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首页> 外文期刊>Crop Protection >Development of a colloidal-gold based lateral-flow immunoassay kit for 'quality-control' assessment of pyrethroid and endosulfan formulations in a novel single strip format
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Development of a colloidal-gold based lateral-flow immunoassay kit for 'quality-control' assessment of pyrethroid and endosulfan formulations in a novel single strip format

机译：基于胶体金的侧向流免疫分析试剂盒的开发，用于以新型单试纸形式对拟除虫菊酯和硫丹制剂进行“质量控制”评估

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摘要

A simple lateral-flow immuno-chromatographic 'dipstick' kit was developed in a novel single strip format to test/verify the label claim of active ingredient in commercial insecticide formulations of endosulfan and the type-II synthetic pyrethroids, cypermethrin, deltamethrin and fenvalerate. Polyclonal antisera were raised in rabbits against immunogens developed by conjugating proteins (bovine serum albumin, BSA and keyhole limpet hemocyanin, KLH) to endosulfan-diol, 4'-hydroxy-cypermethrin and 2'-hydroxy-cypermethrin using an activated ester method through a spacer attachment. Immunoglobulin (IgG) was purified through protein-A affinity chromatography and conjugated to horse radish peroxidase (HRP) and colloidal gold. A competitive ELISA was designed to determine the ability of the test insecticides to compete with the relevant hapten-protein conjugate. A combination of antiserum-4'-OH-Cyper-KLH with the 4'-OH-Cyper-BSA as coating hapten resulted in the highest absorbance (Amax: 1.32) low background value (Amin: 0.05) and good competitive inhibition with cypermethrin (IC"9"9: 2034@mg/L), deltamethrin (IC"9"9: 2297@mg/L) and fenvalerate (IC"9"9: 3546@mg/L). For endosulfan, a combination of antiserum raised against endosulfan-KLH with endosulfan-BSA as the coating hapten gave the highest absorbance (Amax: 1.42), low background value (Amin: 0.05) and good competitive inhibition with endosulfan (IC"9"9: 2378@mg/L). A novel format was designed using the combinations for development of the lateral-flow strip, so as to enable the detection of pyrethroids and endosulfan on a single strip. The strips were designed such that two intensely coloured purple lines appeared when the strip is dipped in a solution devoid of any insecticide. The minimum concentration of the test insecticides, endosulfan (1800@mg/l), cypermethrin (800@mg/l), deltamethrin (1000@mg/l) and fenvalerate (1400@mg/l) was determined, which would be adequate to inhibit the appearance of either of the two lines, when the strip is dipped in a solution containing any one of the diluted insecticides prepared from authentic insecticide formulations. When samples of substandard insecticide formulations are diluted as per the pre-determined dilutions, the aliquots are expected to contain less than the quantity specified above, and would result in the appearance of two purple lines, with one of them at the sample line region of the membrane, the intensity of which would be inversely proportionate to the concentration of the test analyte. The strip can be considered as suitable for on-the-spot analysis of test sample formulations either by farmers, extension workers, quality control officials etc., to generate preliminary prima facie evidence in case of doubtful samples of substandard insecticide formulation.
机译：开发了一种简单的单侧流免疫色谱“试纸”试剂盒，以新颖的单试纸形式测试/验证硫丹和II型合成拟除虫菊酯，氯氰菊酯，溴氰菊酯和灭草菊酯的商业杀虫剂制剂中活性成分的标签要求。在兔中产生多克隆抗血清，以抵抗通过将蛋白质（牛血清白蛋白，BSA和匙孔血蓝蛋白，KLH）与硫丹二醇，4'-羟基-氯氰菊酯和2'-羟基-氯氰菊酯结合而产生的免疫原所产生的免疫原。垫片附件。免疫球蛋白（IgG）通过蛋白A亲和色谱纯化，并与辣根过氧化物酶（HRP）和胶体金结合。设计竞争性ELISA，以确定测试杀虫剂与相关半抗原-蛋白质缀合物竞争的能力。将抗血清4'-OH-Cyper-KLH与4'-OH-Cyper-BSA结合在一起作为涂层半抗原可产生最高的吸光度（Amax：1.32），低背景值（Amin：0.05）和对氯氰菊酯的良好竞争抑制（IC“ 9” 9：2034 @ mg / L），溴氰菊酯（IC“ 9” 9：2297 @ mg / L）和氰戊菊酯（IC“ 9” 9：3546 @ mg / L）。对于硫丹，将针对硫丹-KLH产生的抗血清与硫丹-BSA作为涂层半抗原的组合可获得最高的吸光度（Amax：1.42），较低的背景值（Amin：0.05）和与硫丹的良好竞争抑制作用（IC“ 9” 9） ：2378 @ mg / L）。使用这些组合设计了一种新型格式，用于开发侧流试纸，以便能够在单个试纸上检测拟除虫菊酯和硫丹。试纸条的设计使得当将试纸条浸入不含任何杀虫剂的溶液中时，会出现两条深紫色的线条。确定了测试杀虫剂，硫丹（1800 @ mg / l），氯氰菊酯（800 @ mg / l），溴氰菊酯（1000 @ mg / l）和氰戊菊酯（1400 @ mg / l）的最低浓度，这是足够的为了防止两条线中任何一条的出现，将试纸条浸入含有由真实杀虫剂配方制备的任何稀释杀虫剂的溶液中。当按照预定的稀释度稀释次标准杀虫剂制剂的样品时，这些等分试样的含量应少于上述规定的数量，并会导致出现两条紫色线条，其中一条出现在样品的紫色区域。膜的强度与测试分析物的浓度成反比。农民，推广人员，质量控制人员等都可以认为该试纸适合于现场分析测试样品制剂，以防在不合格的杀虫剂制剂样品中产生初步的初步证据。

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