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1、细心整理本试剂盒只能用于科学探究，不得用于医学诊断大肠杆菌vero毒素(E.coli Verotoxin) ELISA检测试剂盒运用说明书检测原理试剂盒接受双抗体一步夹心法酶联免疫吸附试验ELISA。往预先包被大肠杆菌vero毒素E.coli Verotoxin抗体的包被微孔中，依次参与标本、标准品、HRP标记的检测抗体，经过温育并彻底洗涤。用底物TMB显色，TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的大肠杆菌vero毒素E.coli Verotoxin呈正相关。用酶标仪在450nm 波长下测定吸光度OD 值，计算样品浓度。样品收集、处理及保存方法
2、1. 血清：运用不含热原和内毒素的试管，操作过程中幸免任何细胞刺激，收集血液后，3000转离心10分钟将血清和红细胞快速当心地分别。2. 血浆：EDTA、柠檬酸盐或肝素抗凝。3000转离心30分钟取上清。3. 细胞上清液：3000转离心10分钟去除颗粒和聚合物。4. 组织匀浆：将组织参与适量生理盐水捣碎。3000转离心10分钟取上清。5. 保存：假如样本收集后不刚好检测，请按一次用量分装，冻存于-20，幸免反复冻融，在室温下解冻并确保样品匀整地充分解冻。自备物品1. 酶标仪450nm2. 高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL3. 37恒温箱
3、操作留意事项1. 试剂盒保存在2-8，运用前室温平衡20分钟。从冰箱取出的浓缩洗涤液会有结晶，这属于正常现象，水浴加热使结晶完全溶解后再运用。2. 试验中不用的板条应立刻放回自封袋中，密封低温枯燥保存。3. 浓度为0的S0号标准品即可视为阴性参照或者空白；遵照说明书操作时样本已经稀释5倍，最终结果乘以5才是样本实际浓度。4. 严格遵照说明书中标明的时间、加液量及依次进展温育操作。5. 全部液体组分运用前充分摇匀。试剂盒组成名称96孔配置48孔配置备注微孔酶标板12孔8条12孔4条无标准品0.3mL*6管0.3mL*6管无样本稀释液6mL3mL无检测抗体-HRP10mL5mL无20洗涤缓冲液25
4、mL15mL按说明书进展稀释底物A6mL3mL无底物B6mL3mL无终止液6mL3mL无封板膜2张2张无说明书1份1份无自封袋1个1个无注：标准品S0-S5浓度依次为：0、5、10、20、40、80 ng/mL试剂的准备 20洗涤缓冲液的稀释：蒸馏水按1：20稀释，即1份的20洗涤缓冲液加19份的蒸馏水。洗板方法1. 手工洗板：甩尽孔内液体，每孔加满洗涤液，静置1min后甩尽孔内液体，在吸水纸上拍干，如此洗板5次。2. 自动洗板机：每孔注入洗液350L，浸泡1min，洗板5次。操作步骤1. 从室温平衡20min后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4。2. 设置标准品孔和样本孔，标
5、准品孔各加不同浓度的标准品50L；3. 样本孔先加待测样本10L，再加样本稀释液40L；空白孔不加。4. 除空白孔外，标准品孔和样本孔中每孔参与辣根过氧化物酶HRP标记的检测抗体100L，用封板膜封住反响孔，37水浴锅或恒温箱温育60min。5. 弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次也可用洗板机洗板。6. 每孔参与底物A、B各50L，37避光孵育15min。7. 每孔参与终止液50L，15min内，在450nm波特长测定各孔的OD值。结果判定 绘制标准曲线：在Excel工作表中，以标准品浓度作横坐标，对应OD值作纵坐标，绘制出标准品线
6、性回来曲线，按曲线方程计算各样本浓度值。试剂盒性能1. 精确性：标准品线性回来与预期浓度相关系数R值，大于等于0.9900。2. 灵敏度：最低检测浓度小于1.0 ng/mL。3. 特异性：不与其它可溶性构造类似物穿插反响。4. 重复性：板内、板间变异系数均小于15%。5. 贮藏：2-8，避光防潮保存。6. 有效期：6个月免责声明1. 试剂盒仅供探究运用，不得用于临床试验或人体试验，否那么所产生的一切后果，由试验者担当，本公司概不负责。2. 严格遵照说明书操作，试验者违反说明书操作，后果由试验者担当。FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
7、 PROCEDURES.E.coli Verotoxin ELISA Kit instructionIntended useThis E.coli Verotoxin ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at
8、450 nm using a spectrophotometer. In order to measure the concentration of E.coli Verotoxin in the sample, this E.coli Verotoxin ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve
9、 of Optical Density versus E.coli Verotoxin concentration. The concentration of E.coli Verotoxin in the samples is then determined by comparing the O.D. of the samples to the standard curve.Sample collection and storagesSerum - Use a serum separator tube and allow samples to clot for 30 minutes befo
10、re centrifugation for 10 minutes at approximately 3000g. Remove serum and assay immediately or aliquot and store samples at -20 or -80.Avoid repeated freeze-thaw cyclesPlasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000g at 2-8 within 30 minut
11、es of collection. Store samples at -20or -80. Avoid repeated freeze-thaw cycles.Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20or -80. Avoid repeated freeze-thaw cycles.Note: The samples shoule be c
12、entrifugated dequately and no hemolysis or granule was allowed.Materials required but not supplied1. Standard microplate reader(450nm)2. Precision pipettes and Disposable pipette tips.3. 37 incubatorPrecautions1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates
13、 are matched for optimal performance. Use only the reagents supplied by manufacturer.2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8C in their pouch with the desiccant provided.3. Mix all reagents before using.Remove all kit reagents from refriger
14、ator and allow them to reach room temperature ( 20-25C)Materials suppliedName96 determinations48 determinationsMicroelisa stripplate12*8strips12*4stripsStandard0.3ml*6tubes0.3ml*6tubes Sample Diluent6.0ml3.0mlHRP-Conjugate reagent10.0ml5.0ml20X Wash solution25ml15mlChromogen Solution A6.0ml3.0mlChro
15、mogen Solution B6.0ml3.0mlStop Solution6.0ml3.0mlClosure plate membrane22User manual11Sealed bags11Note: Standard (S0 S5) concentration was followed by:0,5,10,20,40,80 ng/mlReagent preparation20wash solution:Dilute with Distilled or deionized water 1:20.Assay procedure1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples b
