恩诺沙星化学发光免疫分析试剂盒的研制

主要研制一种基于鲁米诺-辣根过氧化物酶体系的化学发光免疫分析试剂盒,用于检测食品中的痕量恩诺沙星残留。反应条件优化结果为:最佳包被抗原浓度为0.5ng/mL,最佳抗体稀释倍数为1:2000,药物稀释液为0.01mol/L的PBST溶液,一抗反应时间为30min。研究结果显示:本文研制的试剂盒的回归曲线方程是y=-22842x+172936,检测线性范围是0.024～2.76ng/mL,IC50为0.106ng/mL,对蜂蜜、牛奶、鱼肉和虾肉样本的添加回收率在96%～116%、90%～97%、92%～112%、86%～112%之间,批内和批间变异系数均小于15%,试剂盒在4℃下有效期为180d。      

对硫磷酶联免疫吸附分析方法的建立和评价

通过制备对硫磷的多克隆抗体,建立了对硫磷的间接竞争酶联免疫吸附分析方法(icELISA)。通过活泼酯法将半抗原分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,获得免疫抗原和包被抗原。使用对硫磷抗血清,经优化条件参数建立了对硫磷的icELISA分析方法。该方法检测线性范围为3.39～533.05ng/mL,最低检测限为0.90ng/mL,对蔬菜样品和水样品的平均添加回收率为75.6%～127.4%,能用于实际样品中对硫磷的检测。     

Development of a direct competitive enzyme-linked immunoassay for carbofuran in vegetables

Three ELISA formats, antigen coated, antibody coated and the second antibody coated for the determination of carbofuran were investigated with conjugations including hapten–BSA, hapten–OVA, hapten–HRP and anticarbofuran IgG–HRP. Results showed that the second antibody-coated method of ELISA had a better performance in the establishment of standard curves and detection of carbofuran residue in vegetables samples. The sensitivity for detection, the I₅₀ value was 36.1 ng/ml at a practical working concentration range from 3.44 to 380.1 ng/ml and the limit of detection for carbofuran was 3.44 ng/ml. The average recoveries of determination for carbofuran spiked in cabbage, lettuce, carrot, winter fragrant-flowered garlic, bamboo shoot and green soy bean were 85.24%, 101.8%, 103.6%, 90.52%, 106.9% and 94.08%, respectively. Additional analyses confirmed that the results given by the ELISA method was in agreement with those of the gas chromatography (GC) method.     

呋喃唑酮代谢物间接竞争化学发光酶免疫法的建立

建立快速检测动物源性食品中呋喃唑酮代谢物AOZ的间接竞争化学发光酶免疫法。利用对醛基苯甲酸（4-CBA）将AOZ衍生化为CPAOZ,利用活化酯法将CPAOZ与卵清蛋白（OVA）偶联,生成完全抗原CPAOZ-OVA。对包被原与抗体的最佳稀释倍数、封闭液浓度、竞争时间、酶标二抗稀释倍数进行优化,建立标准曲线,同时对方法的特异性进行评价。结果表明:包被抗原最佳稀释倍数为2000倍,抗体的最佳稀释倍数为20000倍、封闭液为2%脱脂乳粉、竞争时间为2 h、二抗最佳稀释倍数为4000倍;线性范围是0.075⁷.396 ng/m L,半抑制浓度IC₅₀为0.74 ng/m L,对呋喃唑酮原药的交叉反应率为23.4%,与其他结构类似物及衍生化试剂的交叉反应率均小于0.1%。该方法灵敏度高、特异性好,可为监管部门对于快速检测动物源性食品中AOZ提供依据。      

黄曲霉毒素B1直接竞争化学发光酶免疫分析法的建立

以鲁米诺-辣根过氧化物酶-过氧化氢为检测体系,建立一种检测黄曲霉毒素B1的直接竞争化学发光酶免疫法。经优化,该方法的最佳反应条件为抗体包被为0.0625μg/mL;黄曲霉毒素B1酶标抗原（1mg/mL）稀释10000倍;0.01mol/L磷酸盐缓冲液（pH7.5）。本方法的IC50为0.062ng/mL;检出限为0.01ng/mL;线性范围0.017⁰.215ng/mL,批内和批间相对标准偏差均小于15%。食用油样品的添加回收率为88.7%⁹8%,通过对比实验证实,该方法与ELISA方法相关性良好,可用于实际样品中黄曲霉毒素B1的大规模快速筛查。      

Development of a McAb-based immunoassay for parathion and influence of the competitor structure

A specific monoclonal antibody (McAb) for parathion was produced. Based on this McAb, a battery of competitors as coating antigens were used to develop homologous and heterologous indirect competitive enzyme-linked immunosorbent assays (ELISAs) for parathion. The relationship between the heterology degree of the competitor and the sensitivity of the corresponding immunoassay was investigated. Results showed that, when the specific McAb was used in the ELISA experiment, competitors should have a certain degree of homology with the immunizing hapten for immunoassays, and the best performance occurred when the competitor hapten was highest heterologous to the target analyte. With the most suitable competitor, a sensitive and selective ELISA was developed. The IC₅₀ value of the ELISA was 2.94 ng/ml with a detection limit (IC₂₀) of 0.70 ng/ml. The average recoveries of parathion in spiked water, soil, cucumber and rice were 88.09%, 93.15%, 91.37% and 83.42%, respectively.     

免疫竞争比色传感用于测定小麦中呕吐毒素的方法研究

目的 基于磁分离竞争免疫和酶催化比色传感技术建立一种呕吐毒素快速检测方法。方法 将抗体对呕吐毒素的特异性识别与辣根过氧化物酶催化3,3’,5,5’-四甲基联苯胺/双氧水的比色反应相结合,考察了酶催化显色反应的可行性和线性范围,优化了反应温度和反应时间。呕吐毒素检测过程集成自动化反应装置和高通量终端检测设备,以DON抗体功能化的磁性微粒为固相载体,优化了磁珠用量、酶标抗原浓度和免疫亲和反应时间。结果 呕吐毒素在0~1750ng/mL浓度范围内与催化反应产物的吸光度呈良好的线性关系,线性相关系数为0.98。以该方法对小麦基质进行加标回收,回收率在81.3%~90.3%之间,相对标准偏差小于2.1%,检测结果经液相色谱-质谱联用法验证相关性良好。结论 本方法具有较高的灵敏度和准确度,且自动化程度较高、操作较为便捷,有潜力应用于粮食样品中呕吐毒素的现场检测。     

白酒中邻苯二甲酸二乙酯的化学发光酶联免疫分析方法

以邻苯二甲酸二乙酯(diethyl phthalate,DEP)为研究目标,以4-氨基邻苯二甲酸二乙酯为半抗原,通过重氮法偶联载体蛋白并免疫动物,制备针对DEP的特异性兔多克隆抗体。通过棋盘滴定法和单因素实验确定最佳的实验参数,即包被抗原浓度为50 ng/m L,4℃环境下包被12 h;抗体用T液稀释;药物缓冲液选用p H 5.4、0.005 mol/L的PBS缓冲液;酶标二抗用P液稀释,稀释度为1/3000;反应时间是:一抗∶二抗=30 min:40 min。基于此建立了间接竞争化学发光酶联免疫法检测DEP。该方法对DEP的最低检测限(LOD)为3.09 ng/m L,检测范围(IC20~IC80)为5.93~42.03 ng/m L,半抑制浓度(IC50)值为16.57 ng/m L,与14种结构类似物及功能类似物交叉反应均远低于0.5%,通过对白酒样品添加回收率的测定,证明了该方法的准确性,适用于白酒中DEP的快速检测。     

间接竞争化学发光酶免疫法检测动物源食品中的 呋喃西林代谢物

目的建立动物源食品中呋喃西林代谢物间接竞争化学发光酶免疫检测方法。方法采用活化酯法将衍生后的呋喃西林代谢物半抗原与卵清蛋白(ovalbumin,OVA)偶联成为包被原;其次,对化学发光液体系、包被原和单克隆抗体的最优稀释倍数及其他反应条件进行优化;最后对该法的灵敏度、特异性、精密度及准确度进行评价。结果化学发光液A液为8 mmol/L对碘苯酚溶液和10 mmol/L鲁米诺溶液(1:1,V:V)混合,B液为每10 m L三(羟甲基)氨基甲烷(Tris)-盐酸缓冲液加5μL 30%H_2O_2溶液,A液与B液临用前体积比1:1混匀;最优反应条件是包被原和单抗稀释倍数均为800,封闭液为1%脱脂乳,竞争时间30 min,酶标二抗孵育60 min;该法的线性方程为Y=-0.4654X+0.3768(r~2=0.993),线性范围为0.123~2.398 ng/m L,IC50为0.544 ng/m L,批内和批间变异系数分别为1.9%~4.1%和2.8%~5.3%,空白鸡肉样品添加回收率为89.6%~98.0%。结论该检测方法简单快速,可用于实验室或现场动物源食品中呋喃西林代谢物的筛查。     

直接竞争酶联免疫吸附法检测虾过敏蛋白

目的:建立酶标抗原的直接竞争酶联免疫吸附法(dcELISA)检测食品中虾过敏蛋白,为食品过敏诊断试剂的开发和应用提供理论基础。方法:提取虾主要过敏蛋白,免疫小鼠制备抗虾过敏蛋白多克隆抗体,辣根过氧化物酶(HRP)标记抗原,建立酶标抗原的dcELISA检测虾过敏蛋白。结果:所建立的dcELISA法最低检测限为3.94ng/mL,标准曲线在0.12～128.86ng/mL范围内线性良好,批内和批间变异系数分别为6.16%和2.73%,回收率为82%～98%。结论:该方法具有良好的特异性、敏感性和稳定性,为进一步研制检测虾过敏蛋白的ELISA试剂盒提供有效的方法。     

Fluorescent polarization immunoassay for sulphadiazine using a high specificity antibody

A rapid and sensitive fluorescent‐labelled polarization immunoassay for sulphadiazine (SDZ) is reported. Fluorescein‐labelled tracers were synthesized and a specific SDZ antibody has been used in the development of the method. The influence of the fluorescent label type on the immunoassay sensitivity was investigated. Homologous and non‐homologous tracer – antiserum combinations were varied to provide increased sensitivity of the immunoassay. A detection limit of 0.2 ng mL^?1 for SDZ in 50 μL aqueous sample has been achieved. The cross‐reactivity of other sulphonamide drugs has also been examined.     

磺胺二甲嘧啶间接竞争化学发光酶免疫分析法的建立及应用

建立一种更加灵敏的磺胺二甲嘧啶(SM₂)免疫学检测方法。方法 本研究制备抗SM₂单克隆抗体(SM₂-mAb),用于建立SM₂间接竞争化学发光酶免疫分析法(SM₂-CLEIA),并检测主要动物食品中SM₂残留。结果 本研究制备的SM₂-mAb为IgG_2b亚类,亲和常数为0.12×10⁷ L/mol;SM₂-CLEIA曲线在0.1～1 000 μg/L之间呈现良好的线性关系,相关系数r²=0.990 4,半数抑制浓度(IC₅₀)为4.006 μg/L,检测限为0.174 μg/L,添加回收率为94.41%～104.40%,批内和批间变异系数分别为3.07%和8.22%,与磺胺脒等药物无交叉反应,与SM₂-ELISA试剂盒的阴阳性符合率为100%。结论 建立了灵敏、特异、准确、检测范围宽的SM₂-CLEIA检测方法。     

Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M1 and Escherichia coli O157:H7 in milk

Pathogens, mycotoxins, or antibiotics may exist in a food sample. Micro- and macromolecular substances must be detected quickly. A rapid and convenient lateral flow immunoassay (LFI) integrated with competitive and sandwich models was developed to detect micro- and macromolecular substances. In this study, aflatoxin M₁ (AFM₁) and Escherichia coli O157:H7 were selected as the micro- and macromolecular substances, respectively. Two test lines in the LFI test strip were evaluated to detect AFM1 and E. coli O157:H7 by competitive and sandwich models. Results showed that the limits of detection for detecting AFM1 and E. coli O157:H7 were 50 pg·mL^?1 and 1.58 × 10⁴ cfu·mL^?1, respectively. The whole assay time was 30 min. The recoveries of gold nanoparticle-LFI ranged from 78.0 to 111.6% with coefficients of variation in the range of 3.9 to 8.5% for the detection of AFM₁. For the detection of E. coli O157:H7, the range of recoveries was from 70.1 to 89.6% with coefficients of variation ranging from 4.9 to 13.0%. This study not only tested sensitivity and specificity, but also was a systematic study of location of 2 test lines of the LFI test strip integrated with competitive and sandwich models.     

对硫磷快速ELISA检测方法的建立

由于对硫磷的残留问题及其危害,迫切需要建立一种快速有效的农药检测方法。将对硫磷苯环上的硝基还原为氨基,得到了具有氨基活性的半抗原化合物———氨基对硫磷,然后采用重氮法将氨基-对硫磷与牛血清白蛋白(BSA)或卵清白蛋白(OVA)相偶联,分别合成了免疫原和包被抗原:氨基对硫磷-BSA、氨基对硫磷-OVA。对合成的抗原进行紫外可见扫描,确证偶联成功。用合成的免疫抗原免疫实验用大白兔,制备对硫磷的多克隆抗血清,效价达到1∶1×106以上。对硫磷抗体除了与甲基对硫磷具有极其微弱的交叉反应以外,与甲胺磷和毒死蜱交叉反应低于0.1%,最低检测限2.0ngg,回收率为93.3%。该方法简便、快速、灵敏,适用性强。     

Determination of folic acid in milk, milk powder and energy drink by an indirect immunoassay

BACKGROUND: Folic acid (FA) is essential for healthy people (reference daily intake 400 µg day^?1) and pregnant women (600 µg day^?1). Insufficient intake of FA will increase the risk of neural tube defects in newborns. In this study an indirect enzyme‐linked immunosorbent assay was developed for rapid and convenient detection of FA in vitamin‐fortified foods. RESULTS: A carbodiimide‐modified active ester method was used to synthesise the immunogen (FA–bovine serum albumin (BSA) conjugate) to raise polyclonal antibodies for FA. The coupling ratio of FA with BSA was determined to be 14:1 (molar ratio). The detection limit of the immunoassay was 3.0 ng mL^?1 in buffer, 3.52 ng mL^?1 in energy drink, 11.91 ng mL^?1 in milk and 16.50 ng mL^?1 in milk powder. Intra‐ and inter‐assay variability ranged from 6.6 to 15.1%. Analytical recoveries of FA‐spiked samples were 88.3–108.9%. CONCLUSION: The immunoassay developed in this study can be used as a simple, rapid and accurate method for fast semi‐quantitative and quantitative on‐site analysis of FA in food products. Copyright © 2012 Society of Chemical Industry     

小分子物质酶联免疫分析方法的研究进展

自免疫分析技术问世以来,出现了多种快速检测产品如ELISA试剂盒、免疫层析试纸条,但是目前的快速检测试剂盒普遍存在检测灵敏度低、线性范围窄、易出现假阳性等问题。为解决这些问题,科研工作者发现小分子物质在检测中显示出许多特点,而这些特点正是研究开发酶联免疫检测技术的关键,小分子物质的快速检测已占据越来越重要地位。本文归纳国内外小分子物质酶联免疫分析技术研究成果,总结小分子物质酶联免疫分析方法的内容及特点,介绍小分子物质酶联免疫分析方法中抗原制备特点,分析小分子物质检测中抗体种类及特性,重点比较酶联免疫分析方法中直接和间接竞争法,并分析非竞争法在小分子物质检测中优势,旨在为今后免疫学快速检测技术的研究与开发提供帮助。     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for monitoring quinoxaline-2-carboxylic acid in the edible tissues of animals

The feed drug additive carbadox is a suspected carcinogen and mutagen. To monitor effectively residues of carbadox in the edible tissues of food-producing animals, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect quinoxaline-2-carboxylic acid, the marker residue of carbadox, was developed. Several haptens were synthesised and conjugated to the carrier protein. Nine female New Zealand white rabbits were immunised with the immunising conjugates to produce polyclonal antibodies according to the designed schemes of immunisation. The highly specific antibody that was very sensitive to N-butylquinoxaline-2-carboxamide with an IC₅₀ value of 7.75?µg?l^?1 was selected for the development of an ic-ELISA. The standard curves based on the N-butylquinoxaline-2-carboxamide matrix calibration ranged from 0.2 to 51.2?µg?l^?1. The decision limit and detection capability of the ic-ELISA were 0.60 and 0.83?µg?kg^?1 for liver and 0.68 and 0.79?µg?kg^?1 for muscle of swine, respectively. The recoveries were 57–108% with coefficients of variation of less than 20% when the quinoxaline-2-carboxylic acid was spiked into liver and muscle with the concentrations of 1.0–20.0?µg?kg^?1. Excellent correlations between the results of the ic-ELISA and an HPLC method (r?=?0.9956???0.9969) were observed for incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for screening residues of carbadox in the edible tissues of food-producing animals.     

乳制品中牛IgG含量间接竞争性ELISA检测方法的研究

建立了牛初乳制品和添加了牛初乳成分的乳制品中牛IgG含量间接竞争性酶联免疫吸附测定方法,主要研究步骤包括:制备牛IgG保守区(Fc段),以Fc段为免疫原免疫Balb/C小鼠,取其脾细胞进行细胞融合,制备特异性单克隆抗体及合成适合实际测定用途的ELISA试剂盒。应用本研究建立的方法对标准品和实际样品进行牛IgG含量检测的结果表明,该方法回收率在78.9%～117.5%,批内变异系数小于10%,批间变异系数小于15%,检测结果稳定可靠,可满足目前国内牛初乳制品和添加了牛初乳成分的乳制品品质监控的需要。     

新型高灵敏赭曲霉毒素A间接竞争化学发光免疫分析法

建立了一种测定赭曲霉毒素A的新型高灵敏化学发光间接竞争酶联免疫分析方法。以酶标赭曲霉毒素A二抗上的辣根过氧化物酶催化过氧化脲氧化3-(4-羟苯基)丙酸,生成具有荧光的3-(4-羟苯基)丙酸二聚体。并利用乙腈介质中双[2,4,6-三氯苯基]草酸酯和过氧化脲在增强剂咪唑的作用下反应产生强化学发光,以发光强度确定待检物中赭曲霉毒素A含量。结果表明,在最佳条件下IC50为0. 55 ng/m L,在0. 05～6. 08 ng/m L范围内有良好的线性关系,最低检出限为0. 01 ng/m L。样品加标回收实验显示葡萄干和葡萄汁样品的平均回收率分别为84. 55%～91. 36%和73. 32%～87. 64%,批内与批间变异系数均小于10%,精密度良好。该新型化学发光方法检测赭曲霉毒素A时发光强度更大、发光时间更长,可用于食品中赭曲霉毒素A的高灵敏度痕量检测。     

Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology

We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.