动物性食品中盐酸克伦特罗ELISA检测方法的建立及应用

目的:建立动物性食品中盐酸克伦特罗残留快速检测技术,确保食品安全.方法:以盐酸克伦特罗重氮化后分别连接到牛血清蛋白(BSA)和卵清蛋白(OVA)上制得免疫原BSA-CL和包被抗原OVA-CL.通过免疫得到多抗血清,经硫酸铵沉淀,Sephrose 4B-proteinA对抗体进行纯化,在此基础上建立间接竞争ELISA方法.结果表明:包被抗原的最适质量浓度为10 μg/mL,抗体的最佳稀释度为1∶1000;该方法的IC50为2.18 ng/mL,最低检测限达0.05 ng/mL,与沙丁胺醇和妥洛特罗的交叉反应率分别为8.28％和7.75％,平均加标回收率为90.78％.结论:该法灵敏度高,样品前处理简便、快速,适用于动物源性食品中盐酸克伦特罗残留的大批量现场检测.     

超高效液相色谱——串联质谱法测定动物源性食品中利巴韦林

通过对样品净化方式和仪器条件的优化,建立动物源性食品中利巴韦林的检测方法。样品经水溶液提取,经过HyperSep HyperCarb固相萃取柱净化,采用同位素内标法定量,经超高效液相色谱-串联质谱联用仪进行检测。结果表明,利巴韦林在0.50～10.0μg/L范围内线性关系良好,相关系数大于0.999,样品加标平均回收率在89.9%～102.7%之间,相对标准偏差为3.84%～8.09%。该方法净化效果好,回收率稳定、精密度良好,灵敏度高,适用于动物源性食品中利巴韦林的定量分析。     

高效液相色谱-串联质谱法测定动物源食品中氯霉素残留

目的建立高效液相色谱-串联质谱法测定几种动物源食品中氯霉素残留的方法。方法对《动物源食品中氯霉素残留量的测定高效液相色谱-串联质谱法》(农业部第781号公告)中的测定方法进行优化,样品经提取后经HLB固相萃取小柱净化,采用甲醇-水进行梯度洗脱,经Agilent Eclipse-plus C₁₈色谱柱(3.0 mm×100 mm,1.8μm)分离,采用多反应检测负离子模式进行定性及定量分析。结果在0.5~10.0 ng/mL范围内,氯霉素的浓度和色谱峰面积线性关系良好(r=0.9998),方法检出限为0.03μg/kg,定量限为0.1μg/kg,在1.0、2.0和5.0 ng/mL 3个水平的加标回收率为91.58%~109.52%,相对标准偏差小于7.5%。结论该方法快速、准确、灵敏,能满足几种动物源食品中氯霉素残留的测定。     

超高效液相色谱-串联质谱法检测蛋鸡体内抗病毒药物利巴韦林

为研究禁用抗病毒药物利巴韦林在蛋鸡体内的残留性,对11 只京红蛋鸡以30 mg/kg体质量单次口服灌喂利巴韦林药物,采集给药后不同时间的静脉血,以超高效液相色谱-串联质谱法检测利巴韦林,多反应监测模式和同位素内标法定量。利巴韦林线性范围为5～5 000 ng/mL,检出限为1 ng/mL。在10～1 000 ng/mL范围内样品添加回收率为86.4%～97.1%,批内、批间相对标准偏差均满足定量要求。结果表明,给药后血浆中利巴韦林质量浓度迅速升高,随着代谢消除进行,血浆中利巴韦林质量浓度逐渐下降,至24 h血浆中利巴韦林质量浓度低于方法检出限。蛋鸡口服给药利巴韦林后吸收迅速,到达峰值时间短,体内分布广,消除速度快,残留性不高。     

食品中四环素类残留的酶联免疫检测试剂盒的研制

目的：基于定量检测动物源性食品中四环素类抗生素的残留量,开发研制四环素类抗生素竞争酶联免疫(ELISA)检测试剂盒。方法：采用杂交瘤技术,筛选并克隆稳定分泌抗四环素(CEL)的杂交瘤细胞株,利用免疫BALB/c 小白鼠制备抗四环素单克隆抗体。结果：logit/log 拟合标准曲线为y= － 2.3x ＋ 2.79,相关系数r=0.9971,线性检测范围为0.05～4.05ng/mL,半数抑制剂量(IC50)为0.293ng/mL,灵敏度为0.05ng/mL,检测限为3～5ng/mL;组织、牛奶、蜂蜜的添加回收率分别为(90 ± 10)%、(85 ± 10)%、(90 ± 10)%;平均批内和批间变异系数均小于15%;四环素与金霉素等同类抗生素的交叉反应率大于60%,与其他类抗生素无交叉反应;此试剂盒在4℃可保存180d 以上。结论：建立的试剂盒是符合技术指标的要求,可用于动物源性食品中四环素类抗生素残留的定量检测。     

动物性食品中氨苯砜dcELISA的检测

建立了动物性食品中氨苯砜残留快速检测技术。将氨苯砜重氮化后连接到牛血清蛋白(BSA)上制得免疫原BSA-DDS,免疫新西兰大耳白兔获得多克隆抗体,经Protein A-Sephros 4B对抗体进行纯化,在此基础上建立直接竞争ELISA方法。结果显示:该方法可制备目标抗原BSA-DDS和抗体,氨苯砜与载体蛋白偶联比可达到1∶10;建立的直接竞争ELISA方法的IC50为4.78 ng/m L,最低检测限达0.03 ng/m L,加标回收率为75.45%~91.75%。     

动物源性食品中兽药残留的快速筛查

建立同时快速测定动物源性食品中喹诺酮类药物、β-受体激动剂类及硝基咪唑类药物残留的超高效液相色谱-串联质谱的检测方法 (UPLC-MS/MS)。不同基质样品经粉碎后采用乙腈一次性提取后用高效液相色谱-串联质谱/质谱测定。测定结果分别采用内标法和外标法定量。试验结果表明:三类兽药残留在1～100 ng/mL浓度范围内线性关系良好,相关系数R2均大于0.999;在较低浓度(1μg/kg)添加水平下具有较好的回收率;不同基质中不同添加水平下,平均回收率均可满足大于60%的要求(GB/T 27407), SRSD<10%。该方法操作简便,分析速度快,回收率高,稳定性好,可用于常规动物源性食品中喹诺酮类、β-受体激动剂类以及硝基咪唑类的残留量的同时检测。     

胶体金免疫层析法快速检测动物源性食品中土霉素残留

目的 建立用于快速检测动物源性食品中土霉素残留的胶体金免疫层析试纸条.方法 采用柠檬酸三钠还原法制备胶体金,胶体金粒径选用20 nm,将经鉴定制备成功的胶体金溶液与土霉素多克隆抗体结合得到金标抗体.优化金标抗体的制备条件,将包被抗原(1.0 mg/mL)和羊抗鼠二抗(0.75 mg/mL)分别作为检测线和质控线包被在硝...     

胭脂红酸人工抗原合成及多克隆抗体的制备

为了快速高效检测食品中胭脂红酸含量,本研究通过羟基二咪唑法制备了胭脂红酸人工抗原;通过免疫新西兰大耳白兔获得了胭脂红酸抗血清,经硫酸铵沉淀法分离纯化获得抗胭脂红酸多克隆抗体;利用棋盘滴定法检测了抗体效价,并探究了溶液pH值、离子强度和有机溶剂对ELISA反应的影响,优化出最佳ELISA反应条件;在最佳反应条件下,建立了胭脂红酸的间接竞争ELISA抑制曲线。紫外光谱扫描结果显示,免疫抗原(CA-BSA)和检测抗原(CA-OVA)均与载体蛋白成功偶联;经分离纯化获得的胭脂红酸多克隆抗体效价为1:16000;检测抗原最佳浓度为1μg/m L、ELISA反应最适缓冲液为pH7.4、含50mmol/L Na~+且无甲醇的磷酸盐缓冲液;间接竞争ELISA标准曲线的线性范围为7.8～1150ng/mL,检测限为1ng/mL,灵敏度IC_(50)值为95.2ng/mL。本研究为开发CA快速检测试剂盒对食品中胭脂红酸含量的大量快速测定奠定了基础。     

动物源性食品中65种兽药残留的快速检测方法

建立了动物源性食品中65种兽药残留的快速提取、净化及超高效液相色谱-串联质谱(UHPLC-MS/MS)检测方法。采用乙腈-甲酸溶液为提取溶剂。通过固相萃取柱(ProElut PLS-A)对提取液进行净化,采用多反应监测模式检测。65种目标化合物呈现良好的线性关系,定量限为0.1～1.6μg/kg,大部分兽药残留的回收率在67. 1%～100. 2%之间,相对标准偏差小于7.8%(n=3)。建立的方法适用于动物源性食品中65种兽药残留快速检测分析。     

Preparation of high affinity antibody for ribavirin with new haptens and residue analysis in chicken muscle,eggs and duck muscle

In this work, high affinity polyclonal antibodies for ribavirin (RBV) from new haptens were prepared and were used to analyse RBV residues in chicken muscle, eggs and duck muscle. The new haptens were synthesised with different spacers, and the best antibody was obtained with an IC₅₀ value as low as 0.61 ng/mL in indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivities with another five antiviral drugs including amantadine, rimantadine, moroxydine, zanamivir and oseltamivir were less than 0.1%, which indicated the good specificity of the antibody. An ELISA was developed based on the antibody and applied to detect RBV in multi-food matrices. The sample preparation prior to detection only needed simple dilution after trichloroacetic acid extraction. The limits of detection were 1.07, 1.18 and 1.03 μg/kg in chicken muscle, eggs and duck muscle, respectively. Recoveries ranged from 89.0% to 112.7% with coefficients of variation below 13.0%. Ten blind samples of chicken muscle were analysed simultaneously by ELISA and liquid chromatography-tandem mass spectrometry, and a good correlation between the methods was observed. The results indicated that the high affinity antibody could be applied for the simple and fast detection of RBV in multi-food matrices.     

动物性食品中磺胺二甲嘧啶快速检测方法

根据竞争抑制免疫层析原理,应用胶体金免疫层析技术进行磺胺二甲嘧啶在鸡肉和鲫鱼中的残留检测研究。磺胺二甲嘧啶半抗原-OVA和羊抗兔二抗分别作为检测带(T线)和质控带(C线)包被在硝酸纤维素膜上,金标抗体喷涂在玻璃纤维垫上,制成免疫层析快速检测试纸条。所建立的检测体系对标准溶液的灵敏度达到1μg/L。成功检测了鸡肉和鲫鱼中的磺胺二甲嘧啶残留。该方法灵敏度高,所用试剂无毒、无污染,无需借助仪器,样品前处理简便,快速,检测过程可在10 min内完成,可以作为现场快速检测动物源性食品中磺胺二甲嘧啶残留的有效手段。     

Indirect competitive enzyme-linked immuno-sorbent assay (ELISA) for nitroimidazoles in food products

Ronidazole was used as the starting material to prepare an immunogen and coating antigen. An anti-nitroimidazole monoclonal antibody was produced and an indirect competitive ELISA was established to detect nitroimidazole compounds in food products. The IC(50) values were determined to be 0.20 ng/ml for metronidazole, 4.0 ng/ml for tinidazole, 0.17 ng/ml for dimetridazole and 0.24 ng/ml for ornidazole. Considering that nitroimidazoles were commonly used as veterinary drugs, nitroimidazole residues in food products of animal origin were detected by the method. The coefficient of variation for nitroimidazoles determination in contaminated chicken, chicken liver and shrimp were all <14% and the recovery rate was in the range 74.0-90.6%. The results proved that the developed method was successful in detecting nitroimidazoles in food products.     

Indirect competitive enzyme-linked immuno-sorbent assay (ELISA) for nitroimidazoles in food products

Ronidazole was used as the starting material to prepare an immunogen and coating antigen. An anti-nitroimidazole monoclonal antibody was produced and an indirect competitive ELISA was established to detect nitroimidazole compounds in food products. The IC₅₀ values were determined to be 0.20?ng/ml for metronidazole, 4.0?ng/ml for tinidazole, 0.17?ng/ml for dimetridazole and 0.24?ng/ml for ornidazole. Considering that nitroimidazoles were commonly used as veterinary drugs, nitroimidazole residues in food products of animal origin were detected by the method. The coefficient of variation for nitroimidazoles determination in contaminated chicken, chicken liver and shrimp were all <14% and the recovery rate was in the range 74.0–90.6%. The results proved that the developed method was successful in detecting nitroimidazoles in food products.     

氯丙嗪抗体制备与酶联免疫吸附测定方法的建立

本研究针对动物性食品中违禁药物氯丙嗪残留问题,建立了针对氯丙嗪的酶联免疫吸附测定方法。首次通过以氯丙嗪为原料,先去甲基化合成N-甲基氯丙嗪,再与丙烯酸叔丁酯反应及水解等步骤成功合成了氯丙嗪半抗原（CPZ-H）。半抗原CPZ-H分别与OVA和BSA通过活泼酯法偶联合成包被原与免疫原,采用免疫原CPZ-H-BSA免疫新西兰大白兔成功制备了特异性的抗氯丙嗪的多克隆抗体。基于该抗体建立了快速测定氯丙嗪的间接竞争酶联免疫吸附测定方法（ic-ELISA）。通过优化ic-ELISA方法的最佳工作条件,确定包被原和抗体稀释倍数均为1:8000时效果最好,此时标准曲线的IC50为1.1 ng/mL,线性范围为：0.13 ng/mL~8.8 ng/mL。制备的抗氯丙嗪的多克隆抗体与其它氯丙嗪类似物无交叉反应,特异性好。本研究为今后高特异性氯丙嗪快速检测试剂盒的制备提供依据。     

酶标抗原直接竞争ELISA检测食品中氟喹诺酮类药物多残留

固相包被恩诺沙星抗体,辣根过氧化物酶标记的抗原与标准品（或样品）中氟喹诺酮药物竞争结合抗体,建立了高效、高灵敏的氟喹诺酮药物直接竞争酶联免疫吸附分析检测方法。优化反应条件后,得到方法的IC50为2.04 μg/L,灵敏度为0.15 μg/L,线性范围0.3～15 μg/L;方法可以检测12 种氟喹诺酮药物,在生乳、鸡肉、鱼肉和虾肉4 种样品中12 种药物的回收率为70%～121.5%。     

竞争化学发光酶免疫检测动物源性食品中四环素残留

目的:建立一种准确度高、重复性好的检测四环素(tetracycline,TC)残留的竞争化学发光酶免疫测定法(competitive chemiluminescence enzyme immunity,CLEIA)。方法:通过选择适宜的偶联载体蛋白,优化反应温度、时间等条件,建立了一种测定四环素的竞争化学发光酶免疫测定法(TC-CLEIA)。结果:该方法的检测范围为0.0259~95.9478 μg/L,相关系数r为0.9998,检测限为0.0133 μg/L,准确度为99.17%,灵敏度为0.7305 μg/L,变异系数<10%,对氯霉素、青霉素具有极低的交叉反应;向空白猪肉、鱼肉、鸡肝中添加三种不同浓度的四环素标准品时,检测结果具有较高的准确度和重复性;建立的TC-CLEIA测定法检测了四环素小鼠中毒模型中肝脏、肾脏和肌肉中四环素残留的浓度,浓度大小依次为:肾脏 > 肝脏 > 肌肉。结论:本研究建立的TC-CLEIA检测法简便,检测限低,准确度、重复性、特异性均很好,适用于动物源性食品中四环素残留的检测。     

基于金纳米颗粒信号放大ELISA检测食品中恩诺沙星

以金纳米颗粒（gold nanoparticles,AuNPs）为信号放大载体,利用辣根过氧化物酶（horseradish peroxidase,HRP）标记的二抗为信号探针,建立一种基于AuNPs的信号放大酶联免疫检测方法（AuNPs signal amplification enzyme-linked immunosorbent assay,AuNPs-HRP-IgG ic-ELISA）,检测食品中恩诺沙星（enrofloxacin,ENR）。该方法对ENR的检测限（IC15）为5×10－4?ng/mL,半数抑制浓度（IC50）为0.24?ng/mL,检测范围为0.16～500?ng/mL,与建立的传统ELISA方法（IC50=8.76?ng/mL）相比,显著提高了检测灵敏度,并且该方法与环丙沙星、氧氟沙星、诺氟沙星交叉反应率均小于0.1%,具有良好的特异性。该AuNPs-HRP-IgG?ic-ELISA方法的实用性得到了样品添加回收实验和商业化ELISA检测试剂盒的验证,其在牛奶样品中的加标回收率可达80.52%～102.66%,适用于实际牛奶样本中ENR的快速灵敏检测,也为建立其他食品危害物质的精准检测技术开发提供参考。     

Preparation of anti‐danofloxacin antibody and development of an indirect competitive enzyme‐linked immunosorbent assay for detection of danofloxacin residue in chicken liver

BACKGROUND: Danofloxacin is used widely as both a clinical medicine for humans and a veterinary drug in animal husbandry. In this study a polyclonal anti‐danofloxacin antibody was prepared for the first time and a simple and rapid indirect competitive enzyme‐linked immunosorbent assay (cELISA) method based on the antibody was developed to monitor danofloxacin residue in chicken liver. RESULTS: The prepared antibody showed high sensitivity, with an IC₅₀ value of 2.0 ng mL^?1 towards danofloxacin, and good specificity, with significant cross‐reactivity only towards pefloxacin (22%) and fleroxacin (21%) among commonly used (fluoro)quinolones evaluated in the study. The developed cELISA test kit had a detection limit of 0.8 ng mL^?1, and satisfactory results were obtained when it was applied to chicken liver spiked with various levels of danofloxacin. The cELISA test kit was also used to detect danofloxacin in chicken liver samples purchased from a local food market, and the results were confirmed by liquid chromatography/mass spectrometry. CONCLUSION: The anti‐danofloxacin antibody prepared in this study exhibits excellent quality, with high sensitivity and good specificity. The cELISA test kit based on the antibody has a very low detection limit and is suitable for use as an efficient screening method to detect danofloxacin residue in foods and food products. Copyright © 2009 Society of Chemical Industry