对氯甲基苯甲酸链接的莱克多巴胺人工抗原的合成与鉴定

采用对氯甲基苯甲酸(CBA)为链接臂,用混合酸酐法将莱克多巴胺(RAC)与牛血清白蛋白(BSA)偶联制备人工抗原(RAC-CBA-BSA),通过紫外、红外、电泳鉴定抗原合成成功;再以卵清蛋白(OVA)为载体蛋白合成包被抗原(RAC-CBA-OVA),测定其与抗体的亲和力和抑制率。间接竞争ELISA结果以对CBA为链接臂合成的包被抗原对应的抗体滴度为7047.3、IC50为17.05ng/mL,结果表明,CBA可以用于RAC人工抗原的合成,使用RAC-CBA-OVA作为包被抗原可以提高ELISA检测灵敏度。     

土霉素完全抗原的制备与鉴定

为了建立土霉素完全抗原的制备方法,实验以土霉素为半抗原,牛血清白蛋白(BSA)为载体蛋白,利用N,N,-二环己基碳化二亚胺(DCC)、邻联甲苯胺(o-tolidine)为偶联剂,将牛血清白蛋白与土霉素偶联制备土霉素人工完全抗原。通过偶联率的计算确定邻联甲苯胺为最佳偶联剂。随后利用红外光谱分析和免疫原性测定实验鉴定了土霉素、牛血清白蛋白和邻联甲苯胺的偶联情况。结果表明,制得的土霉素完全抗原在免疫健康的Balb/c小鼠体内能刺激小鼠机体产生最高效价为25600(ELISA)的抗血清。     

杀草丹半抗原及其人工抗原的制备与鉴定

以乙基羟乙胺、升华硫、一氧化碳气体和对氯氯苄为原材料,合成羟基化杀草丹;经纯化后与琥珀酸酐反应,合成杀草丹半抗原,采用薄层层析色谱法、质谱法和红外光谱法分析鉴定为预期产物;利用碳二亚胺法将半抗原与牛血清蛋白BSA偶联制备杀草丹人工抗原,应用紫外光谱和荧光光谱法分析鉴定。结果表明,羟基化杀草丹经薄层层析展开效果良好,杀草丹半抗原经红外光谱分析具有明显的羧酸基团,有利于后续偶联操作;在26 ℃室温条件下制备的杀草丹人工抗原,其半抗原与载体蛋白偶联比为6.59:1。杀草丹半抗原与杀草丹人工抗原的成功制备,可为进一步研制杀草丹抗体奠定基础。     

强力霉素人工抗原的合成与抗体制备

采用改进的碳二亚胺两步法将强力霉素半抗原与载体蛋白BSA连接制备强力霉素-牛血清白蛋白(BSA-DC)人工免疫抗原,并用同样方法将强力霉素与载体蛋白OVA连接制备人强力霉素-卵清白蛋白(OVA-DC)人工包被抗原。经紫外扫描分析和动物免疫试验证实:强力霉素人工抗原合成成功,强力霉素与BSA的结合比为3∶1,经动物免疫试验所得抗血清效价为2.048×106,完全能够满足强力霉素残留的ELISA检测要求。     

三聚氰胺人工抗原化合物的合成及鉴定

目的：建立三聚氰胺残留的免疫分析方法。方法：以三聚氰胺结构类似物2-氯-4,6-二氨基-1,3,5-三嗪为原料,分别与6-氨基己酸和对氨基苯甲酸,各经两步化学反应合成两种具有不同长度间隔臂的半抗原MEA和MEB,并分别通过碳二亚胺法和混合酸酐法将MEA和MEB与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联,制备三聚氰胺的免疫原(MEA-BSA)和包被原(MEB-OVA)。结果：经质谱和紫外光谱表征,证明所合成的产物为目标半抗原MEA和MEB,并且与载体蛋白偶联比可达到25.28:1(MEA-BSA)和13.11:1(MEA-OVA)。结论：半抗原MEA和MEB及人工抗原合成成功。     

抗莱克多巴胺单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定

莱克多巴胺(RAC)经化学修饰引入功能基团羧基,合成半抗原RAC- 戊二酸酐半醛化合物(RAC-SA),用混合酸酐法(MA)将RAC-SA 偶联于牛血清白蛋白(BSA),合成人工抗原BSA-RAC,用红外(IR)、紫外(UV)和凝胶电泳(SDS-PAGE)对其进行鉴定;用BSA-RAC免疫Balb/C 小鼠,细胞融合技术建立抗RAC的单克隆抗体(RAC mAb)杂交瘤细胞株,体内诱生腹水法制备RAC mAb,并鉴定其免疫学特性。结果表明,BSA-RAC 偶联成功,偶联率为24.5:1;筛选出3F10、3H12、4D8 共3 株杂交瘤细胞,其中最好的4D8 株间接ELISA 效价细胞培养上清为1:1.28 × 103,腹水为1:6.4 × 105,同种型为IgG1/κ,亲和常数(Ka)为1.65 × 1010L/mol,对RAC 的半数抑制浓度(IC50)为5.7ng/ml,与多巴酚丁胺的交叉反应率(CR)为22.3%,与其他化合物无CR。     

酸性橙Ⅱ半抗原及人工抗原的合成与鉴定

目的建立酸性橙Ⅱ半抗原及人工抗原的合成及鉴定方法。方法将重氮化后的2-氨基-5-磺基苯甲酸与2-萘酚经偶氮反应合成带羧基基团酸性橙Ⅱ,通过碳二亚胺法将其与载体蛋白(BSA)偶联制备酸性橙Ⅱ人工抗原。采用核磁共振碳谱和氢谱对合成的酸性橙Ⅱ半抗原进行结构分析,并用紫外光谱扫描法和蛋白质电泳法鉴定酸性橙Ⅱ人工抗原并计算偶联比。结果通过偶氮反应及碳二亚胺法最终得到纯化后的酸性橙Ⅱ半抗原约47 mg,核磁结果显示成功合成酸性橙Ⅱ半抗原。经SDS-PAGE、N-PAGE和紫外光谱扫描结果均显示酸性橙Ⅱ人工抗原蛋白分子质量大于BSA,所带负电荷大于BSA,且合成后的酸性橙Ⅱ人工抗原具有吸收峰叠加效应,均表明酸性橙Ⅱ人工抗原合成成功,偶联比为20.9:1。结论此方法能够成功合成酸性橙Ⅱ半抗原和人工抗原,为进一步制备特异性酸性橙Ⅱ抗体和建立酸性橙Ⅱ的免疫检测方法奠定基础。     

菜克多巴胺人工抗原的光谱表征及免疫鉴定研究

盐酸菜克多巴胺(Ractopamine hydrochloride,RCT)是一种β-肾上腺素受体激动剂,即使极低剂量残留对人类健康也有危害.为了建立简单、快速并适应现场检测的免疫分析方法,必须先合成它的人工抗原并进行鉴定.通过对莱克多巴胺进行结构修饰,用碳二亚胺将结构修饰了的莱克多巴胺与牛血清蛋白相偶联,经透析得到纯的莱克多巴胺人工抗原并冷冻干燥备用.结果表明,紫外光谱图中合成的人工抗原的特征吸收峰介于蛋白质和莱克多巴胺的特征吸收峰之间;人工抗原红外光谱图中呈现莱克多巴胺和牛血清蛋白的红外特征吸收峰;通过竞争ELISA实验表明用合成的人工抗原免疫的Balb/c小鼠血清中产生了莱克多巴胺的抗体,三种方法综合鉴定结果表明,莱克多巴胺人工抗原合成成功,该人工抗原可以进一步用于制备抗莱克多巴胺的单克隆抗体.     

特布他林人工抗原的制备

特布他林(Terbutaline,TBL)又名特布特罗,属于小分子化合物,其分子量为225.29,本身不具有免疫原性,需要和载体蛋白偶联后方可进行抗体的制备。本试验以1,4-丁二醇二缩水甘油醚将半抗原与载体蛋白牛血清白蛋白(BSA)偶联,并运用碳二亚胺法将半抗原与卵清白蛋白(OVA)进行偶联,分别制备出免疫抗原TBL-cBSA与包被抗原TBL-cOVA。紫外分光光度法及SDS-PAGE进行鉴定。经紫外扫描,证明其偶联比分别为7.60∶1、7.65∶1,此特布他林完全抗原可以免疫动物,本研究成果为特布他林抗体的制备奠定了坚实基础。     

黄曲霉毒素B1完全抗原构建中结合位点研究

通过衍生化反应,合成了AFB1羧甲基活化物,然后利用碳二亚胺法合成AFB1-O-BSA偶联物,构建AFB1完全抗原,并通过多种光谱和质谱对合成完全抗原过程中偶联比和结合位点进行研究。通过荧光光谱在分子水平上探讨AFB1与BSA载体蛋白的偶联机制及偶联反应对BSA的构象影响,推测黄曲霉毒素和牛血清白蛋白反应的结合部位,同时发生在BSA的酪氨酸残基和色氨酸残基上,使得BSA疏水性增加,肽链伸展程度降低。     

时间分辨荧光免疫分析法测定莱克多巴胺

目的 用时间分辨荧光免疫分析( TRFIA)方法测定莱克多巴胺(ractopamine,RAC).方法 采用RAC与牛血清白蛋白( BSA)的偶联物(RAC-BSA)包被96孔板为固相抗原,与样品中游离的RAC共同竞争Eu3+标记的抗RAC抗体,用直接竞争法检测尿样中的RAC,并对此方法进行方法学评价.结果 分析灵敏度为0.01 ng/ml;剂量反应曲线线性相关系数绝对值|r| =0.998 5;回收率为91.6％ ～ 110.0％;与多巴酚丁胺、异舒普林、沙美特罗、非诺特罗、克伦特罗、特布他林和沙丁胺醇7种药物的交叉反应率分别小于0.8％、0.1％、0.1％、0.05％、0.01％、0.01％和0.01％;分析内相对标准偏差为2.38％ ～ 3.90％;分析间相对标准偏差为3.68％ ～ 5.43％;与进口ELISA试剂比较,检测结果相关系数r=0.928.结论 莱克多巴胺时间分辨荧光免疫分析法灵敏度比ELISA分析法更高、稳定性更好,具有良好的应用前景.     

Determination of ractopamine residue in tissues and urine from pig fed meat and bone meal

In many countries, ractopamine hydrochloride (RAC) is allowed to be used in animal production as a β-agonist, which is an energy repartitioning agent able to offer economic benefits such as increased muscle and decreased fat deposition, feed conversion improvement and an increase in average daily weight gain. However, some countries have banned its use and established strict traceability programmes because of pharmacological implications of β-agonist residues in meat products. In Brazil, commercial RAC is controlled (5–20 mg kg^?1) and only added to pig diet during the last 28 days before slaughter. However, the control is more difficult when co-products, like meat and bone meal (MBM), which can be produced from RAC treated animals, are part of the feed composition. Therefore, a study was undertaken to evaluate the presence of RAC residue concentrations in urine and tissues of gilts (n = 40) in four dietary groups: 0%, 7%, 14% and 21% (w/w) of MBM-containing RAC (53.5 µg kg^?1). The concentration of RAC residues in MBM, pig tissues and urine was determined by LC–MS. Low RAC concentrations were detected in muscle, kidney, liver and lungs (limit of detection = 0.15, 0.5, 0.5 and 1.0 µg kg^?1, respectively); however, no RAC residues were quantified above the limit of quantification (0.5, 2.5, 2.5 and 2.5 µg kg^?1, respectively). In urine, the RAC concentration remained below 1.35 µg L^?1. These data suggest that MBM (containing 53.5 µg kg^?1 RAC) added to diet up to 21% (w/w) could hamper the trade where RAC is restricted or has zero-tolerance policy.     

基于PtNPs-PV的双重放大免疫比色法检测肉中莱克多巴胺痕量残留的研究

本文提出了一种免疫比色方法用于检测猪肉中莱克多巴胺的残留。该方法结合了抗原抗体特异性结合的高选择性特点和胶体铂颗粒(Pt NPs)与多聚酶螯合物复合物(Pt NPs-PV)的信号放大效应,能特异、灵敏地检测样品中痕量的莱克多巴胺(Ractopamine,RAC)残留。多聚酶螯合物(Power Vision,PV)上含有二抗和大量的辣根过氧化物酶(Horseradish Peroxidase,HRP),与抗体特异结合的同时催化底物二氨基联苯胺(3,3’-diaminobenzidine,DAB)产生显色沉淀,并且胶体铂颗粒(Pt NPs)也能催化DAB产生显色沉淀与复合物,从而达到双重放大效果。通过测定DAB沉淀吸光度的变化能对应计算样品中莱克多巴胺的残留量。结果表明,在优化的测定条件下,该方法具有较高的检测灵敏度并在0.05~20 ng/m L范围内具有良好的相关性,R2=0.992,检测下限是0.025 ng/m L。所提出的方案具备灵敏度高、选择性强、准确性高且检测速度快等优点,可以适应猪肉中莱克多巴胺残留的现场检测需求。     

环丙沙星单克隆抗体的制备及鉴定

通过改进的碳二亚胺法制备环丙沙星- 牛血清白蛋白偶联物(CIP-BSA),用紫外扫描、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)方法鉴定偶联物。利用制备的CIP-BSA 偶联物作为免疫原,免疫Balb/c 小鼠,用杂交瘤技术制备环丙沙星单克隆抗体,并对其效价、亲和力和特异性进行鉴定。结果表明：CIP-BSA 的偶联比为30:1。经间接酶联免疫吸附(ELISA)法筛选,共筛出两株敏感、特异的杂交瘤细胞(JY-1、JY-2)。这两株细胞的腹水经纯化后的抗体效价分别为5.12 × 106、2.56 × 106,亲和力常数分别为2.88 × 109、2.46 × 109L/mol,两抗体均为IgG1a,取亲和力高的JY-1 进行竞争抑制测定,IC50 值可以达到9.83ng/mL。该单抗仅与恩诺沙星存在交叉反应,交叉反应率为95%。通过验证得到的JY-1 细胞株分泌的单克隆抗体可用于水产品中环丙沙星残留的快速检测。     

基于智能手机同时检测4 种兽药残留的免疫芯片技术

建立一种基于智能手机同时检测猪尿中莱克多巴胺、盐酸克仑特罗、氯霉素、氟苯尼考及其代谢物氟苯尼考胺多种兽药残留的免疫蛋白芯片方法。以硝酸纤维素膜（NC膜）为固相载体固定4 种药物抗原,基于分析物与固相抗原竞争抗体的反应,利用不同质量浓度的药物标准品与芯片显色斑点检测强度作标准曲线,建立智能手机检测方法。该方法对莱克多巴胺、盐酸克仑特罗、氯霉素、氟苯尼考的检出限分别为0.146、0.137、0.035、3.73 ng/mL,线性范围分别为0.353～1.017、0.309～0.897、0.082～0.249、9.424～35.594 ng/mL,与氟苯尼考胺的交叉反应率为81.5%。同时将本方法与胶体金免疫层析法、酶联免疫吸附法进行对比,检测结果基本一致。本方法具有低成本、高通量、操作方便等特点,适用于现场快速大量筛选。     

Automated determination of oxytetracycline residues in muscle, liver, milk and egg by on-line dialysis and post-column reaction detection HPLC.

An automated method for control of oxytetracycline (OTC) residues in chicken and bovine muscle, salmon liver, bovine milk and hen egg has been developed. Tissue homogenate, decreamed milk or whole egg solution was dialysed and the dialysate enriched on a small polystyrene column on-line to HPLC. OTC and the internal standard (tetracycline) were separated on a polystyrene column by ion-pair chromatography. The column effluent was mixed with sodium hydroxide and irradiated at 366 nm. Monitoring the resulting derivatives with a fluorescence detector (excitation: 358 nm, emission: 460 nm), OTC could be detected at 1 ng/ml in milk, 1 ng/g in egg, 3-4 ng/g in muscle and 8 ng/g in liver. Relative standard deviations at 50 and 200 ng/g (milk: 20 and 100 ng/ml) ranged from 1.6 to 3.1%.     

Oxytetracycline and oxolinic acid residues in kuruma prawn (Penaeus japonicus) and the effect of cooking procedures on the residues

Tissue distribution and residue depletion of oxytetracycline (OTC) and oxolinic acid (OA) were studied in the kuruma prawn (Penaeus japonicus). The prawn were kept in tanks with recirculated artificial seawater at a salinity of 22-23@1000. The water temperature was maintained at 25 degrees C. The average body weight was 22.9 +/- 4.9 g for OTC and 22.5 +/- 3.6 g for OA. The drug was mixed with the diet and orally administered through a catheter to the prawn. The doses of OTC and OA, respectively, were 50 mg/kg body weight. At each sample time, four prawns were sacrificed and tissues were sampled. OTC and OA levels were determined by high-performance liquid chromatography. At the highest levels, the concentrations of OTC were in the other: shell (13.57 micrograms/g) > hemolymph (12.20 micrograms/mL) > muscle (8.30 micrograms/g). For OA, the order was: shell (20.74 micrograms/g) > hemolymph (7.06 micrograms/mL) > muscle (2.05 micrograms/g). The elimination half-lives of hemolymph and muscle were 44.7 and 46.8 hours for OTC and 55.0 and 107.9 hours for OA, respectively. Residual OTC could not be detected in hemolymph and muscle at 20 days after dosing. Residual OA disappeared from hemolymph and muscle at 25 days after dosing. A 25-day period for OTC and 30-day period for OA could be regarded as the proper withdrawal time established for kuruma prawn by the Pharmaceutical Law in Japan. However, the elimination half-lives of shell for OTC and OA could not be calculated because both drug residues persisted in shell tissues, and the elimination phase was not completed during the experimental period. Residual OTC (14.10 +/- 2.26 micrograms/g, n = 6) and OA (0.32 +/- 0.06 microgram/g, n = 7) were detected in exuviae at 3 days and 4 days after dosing, respectively. Residual OTC was reduced to 50-70% in muscle by the usual methods of cooking (boiling, baking at 200 degrees C and frying at 180 degrees C), whereas reduction levels in shell were only 20-30%. Residual OA was reduced to 20-30% in muscle and shell by the cooking. These results confirm that the cooking procedures could only reduce but not completely eliminate these drug residues in prawn.     

格列本脲人工抗原的合成与鉴定

目的：合成格列本脲人工抗原。方法：采用对氨基苯甲酸法制备半抗原,将半抗原分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)通过碳二亚胺法偶联制备免疫原(Gli-BSA)和包被原(Gli-OVA),利用紫外扫描进行抗原的化学鉴定,通过免疫原免疫Balb/c小鼠,间接酶联免疫吸附法测定抗血清进行生物鉴定。结果：制备了Gli-BSA、Gli-OVA的人工抗原,经紫外光谱扫描,偶联比分别为4:1和17.7:1。免疫小鼠后获得抗血清的效价均达到32000以上,半抑制质量浓度为10μg/mL。结论：成功合成了格列本脲人工抗原,并获得了格列本脲抗体,为格列本脲的免疫学检测方法进一步研究提供了实验基础。     

Laboratory validation of an LC-MS/MS method for the detection of ractopamine,clenbuterol and salbutamol in bovine and swine muscle at sub-μg kg–1 regulatory limits

ABSTRACT

Ractopamine (RAC), is a β-adrenergic agonist increasingly used in the swine and cattle industry. This compound redirects nutrients to favour leanness rather than fat deposition, improves growth and carcass traits gaining higher economic benefit to producers. Countries around the world are split over whether to allow the use of RAC in meat production. Clenbuterol (CLB) and salbutamol (SLB) are anillinic and phenolic β-agonists, respectively, with the same capacity of producing economic benefits for the meat sector. However, they are prohibited because of the potentially adverse reactions they can cause in consumers. The three β-agonist compounds have been included in the Brazilian National Regulatory Survey and consequentially there is an eminent need for reliable methods capable of detecting those substances at the same time and reduce analytical costs. Therefore, an LC-MS/MS method for the simultaneous determination of residual RAC, CLB and SAL in swine and cattle muscle was developed and validated with quantification levels respecting the action levels established for Brazil which are 0.1, 0.2 and 5 µg kg^–1 for RAC, CLB and SAL, respectively. Samples were quantified using RAC-d₅, CLB-d₉ and SLB-d₆ as internal standards. The validation was performed according to European Union Decision 2002/657, which includes criteria (CCα, CCβ, recovery, repeatability, reproducibility and calibration curve). The method meets the Brazilian regulatory requirement that establishes criteria and procedures for the determination of parameters such as CCα, CCβ, precision and recovery. CCα values were 0.02, 0.21 and 5.42 µg kg^–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples; CCβ values were 0.03, 0.22 and 5.8 µg kg^–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples. Average recoveries fortified with 0.05–7.5 µg kg^–1 of the studied β-agonist leads around 95%. The method was demonstrated to be suitable for the determination of RAC, CLB and SLB in swine and cattle muscle samples.