD1 8 T载体连接 ,转化至JM1 0 9大肠埃希氏菌感受态细胞 ,筛选阳性克隆 ,测序。序列分析结果表明 ,低水平 (MIC≤ 1 6μg/ml)的耐药突变株只发生grlASer80 (TCC)→Phe (TGC)或grlA ,Ser80 (TCC)→Tyr (TAC)点突变 ,高水平 (MIC≥1 2 8μg/ml)的耐药突变株需要grlASer80 (TCC)→Phe (TGC)和gyrASer84(TCA)→Leu (TTA)等两个位点的突变。三株耐药株均发生grlASer80→Tyr (Phe)点突变 ,说明恩氟沙星作用于金黄色葡萄球菌的首要靶酶是拓扑异构酶IV     

Monoclonal antibody for the development of specific immunoassays to detect Enrofloxacin in foods of animal origin

The carbodiimide active ester method was employed to synthesise the antigen of 1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid [Enrofloxacin (ENFX)], and male Balb/c mice were used to produce anti-ENFX monoclonal antibody. Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay standard curve was established. This assay was sensitive and highly specific to ENFX with the half maximal inhibitory concentration and limit of detection values of 0.15?ng?mL^?1 and 0.028ng?mL^?1, respectively. Similarly, the antibody had a high affinity as seen in A_max values. Spiked cow milk beef liver and fish samples analysed using this kit had recoveries in the range of 90.2–110.2%, 70.7–81.5% and 89.1–101%, respectively, showing satisfactory results. The results suggest that this ELISA kit could be applied as a screening method to detect and control the illegal content of ENFX in food products.     

Influence of enrofloxacin traces in drinking water to doxycycline tissue pharmacokinetics in healthy and infected by Mycoplasma gallisepticum broiler chickens

Most of antibiotics, administrated in the treatment of poultry diseases are dissolved in drinking water, and it can lead to water supply systems contamination, especially when the regular cleaning is not using. This situation can lead to unconscious administration of low doses of antibiotics to untreated animals. The aim of this study was to clarify the impact of the exposure of enrofloxacin traces (500 μg l⁻¹) to doxycycline pharmacokinetics in healthy and experimentally Mycoplasma gallisepticum infected broiler chickens., Two experimental groups, received of enrofloxacin in water and all groups, received 20 mg kg⁻¹ bw of doxycycline. The compounds concentrations in muscles and livers were determined by LC-MS/MS. The maximum drug tissue concentration (C_max) of doxycycline was highest in liver obtained from infected chickens which, received enrofloxacin traces (ENR + DC/MG). It was about 40% higher than in healthy chickens from group I which received only doxycycline. It was found that the concentration–time curve AUC_(0-t) values in group ENR + DC/MG were almost 75% higher than in the group (DC) and 35% higher than in group (ENR + DC) which also received enrofloxacin traces.The constant exposure of broiler chickens on enrofloxacin traces as well as infection, may significantly influenced on doxycycline tissue pharmacokinetic profile.     

Effects of some quinolone antibiotics on malondialdehyde levels and catalase activity in chicks

In this study, the effects of enrofloxacin, ciprofloxacin and norfloxacin at therapeutic doses and over therapeutic periods on plasma malondialdehyde (MDA) levels and erythrocyte catalase (CAT) activity were investigated. For this purpose, a hundred and sixty 3-day-old female Ross breed broiler chicks were used. Four groups, each comprising 40 were established. While group 1 was maintained as control, groups 2, 3 and 4 were administered enrofloxacin, ciprofloxacin and norfloxacin, respectively, at 10 mg/kg bw/day in drinking water. On days 1, 3, 5 and 7, ten animals were killed from each group, and plasma MDA levels and erythrocyte CAT activity were measured spectrophotometrically. The data obtained demonstrated that CAT activity was statistically decreased in groups 2 and 4, and increased in group 3 on day 1. In conclusion, it was determined that neither of the three drugs generated free radicals, in other words, caused lipid peroxidation physiologically when given at therapeutic dosage.     

Development and application of an immunoaffinity column clean-up for enrofloxacin determination in food samples

Enrofloxacin (ENR) is a synthetic fluoroquinolone antibiotic used for the treatment of infection in animals. The use of ENR has been strictly regulated because of its harmful effect on consumers of animal products. In this study the generation of polyclonal antibody (pAb) against ENR, the preparation of an immunoaffinity chromatography column and its potential application to the selective extraction of ENR residues from food samples were described. The produced pAb exhibited good sensitivity to ENR with an IC₅₀ value of 5.4?ng/mL. The cross-reactivity values of the antibody with ENR structurally related compounds were lower than 0.5%. An immunoaffinity column was constructed by coupling antibody covalently with chitosan microspheres. 0.8% NaOH/0.01?mol/L phosphate-buffered saline (PBS) (pH 7.4) and 85% methanol were selected as loading and eluting solution by careful optimization. Further characterization indicated that the binding capacity of the column for ENR was approximately 3780?ng/mL gel. The immunoaffinity columns were then challenged with ENR-fortified pork, and the recoveries of ENR were found to be in the range of 79.8–94.9%, demonstrating the feasibility of the prepared immunoaffinity column for sample clean-up in ENR residue determination.     

Various Solvent Systems for Solubility Enhancement of Enrofloxacin

Solubility enhancement of antimicrobial drug enrofloxacin has been studied using a series of co-solvents and surfactants. Aqueous solubility of enrofloxacin could be increased up to 26 times. Co-solvents alone produced only small increase in solubility. However, the combined effect of co-solvents and buffer was synergistic and a large increase in solubility could be attained. Ionic surfactants were found to be much better solubilizing agents than non-ionic surfactant. Amongst ionic surfactants, solubility was found to be very high in anionic surfactant, sodium dodecylsulphate as compared to the cationic surfactant, cetyltrimethylammonium bromide. Up to 3.8 mg/ml of enrofloxacin could be dissolved in sodium dodecylsulphate. Mechanism of solubilization has been proposed and surfactant solubilization parameters have been calculated.     

Physicochemical properties of enrofloxacin

The physicochemical properties of enrofloxacin, a fluoroquinolone that inhibits the activity of bacterial DNA gyrase, are described. Its spectral, solubility and related physicochemical characteristics are discussed. The dissociation behaviour of enrofloxacin was examined by UV spectrophotometry at 25°C in a series of buffers ranging from pH 1 to 10. The corresponding macro- and microscopic dissociation constants were calculated. The apparent n-octanol-water partition coefficients were measured from pH 2 to 10.     

The effects of enrofloxacin on decorin and glycosaminoglycans in avian tendon cell cultures

Tendonitis and tendon rupture have been reported to occur during or following therapy with fluoroquinolone antibiotics. Though the pathogenesis is unknown, several studies suggest that fluoroquinolone antibiotics alter proteoglycan content in soft tissues, including tendons, and thereby alter collagen fibrillogenesis. To better understand the mechanism of action of fluoroquinolones, we studied the effects of enrofloxacin, a widely used fluoroquinolone in veterinary medicine, on avian tendon cell cultures established from gastrocnemius tendons from 18-day-old chicken embryos. We found that cell proliferation was progressively inhibited with increasing concentrations of enrofloxacin. This was accompanied by changes in morphology, extracellular matrix content and collagen fibril formation as detected by electron microscopy. We also observed a 35% decrease in the content of total monosaccharides in enrofloxacin-treated cells. The ratio of individual monosaccharides was also altered in enrofloxacin-treated cells. Enrofloxacin also induced the synthesis of small amounts of keratan sulfate in tendon cells. Moreover we observed enrofloxacin-induced changes in glycosylation of decorin, the most abundant tendon proteoglycan, resulting in the emergence of multiple lower molecular bands that were identifiable as decorin after chondroitinase ABC and N-glycanase treatment of extracts from enrofloxacin-treated cells. Medium conditioned by enrofloxacin-treated cells contained less decorin than did medium conditioned by control cells. We hypothesize that enrofloxacin induces either changes in the number of N-linked oligosaccharides attached to the core protein of decorin or changes in decorin degradation process. In conclusion, our data suggest that enrofloxacin affects cell proliferation and extracellular matrix through changes in glycosylation.