发展的一个特定职业的免疫反应.doc

开发类型1拟除虫菊酯杀虫剂的特效的免疫检测Development of a class-specic immunoassay for the type I pyrethroid insecticides一般的酶联免疫吸附测定法建立了型拟除虫菊酯杀虫剂,如氯菊酯,phenothrin,resmethrin和bioresmethrin。 通过immunizing多克隆抗体生成与氯菊酯衍生物,二、2-dimethyl-3 -(5-carboxy-pent-1-en-yl)cyclopropanecarboxylic酸-(3-phenoxybenzyl)酯与thyroglobu共轭-林、牛serumalbumin或以便。 对8个不同的涂层Antiserawere筛选抗原。 antibody-antigen的结合最低的背景、氯菊酯的敏感度最高为进一步优化和测试,为的溶剂及清洁剂宽容。这I50优化免疫的30克/升为氯菊酯和20克/升为phenothrin,分别。测量不cross-reactivities对II型拟除虫菊酯,例如esfenvalerate、氟氯氰菊酯、氯氰菊酯 酯和fluvalinate氰戊菊酯。 这篇文章可用于监测研究区分类型I和II栏目中拟除虫菊酯。引言The synthetic pyrethroids, such as permethrin, phe-nothrin, bioresmethrin, fenvalerate and deltamethrin differ in their toxicity, photostability and chemical structure 1. They have been widely used to control insect pests in agriculture. Among type I pyrethroids,permethrin has been widely used for the control of insect disease vectors and as a moth-proong agent by the textile industry 2,3 because it is less toxic to sh than the type II pyrethroids. The use of pyrethroid compounds has recently increased due to the low 拟除虫菊酯的合成,如氯菊酯、苯丙氨酸-nothrin,bioresmethrin、氰戊菊酯和溴氰菊酯不同的毒性、光化学稳定性和化学物质结构1。他们已经被广泛地应用于控制害虫在农业生产上。在型拟除虫菊酯,氯菊酯广泛用于控制的疾病和昆虫向量防蛀的代理通过纺织行业(2、3)因为它是毒性小的鱼比II型拟除虫菊酯。利用拟除虫菊酯类最近又增加了化合物由于低哺乳动物的毒性。因此,污染生态系统元件和食品来自农业也可能发生。因此,一个敏感的,选择性监测和快速的方法拟除虫菊酯残留物是必需的。There are many methods for the detection of pyrethroids, such as high-performance liquid chro-matography (HPLC), gas chromatography with electron capture detector (GCECD) and with mass spectrometry (GCMS). Though capable of good sensitivity, the procedure for sample preparation in such methods is very complicated, and highly skilled operations are required. Therefore, we need another effective and rapid method for analysis of many pesticides at residue levels. Hammock and Mumma reported that immunoassay could be used for pesticide residue analysis and screening. Due to the lower cost and higher sample throughput achievable compared to instrumental analyses, ELISA can be an alternative or has the potential for use as a rst screen to reduce the numbers of samples requiring subsequent instrumental analyses. It is easy to quantify pesticide residues in complex matrices, such as sediments and soils or animal or plant tissues, and it may be possible to reduce the harmful organic solvents used for extraction. With this background, Stanker et al. reported the production of monoclonal antibodies against an immunogen containing the phenoxybenzyl moiety and a cyclopropane ring, and applied this to detect permethrin in meat extracts. Skerritt et al. described the ELISA format using the same antibodies to detect permethrin in grain and our extracts. To study pyrethroid fate in the aquaticecosystem, methods for analysis of large numbers of samples is necessary. In addition, in situation such as analysis of stormwater runoff, the ability to determine which pyrethroid is present is important. Thus,a series of assays beginning with broad, then narrowing specicities would be useful tools. Consequently,a class-specic immunoassay that can distinguish between types I and II pyrethroid is desirable. This goal necessitates careful hapten design. Therefore, we produced a polyclonal antibody against a permethrin analogue with a spacer of three carbons as an immunogen, and describe here the development of a class-specic immunoassay for the type I pyrethroids.有许多方法拟除虫菊酯的检测,如高效液相chromatography(HPLC)、气相色谱法,以电子捕获检测器(GC-ECD)和质谱(GC-MS)。虽然能很好的感知的过程,在这样的样品制备方法是非常复杂的,技艺精湛的操作是必要的。因此,我们需要一个有效、快速的方法,在分析多种农药残留水平。报道说,免疫软床与姆马可用于农药残留分析和筛选。由于低成本,高吞吐量达到样品相比,仪器分析、ELISA法,可以替代或有可能利用作为一个开始了第一个银屏减少的数量需要后续仪器分析样本。很容易量化农药残留在复杂的矩阵,如沉积物和土壤或动物或植物的组织,可减少有害的有机溶剂用于提取。在这一时代背景下,生产等问题Stanker报道单克隆抗体的攻击immunogen含有phenoxybenzyl半族和环丙烷戒指,并以此来检测肉类香精。氯菊酯Skerritt等问题的描述格式使用相同的抗体ELISA法检测粮食和面粉氯菊酯提取物。拟除虫菊酯的命运,研究水栖动物生态系统,通过对大量样品分析是必要的。此外,在形势分析雨水径流的能力,来确定哪些拟除虫菊酯是目前是很重要的。因此,一系列的检测具有宽广的开始,然后将缩小性质有用的工具。因此,一个职业专用免疫法能分辨类型I及II型拟除虫菊酯是可取的。这个目标必须予以hapten设计。因此,我们制作了多克隆抗体对氯菊酯与间隔垫片的模拟三碳作为一个immunogen,并给出了h2.实验2.1试剂Permethrin (3-phenoxybenzyl-(RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxyl-ate) (cis:trans = 25:75), cis- and trans-permethrin were obtained from Reidel de Haen (Seelze, Ger-many). The immunogen hapten I (2,2-dimethyl-3-(5-carboxy-pent-1-en-yl)cyclopropanecarboxylic acid-(3-phenoxybenzyl)ester) (Fig. 1) was provided by Bayer AG (Leverkusen, Germany). Esfenvalerate (S)-cyano-3-phenoxybenzyl-(S)-2-(4-chlorophenyl)-3-methylbutyrate), fenvalerate (RS)-cyano-3-pheno-xybenzyl-(RS)-2-(4-chlorophenyl)-3-methylbutyrate),cypermethrin (RS)-cyano-3-phenoxybenzyl-(RS)-Fig. 1. Structure of permethrin and permethrin hapten.cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopro-panecarboxylate), cyuthrin (RS)-cyano-4-uoro-3-phenoxybenzyl-(RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate), deltamethrin (S)-cyano-3-phenoxybenzyl-(R)-cis-3-(2,2-dibrom ovinyl)-2,2-dimethylcyclopropanecarboxylate), uva-linate (RS)-cyano-3-phenoxybenzyl-N-(2-chloro-,-triuoro-p-tolyl)-d-valinate), phenothrin (3-ph-enoxybenzyl-(RS)-cis,trans-2,2-dimethyl-3-(2-methy-lprop-1-enyl)cyclopropanecarboxylate), resmethrin (5-benzyl-3-furylmethyl-(RS)-cis,trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate) and bioresmethrin (5-benzyl-3-furylmethyl-(R)-trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopro-panecarboxylate) were purchased fromReidel de Haen (Seelze, Germany) (Fig. 2). The eight coating antigen haptens used are shown in Table 1. Methanol was of GC Resolve grade and obtained from Fisher Scien-tic (Pittsburgh, PA), and dimethylsulfoxide (99.8%)was from Aldrich (St. Louis, MO). 1-3-(Dimethyl-amino)propyl-3-ethylcarbodiimide hydrochloride(EDC) (Aldrich), N,N-dimethylformamide (Aldrich) and N-hydroxysulfosuccinimide sodium salt (Fluka,Buchs, Switzerland) were used for the production of conjugate to protein. Thyroglobulin, bovine serum al-bumin and ovalbumin were obtained from Sigma (St.Louis,MO) as carrier proteins, and goat anti-rabbit immunoglobulin conjugated to horseradish-peroxidase(HRP) and 3,3,5,5-tetramethylbenzidine (TMB)were also purchased from Sigma. Water used was puried by a NANOpure II system (Barnstead, Newton,MA).2.2仪器ELISA experiments were performed in 96-well microplates (Nunc, Roskilde, Denmark) and the absorbances were read with a Vmax microplate reader(Molecular Devices, Menlo Park, CA) in dual-wavelength mode (450650 nm). The characterization of hapten to protein conjugate was done with a Shimadzu UV-2101PC UVVIS scanning spectrophotometer (Shimadzu, Kyoto, Japan)酶联免疫吸附试验对96 -哦microplates(Nunc,Roskilde、丹麦)和ab -2正念到sorbances酶与空间的读者(分子器件、摩洛公园,CA)在双-波长模式(450 - 650海里)。这characteriza -概括了蛋白质hapten共轭做是有一个岛津万能试验机UV - 2101 - PC紫外可见光谱扫描规格-trophotometer(岛津万能试验机、京都、日本)酶联免疫吸附试验分别在96孔微孔板（情报，罗斯基勒，丹麦）和吸光度的读取速度酶标仪（分子器件，门罗公园，加利福尼亚州）在双波长模式（450650纳米）。表征抗原蛋白共轭是岛津uv-2101pc紫外可见扫描分光光度计（岛津，京都，日本）Homogeneous enzyme均相酶免疫法A simple glutathione transferase-based colorimetric endpoint assay for insecticide detection一个简单的谷胱甘肽转移酶-based分光光度测定杀虫剂端点检测摘要：The natural ability of the detoxication enzymes glutathione transferases (GSTs) to interact with xenobi-otics can be used for the production of colorimetric assays. Detection is usually based on the inhibition ofthe GST-catalysed reaction, with detection achieved spectrophotometrically or electrochemically. Herewe have adopted a chromogenic (visual) activity assay for screening GSTs with alkyltransferase activityfor iodoalkene substrates for detection of insecticides. We screened a number of GSTs from insecticideresistant mosquito species for their ability to catalyse iodoalkane biotransformation reactions. AaGSTE2was found tometabolise iodoethanewith high turnover,which resulted in a dark blue colour in the enzy-matic reaction. Following assay optimisationwe exploited the high recognition afnity of the AgGSTE2 forinsecticides to develop a novel colorimetric detection assay for organochlorine and pyrethroid quantica-tion. Calibration curves were obtained for permethirn, deltamethrin, -cyhalothrin and DDT, with usefulconcentration ranges of 040g/ml (0100M), 050g/ml (0100M), 0100g/ml (0220M), and050g/ml (0140M), respectively. The assaywas validatedwith extracts frominsecticide sprayed sur-faces and found to be reproducible and reliable compared with HPLC. The assay is therefore suitable formonitoring insecticide residues in insecticide treated materials, and therefore has potential for insectvector control operations自然能力的排毒阳离子酶谷胱甘肽转移酶（酶）的互动与xenobi病病人可用于生产的比色法。检测通常是基于抑制该gst-catalysed反应，经检测达到分光或电化学。在这里我们采用显色（视觉）活动法筛选酶与烷基活动为iodoalkene基板检测杀虫剂。我们筛选了一些酶从杀虫剂抗蚊种的能力，催化碘代烷烃转化反应。aagste2被发现tometaboliseiodoethanewith更替率高，导致在一个深蓝色的颜色在酶自动反应。下列检测optimisationwe利用高识别自动对焦仁慈的aggste2为杀虫剂开发一种新的比色检测法有机和拟除虫菊酯类定量钙问题。校准曲线获得了permethirn，溴氰菊酯，-氯氟氰菊酯、滴滴涕，有用的浓度范围为040克/毫升（0100米），050克/毫升（0100米），0100克/毫升（0220米），和050克/毫升（0140米），分别。该assaywasvalidatedwith提取物frominsecticide喷上脸和发现是重复性和可靠的含量比较。该法适合监测农药残留在经杀虫剂处理的材料，因而具有潜在的昆虫矢量控制操作The glutathione transferases (GSTs, EC 2.5.1.18) are a large family of enzymes that catalyse the nucleophilic addition of the thiol of reduced glutathione (GSH) to a wide range of molecules.This conjugation reaction is a critical step in cellular detoxication, and cytosolic GSTs represent a large pool of proteins with good binding afnity for a variety of diverse endogenous and exogenous compounds. The broad substrate specicity coupled with the general stability and ease of production of recombinant GSTs have prompted the use of these enzymes for the detection of xenobiotics. Notably, GSTs fromdifferent insect species of agricultural andmedical importance with high afnity for insecticides have been employed for detecting insecticides. These systems, along with immunological techniques, have potential advantages over bioassays and laboratory machine-based analytical methods (HPLC, GC) in terms of lower cost and technical complexity, coupled with high specicity and reasonable sensitivity for certain applications, such as the determination of insecticide residues on treated material. Given the current expansion of DDT and pyrethroid residual spraying formalaria control, this ismostuseful as aprocurement and quality control tool for vector control interventions in developing countries across the world.谷胱甘肽转移酶（酶，欧共体2.5.1.18）是一个大家庭的酶催化亲核加成的巯基的还原性谷胱甘肽（谷胱甘肽）对各种分子。这共轭反应的一个关键步骤是在细胞排毒阳离子，和细胞内的酶是一个大游泳池的蛋白质具有很好的结合自动对焦，为各种不同的内源性和外源性化合物。广泛的底物特异市加上一般的稳定性和易生产的重组酶提示使用这些酶检测外源性化学物质。值得注意的是，酶从不同的昆虫物种的农业、医疗重要性高自动对焦可能杀虫剂已用于检测杀虫剂。这些系统，随着免疫学技术，具有潜在的优势，生物测定和室内机的分析方法（高效液相色谱，气相色谱）在较低的成本和技术复杂性，加上特殊的城市合理的敏感性的某些应用，如杀虫剂残留量的测定处理材料。鉴于目前的扩张滴滴涕和拟除虫菊酯残留喷洒疟疾控制，这ismostuseful作为aprocurement和质量控制工具，矢量控制措施在全世界的发展中国家。谷胱甘肽的transferases(表、欧共体2.5.1.18)是一个大的家族,亲核酶的催化作用的加入巯基共同还原型谷胱甘肽(GSH)的一个广泛的分子。这个接合反应是细胞解毒中一个重要的步骤,表代表一个大水坑里细胞的蛋白质具有良好的亲和力为多种内源性和外源性化合物。广阔的底物特异性加上总体稳定,减少生产重组表促使这些酶的使用xenobiotics检测。值得注意的是,昆虫种类表断然农业andmedical高亲和力的重要性对杀虫剂一直受雇于检测杀虫剂。这些系统,随着免疫学技术,具有潜在的优势和实验室machine-based生物实验分析方法(高效液相色谱、气相色谱的角度和技术复杂性低成本,加上合理的灵敏度较高的特异性和一定的应用,如杀虫剂残留量的测定在处理材料。鉴于当前的扩张formalaria拟除虫菊酯类残留喷洒DDT和控制,这ismostuseful作为aprocurement和质量控制工具,在发展中国家的矢量控制干预世界各地。With the exception of the GSTE2 DDT dehydrochorinase assay7, the GSTmethodologies described to-date are based on the inhi-bition of GST activity by the insecticides present in the reactionmixture 58. The detection and quantication of xenobiotics aretypically achieved spectrophotometrically 5, or electrochemically(e.g., pH- or ion-selective electrodes) 8. Enayati et al. developeda spectrophotometric assay to measure the amount of pyrethroidinsecticides present in the reactionmixture frominhibition of GST-catalysed 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH)conjugation 5. The strong binding of the organophosphatemalathion with maize GST coupled with its inhibitory effect onproton release during the CDNB/GSH conjugation reaction, wasutilised to produce a pH electrode-based detection assay除了GSTE2 DDT dehydrochorinase检测7的基础上,to-date GSTmethodologies描述是基于inhi -bition GST活动的杀虫剂在目前的反应8.混合物xenobiotics检测和量化通常取得了spectrophotometrically5,或者用电(例如,pH值-或离子选择性电极)8。Enayati发展等问题一个分光光度分析测定菊酯类的数量在目前的reactionmixture杀虫剂的frominhibition GST -催化1-chloro-2,4-dinitrobenzene(CDNB)/谷胱甘肽(GSH)接合5。有强烈的有机磷用黄色GST加上喷雾的抑制作用质子释放CDNB /谷胱甘肽在接合反应,使用酸碱度的electrode-based生产检测分析方法虽然pH-change出现,一种相对简单的检测计划改变以较低的缓冲性能的影响介质。因此,申请以天然萨姆-测量ples可能造成麻烦。An alternative colorimetric detection assay was previously described for the quantication of pyrethroid insecticides, where detection of the GST-catalysed CDNB/GSH conjugation reactionrate and its inhibition by pyrethroids was determined by iodometric titration of the non-conjugated substrate GSH 5,13. Although detection in that system is visual, the assay provides moderate accuracy, as it relies on measurement of GSH substrate depletion where only a small fraction of the substrate is actually utilised in the enzymatic reaction. Thus, a method for more direct detection of enzymatic activity/inhibition such as monitoring the formation of a colour reaction product, particularly if catalysed by GSTs with high afnity for insecticides, would be of particular interest for the development of more practical quantication assays另一个色度检测化验先前的定量分析了拟除虫菊酯杀虫剂,在检测的CDNB /谷胱甘肽GST-catalysed接合反应它由拟除虫菊酯抑制率和确定了iodometric滴定的谷胱甘肽non-conjugated基质5条,第13条。虽然该系统检测视觉、检测精度提供了适度,因为它依赖于谷胱甘肽的耗竭测量在基质只是一小部分的基质在实际上是使用酶促反应。因此,一个更直接的方法检测/抑制酶活性的监测形成颜色反应产品,特别是催化表与高亲和力的杀虫剂,会特别感兴趣的发展的量化分析更实际A robust colorimetric endpoint assay for GSTs with high alkyl transferase activity capable of catalysing the release of iodine from haloalkene substrates has recently been described.The detection is based on the classical reaction of iodine with starch amylose producing a blue colour, which can be measured spectrophotometrically at 610 nm or visually. The reactiondepends on the release of iodide from the substrate as a consequence of its conjugation with glutathione catalysed by GST, which is subsequently oxidized to iodine by the addition of acidied hydrogen peroxide. Th