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1.
莱克多巴胺胶体金免疫层析法快速检测试纸条的研制   总被引:1,自引:0,他引:1  
为研制一种快速、简便的基于胶体金免疫层析法的试纸条,快速检测猪尿中莱克多巴胺(ractopamine,RAC药物残留),采用柠檬酸钠还原法制备胶体金颗粒,标记莱克多巴胺单克隆抗体并喷于玻璃纤维上制备胶金垫,RAC-BSA偶联抗原和羊抗鼠IgG分别结合于硝酸纤维膜上,依次将样品垫、胶体金垫、硝酸纤维素膜和吸水纸组装并切割成莱克多巴胺胶体金免疫层析快速检测试纸条。试验结果表明该快速检测试纸条的检测限为5 ng/mL,可在8~10 min内完成测试,肉眼即可判断,无需其他检测设备。该试纸条灵敏度、特异性、稳定性和重复性均达到临床使用要求,适用于现场快速检测和筛选工作。  相似文献   

2.
为了现场灵敏、特异、快速地检测猪尿中莱克多巴胺(RAC)的残留情况,试验用胶体金标记RAC单克隆抗体构建一种能够快速检测猪尿中RAC的免疫层析试纸条,通过正交试验优化试纸条中标记抗体的量、胶体金结合垫上胶体金单克隆抗体(CG-mAb)溶液的体积和牛血清白蛋白封闭的莱克多巴胺(RAC-BSA)的浓度得到最优组合,并测定试纸条的特异性、稳定性、检测时间和检测限。结果表明:经过优化后得到的最优组合为标记抗体的量1.5μg/mL,胶体金结合垫上CG-mAb溶液的体积1.5μL,RAC-BSA的浓度0.2 mg/mL;该试纸条可在3 min内特异性地检测RAC,检测限为3 ng/mL,在室温条件下能保存12个月。说明试验构建的快速检测猪尿中RAC试纸条能达到现场使用的要求,适用于快速筛查猪尿中RAC的残留。  相似文献   

3.
为了验证ELISA方法检测猪尿中莱克多巴胺残留量的可行性,采用ELISA方法对试剂盒的校正曲线、检测限、加标回收率试验等方面进行了验证。结果为试剂盒校正曲线的线性相关系数为0.9913;样品的定性检出限为0.087ng/mL,定量检出限为0.29ng/mL;添加1.1ng/mL、2.2ng/mL、4.4ng/mL、6.6ng/mL和11.0ng/mL的莱克多巴胺标准液时,变异系数分别为3.06%、8.55%、4.41%、9.92%、8.19%;平均回收率分别为93%、113%、103%、96%、91%。结果表明,应用ELISA方法检测猪尿中莱克多巴胺残留量符合要求,适合检测猪尿中莱克多巴胺残留量的初筛。  相似文献   

4.
化学发光直接竞争免疫分析法检测猪尿中莱克多巴胺残留   总被引:1,自引:1,他引:0  
本试验研究建立了猪尿中莱克多巴胺(Ractopamine, RAC)残留的直接竞争化学发光免疫检测方法。合成了莱克多巴胺和辣根过氧化物酶的偶联物(RAC-HRP),方法的检测限为0.09 ng/mL,空白猪尿中添加浓度为0.5、10和100 ng/mL RAC时,检测范围为0.3~157.0 ng/mL;回收率为76.3%~87.6%,批内变异系数为10.0%~12.2%,批间变异系数为14.2%~15.2%。  相似文献   

5.
化学发光免疫分析法检测猪尿中莱克多巴胺残留   总被引:4,自引:1,他引:3  
本试验运用化学发光免疫分析法(chemiluminescence immunoassay,CLIA)检测猪尿中莱克多巴胺(Ractopamine,RAC)的残留。检测限为0.01 ng/mL,检测范围为0.036~32.2 ng/mL。添加浓度分别为0.1、1、10 ng/mL,平均回收率为73.2%,批内平均变异系数为9.7%,批间平均变异系数为11.3%。而本研究所建立的化学发光免疫检测方法(CLISA)可以达到检测RAC残留的要求。  相似文献   

6.
用混合酸酐法将莱克多巴胺(Rac)与匙孔蓝蛋白(KLH)、牛血清白蛋白(BSA)偶联,经紫外光谱扫描确定偶联成功,测得偶联物Rac-KLH浓度为3.6 mg/mL,Rac-BSA浓度为6.2 mg/mL。以偶联物Rac-KLH作为免疫原,免疫6~8周龄雌性BALB/c小鼠,取小鼠脾细胞和骨髓瘤细胞SP2/O-Ag-14进行融合。以Rac-BSA作为包被抗原,经间接ELISA筛选出阳性细胞株,再用有限稀释法进行多次亚克隆,获得稳定分泌单克隆抗体的杂交瘤细胞株1D9、1F3、2C11、5C3、3A10、4A8、6B7、6D5、7C11、7D3。其中单克隆抗体5C3和3A10间接ELISA效价1∶500 000,对莱克多巴胺的半数阻断浓度(IC50)均为3.98 ng/mL,对盐酸克伦特罗和沙丁醇胺的IC50均大于2 000 ng/mL;对盐酸克伦特罗和沙丁醇胺的交叉反应率均小于0.2%。间接竞争ELISA检测莱克多巴胺在0.5~100 ng/mL范围内为线性分布,确定的最低检测限为0.5 ng/mL。  相似文献   

7.
直接竞争酶联免疫法检测菜克多巴胺研究   总被引:1,自引:1,他引:0  
莱克多巴胺(RAC)是一种β2激动剂,人类食入高残留量莱克多巴胺的食物后会产生严重的毒副作用,研究建立了直接竞争酶联免疫法用于莱克多巴胺的快速检测。用合成的免疫抗原RAC-BSA免疫大白兔,获得了效价为1.6×104的多克隆抗体,用1,4-丁二醚法将RAC与辣根过氧化物酶偶联合成酶标记物,建立了RAC直接竞争酶联免疫吸附法。经质量评价,直接竞争酶联免疫吸附法的检测限为0.1 ng/mL,半数抑制浓度IC50为1.07 ng/mL,批内和批间变异系数均小于11%,抗体与多巴酚丁胺的交叉反应率为8.3%,与RAC的其他类似物几乎不发生交叉反应,猪尿样品中平均添加回收率为104%,表明建立的直接竞争酶联免疫吸附法可用于对莱克多巴胺的残留检测。  相似文献   

8.
肉品中莱克多巴胺残留的酶联免疫快速检测方法的建立   总被引:1,自引:0,他引:1  
用自主制备的莱克多巴胺特异性抗体建立了猪肉样品中莱克多巴胺药物残留的间接竞争酶联免疫检测方法,对该检测体系的灵敏度、准确度、精密度、特异性等性能进行了测定,并将该检测系统与高效液相色谱法(HPLC)进行了对比分析。结果显示:莱克多巴胺药物残留的酶联免疫检测体系的检测范围为1~100 ng,灵敏度为0.12 ng/mL,检测限为1 ng/mL,回收率为80%~100%,与同类药物如盐酸克仑特罗、硫酸特布他林及硫酸克仑特罗的交叉反应率均小于0.01%;采用HPLC方法进行莱克多巴胺药物残留的检测时,其检测范围为10~200μg/mL,检测限为1μg/mL,回收率为83%~100%。该方法与高效液相色谱法相比灵敏度较高,特异性强,检测结果准确度相近,但是在检测结果稳定性方面逊于HPLC方法。  相似文献   

9.
本试验旨在探讨一种能够快速、简便及特异检测MHV病毒的金标检测方法。采用柠檬酸三钠还原法制备胶体金颗粒,标记抗小鼠肝炎病毒(MHV)抗体,选择优化层析条件,建立双抗体夹心模式的免疫层析法试纸条对MHV进行检测。20 nm左右的胶体金颗粒、5.05 μg为最低稳定抗体量(稳定0.5 mL胶体金)、M135层析材料、3%BSA是最佳封闭液。640 μg/mL抗体蛋白为最适检测线包被浓度,而800 μg/mL抗体蛋白为质控线最适包被浓度。此试纸条能10 min快速特异地检测出MHV抗原,阳性时检测线和质控线均显示红色,阴性时则只有质控线显示红色。结果表明,胶体金免疫层析方法能快速、特异、稳定地检测MHV抗原。  相似文献   

10.
采用胶体金试纸卡法和LC-MS/MS法分别对猪肉、猪肝样品进行检测,鉴定胶体金试纸卡法和LC-MS/MS法检测结果的符合率。结果表明:应用胶体金试纸卡法检测样品中莱克多巴胺的最低检测限为5.0μg/kg,LC-MS/MS检测方法的检测限为0.25μg/kg;2种方法的检测结果一致。胶体金试纸卡法特异性较高,样品前处理简单,检测快速,成本低,适用于动物组织中莱克多巴胺残留的定性筛查;LC-MS/MS灵敏度高,适合于阳性样品的确证和精确定量。  相似文献   

11.
猪肝用乙酸乙酯、盐酸溶液提取后,经SCX固相萃取柱净化、BSTFA衍生化,定容后用气相色谱-质谱(GC/MS)外标法测定。测定结果表明:采用该方法,猪肝中盐酸克伦特罗和莱克多巴胺的检出限分别为0.3、1.0ng/g。盐酸克伦特罗在3.0~300ng/mL范围内与峰面积具有良好的线性关系,平均回收率为85.6%;莱克多巴胺在10~1000ng/mL范围内与峰面积具有良好的线性关系,平均回收率为76.6%。该方法简便可靠、干扰小、灵敏度高,可用作猪肝中克伦特罗和莱克多巴胺残留的同时测定。  相似文献   

12.
建立了高效液相色谱-串联质谱(HPLC-MS/MS)同时检测猪毛发中克仑特罗和莱克多巴胺残留的方法。猪毛发经1 mol/L氢氧化钠溶液水解、乙酸乙酯萃取、MCX固相萃取柱净化,流动相溶解后用HPLC-MS/MS进行检测。克仑特罗在1.9~463.2 ng/mL浓度范围内线性关系良好(R2=0.999 0);回收率为83.3%~86.6%,相对标准偏差为6.7%~9.2%;检出限为0.3μg/kg,定量限为0.8μg/kg。莱克多巴胺在2.8~562.9 ng/mL浓度范围内线性关系良好(R2=0.999 2);回收率为83.4%~88.4%,相对标准偏差为7.2%~9.6%;检出限为0.4μg/kg,定量限为1.2μg/kg。应用该方法研究了克仑特罗、莱克多巴胺在猪毛发中的代谢规律并进行了猪毛发样品检测。结果喂药7 d至停药21 d猪毛发中均检出克仑特罗、莱克多巴胺残留;检测猪毛发样品25份,1份样品检出克仑特罗,残留量为58.68μg/kg。  相似文献   

13.
本研究依据直接竞争ELISA原理建立了齐帕特罗一步法ELISA试剂盒。以齐帕特罗与载体蛋白偶联物作为免疫原免疫新西兰大白兔制得齐帕特罗多克隆抗体,棋盘包被法确定其最佳抗体包被浓度、酶标抗原工作浓度、包被条件、反应时间、底物显色时间等,并对试剂盒的各项技术指标进行确认。结果表明,成功组装了齐帕特罗一步法ELISA试剂盒,并建立了尿液、饲料、奶粉和奶汁,以及组织等样品的前处理方法,检测限均远低于1 μg/kg。该试剂盒线性检测范围为0.15~10 ng/mL,IC50浮动范围0.43~0.79 ng/mL,样品板内、批内、批间的变异系数均小于15%,平均回收率在70%~110%之间,与其同类药物的交叉反应率均小于0.1%。提示,本试验研制的试剂盒重复性、特异性、稳定性等各项指标均符合技术要求,可用于动物源性食品中齐帕特罗药物残留的检测。  相似文献   

14.
Insulin binding to mouse adipocytes was measured after in vitro (30 min) and in vivo (5 days) exposure to clenbuterol and ractopamine. At 10(-6) M, both agonists decreased insulin binding by 20-30% after a 30 min preincubation at each insulin concentration between 1 and 25 ng/ml. Binding was not decreased if propranolol was present. Scatchard plots suggested that decreased binding was due to a decrease in insulin receptor concentration. Insulin binding was decreased approximately 10% at agonist concentrations as low as 10(-13) M, but binding was not further decreased until concentrations exceeded 10(-9) M. Rate of gain was increased 2-fold by clenbuterol (10 mg/liter of drinking water) and 50% by 500 mg ractopamine/liter, but not by 50 mg ractopamine/liter. Clenbuterol and ractopamine (500 mg/liter) decreased fat pad weight but only clenbuterol increased hind limb muscle mass. Insulin binding following in vivo administration was not influenced by ractopamine at 50 mg/liter, but tended to be increased by clenbuterol and ractopamine at 500 mg/liter. The disparity in results between administering the beta-agonists in vitro or in vivo suggests that counter regulatory factors influenced insulin binding capacity in vivo. Results indicate that ractopamine and clenbuterol can decrease insulin binding to adipocytes but the relevance of this response to decreased fat accretion is not clear.  相似文献   

15.
Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 µg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 µg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.  相似文献   

16.
参照GenBank发表的序列,在金黄色葡萄球菌、无乳链球菌和停乳链球菌16SrRNA与23SrRNA之间的区域设计了3对引物,参照念珠菌和隐球菌的18SrRNA的序列设计1对引物,建立了检测金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母真菌4种乳腺炎主要致病菌的多重PCR方法。参照Skladny的方法制备模拟了细菌感染l临床标本。结果表明:本试验建立的多重PCR方法具有较好的特异性,多重PCR方法检测乳样中的金黄色葡萄球菌的细菌最小浓度为10^4CFU/mL,检测无乳链球菌、停乳链球菌和酵母真菌的细菌最小浓度分别为10^4CFU/mL、10^3CFU/mL和10^3CFU/mL。通过对采自临床型乳腺炎(46个)和隐性乳腺炎(167个)动物共计213个乳样分别用传统细菌学培养法和多重PCR方法进行检测,多重PCR对金黄色葡萄球菌和酵母真菌的检测具有更高的检出率(P〈0.01),但该方法对无乳链球菌和停乳链球菌的检出率与培养法差异不显著(P〉0.05)。  相似文献   

17.
Objective of the study was to develop a preparation treatment for salbutamol in feed by enzyme-linked immunosorbent assay (ELISA).The assay optimized the influence factor of the preparation and used to extract salbutamol residues in feed.The results showed that the inhibition concentration (IC50) for the developed ELISA was 0.606 ng/mL with the detection range of 0.221 to 1.658 ng/mL and R2 was 0.9998.After optimization,pH 7.5,0.06 mol/L PBS with the dilution ratio of 10 was selected to extract salbutamol residues in feed and the limit of detection (LOD) was 5.0 ng/mL in feed.The recoveries ranged from 77% to 110% for the spiked samples (5 and 10 μg/kg),and RSD was less than 8%.  相似文献   

18.
Satellite cells were isolated from 20-d embryonic chick breast muscle via a Percoll density gradient fractionation technique. Culturing of these cells gave rise to at least 89% fusion (myotube nuclei number/total nuclei number). Proliferation of cultured satellite cells (indicated by myotube nuclei number) was increased in a dose-dependent manner when fibroblast growth factor (FGF) was included in the medium (25 to 200 ng/ml). Similar cultures were used to examine the effects of ractopamine and isoproterenol on satellite cell proliferation. Ractopamine and isoproterenol were added to culture medium (10(-11) to 10(-4) M) 24 h after initial plating. After a 72-h treatment period, the treatments were removed and replaced with a medium to promote fusion for 48 h. Cells then were fixed and stained, and myotube and total nuclei were counted. In later experiments, ractopamine and isoproterenol each increased (P less than .01) myotube nuclei number vs that observed in control cultures by 2.3 and 2.1 times, respectively. Similar differences were observed with total nuclei number. The number of myotube nuclei observed in cultures treated with 10(-6) M ractopamine or isoproterenol was reduced (P less than .01) by 25.4 and 23.6%, respectively, when propranolol, a beta adrenergic antagonist, was included at 10(-5) M with the respective agonist. These results indicate that ractopamine and isoproterenol each enhance the proliferative activity of chick satellite cells in culture and that the beta adrenergic receptor mediates this proliferative effect.  相似文献   

19.
根据GenBank公布的猪博卡病毒(PBoV)序列,通过VP1/2基因设计引物和Taq Man探针建立实时荧光定量PCR检测方法。建立的方法与PPV、PRRSV及PCV均无交叉反应,具有较高特异性,在107 copies/mL~101 copies/mL模板范围内具有良好的线性关系,所制作的标准曲线相关系数为0.997,最低可检测到101copies/mL的阳性质粒。说明所建立的PBoV实时荧光定量PCR检测方法具有灵敏度高、特异性好和精确性高等优点。  相似文献   

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