發(fā)展的一個特定職業(yè)的免疫反應.doc

開發(fā)類型1擬除蟲菊酯殺蟲劑的特效的免疫檢測Development of a class-specic immunoassay for the type I pyrethroid insecticides一般的酶聯(lián)免疫吸附測定法建立了型擬除蟲菊酯殺蟲劑,如氯菊酯,phenothrin,resmethrin和bioresmethrin。 通過immunizing多克隆抗體生成與氯菊酯衍生物,二、2-dimethyl-3 -(5-carboxy-pent-1-en-yl)cyclopropanecarboxylic酸-(3-phenoxybenzyl)酯與thyroglobu共軛-林、牛serumalbumin或以便。 對8個不同的涂層Antiserawere篩選抗原。 antibody-antigen的結合最低的背景、氯菊酯的敏感度最高為進一步優(yōu)化和測試,為的溶劑及清潔劑寬容。這I50優(yōu)化免疫的30克/升為氯菊酯和20克/升為phenothrin,分別。測量不cross-reactivities對II型擬除蟲菊酯,例如esfenvalerate、氟氯氰菊酯、氯氰菊酯 酯和fluvalinate氰戊菊酯。 這篇文章可用于監(jiān)測研究區(qū)分類型I和II欄目中擬除蟲菊酯。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 擬除蟲菊酯的合成,如氯菊酯、苯丙氨酸-nothrin,bioresmethrin、氰戊菊酯和溴氰菊酯不同的毒性、光化學穩(wěn)定性和化學物質結構1。他們已經(jīng)被廣泛地應用于控制害蟲在農業(yè)生產(chǎn)上。在型擬除蟲菊酯,氯菊酯廣泛用于控制的疾病和昆蟲向量防蛀的代理通過紡織行業(yè)(2、3)因為它是毒性小的魚比II型擬除蟲菊酯。利用擬除蟲菊酯類最近又增加了化合物由于低哺乳動物的毒性。因此,污染生態(tài)系統(tǒng)元件和食品來自農業(yè)也可能發(fā)生。因此,一個敏感的,選擇性監(jiān)測和快速的方法擬除蟲菊酯殘留物是必需的。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.有許多方法擬除蟲菊酯的檢測,如高效液相chromatography(HPLC)、氣相色譜法,以電子捕獲檢測器(GC-ECD)和質譜(GC-MS)。雖然能很好的感知的過程,在這樣的樣品制備方法是非常復雜的,技藝精湛的操作是必要的。因此,我們需要一個有效、快速的方法,在分析多種農藥殘留水平。報道說,免疫軟床與姆馬可用于農藥殘留分析和篩選。由于低成本,高吞吐量達到樣品相比,儀器分析、ELISA法,可以替代或有可能利用作為一個開始了第一個銀屏減少的數(shù)量需要后續(xù)儀器分析樣本。很容易量化農藥殘留在復雜的矩陣,如沉積物和土壤或動物或植物的組織,可減少有害的有機溶劑用于提取。在這一時代背景下,生產(chǎn)等問題Stanker報道單克隆抗體的攻擊immunogen含有phenoxybenzyl半族和環(huán)丙烷戒指,并以此來檢測肉類香精。氯菊酯Skerritt等問題的描述格式使用相同的抗體ELISA法檢測糧食和面粉氯菊酯提取物。擬除蟲菊酯的命運,研究水棲動物生態(tài)系統(tǒng),通過對大量樣品分析是必要的。此外,在形勢分析雨水徑流的能力,來確定哪些擬除蟲菊酯是目前是很重要的。因此,一系列的檢測具有寬廣的開始,然后將縮小性質有用的工具。因此,一個職業(yè)專用免疫法能分辨類型I及II型擬除蟲菊酯是可取的。這個目標必須予以hapten設計。因此,我們制作了多克隆抗體對氯菊酯與間隔墊片的模擬三碳作為一個immunogen,并給出了h2.實驗2.1試劑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儀器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)酶聯(lián)免疫吸附試驗對96 -哦microplates(Nunc,Roskilde、丹麥)和ab -2正念到sorbances酶與空間的讀者(分子器件、摩洛公園,CA)在雙-波長模式(450 - 650海里)。這characteriza -概括了蛋白質hapten共軛做是有一個島津萬能試驗機UV - 2101 - PC紫外可見光譜掃描規(guī)格-trophotometer(島津萬能試驗機、京都、日本)酶聯(lián)免疫吸附試驗分別在96孔微孔板（情報，羅斯基勒，丹麥）和吸光度的讀取速度酶標儀（分子器件，門羅公園，加利福尼亞州）在雙波長模式（450650納米）。表征抗原蛋白共軛是島津uv-2101pc紫外可見掃描分光光度計（島津，京都，日本）Homogeneous enzyme均相酶免疫法A simple glutathione transferase-based colorimetric endpoint assay for insecticide detection一個簡單的谷胱甘肽轉移酶-based分光光度測定殺蟲劑端點檢測摘要：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自然能力的排毒陽離子酶谷胱甘肽轉移酶（酶）的互動與xenobi病病人可用于生產(chǎn)的比色法。檢測通常是基于抑制該gst-catalysed反應，經(jīng)檢測達到分光或電化學。在這里我們采用顯色（視覺）活動法篩選酶與烷基活動為iodoalkene基板檢測殺蟲劑。我們篩選了一些酶從殺蟲劑抗蚊種的能力，催化碘代烷烴轉化反應。aagste2被發(fā)現(xiàn)tometaboliseiodoethanewith更替率高，導致在一個深藍色的顏色在酶自動反應。下列檢測optimisationwe利用高識別自動對焦仁慈的aggste2為殺蟲劑開發(fā)一種新的比色檢測法有機和擬除蟲菊酯類定量鈣問題。校準曲線獲得了permethirn，溴氰菊酯，-氯氟氰菊酯、滴滴涕，有用的濃度范圍為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），分別。該assaywasvalidatedwith提取物frominsecticide噴上臉和發(fā)現(xiàn)是重復性和可靠的含量比較。該法適合監(jiān)測農藥殘留在經(jīng)殺蟲劑處理的材料，因而具有潛在的昆蟲矢量控制操作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.谷胱甘肽轉移酶（酶，歐共體2.5.1.18）是一個大家庭的酶催化親核加成的巰基的還原性谷胱甘肽（谷胱甘肽）對各種分子。這共軛反應的一個關鍵步驟是在細胞排毒陽離子，和細胞內的酶是一個大游泳池的蛋白質具有很好的結合自動對焦，為各種不同的內源性和外源性化合物。廣泛的底物特異市加上一般的穩(wěn)定性和易生產(chǎn)的重組酶提示使用這些酶檢測外源性化學物質。值得注意的是，酶從不同的昆蟲物種的農業(yè)、醫(yī)療重要性高自動對焦可能殺蟲劑已用于檢測殺蟲劑。這些系統(tǒng)，隨著免疫學技術，具有潛在的優(yōu)勢，生物測定和室內機的分析方法（高效液相色譜，氣相色譜）在較低的成本和技術復雜性，加上特殊的城市合理的敏感性的某些應用，如殺蟲劑殘留量的測定處理材料。鑒于目前的擴張滴滴涕和擬除蟲菊酯殘留噴灑瘧疾控制，這ismostuseful作為aprocurement和質量控制工具，矢量控制措施在全世界的發(fā)展中國家。谷胱甘肽的transferases(表、歐共體2.5.1.18)是一個大的家族,親核酶的催化作用的加入巰基共同還原型谷胱甘肽(GSH)的一個廣泛的分子。這個接合反應是細胞解毒中一個重要的步驟,表代表一個大水坑里細胞的蛋白質具有良好的親和力為多種內源性和外源性化合物。廣闊的底物特異性加上總體穩(wěn)定,減少生產(chǎn)重組表促使這些酶的使用xenobiotics檢測。值得注意的是,昆蟲種類表斷然農業(yè)andmedical高親和力的重要性對殺蟲劑一直受雇于檢測殺蟲劑。這些系統(tǒng),隨著免疫學技術,具有潛在的優(yōu)勢和實驗室machine-based生物實驗分析方法(高效液相色譜、氣相色譜的角度和技術復雜性低成本,加上合理的靈敏度較高的特異性和一定的應用,如殺蟲劑殘留量的測定在處理材料。鑒于當前的擴張formalaria擬除蟲菊酯類殘留噴灑DDT和控制,這ismostuseful作為aprocurement和質量控制工具,在發(fā)展中國家的矢量控制干預世界各地。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檢測7的基礎上,to-date GSTmethodologies描述是基于inhi -bition GST活動的殺蟲劑在目前的反應8.混合物xenobiotics檢測和量化通常取得了spectrophotometrically5,或者用電(例如,pH值-或離子選擇性電極)8。Enayati發(fā)展等問題一個分光光度分析測定菊酯類的數(shù)量在目前的reactionmixture殺蟲劑的frominhibition GST -催化1-chloro-2,4-dinitrobenzene(CDNB)/谷胱甘肽(GSH)接合5。有強烈的有機磷用黃色GST加上噴霧的抑制作用質子釋放CDNB /谷胱甘肽在接合反應,使用酸堿度的electrode-based生產(chǎn)檢測分析方法雖然pH-change出現(xiàn),一種相對簡單的檢測計劃改變以較低的緩沖性能的影響介質。因此,申請以天然薩姆-測量ples可能造成麻煩。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另一個色度檢測化驗先前的定量分析了擬除蟲菊酯殺蟲劑,在檢測的CDNB /谷胱甘肽GST-catalysed接合反應它由擬除蟲菊酯抑制率和確定了iodometric滴定的谷胱甘肽non-conjugated基質5條,第13條。雖然該系統(tǒng)檢測視覺、檢測精度提供了適度,因為它依賴于谷胱甘肽的耗竭測量在基質只是一小部分的基質在實際上是使用酶促反應。因此,一個更直接的方法檢測/抑制酶活性的監(jiān)測形成顏色反應產(chǎn)品,特別是催化表與高親和力的殺蟲劑,會特別感興趣的發(fā)展的量化分析更實際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