氟喹诺酮药物格林沙星抗体制备及其icELISA方法建立

目的:制备格林沙星多克隆抗体并建立间接竞争酶联免疫吸附分析方法(icELISA)。方法:采用碳二亚胺法将格林沙星偶联于牛血清白蛋白(BSA)作为免疫原,免疫新西兰大白兔获多克隆抗体;采用活泼脂法分别将格林沙星、克林沙星、加替沙星、培氟沙星和沙拉沙星偶联于卵清白蛋白(OVA),制备5种包被抗原并做性能比较;建立icELISA方法并对其灵敏度和特异性进行评价。结果:成功制备了免疫原和包被抗原及抗体,抗体效价最高达64 000倍。以克林沙星-OVA作异源包被,建立的icELISA方法灵敏度最高,半抑制药物浓度(IC50)为9.14 ng/mL,检测限(IC10)为0.9 ng/mL,检测范围(IC20~IC80)为2.3~36.4 ng/mL,与其他喹诺酮类药物无明显交叉反应。结论:首次制备出格林沙星抗体并建立间接竞争酶联免疫吸附检测方法,该法灵敏度高、特异性强,为分析生物样品中格林沙星含量的测定提供了1种新的方法。     

恩诺沙星快速酶联免疫吸附法检测试剂盒的研制

目的：研制恩诺沙星酶联免疫吸附法检测试剂盒。方法：采用EDC-NHS 法制备ENR-M-BSA 免疫原和ENR-OVA 检测抗原,用ENR-M-BSA 免疫BALB/C 小鼠,采用甲基纤维素半固体培养基法筛选单克隆抗体并采用间接竞争ELISA 对其进行分析。结果：通过公式计算ENR-M-BSA 摩尔偶联比为15:1,ENR-OVA 摩尔偶联比为7.2:1。筛选出1 株特异性分泌株3E9,其腹水纯化后间接竞争ELISA 测其效价为107,分型试剂盒测试显示此抗体属于IgG1 型;检测限为1ng/mL,线性范围在1～100ng/mL 之间,半量抑制浓度(IC50 值)为10ng/mL,与4 种结构类似物交叉反应均小于1%。结论：此株单克隆抗体可以用来建立恩诺沙星ELISA 检测试剂盒。     

化学发光酶联免疫法检测苏丹红Ⅰ

为检测食品中苏丹红Ⅰ残留,建立间接竞争化学发光酶联免疫分析（chemi luminescent enzymeimmunoassay,CLEIA）法。通过优化包被抗原中本抗原与载体物质的量比、包被抗原质量浓度、抗体稀释比例,建立竞争抑制曲线。线性范围为0.156～5 ng/mL,最低检测限为0.078 9 ng/mL,IC50为0.679 ng/mL。CLEIA回收率为75.08%～112.18%,变异系数为8.89%～15.61%;通过与酶联免疫吸附（enzyme-linked immunosorbent assay,ELISA）法进行比较,在相同抗原抗体质量浓度条件下,CLEIA法测定的IC50较ELISA方法降低30%,具有较高的灵敏度。     

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。     

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。     

基于Arah6的花生间接竞争ELISA检测方法的建立

建立了基于花生主要过敏原蛋白Arah6的间接竞争酶联免疫检测法,实现了对食物中花生的定量检测。利用花生过敏原Arah6纯品免疫新西兰大白兔,制得兔抗Arah6的多克隆抗体,以Arah6纯品作为包被抗原、自制抗体为一抗、辣根过氧化物酶标记的羊抗兔IgG为二抗,确定了包被抗原质量浓度为1μg/mL,一抗最佳稀释度为1:50000,酶标二抗稀释度为1:5000。此检测方法对Arah6的定量检测范围为16.5～10000ng/mL(折合成含花生的含量,约为165ng/mL～100μg/mL),抑制方程为抑制率I=21.418lgC-6.0633(C为Arah6的质量浓度,单位ng/mL),IC50为414.6ng/mL,相关系数R2=0.9989。该检测方法灵敏度高,检测范围广,适用于食品中花生现场快速检测。     

莱克多巴胺抗体的制备与评价

本文以RAC-linker-BSA为免疫抗原获得了莱克多巴胺的多克隆抗体,用混合酸酐法合成包被抗原,建立了莱克多巴胺的间接竞争酶联免疫检测方法(ELISA),并对抗体性质进行了评价.结果表明,制备出的莱克多巴胺抗体具有较高的灵敏度,IC50为0.9 ng/mL,检出限(IC15)为0.1 ng/mL,低于同类文献的报道结果;除了与多巴酚丁胺的交叉反应为7.5 %外,与克伦特罗、沙丁胺醇、特步他林、异丙肾上腺素和异奎胍均无交叉现象,说明抗体特异性较高;此外,抗体具有很好的稳定性,可在4 ℃冰箱中储存半年以上.为建立酶联免疫快速检测方法,实现对莱克多巴胺的有效监控奠定了良好的基础.     

雌二醇间接竞争酶联免疫吸附方法的建立

建立食品中雌二醇免疫分析方法。将雌二醇进行结构改造合成半抗原并经过核磁氢谱和红外光谱验证,再采用活泼酯法与载体BSA和OVA偶联制备人工抗原,免疫兔子制备多克隆抗体,经过反应条件优化建立间接竞争酶联免疫吸附检测方法。结果表明：IC50为3.9ng/mL,检测限为0.3ng/mL,交叉反应率均小于1.21%。检测方法具有高灵敏度和特异性,可应用于食品中雌二醇检测试剂盒的研制。     

沙丁胺醇直接酶联免疫快速检测的方法

沙丁胺醇与瘦肉精同属于禁用的饲料添加剂,可以在动物体内大量残留,人体过量摄入会引起中毒反应。为了建立沙丁胺醇的快速检测方法,从硫酸沙丁胺醇出发制备了半抗原沙丁胺醇丁二酸衍生物,用混合酸酐法将半抗原与载体蛋白-钥孔嘁血蓝蛋白(KLH)偶联作为免疫抗原制备了沙丁胺醇的多克隆抗体,建立了沙丁胺醇直接竞争酶联免疫检测方法,该方法抗体最佳包被抗体量为1μg/孔,酶标抗原稀释比例为1∶16000,掩蔽剂采用脱脂奶粉。所建立的方法具有很高的灵敏度和特异性I,C50为0.90ng/mLI,C15达到了0.05ng/mL,远低于国家残留限量标准,与沙丁胺醇的结构类似物基本没有交叉反应。     

百菌清酶联免疫吸附检测方法的影响因素

以方阵滴定法筛选适合百菌清酶联免疫吸附检测的包被抗原和抗体浓度,同时研究反应缓冲体系和pH值等因素对酶联免疫吸附方法的影响以及百菌清多克隆抗体的亲和性和特异性,建立蔬菜中百菌清残留检测的间接竞争酶联免疫吸附法。结果表明:方法最低检测限(IC20)为0.0123ng/mL,回收率为84.0%～89.8%,变异系数为3.0%～7.6%,与百菌清结构类似化合物交叉反应率均小于0.01%,可在试剂样品检测中应用。     

Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Dibutyl Phthalate in White Wine,Compared With GC-MS

Plasticizer has attracted more and more attention in China for the past 3 years, especially in Taiwan district. In this study, an indirect competitive enzyme-linked immunosorbent assay (icELISA) has been developed for the determination of a plasticizer dibutyl phthalate (DBP) in white wine. Dibutyl 4-aminophthalate coupled with OVA was synthesized as an immunogen to produce polyclonal antibodies against DBP. The antibody exhibited negligible cross-reactivity with other related compounds. The influence of several physicochemical parameters, such as coating procedure, organic solvent, competitive reaction time, and pH was investigated. The limit of detection was 64.5 ng/mL, which was sensitive enough for a screening assay. The linear range was 64.5–1,606.2 ng/mL with a correlation coefficient (R ²) of 0.996. The method was successfully applied to the determination of DBP in white wine. Recoveries were between 83.1 and 101.7 %. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DBP monitoring. The results obtained were compared with those obtained using gas chromatography–mass spectrometry (GC-MS), and a satisfied correlation coefficient of 0.928 was obtained by real sample detection.     

间接竞争ELISA法检测呋喃唑酮代谢物

以人工抗原和特异性抗体为基础,建立了以2-硝基苯甲醛为衍生试剂检测呋喃唑酮代谢物3-氨基-2-恶唑烷酮(AOZ)间接竞争ELISA方法。通过方阵滴定和间接竞争法确定ELISA方法的抗原抗体的最佳工作浓度;得到标准曲线的线性范围为0.25~10ng/mL,线性关系良好;根据标准曲线计算IC50为0.5881~1.1333 ng/mL。此外,检测了与其他类似物的交叉反应率,显示了很好的特异性;并且通过对虾样品添加回收率的测定,证明了该方法的准确性。高温短时间衍生化处理可达到与37℃孵育过夜同样的衍生效果。     

酶联免疫法检测食品中的双酚A残留

建立检测双酚A(BPA)的间接竞争酶联免疫检测法(icELISA),探讨其在实际样品检测中的灵敏性及准确性。以抗BPA的腹水单抗建立BPA的icELISA法,利用该法对市面上聚碳酸酯(PC)制包装材料中BPA向食物的迁移量进行检测,并与高效液相色谱法检测结果相比较。BPA的icELISA法检测范围为2.817 ng/mL～1.212×106ng/mL,IC50为1 847.9 ng/mL,检测限为0.324 ng/mL,该法测定罐装鱼肉和蔬菜中BPA分别为1 015.25 ng/mL和207.22 ng/mL。     

基于单链抗体的呋喃唑酮酶联免疫检测方法的建立

目的:为快速检测呋喃唑酮(furazolidone,FZD)在动物性食品中的残留量。方法:基于单链抗体的间接竞争酶联免疫吸附实验法建立了FZD检测方法。结果:最佳抗原工作质量浓度为2μg/m L,最佳抗体稀释倍数为1∶500,一抗最佳反应时间为60 min,二抗最佳反应时间为45 min,四甲基联苯胺最佳显色时间为20 min。FZD检测试剂盒在10~100 ng/m L范围具有较好的线性关系,IC50值为13.01 ng/m L,检出限为1.28 ng/m L,回收率为73.38%~84.52%。结论:与抗FZD单克隆抗体相比,所建立的检测试剂盒检测范围更广,且具有很高的灵敏度以及很好的特异性和稳定性。     

氟乐灵抗体的制备及其酶联免疫分析方法的建立

该研究针对农(水)产品中氟乐灵农药残留的问题,建立了间接竞争酶联免疫分析方法,快速检测氟乐灵农药.利用3,5-二硝基-4-氯-三氟甲苯与仲胺侧链氨基反应合成半抗原2C,采用碳二亚胺法将半抗原2C与牛血清白蛋白/卵清蛋白偶联合成完全抗原.通过免疫新西兰大白兔获得多克隆抗体PcAb-2C,基于间接竞争酶联免疫分析方法原理对...     

Development of a monoclonal antibody-based competitive ELISA for detection of β-conglycinin,an allergen from soybean

Soybean β-conglycinin is one of the major food allergies for children and young animals. In order to detect immunoreactive β-conglycinin from soybean and soybean products, monoclonal antibodies against β-conglycinin were prepared using a conjugated chicken ovalbumin with a synthetic peptide that corresponded to one epitope sequence of β-conglycinin as the immunogen. The generated monoclonal antibodies, named as 6G₄, 3B₇, and 5F₁₀, were identified as being of IgG₁ isotype and exhibited high specificity to β-conglycinin. A competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody 6G₄ was established to determine β-conglycinin and showed an IC₅₀ value of 4.7 ng/mL with a detection limit of 2.0 ng/mL. The recovery tests of β-conglycinin indicated that the monoclonal antibody 6G₄-based competitive ELISA gave reliable reproducibility. Therefore, the produced monoclonal antibody 6G₄ and the developed competitive ELISA could provide a valuable tool for sensitive determination of β-conglycinin and for future studies on food allergies related to soybean β-conglycinin.     

应用抗黄曲霉毒素单链抗体检测酱油中黄曲霉毒素B1

利用本实验室制备的抗黄曲霉毒素B1的单链抗体(ScFv),通过棋盘实验确定了抗原抗体的最适工作浓度,在此之上根据间接竞争酶联免疫法(ELISA)绘制标准曲线,检测酱油中AFB1的含量;通过改变样品的盐浓度及pH来确定其对ELISA检测结果的影响。研究结果表明,利用ScFv检测黄曲霉毒素的最小检测值为0.10ng/mL,平均加标回收率在84%～109%之间,本文建立的ELISA方法在pH5～8,盐浓度小于10%时较稳定。本文建立的利用抗AFB1的ScFv检测黄曲霉毒素含量的方法方便快捷,稳定性较好,并且成本较低,适合于食品中黄曲霉毒素的检测。     

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.