拟除虫菊酯类农药人工抗原的合成与鉴定

目的 合成拟除虫菊酯类农药人工抗原并对其进行鉴定.方法 采用功夫菊酸和3-间苯氧甲酸2种拟除虫菊酯类农药中间体,与四碳"间隔臂"--γ-氨基丁酸反应合成2种半抗原,经质谱鉴定合成成功.以牛血清蛋白为载体,采用活化酯法制备拟除虫菊酯的2种免疫原;以卵清蛋白为载体,采用混合酸酐法制备了3种包被原;经紫外光谱鉴定,粗略计算了...     

盐酸西布曲明人工抗原的合成与鉴定

目的：建立盐酸西布曲明的免疫分析方法。方法：4-氯苯乙腈和1, 3-二溴丙烷为原料合成与盐酸西布曲明具有相同母核结构的小分子双去甲基西布曲明(M2),以双去甲基西布曲明为半抗原,并分别通过活泼酯法、戊二醛法和混合酸酐法将半抗原与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备免疫原(M2-BSA)和包被抗原(M2-OVA)。结果：紫外光谱扫描证明半抗原M2与载体蛋白偶联比为24.6:1(M2-BSA)和16.2:1(M2-OVA),抗血清ELISA效价均达到1:8000以上,IC50=0.42μg/mL。结论：半抗原M2与载体蛋白均已成功偶联,其中活泼酯法对半抗原活性基团的影响最小,合成人工抗原的特异性最强。     

呋喃唑酮代谢物人工抗原的制备及鉴定

在催化剂条件下,以碳酸二甲酯和乙醇肼为原料,四氢呋喃为溶剂,合成了高收率的呋喃唑酮代谢物3-氨基-2-恶唑烷酮(AOZ)。实验结果表明,得到的AOZ收率可达到82.7%,此时的最佳反应条件如下:催化剂用量12%,反应温度75℃,反应时间3h。随后对AOZ进行结构改造,得到AOZ半抗原衍生物,同时采用戊二醛法将AOZ及其半抗原衍生物与活化的卵血清蛋白(cOVA)和牛血清白蛋白(cBSA)交联,成功制备了免疫原和包被原;通过气相质谱联用仪(GC-MS)、元素分析、紫外分光光度计等手段对半抗原和完全抗原进行鉴定。GC-MS结果证明了AOZ的成功合成;元素分析数据表明,半抗原顺利合成;紫外扫描数据显示,半抗原与cOVA、cBSA偶联成功,免疫原I、免疫原Ⅱ的偶联比分别为16.74和24.05。     

多效唑人工抗原的合成与鉴定

为合成新的、有效的多效唑人工抗原,采用琥珀酸酐法对多效唑小分子进行结构修饰,以获得含羧基的多效唑半抗原。将纯化后的半抗原分别与牛血清白蛋白和卵清蛋白经N-羟基琥珀酰亚胺活泼酯法偶联制备免疫原和包被原。采用质谱、红外光谱、核磁共振氢谱对多效唑半抗原的结构进行鉴定;采用紫外光谱及高性能基质辅助激光解吸电离-飞行时间质谱对偶联物的结构进行鉴定。结果显示成功合成出多效唑人工抗原,为其抗体的制备和免疫学方法的构建奠定了前期研究基础。     

泰拉霉素完全抗原的合成和鉴定

利用丁二酸酐法对泰拉霉素的羟基衍生化,引入游离羧基,合成泰拉霉素半抗原。采用活泼酯法将泰拉霉素半抗原分别与牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)偶联,合成人工完全抗原泰拉霉素-BSA和泰拉霉素-OVA。利用LC-MS鉴定衍生化后半抗原的合成,利用荧光光谱、考马斯亮法鉴定完全抗原的合成。用泰拉霉素-BSA免疫小鼠,采用间接ELISA法测定小鼠抗血清效价。泰拉霉素半抗原和BSA的偶联比为17∶1。三免后小鼠抗血清效价可达1∶8 000。     

玉米赤霉烯酮多克隆抗体的制备及特性分析

合成玉米赤霉烯酮半抗原,应用液相色谱-质谱联用法进行鉴定,并采用活泼酯法将半抗原与载体蛋白OVA或BSA偶联,分别作为免疫原或包被原,并通过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和紫外扫描法鉴定偶联效果,免疫3只BALB/c小鼠,制备玉米赤霉烯酮多克隆抗体。结果表明:抗血清效价最高达1∶32 000,以此多抗建立的玉米赤霉烯酮间接竞争标准曲线IC50为39.8ng/mL,IC10为0.71ng/mL,多抗与玉米赤霉烯酮类似物β-zearalenol、zear-alanone、α-zearalanol、β-zearalanol交叉反应率分别为4.80%,3.07%,0.96%,0.09%。说明试验成功制备了玉米赤霉烯酮人工抗原及高特异性多克隆抗体。     

百菌清人工抗原的制备与鉴定

以杀菌剂百菌清为起始反应物,通过两步化学反应合成百菌清的衍生物(半抗原);采用活化酯法与混合酸酐法分别将半抗原与载体蛋白(牛血清蛋白、卵清蛋白)进行偶联,制备百菌清的人工抗原,同时通过免疫新西兰大白兔获得抗血清,用酶联免疫吸附法(ELISA)测定其效价。利用红外光谱(IR)、质谱(MS)、核磁共振波谱(NMR)对百菌清半抗原进行表征,并用紫外扫描的方法对人工抗原进行鉴定。结果表明,成功制备出百菌清半抗原及人工抗原,并且半抗原与载体蛋白的偶联比分别为27.86:1 和12.32:1,抗血清效价约为1:1.28 × 104。     

3-氨基-2-嗯唑烷酮半抗原、人工抗原的合成及鉴定

以2,4-二硝基氟苯为衍生剂,建立了3-氨基-2-噁唑烷酮(AOZ)的高效衍生方法,成功获得了衍生物F-AOZ,并进一步通过硝基的选择性还原合成了新的半抗原AM-AOZ,经电喷雾电离质谱(ESI-MS)和核磁共振(NMR)鉴定F-AOZ与AM-AOZ合成成功;采用重氮化法和戊二醛交联法将半抗原AM-AOZ分别与牛血清蛋白BSA和卵清白蛋白OVA进行偶联,合成了AOZ免疫原和包被原,经紫外吸收法进行成功鉴定;同时,分别采用紫外扫描法和三硝基苯磺酸法(TNBS)测定偶联比。另外,采用分子模拟手段对抗原表面半抗原决定簇的前线分子轨道分布情况进行了模拟分析,讨论了前线轨道的分布对可能获得的抗体特性的影响,为进一步制备高质量抗体提供理论支持。     

格列本脲人工抗原的合成与鉴定

目的：合成格列本脲人工抗原。方法：采用对氨基苯甲酸法制备半抗原,将半抗原分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)通过碳二亚胺法偶联制备免疫原(Gli-BSA)和包被原(Gli-OVA),利用紫外扫描进行抗原的化学鉴定,通过免疫原免疫Balb/c小鼠,间接酶联免疫吸附法测定抗血清进行生物鉴定。结果：制备了Gli-BSA、Gli-OVA的人工抗原,经紫外光谱扫描,偶联比分别为4:1和17.7:1。免疫小鼠后获得抗血清的效价均达到32000以上,半抑制质量浓度为10μg/mL。结论：成功合成了格列本脲人工抗原,并获得了格列本脲抗体,为格列本脲的免疫学检测方法进一步研究提供了实验基础。     

沙丁胺醇人工抗原合成及鉴定研究

用琥珀酸酐将沙丁胺醇活化后制备出半抗原沙丁胺醇半琥珀酸,经电喷雾电离质谱(ESI-MS)、红外光谱(IR)和核磁共振(1H-NMR)鉴定合成成功,同时证实琥珀酸酐连结在沙丁胺醇乙醇胺的羟基处,且半抗原以内胺盐形式存在。用混合酸酐、活泼酯方法将沙丁胺醇琥珀半酸分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联制备免疫抗原和包被抗原,经紫外吸收法鉴定偶联成功,偶联比分别为12.8:1、23:1。本研究为进一步制备沙丁胺醇抗体奠定了基础。     

Phage-borne peptidomimetics accelerate the development of polyclonal antibody-based heterologous immunoassays for the detection of pesticide metabolites

Competitive immunoassays for the detection of small analytes, such as pesticides and their metabolites, use haptens that compete with the target compounds for binding to the antibody. This competing hapten can be either the same as the immunizing hapten (homologous assay) or structurally modified mimics of the immunizing hapten (heterologous assay). Polyclonal antibody-based heterologous immunoassays have shown superior sensitivities to homologous ones, butthe synthesis of heterologous haptens may be time-consuming, requiring expertise in synthetic chemistry. In this work we demonstrate that phage display peptide libraries can be used as a source of phage-borne peptidomimetics to facilitate the development of sensitive heterologous assays. Different strategies for the isolation of these peptides were explored using two metabolites of pyrethroid insecticides. The sensitivities of the best competitive phage heterologous enzyme-linked immunosorbent assays were 13 fold and 100 fold better than the homologous assay, for the glycine conjugate of trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid and 3-phenoxybenzoic acid, respectively. The phage particles were highly versatile as tracer reagents, allowing the use of enzymatic, chemiluminescent, or immuno-polymerase chain reaction detection. The data presented here shows a new systematic procedure that enables the fast generation of several competing haptens for the rapid development of sensitive heterologous immunoassays.     

Determination of Pyrethroid Insecticides in Crude and Canned Vegetable Samples by Supercritical Fluid Chromatography

A supercritical fluid chromatography method for the determination of seven pyrethroid insecticides (allethrin, resmethrin, phenothrin, permethrin, tetramethrin, cypermethrin, deltamethrin) and one of their common metabolites, phenoxybenzyl alcohol, in whole and peeled potatoes and mixed vegetables was developed. The efficiencies of the two extraction techniques, supercritical fluid extraction and microwave-assisted extraction, for the extraction of pyrethroids from vegetable samples were also compared. The retention times of various pyrethroids ranged from 8.4 to 22.9 min, while all of the peaks were well-resolved and distinctly identified. The limits of detection of pyrethroid insecticides ranged between 0.31 and 0.54 ppm, whereas the limits of detection of phenoxybenzyl alcohol was 0.62 ppm. The recoveries of pyrethroid insecticides from whole potatoes, peeled potatoes, and mixed vegetables ranged as 93.83–99.8%, 92.3–105.8%, and 93.67–102.7%, respectively, with the use of supercritical fluid extraction. The corresponding recovery ranges while using microwave-assisted extraction were 94.2–102%, 96.6–101.2%, and 96–103.2%. These findings suggested that supercritical fluid chromatography was a sensitive and rapid technique for the analysis of pyrethroids in complex matrices, such as vegetables, fruits, and other agricultural products.     

基于电化学技术的Ⅱ型拟除虫菊酯分子印迹膜的研制

拟除虫菊酯类农药残留造成的安全问题已受到越来越广泛的关注,开发快速检测技术可为农副产品中拟除虫菊酯类农药残留的监控提供技术支持。本实验以邻氨基酚为功能单体,以Ⅱ型拟除虫菊酯农药结构类似物间苯氧基苯甲酸为模板分子,采用循环伏安法电聚合技术在玻碳电极表面聚合形成可检测含间苯氧基苯甲酸结构的Ⅱ型拟除虫菊酯农药的分子印迹膜,用于Ⅱ型拟除虫菊酯类农药的检测研究。5 min可完成对农药的响应,其响应电流变化值与一定浓度范围内的溴氰菊酯、氰戊菊酯、甲氰菊酯、氯氟氰菊酯呈良好的线性关系,相关系数r分别为0.991、0.992、0.992、0.990,精密度分别为2.20%、2.80%、2.50%、2.12%,检出限在0.01mg/kg~0.30 mg/kg之间。对加标红桔、贡桔和椪柑样品进行检测,准确性高。     

Occurrence and potential sources of pyrethroid insecticides in stream sediments from seven U.S. metropolitan areas

A nationally consistent approach was used to assess the occurrence and potential sources of pyrethroid insecticides in stream bed sediments from seven metropolitan areas across the United States. One or more pyrethroids were detected in almost half of the samples, with bifenthrin detected the most frequently (41%) and in each metropolitan area. Cyhalothrin, cypermethrin, permethrin, and resmethrin were detected much less frequently. Pyrethroid concentrations and Hyalella azteca mortality in 28-d tests were lower than in most urban stream studies. Log-transformed total pyrethroid toxic units (TUs) were significantly correlated with survival and bifenthrin was likely responsible for the majority of the observed toxicity. Sampling sites spanned a wide range of urbanization and log-transformed total pyrethroid concentrations were significantly correlated with urban land use. Dallas/Fort Worth had the highest pyrethroid detection frequency (89%), the greatest number of pyrethroids (4), and some of the highest concentrations. Salt Lake City had a similar percentage of detections but only bifenthrin was detected and at lower concentrations. The variation in pyrethroid concentrations among metropolitan areas suggests regional differences in pyrethroid use and transport processes. This study shows that pyrethroids commonly occur in urban stream sediments and may be contributing to sediment toxicity across the country.     

Residue analysis of pyrethroid insecticides in cereal grains, milled fractions and bread

A procedure is described for the residual analysis of eight pyrethroid insecticides at levels of 2-5 micrograms/kg. Residues are extracted from cereal grains, flour or bread with a mixture of acetonitrile/water (2 + 1) and are partitioned into petroleum ether. Clean-up is performed by gel permeation chromatography on Bio-Beads S-X3 and an additional Florisil column. Electron capture gas liquid chromatography on a non-polar SE-30 capillary column separates the isomers of the compounds. Under the conditions used in the experiments with cyfluthrin, cypermethrin, deltamethrin, fenvalerate, and permethrin, the compound levels in rye and wheat did not decrease significantly. Losses during milling and baking were small. Consequently, pyrethroid insecticides remain effective in grain stored over a long period of time. However, considerable residues result in the milled fractions as well as in different types of bread.     

Insecticide resistance and synergism in Brazilian populations of Sitophilus zeamais (Coleoptera: Curculionidae)

Insecticide resistance to three organophosphate (chlorpyrifos-methyl, malathion and pirimiphos-methyl) and three pyrethroid (cypermethrin, deltamethrin and permethrin) insecticides was surveyed in nine field-collected populations of the maize weevil from six Brazilian States and contrasted with a standard susceptible population and a DDT and pyrethroid resistant population. Bioassay tests were carried out in glass vials (20 ml) impregnated with dried insecticide residue and the resistant populations were recognized by using discriminating concentrations established from LC₉₅s estimated for a standard susceptible population. Four field-collected populations had reduced susceptibility to cypermethrin (64-77% mortality) and to chlorpyrifos-methyl (76-77% mortality). All populations were susceptible to malathion and pirimiphos-methyl and only the DDT and pyrethroid resistant check showed resistance to all pyrethroids. Concentration-mortality curves showed non-significant levels of insecticide resistance in the field-collected populations and moderate (10-100-fold) to very high levels (>1000-fold) of resistance to pyrethroids in the DDT and pyrethroid resistant population. Results from insecticide bioassays with synergists (diethyl maleate, piperonyl butoxide, and triphenyl phosphate) suggested a major involvement of esterase as the cypermethrin resistance mechanism, while no clear result was obtained for chlorpyrifos-methyl. Synergism results provide support for target site resistance to pyrethroids in the DDT and pyrethroid resistant population used in this study.     

Selective treatments,including combinations of pyrethroid and organophosphorus insecticides. For control of stored product Coleoptera at two temperatures

Selective treatments, appropriate to simulated storage conditions, reinfestation pressures and species or strains of insects present in the wheat, required less insecticide than any “blanket” or all-purpose treatment. In particular, the pyrethroids bioresmethrin, synergized bioresmethrin or synergized pyrethrins were especially effective where Rhyzopertha dominica and especially a strain resistant to organophosphorus insecticides, was the only species present. Conversly, the organophosphorus insecticides dichlorvos, malathion, fenitrothion and pirimiphos-methyl were especially effective against Tribolium and Sitophilus species. There was no antagonism between any of the pyrethroids and any of the organophosphorus insecticides when they were applied in combination, in that the period of protection given by combinations was the period of protection given by the pyrethroid component against R. dominica and the organophosphorus component against Tribolium and Sitophilus species. As an example of the data required for selective treatments, bioassays were conducted at 20 and 30°C and it was found that the net effect of the lower temperature in reducing toxicity but increasing persistence was to increase the period of protection given by insecticides in cooled storages. It is suggested that the amounts of insecticides applied to grain could be considerably reduced if more consideration was given to grain conditions, if reinfestation pressures were more carefully examined and if less attention were given to surviving adults under conditions where progeny do not develop.     

吉林省5种食用菌中杀虫剂残留量的检测及其风险评价

目的 了解吉林省市售食用菌杀虫剂残留污染水平, 评价其膳食暴露健康风险。方法 采集吉林省市售食用菌样本116份, 按照GB 23200.113-2018《食品安全国家标准植物源性食品中208种农药及其代谢物残留量的测定 气相色谱-质谱联用法》规定的标准操作程序进行32种杀虫剂检测, 运用食品安全指数法对5种食用菌(金针菇、木耳、平菇、香菇、杏鲍菇)中的杀虫剂残留所带来的风险进行评估。结果 吉林省5种食用菌所检测的32种杀虫剂中有11种有检出, 分别为5种氨基甲酸酯类杀虫剂、5种菊酯类杀虫剂和1种有机磷杀虫剂, 其中氰戊菊酯、氯氟氰菊酯和氯氰菊酯3种杀虫剂污染程度最为严重, 检出率分别为18.97%、18.10%、11.21%。食用菌中有检出的11种杀虫剂的IFS值与5种食用菌的 值均远远小于1。结论 所检测的32种杀虫剂的残留量对吉林省5种食用菌产品的食用安全没有影响, 5种食用菌的安全状态都处在可接受的安全水平。     

Residues of insecticides, and fungicides in fruit produced in Ontario, Canada, 1986-1988

Between 1986 and 1988, 306 composite samples of fruit representing eight commodities were collected from farm deliveries to the marketplace in Ontario, Canada. All samples were analysed for insecticides and fungicides. The analysis procedure included tests for organochlorine, organophosphorus, synthetic pyrethroid and methylcarbamate insecticides and dithiocarbamate, dicarboximide and organochlorine fungicides. The commodities tested included apples, blueberries, cherries, grapes, peaches, pears, raspberries and strawberries. In 14% of all fruit samples, pesticide residues were below the detection limits, which ranged between 0.005 and 0.02 mg/kg. A further 14% had total combined pesticide residues below 0.1 mg/kg. Total combined fungicide and insecticide residues ranged from 0.1 to 11 mg/kg in 72% of samples. Six different pesticides were in violation of maximum residue limits (MRL) on 11 (3.6%) of fruit samples. Captan exceeded the 5 mg/kg MRL in five samples and EBDC exceeded the 7 mg/kg MRL in two. Other violations included single fruit samples with dicofol, endosulphan, phosalone and iprodione above the MRL. Raw grapes harvested for wine contained residues of 10 pesticides and the number changed little following the crushing of the grapes; however, fermentation into wine significantly reduced residues. Six insecticides and four fungicides were present on the raw grapes and 4/105 were above the MRL. Following crushing, four insecticides and five fungicides were identified and 4/40 were above the MRL. In wine only three insecticides were identified and all were well below the MRL. Carbaryl appeared to be the most persistent, declining very little between raw grapes and wine.     

Aquatic toxicity due to residential use of pyrethroid insecticides

Pyrethroids are the active ingredients in most insecticides available to consumers for residential use in the United States. Yet despite their dominance in the marketplace, there has been no attempt to analyze for most of these compounds in watercourses draining residential areas. Roseville, California was selected as a typical suburban development, and several creeks that drain subdivisions of single-family homes were examined. Nearly all creek sediments collected caused toxicity in laboratory exposures to an aquatic species, the amphipod Hyalella azteca, and about half the samples caused nearly complete mortality. This same species was also found as a resident in the system, but its presence was limited to areas where residential influence was least. The pyrethroid bifenthrin is implicated as the primary cause of the toxicity, with additional contributions to toxicity from the pyrethroids cyfluthrin and cypermethrin. The dominant sources of these pyrethroids are structural pest control by professional applicators and/ or homeowner use of insecticides, particularly lawn care products. The suburbs of Roseville are unlikely to be unique, and similar sediment quality degradation is likely in other suburban areas, particularly in dry regions where landscape irrigation can dominate seasonal flow in some water bodies.