中文摘要：本发明公开了定量检测转基因玉米中抗虫蛋白Vip3Aa20的酶联免疫试剂盒及其检测方法，所述酶联免疫试剂盒由包被捕获抗体的微孔反应板、样品处理液、生物素标记的检测抗体、标准品、辣根过氧化物酶标记的亲和素、生物素标记的抗体稀释液、辣根过氧化物酶标记的亲和素稀释液、浓缩洗涤液、底物溶液、终止液和板贴组成，从而具有操作简便、能同时快速检测大批样品，具有高灵敏度、高特异性的特点。
外文摘要：   NOVELTY - Kit comprises micro-porous reaction plate coated capturing antibody, sample processing solution, biotin-labeled detection antibody, standard substance, horseradish peroxidase labeled avidin, biotin-labeled antibody diluent, horseradish peroxidase labeled avidin diluent, concentrated cleaning solution, substrate solution, stopping solution and plate.    USE - The kit is useful for quantitatively detecting insect-resistant protein Vip3Aa20 in transgenic maize.    ADVANTAGE - The kit is easy to operate, enables rapid detection of large number of samples with high sensitivity and specificity.    DETAILED DESCRIPTION - Kit comprises micro-porous reaction plate coated capturing antibody, sample processing solution, biotin-labeled detection antibody, standard substance, horseradish peroxidase labeled avidin, biotin-labeled antibody diluent, horseradish peroxidase labeled avidin diluent, concentrated cleaning solution, substrate solution, stopping solution and plate. The sample processing solution is prepared by diluting 500 mu l 1M tris in pH of 7.5, 1.5 ml 1M sodium chloride, 20 mu l 0.5 M EDTA, 2 ml 50% glycerol, 1 ml 10% sodium dodecyl sulfate with 10 ml double distilled water, adding protease inhibitor and 50 mu l 1 mM phenylmethylsulfonyl fluoride. The biotin-labeled detection antibody is 100 mu g/ml biotin-labeled Vip3Aa201F9-1F5 mousemonoclonal antibody ( phosphate-buffered saline (PBS), 50% glycerol) solution. The standard product is 2 ng His-Vip3Aa20 recombinant protein. The horseradish peroxidase labeled avidin is 1:40 horseradish peroxidase labeled avidin solution. The biotin-labeled antibody diluent and the horseradish peroxidase-labeled avidin dilent are 1 g 1% bovine serum albumin (BSA), 0.05 ml 0.05% Tween20 (RTM: polysorbate 20), 0.1 ml 0.1% sodium azide and 1x PBS for making volume to 100 ml. The concentrated cleaning solution is 10X PBS containing 1.0% Tween20 (RTM: polysorbate 20). The substrate solution is 0.5 ml 2 mg/ml tetramethylbenzidine (TMB) in absolute ethanol, 10 ml substrate buffer and 32 mu l 30% hydrogen peroxide and 1 M sulfuric acid. An INDEPENDENT CLAIM is included for method of quantitatively detecting the insect-resistant protein Vip3Aa20 in transgenic maize using kit which involves (a) equilibrating sample processing solution, a biotin-labeled detection antibody, a standard, horseradish peroxidase-labeled avidin, biotin-labeled antibody diluent, horseradish peroxidase-labeled avidin diluent, concentrated cleaning solution, substrate solution, stopping solution at 18-25 degrees C for at least 30 minutes, (b) setting up standard sample wells and sample wells, adding standard samples or test samples for incubation in each well, and then discarding the liquid, drying and adding biotin-labeled detection antibody working solution for incubation, then discarding the liquid in the well, washing and drying well, adding each horseradish peroxidase-labeled avidin working solution for incubation, then discarding fluid in the hole, washing and drying content, adding substrate solution to each well, incubating plate in dark, then adding stopping the solution after color development in each well, mixing and measuring the optical density OD value of each well with a microplate reader at a wavelength of 450 nm within 5 minutes after the termination of the reaction and (c) processing data by subtracting the S0 hole value from the standard and sample values, drawing the curve, if you set a duplicate hole, calculating the average value, the standard concentration is the ordinate, OD is abscissa, drawing the standard curve in accordance to the sample OD value and identifying corresponding concentration from the standard curve. 
主权项：一种定量检测转基因玉米中抗虫蛋白Vip3Aa20的酶联免疫试剂盒，其特征在于，所述酶联免疫试剂盒由包被捕获抗体的微孔反应板、样品处理液、生物素标记的检测抗体、标准品、辣根过氧化物酶标记的亲和素、生物素标记的抗体稀释液、辣根过氧化物酶标记的亲和素稀释液、浓缩洗涤液、底物溶液、终止液和板贴组成； 其中，样品处理液的配方为：1M Tris,pH 7.5 500uL，1M NaCL 1.5mL，0.5M EDTA20uL，50％甘油 2mL，10％SDS 1mL，用双蒸水配成10ML，使用时添加1片蛋白酶抑制剂以及50uL的1mM苯甲基磺酰氟； 生物素标记的检测抗体为100μg/mL生物素标记的Vip3Aa20 1F9-1F5鼠单抗(PBS,50％甘油)溶液； 标准品为2ng的His-Vip3Aa20重组蛋白； 辣根过氧化物酶标记的亲和素为1:40辣根过氧化物酶标记的亲和素溶液； 生物素标记的抗体稀释液以及辣根过氧化物酶标记的亲和素稀释液配方为1％BSA1g，0.05％的吐温20 0.05mL，0.1％的NaN3 0.1mL，最终用1×PBS定容至100mL； 浓缩洗涤液为含有1.0％Tween-20的10×PBS； 底物溶液为0.5mL 2mg/mL TMB无水乙醇溶液，10mL底物缓冲液，32μL 30％H2O2混合，现用现配； 终止液为1M H2SO4.
申请号：CN201711329696.2
专利号：CN107894510A
公开/公告号：CN107894510A
申请/专利权人：中国农业科学院生物技术研究所;
发明/设计人：刘卫晓; 李亮; 金芜军; 宛煜嵩; 黄卫红; 苗朝华; 高进; 董美; 王迪; 胡晓颖; 安娜
分类号：G01N33/68
主分类号： G01N33/6
法律状态：实质审查
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