动物性食品中替米考星酶联免疫检测方法的研究

通过对替米考星分子结构进行改造,制备替米考星半抗原及人工抗原,通过免疫动物得到替米考星的单克隆抗体。该抗体灵敏度为0.5μg/L,对泰乐菌素的交叉反应率小于1%。基于该抗体建立了替米考星间接竞争酶联免疫检测方法检测动物组织、肝脏、蜂蜜、鸡蛋、牛奶的检测限分别为0.5、10、1、5、5μg/kg,添加水平回收率为70%~110%,变异系数小于15%。     

动物性食品中诺氟沙星残留的荧光免疫分析方法研究

利用杂交瘤技术制备了诺氟沙星的单克隆抗体,建立了以抗诺氯沙星单克隆抗体为基础的荧光免疫分析方法,工作曲线表明在10-500μg／L浓度范围内呈良好的线性关系,回归方程为：I=31．9210gC-5．0083,相关系数r=0．9965,对诺氟沙星最低检出限迭6．09μg／L,抑制中浓度为52．88μg／L,且对其它结构类似喹诺酮类药物均有特异性识别。本法可应用于检测动物性食品中残留的微量诺氟沙星。     

双酚A单克隆抗体的制备及酶联免疫分析方法的建立

采用活泼酯法将双酚A的结构类似物双酚酸与载体蛋白偶联制备人工抗原,用制备的人工抗原免疫BALB/c小鼠,采用聚乙二醇法进行细胞融合制备双酚A单克隆抗体,成功获得一株分泌抗双酚A单克隆抗体的细胞株3H1,经鉴定抗体属于IgG1亚型,轻链为κ,并建立了间接竞争酶联免疫分析法。线性范围为1～50 ng/mL,最低检测限为0.43 ng/mL,半数抑制浓度为6.56 ng/mL。回收率为82.83%～101.94%,变异系数为2.94%～12.95%。该方法具有较高的灵敏度和特异性,具有良好的应用前景。     

双抗体夹心酶联免疫方法检测丁香假单胞杆菌斑点致病变种病菌

丁香假单胞杆菌斑点致病变种病菌可以引起十字花科细菌性黑斑病,是侵染萝卜、白菜、花椰菜等农作物的重要病害之一。以灭活的丁香假单胞杆菌斑点致病变种病菌(NCPPB1820)为免疫原免疫小鼠,通过杂交瘤细胞技术,筛选最终获得6株产生特异性单克隆抗体的杂交瘤细胞。将辣根过氧化物酶(horseradish peroxidase,HRP)与抗体偶联,作为检测探针,分别将6种单克隆抗体作为包被抗体,进行两两配对筛选。结果表明,所制备的抗体特异性较好,对水稻细菌性谷枯病菌、水稻细菌性条斑病菌、玉米细菌性枯萎病菌、丁香假单胞菌丁香致病变种均没有交叉反应。以6号抗体作为检测抗体(6-HRP),以1号抗体作为包被抗体,建立酶联免疫分析法获得了最佳的敏感度。检测丁香假单胞杆菌斑点致病变种的最低检测限为1.5×105cfu/m L,定量限为4.12×105cfu/m L。基于双抗体夹心的酶免疫方法特异性好,便捷,可以实现对丁香假单胞杆菌斑点致病变种病菌高通量测定。     

Simultaneous determination of sulphamerazine,sulphamethazine and sulphadiazine in honey and chicken muscle by a new monoclonal antibody-based fluorescence polarisation immunoassay

A new monoclonal antibody (Mab) against sulphamerazine (SMR) was produced and a fluorescence polarisation immunoassay (FPIA) based on the Mab was developed and optimized for the simultaneous qualitative screening of SMR, sulphamethazine (SMZ) and sulphadiazine (SDZ). The Mab, raised from mice immunized with SMR, was bound to bovine serum albumin (BSA) using glutaraldehyde as the coupling reagent. Fluorescein-labelled SMR and SMZ (tracer) were synthesized and purified by thin layer chromatography (TLC). Cross-reactivities below 3.6% were displayed in the optimized FPIA for another 14 sulphonamides when both tracers were employed. The limits of detection (LOD) were 0.9 ng g^?1 for SMR, 2 ng g^?1 for SMZ and 3.1 ng g^?1 for SDZ. Analysis of SMR, SMZ and SDZ fortified chicken muscle and honey samples by the FPIA showed average recoveries of 86–131% with a standard deviation (SD) of 4.6–32. Comparative analyses of a SMZ-treated chicken muscle sample by both FPIA and high performance liquid chromatograph (HPLC) showed a good correlation (r?=?0.9991). The study demonstrates the practical application of FPIA in screening chicken muscle and honey samples for sulphonamides residues.     

杂交-杂交瘤技术制备BsMAb的研究进展及其在食品安全检测中的应用

双特异性单克隆抗体（bispecific monoclonal antibody,BsMAb）是指具有两个不同特异性抗原结合位点的单克隆抗体分子。杂交-杂交瘤技术因其简便易行、制备周期短,在制备BsMAb方面有较大优势。基于杂交-杂交瘤技术能制备出可以特异性识别两种或者两类小分子药物的BsMAb,在食品安全多残留免疫分析检测中具有较好的应用价值。本文综述了杂交-杂交瘤技术制备BsMAb的进展,探讨了基于该技术的BsMAb生成机制,讨论分析了杂交-杂交瘤及BsMAb筛选制备的关键技术要点,并归纳了BsMAb在检测食品中小分子污染物方面的应用进展,旨在为食品安全多残留免疫分析技术的发展应用提供参考。     

基于单克隆抗体的双酚A间接竞争酶联免疫分析法的建立

为建立一种简便、快速、准确的双酚A检测方法,采用活性酯法将双酚酸与牛血清蛋白、卵清蛋白偶联制备人工抗原免疫BALB/c小鼠。选择抗血清效价和灵敏度高的BALB/c小鼠,采用聚乙二醇法制备杂交瘤细胞,获得一株可分泌双酚A单克隆抗体的杂交瘤细胞株。通过方阵滴定和反应条件优化,建立一种基于单克隆抗体的双酚A间接竞争酶联免疫分析法。该检测方法在0.5 ng/mL～50 ng/mL内有良好的线性关系,最低检测限IC10为0.5 ng/mL,半数抑制率IC50为16.65 ng/mL,水样中加标回收率为89.72%～105.25%。该单克隆抗体效价高、特异性强,该检测方法灵敏度高。     

呋喃西林代谢物半抗原的制备及其免疫检测中的应用

本研究为了克服现有SEM半抗原结构不含硝基端基团的不足,将SEM与4-(3-醛基-4-硝基-苯氧基)-丁酸衍生制备半抗原,保留了硝基端基团。然后,将制备的半抗原分别与BSA和OVA偶联制备SEM-BSA免疫原和SEM-OVA包被原。免疫原免疫BALB/C小鼠后,结合细胞融合技术制备单克隆抗体,并利用间接竞争酶联免疫分析法(ci ELISA)评价单克隆抗体的效价及特异性。结果显示,单克隆抗体可特异性识别SEM的2-硝基苯甲醛衍生物(2-NPSEM),效价达1:2560000。应用ci ELISA检测法,在0.05~4.05μg/L呈线性关系,检测限高达0.04μg/L,IC_(50)值为0.23μg/L,与结构类似物NPAMOZ、NPAOZ和NPAHD的交叉反应率分别为0.03%、0.02%和0.26%。本论文提供的SEM半抗原的设计和改造方法,为呋喃西林的代谢物SEM的高效检测提供了新思路和方法。     

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15?±?0.02 µg l^?1 and an IC₅₀ value of 1.13?±?0.16 µg l^?1. Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0–909.8 µg kg^?1. Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant® zearalenone kit (r ²?=?0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.     

Monoclonal Antibodies to Porcine Thermal-Stable Muscle Protein for Detection of Pork in Raw and Cooked Meats

Four hybridoma cell lines secreting monoclonal antibodies (MAbs) specific to porcine thermal-stable muscle proteins (TSMPs) were developed. The MAbs reacted with three protein bands (20.5, 22 and 24 kD) from raw pork extract; the protein band (24 kD) which was also present in cooked pork was identified as porcine-specific TSMP. Epitope analysis indicated four MAbs recognized same or closely located antigenic sites on the pork TSMP. The developed MAb-based indirect enzyme-linked immunosorbent assay (ELISA) enabled the detection of pork in raw and cooked heterogeneous meat mixtures as low as 10 g/kg. The curvilinear relations of second-degree polynomial (r² > 0.995) between pork concentrations and ELISA responses enabled quantifying degree of adulteration of pork in meat products.     

噬菌体随机肽库淘选桔霉素模拟表位的研究

目的:从噬菌体随机肽库中淘选模拟桔霉素表位的噬菌体粒子。方法:以抗桔霉素的单克隆抗体为配基,分别免疫亲和淘选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机7肽库和12肽库,以ELISA方法鉴定阳性克隆,同时进行DNA测序以分析插入的7肽和12肽的氨基酸序列。结果:经过3轮淘选,在7肽库中淘选到20株能与该抗体特异性结合的阳性克隆,在12肽库中淘选到33株能与该抗体特异性结合的阳性克隆,且该结合均能被桔霉素阻断,模拟表位的共有序列为X-组氨酸-赖氨酸-X-X-X-X,X为任意氨基酸。以7肽库中亲和力最强的克隆(P10)建立了竞争ELISA检测方法,线性范围为10～325ng/ml,检测下限为10ng/ml;以12肽库中亲和力最强的克隆(P1)建立了竞争ELISA检测方法,线性范围为10～439ng/ml,检测下限为10ng/ml。结论:噬菌体展示技术可成功淘选到桔霉素模拟表位,高度保守的His和Lys的存在,提示His和Lys在CIT与其配体的结合中可能起重要作用。     

雌二醇单克隆抗体制备及胶体金侧流层析免疫分析方法的建立

目的 建立一种牛奶中雌二醇(17β-estradiol, E2)胶体金(colloidal gold, CG)试纸条快速检测方法。方法 从E2的3号位羟基和17号位羟基引入活性基团制备半抗原H1和H2, 偶联载体蛋白制备完全抗原, 经过动物免疫和细胞融合筛选, 制备单克隆抗体, 采用间接竞争酶联免疫吸附实验(indirect competitive enzyme-linked immunosorbent assay, icELISA)对单克隆抗体性能进行评估, 筛选出灵敏度最高的单克隆抗体, 最后采用静电吸附法将胶体金标记抗体为探针, 构建牛奶中E2的胶体金试纸条检测方法。结果 制备了H1-7B7、H1-9C7、H2-4A10、H2-2E2 4种单克隆抗体, 经过评估, 选取H1-9C7和H2-OVA作为最优抗体及抗原, 建立的胶体金试纸条消线(cut-off)值为6.00 ng/mL, 半数抑制浓度(half maximal inhibitory concentration, IC50)为1.54 ng/mL, 与苯甲酸雌二醇和雌三醇的交叉反应率分别为101.31%和2.43%。结论 本研究建立的试纸条具有操作简单、灵敏度高等特点, 能够基本满足牛奶中E2快速检测要求。