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                                        人(Human)副流感病毒?(HPIV) ELISA检测试剂盒
                                        产品编号 HP-E1902P
                                        中文名称: 人(Human)副流感病毒?(HPIV) ELISA检测试剂盒
                                        英文名称: Human parainfluenza virus (HPIV) ELISA Kit
                                        品牌: 赫澎生物
                                        规格型号: 48T 96T
                                        本试剂盒只能用于科学研究,不得用于医学诊断
                                        Human副流感病毒 (HPIV)
                                        ELISA检测试剂盒
                                        使用说明书
                                        检测原理
                                        试剂盒采用双抗一步夹心法酶联免疫吸附试验(ELISA)。往预先包被副流感病毒抗原包被微孔中,依次加入标本、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。用酶标仪在450nm波长下测定吸光度(OD 值),与CUTOFF值相比较,从而判定标本中副流感病毒(HPIV)的存在与否。
                                        样品收集、处理及保存方法
                                        1.  血清:使用不含热原和内毒素的试管,操作过程中避免任何细胞刺激,收集血液后,3000转离心10分钟将血清和红细胞迅速小心地分离。
                                        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:将组织加入适量生理盐水捣碎。3000转离心10分钟取上清。
                                        5.  保存:如果样本收集后不及时检测,请按一次用量分装,冻存于-20℃,避免反复冻融,在室温下解冻并确保样品均匀地充分解冻。
                                        自备物品
                                        1. 酶标仪(450nm)
                                        2. 高精度加样器及枪头:0.5-10uL、2-20uL、20-200uL、200-1000uL
                                        3. 37℃恒温箱
                                        操作注意事项
                                        1.  试剂盒保存在2-8℃,使用前室温平衡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使用。
                                        2.  实验中不用的板条应立即放回自封袋中,密封(低温干燥)保存。
                                        3.  预处理后的样本请按照操作步骤用样本稀释液适当稀释以达到试剂盒的的最佳检测效果。
                                        4.  严格按照说明书中标明的时间、加液量及顺序进行温育操作。
                                        5.  所有液体组分使用前充分摇匀。
                                        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
                                        阴性对照 0.5mL 0.5mL
                                        阳性对照 0.5mL 0.5mL
                                        检测抗原-HRP 10mL 5mL
                                        20×洗涤缓冲液 25mL 15mL 按说明书进行稀释
                                        样本稀释液 6mL 3mL
                                        底物A 6mL 3mL
                                        底物B 6mL 3mL
                                        终止液 6mL 3mL
                                        封板膜 2张 2张
                                        说明书 1份 1份
                                        自封袋 1个 1个
                                         
                                        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350μL,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.  设置阴、阳性对照孔和样本孔,阴、阳性对照孔中加入阴性对照、阳性对照各50μL;
                                        3.  待测样本孔先加待测样本10μL,再加样本稀释液40μL;
                                        4.  随后阴、阳性对照孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗原100μL,用封板膜封住反应孔,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.  每孔加入底物A、B各50μL,37℃避光孵育15min。
                                        7.  每孔加入终止液50μL,15min内,在450nm波长处测定各孔的OD值。
                                        结果判断
                                         1.  试验有效性:阳性对照孔OD值平均值≥1.00;
                                                        阴性对照孔OD值平均值≤0.15。
                                        2.  临界值(Cut off)计算:临界值=阴性对照孔平均值+0.15
                                        3.  阴性判断:样品OD值<临界值(Cut off),样品为阴性
                                        4.  阳性判断:样品OD值>临界值(Cut off),样品为阳性
                                        试剂盒性能
                                        1.  准确性:阳性对照孔OD值平均值≥1.00;阴性对照孔OD值平均值≤0.15,说明试验结果有效。
                                        2.  特异性:不与其它可溶性结构类似物交叉反应。
                                        3.  重复性:板内、板间变异系数均小于15%。
                                        4.  贮藏:2-8℃,避光防潮保存。
                                        5.  有效期:6个月
                                        免责声明
                                        1.   试剂盒仅供研究使用,不得用于临床实验或人体实验,否则所产生的一切后果,由实验者承担,本公司概不负责。
                                        2.   严格按照说明书操作,实验者违反说明书操作,后果由实验者承担。
                                         
                                         
                                         
                                         
                                         
                                         
                                         
                                         
                                         
                                        FOR RESEARCH USE ONLY. 
                                        NOT FOR USE IN DIAGNOSTIC PROCEDURES.
                                         
                                        Human parainfluenza virus (HPIV) ELISA Kit instruction
                                         
                                        Intended use
                                        This HPIV 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 450 nm using a spectrophotometer. In order to measure the concentration of HPIV in the sample, this HPIV 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 cutoff value. The existence or not of HPIV in the samples is then determined by comparing the O.D. of the samples to the CUT OFF.
                                        Sample collection and storages
                                        Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles
                                        Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -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 -20℃or -80℃. Avoid repeated freeze-thaw cycles.
                                        Note:  The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.
                                        Materials required but not supplied
                                        1.  Standard microplate reader(450nm)
                                        2.  Precision pipettes and Disposable pipette tips.
                                        3.  37 ℃ incubator
                                        Precautions
                                        1.  Do not substitute reagents from one kit to another. Standard, conjugate and microplates 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 2-8°C in their pouch with the desiccant provided.
                                        3.  Mix all reagents before using.
                                        Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)
                                        Materials supplied
                                        Name 96 determinations 48 determinations
                                        Microelisa stripplate 12*8strips 12*4strips
                                        Negative control 0.5ml 0.5ml
                                        Positive control 0.5ml 0.5ml
                                        HRP-Conjugate reagent 10.0ml 5.0ml
                                        20X Wash solution 25ml 15ml
                                        Sample Diluent 6.0ml 3.0ml
                                        Chromogen Solution A 6.0ml 3.0ml
                                        Chromogen Solution B 6.0ml 3.0ml
                                        Stop Solution 6.0ml 3.0ml
                                        Closure plate membrane 2 2
                                        User manual 1 1
                                        Sealed bags 1 1
                                        Reagent preparation
                                        20×wash solution:Dilute with Distilled or deionized water 1:20.
                                        Assay procedure
                                        1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.
                                        2.  Separately add Positive control and Negative control 50μl to the Positive and Negative well; Add testing sample 10μl then add Sample Diluent 40μl to testing sample well.
                                        3.  Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C. 
                                        4.  Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
                                        5.  Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
                                        6.  Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not
                                        appear uniform, gently tap the plate to ensure thorough mixing. 
                                        7.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
                                         
                                        Determine the result
                                        1.  Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤0.15.
                                        2.  Calculate Critical (CUT OFF): Critical= the average of Negative control well + 0.15.
                                            Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.
                                            Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.
                                        Storage and validity
                                        Storage:  2-8℃.
                                        validity: six months.
                                         
                                         
                                        FOR RESEARCH USE ONLY;
                                        NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! 
                                        PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
                                         
                                        检测报告(COA)
                                        使用说明
                                        MSDS查询
                                        使用说明
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