二氟沙星残留检测的Ci-ELISA方法建立

以合成的二氟沙星（DIF）人工抗原为基础，制备出了高效价抗DIF血清（1∶29×104），并建立了DIF间接竞争酶联免疫吸附测定法(Ci-ELISA法)。标准曲线的线性回归方程为y=-0.223x+0.9631(r=0.9929)，中值（I50）=119.21 ng/ml，最低检测限（LOD）=1.14 ng/ml，线性范围为1.14~10000 ng/ml，方法的平均批内变异系数（CV）为4.83%,平均批间CV为10.69%。DIF以浓度15~10000 ng/ml在鸡肉中添加时，回收率为81.24%~90.64%，变异系数为8.18%~11.75%。     

醋酸甲孕酮（MPA）间接竞争ELISA检测方法的建立

将半抗原醋酸甲孕酮（MPA）衍生物与牛血清白蛋白(BSA)偶联后，作为包被原与游离的醋酸甲孕酮竞争抗MPA多克隆抗体，通过该模式建立间接竞争ELISA方法检测MPA的残留量。试验结果表明：理想的包被抗原为0.625 μg/ml,抗体的工作浓度为1∶8000，标准曲线在1 ～50 ng/ml之间线性关系较好，最低检测限0.1 ng/ml, 得回归方程y=-1.1479x+0.9426(r2=0.9679),批内和批间变异系数分别为5. 35%、10. 89%。     

达氟沙星间接竞争ELISA检测方法的建立

将达氟沙星（danofloxacin, DFLX）与牛血清白蛋白(BSA)、卵清白蛋白(OVA)偶联分别作为免疫原与包被原, 达氟沙星为竞争的半抗原，建立间接竞争ELISA检测方法。试验结果表明,理想的包被抗原浓度为1.25 μg/L, 多抗(DFLX-PcAb)工作浓度为1∶10000,酶标二抗的工作浓度为1∶4000,最适检测范围为0.1～10 ng/L,最低检测限为0.2 ng/L, 批内和批间变异系数分别为3.81%和6.25%。得到回归方程y=0.7975-0.125x（R2=0.989）和标准曲线，从而建立了DFLX的快速检测残留的间接竞争酶联免疫吸附试验（ci-ELISA）。     

鸡肌肉组织中左氧氟沙星残留ELISA检测方法的研究

用N-羟基琥珀酰亚胺法把左氧氟沙星(LVL)与牛血清白蛋白(BSA)偶联制备免疫抗原,免疫新西兰大白兔得到LVL的多克隆抗体,建立了LVL间接竞争ELISA方法.结果表明:抗LVL血清效价达1: 212以上,得到标准曲线的线性回归方程为y=-0.289 4x+0.049 5(R2=0.987 8),50%抑制浓度(IC50)为64 ng/mL,最低检测限(LOD)为10 ng/mL,标准曲线的线性范围为10～1 000 ng/mL.批内变异系数为3.51%～7.46%,批间变异系数为8.66%～11.89%,鸡肌肉中的LVL添加浓度在10～1 000 ng/ml范围内时,回收率为73.5%～84.36%.本试验建立的ELISA方法能够满足左氧氟沙星兽药残留检测要求.     

鸡肌肉组织中左氧氟沙星兽药残留ELISA检测方法的研究

用N-羟基琥珀酰亚胺法把左氧氟沙星（LVL）与牛血清白蛋白（BSA）偶联制备免疫抗原,免疫新西兰大白兔得到LVL的多克隆抗体,建立了LVL间接竞争ELISA方法。结果表明：抗LVL血清效价达1∶212以上,得到标准曲线的线性回归方程为y=-0.289 4x＋0.049 5（R2=0.987 8）,50%抑制浓度（IC50）为64 ng/mL,最低检测限（LOD）为10 ng/mL,标准曲线的线性范围为10～1 000 ng/mL。批内变异系数为3.51%～7.46%,批间变异系数为8.66%～11.89%,鸡肌肉中的LVL添加浓度在10～1 000 ng/ml范围内时,回收率为73.5%～84.36%。本试验建立的ELISA方法能够满足左氧氟沙星兽药残留检测要求。     

非淀粉多糖酶对蛋鸡玉米——杂粕型日粮离体消化能的影响

选用木聚糖酶(x1)、β-葡聚糖酶(x2)、纤维素酶(x3)和果胶酶(x4)4种非淀粉多糖(NSP)酶制剂,利用胃蛋白酶-胰液素法分别研究其对蛋鸡玉米—杂粕型日粮能量的当量调控。结果表明,4种NSP酶对蛋鸡玉米—杂粕型日粮的离体消化能改进值分别为:木聚糖酶0.5442MJ/kg、β-葡聚糖酶0.4791MJ/kg、纤维素酶0.3345MJ/kg、果胶酶0.4457MJ/kg。4种NSP酶的添加水平与离体消化能(y)进行S曲线拟合,其回归方程分别为:y1=10.6219-0.6354/｛1+exp([x1-446.6123)/146.000](｝R2=0.9961);y2=10.5065-0.5687/｛1+exp[(x2-282.9857)/146.599](｝R2=0.9964);y3=10.3612-0.3464/｛1+exp[(x3-468.4880)/93.931]｝(R2=0.9939);y4=10.4684-0.4669/｛1+exp[(x4-367.4321)/100.142]｝(R2=0.9989)。     

中药复方制剂在鸡体内的药代动力学研究

作者旨在研究中药复方制剂中的白藜芦醇苷和大黄素在鸡体内的药代动力学。采用ZORBAX SB-C18分析柱(4.6 mm×150 mm,5μm),以1~20 min 10∶90~90∶10甲醇∶水为流动相,紫外检测波长为290±30 nm。白藜芦醇苷和大黄素在0.02~8μg/ml范围内均呈良好的线性关系,白藜芦醇苷的标准曲线方程y=0.0648x-0.0079(r=0.9977),平均相对回收率达100.36%,平均日内、日间精密度CV分别为1.74%和1.87%;大黄素的标准曲线为:y=0.0584x-0.0254(r=0.9992),平均相对回收率达98.83%,平均日内、日间变异系数CV分别为2.27%和3.98%。鸡静脉注射中药复方后的白藜芦醇苷的血药浓度-时间曲线符合无吸收三室开放模型;大黄素的血药浓度-时间曲线符合无吸收二室开放模型。     

运用OD值对常见畜禽腹泻病原菌计数的研究

本研究就畜禽常见腹泻病原菌的生长特性、细菌悬液浊度和细菌计数之间的关系进行探讨，通过对常见畜禽腹泻病原菌大肠杆菌(O8型和O139型)、鸡白痢沙门菌、鼠伤寒沙门菌和鸡伤寒沙门菌不同培养时间段吸光度(optical density,OD)值的测定，结合平板活菌计数法来确定这5种细菌的生长曲线，并对细菌浊度和细菌计数相关性进行统计分析，发现极显著相关(P <0.01)，建立了5种细菌在生长期和稳定期的OD值-细菌数对数的曲线回归方程，O8型大肠杆菌：y=2.55x+6.72(r²=0.96);O139型大肠杆菌：y=1.45x+8.77(r²=0.95)；鸡伤寒沙门菌：y=4.50x+7.02(r²=0.95)；鼠伤寒沙门菌：y=5.59x+7.67(r²=0.97)；鸡白痢沙门菌：y=9.72x+6.60(r²=0.98)。利用该回归方程可快速准确地确定菌液浓度，为探讨细菌的培养特性、致病机制及动物实验中攻毒剂量的确定等相关实验提供参考依据。     

数种牧草种子劣变的生活力与膜透性的关系

以3种豆科和2种禾本科牧草包括紫花苜蓿，光叶紫花苕，沙打旺，苏丹草和老芒麦为材料，研究了种子劣变过程中生活力与水浸电导率的关系。结果表明3种豆科种子的生活力（x)与水浸电导率（y)皆呈极显著负相关（P＜0．01）。可用如下回归方程表示：紫花苜蓿：y=-1.866x 288.56(r=-0.982)；光叶紫花茹:y=-1.099x 194.20(r=-0.902)，沙打旺：y=-1.889x 308.22(r=-0.983)。然而，2种禾本科种子生活力却与电导率不相关（P＞0．05），而且电导率的测定值甚小。种子吸水实验证明禾本科与豆科种子在24h内的不同吸水阶段皆表现为高生活力的吸水率显著低于低生活力种子（P＜0．01）。     

兰州大尾羊解偶联蛋白3(UCP3)基因荧光定量PCR检测方法的研究

试验通过提取兰州大尾羊不同组织中总RNA,反转录获得总cDNA样品,建立了用SYBR荧光定量PCR法检测兰州大尾羊解偶联蛋白(UCP3)基因mRNA在不同组织中的相对表达量的检测方法,并进行批内、批间重复性检验。结果表明:UCP3和GADPH基因标准曲线回归方程分别为CtUCP3=-3.2343x+41.6198和CtGADPH=-3.2851x+38.0344,相关系数(r2)分别为0.9958和0.9986,扩增效率分别为103%和97%;批内、批间重复性测定的变异系数分别为小于1.4%和10%。     

二氟沙星残留检测用单克隆抗体的制备及其鉴定

用DFX-BSA免疫BALB/c小鼠,采用显微克隆技术成功筛选出5株特异性分泌细胞株1a6、2b4、4a4、X1和X2。将X1株细胞株直接注入小鼠腹腔获取腹水,腹水纯化后效价达1:1638400。蛋白质含量为6.86mg/ml,单抗X1属IgG2a亚型。X1株单克隆抗体的检测标准曲线方程为Y=0.4813-3.1067X(R=0.9510),最低检测限(LOD)为2ng/ml,线性范围为0.002-5μg/ml,DFX对其IC50为0.085μg/ml。单抗对氟喹诺酮类药物有一定的交叉反应,对沙拉沙星最高可达10.9%,对兽医临床常用抗菌药物氨苄西林、卡那霉素、盐酸四环素、磺胺甲噁唑和氯霉素等没有交叉反应。Ci-ELISA反应中McAb对低浓度的乙腈和丙酮具有良好的耐受性,而低浓度的甲醇对McAb的结合力有一定的影响。     

Preparation of Monoclonal Antibody Against Lasalocid and Deveopment of Indirect Competitive ELISA Detection Method

To prepare monoclonal antibodies (MAb) against lasalocid (LAS) and establish an indirect competitive ELISA (Ci-ELISA) detection method,conjuaction of LAS-BSA and LAS-OVA were synthetized as the immunogen and coating antigen by using active ester method in this experiment. After 6 times of immunization,the spleen cells of mice and myeloma cells were fused. Finally,a hybridoma cell line that could stably secrete specific MAb against LAS was screened.The immunological subtype of the MAb was identified as IgG1,and its light chain was κ type.The titer of the ascites was 1:16 000, which showed no cross activity with monensin sodium, salinomycin sodium,maduramycin,cefalotin sodium and streptomycin sulfate. Ci-ELISA method was established based on the MAb against LAS with the linear equation was y=0.376x-0.2374(R²=0.9914), and the linear range was 5 to 1 000 ng/mL and the IC₅₀ was 90.22 ng/mL. The method was used to detect LAS in spiked samples and the recovery rate was 81.76% to 102.41% within the detection range,indicating that the method was successfully established with good accuracy and high sensitivity. The preparation of MAb against LAS and the establishment of Ci-ELISA method laid the foundation for the development of LAS detection kit.     

检测牛乳中三聚氰胺的间接竞争ELISA方法的建立

利用戊二醛法合成的三聚氰胺—钥孔匙锥蓝蛋白(KLH)为免疫原制得三聚氰胺的多克隆抗体,并在此基础上以重氮法制备的三聚氰胺—牛血清白蛋白为包被抗原建立了检测三聚氰胺的间接竞争ELISA(ciELISA)检测方法。结果表明,理想的包被抗原质量浓度为0.8mg/L,抗三聚氰胺抗血清的稀释倍数为1:400,最适检测范围为0.03-10mg/L,最小检测量为0.01mg/L,批内与批间变异系数分别为1.952%和6.673%,回归方程为y=-0.4239-0.0933。本实验建立的三聚氰胺含量的间接竞争酶联免疫吸附试验(ci-ELISA)方法可用于牛乳样品中三聚氰胺残留的检测。     

恩诺沙星在鸡肉组织中残留的ELISA检测方法研究

分别用N-羟基琥珀酰亚胺法和氯甲酸异丁酯法把恩诺沙星(ENR)与载体蛋白牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联制备免疫抗原和包被抗原,免疫新西兰大白兔得到ENR的多克隆抗体,建立了ENR间接竞争ELISA方法。结果表明:抗ENR血清效价达达1∶212以上,得到标准曲线的线性回归方程为Y=-0.2341X+0.1193(R2=0.9878),中值(IC50)为36 ng/mL,最低检测限(LOD)为10 ng/mL,标准曲线的线性范围为10~1000 ng/mL。批内变异系数为3.18%~7.64%,批间变异系数为9.69%~11.94%,鸡组织中的ENR的回收率为76.5%~89.42%。本试验建立的ELISA方法能够满足恩诺沙星兽药残留检测要求。     

氟喹诺酮类药物胶体金试纸的研制

用针对抗氟喹诺酮类(fluoroquinolones,FQs)药物的广谱性单克隆抗体建立了可以同时检测11种FQs药物的胶体金免疫层析方法。该试纸条用20 nm的胶体金标记单抗,将诺氟沙星－卵清蛋白喷涂在检测线上,羊抗小鼠二抗喷涂在质控线上。该胶体金试纸对猪肉和虾中环丙沙星、恩诺沙星和氧氟沙星的检测限都是30 ng/g,对诺氟沙星、培氟沙星、依诺沙星、麻保沙星、洛美沙星、达氟沙星、沙拉沙星和二氟沙星8种药物在以上两种样品中添加浓度为100 ng/g时都可被检出,整个检测过程包括样品前处理的时间可在20 min内完成。试验结果表明符合对这11种FQs药物在鸡肉和虾中残留现场大量筛查的要求。     

氯霉素间接竞争ELISA(ciELISA)检测方法的建立

以人工合成的氯霉素－牛血清白蛋白（ＣＡＰ－ＢＳＡ）为包被抗原,氯霉素（Ｃｈｌｏｒａｍｐｈｅｎｉｃｏｌ,ＣＡＰ）为竞争的半抗原,两者与一定量的抗ＣＡＰ单抗（ＣＡＰ－ＭｃＡｂ）反应。实验结果表明,理想的包被抗原浓度为１．２５μｇ／ｍｌ,抗ＣＡＰ－ＭｃＡｂ工作浓度为１：１２０００,酶标二抗工作浓度为 １： ５０００,可测最适范围为 １ｎｇ／ｍｌ－１００ｎｇ／ｍｌ,最小检测量为０．１ｎｇ／ｍｌ,批内和批间变异系数分别为３． ６２％和 ５． １９％。得到回归方程 ｙ ＝１．２７３０－ ０．６７４５ｘ（ｒ２＝ ０． ９７７９）和标准曲线,从而建立了快速定量测定 ＣＡＰ含量的间接竞争酶联免疫吸附试验（ＥＬＩＳＡ）。整个测定时间为６小时。     

Pharmacokinetics and milk penetration of difloxacin after intravenous, subcutaneous and intramuscular administration to lactating goats

The single-dose disposition kinetics of difloxacin were determined in clinically normal lactating goats (n = 6) after intravenous (i.v.), subcutaneous (s.c.) and intramuscular (i.m.) administration of 5 mg/kg. Difloxacin concentrations were determined by high performance liquid chromatography with fluorescence detection. The concentration-time data were analysed by compartmental and noncompartmental kinetic methods. Steady-state volume of distribution (V(ss)) and total body clearance (Cl) of difloxacin after i.v. administration were estimated to be 1.16 +/- 0.26 L/kg and 0.32 +/- 0.05 L/h x kg respectively. Following s.c. and i.m. administration difloxacin achieved maximum plasma concentrations of 1.33 +/- 0.25 and 1.97 +/- 0.40 mg/L at 3.37 +/- 0.36 and 1.79 +/- 1.14 h respectively. The absolute bioavailabilities after s.c. and i.m. routes were 90.16 +/- 11.99% and 106.79 +/- 13.95% respectively. Difloxacin penetration from the blood into the milk was extensive and rapid, and the drug was detected for 36 h after i.v. and s.c. dosing, and for 72 h after i.m. administration.     

Analysis of fluoroquinolone residues in edible chicken tissues using supercritical fluid extraction

We evaluated a procedure that uses high-performance liquid chromatography to determine the levels of four fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, and ofloxacin) remaining in chicken breast muscle, liver, and kidneys after the analytes are extracted using supercritical fluid extraction (SFE). These antimicrobial fluoroquinolones were extracted by SFE using supercritical CO2 containing 30% (v/v) methanol. The recovery, limit of detection, accuracy, and precision of this method were evaluated on the basis of fortified matrices at concentrations of 25 to 250 microg/kg. The method is validated and shown to be linear in the range of 2.5-50 microg/kg. Spike recoveries for muscle, liver and kidney samples prepared at 4 spiking levels were ranged from 56.6-104.3%, 51.2-98.4% and 62.2-97.8%, respectively. The coefficient of variation (CV) for recovery as a measure of relative variability was between 3% and 13%, and the relative standard deviation (RSD) was < 20%. The limits of quantitation (LOQ) were 10 microg/kg for enrofloxacin, 12.5 microg/kg each for ciprofloxacin and norfloxacin and 25 microg/kg for ofloxacin; these values were lower than the maximum residue levels (MRLs) authorized by the European Community. The 4 compounds were evaluated simultaneously, and the method was shown to be applicable for analyzing their residues in edible chicken tissues.