人BDNF Elisa试剂盒

www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 人 BDNFBDNFBDNFBDNF定量分析酶联免疫检测试剂盒 本试剂盒仅供科研使用。用于体外定量检测人血清、血浆或细胞培养上清液中的 BDNF 浓度。使用前请仔细阅 读说明 书 关于书的成语关于读书的排比句社区图书漂流公约怎么写关于读书的小报汉书pdf 并检查试剂组分是否完整, 如有疑问请与北京博凌科为生物科技有限公司联系，我们将提供力所能及的帮 助。 如您有其它需求，请登录北京博凌科为生物科技有限公司网站或致电本公司。 BDNFBDNFBDNFBDNF简介： 人脑源性神经营养因子（BDNF）是神经营养因子家族中的一员，这对中枢神经系统和外周神经系统中某些特 定的神经组份的分化和存活是必须的。BDNF 由分泌的前前体蛋白，经裂解成信号肽和前体蛋白后，前体蛋白裂再 裂解成 119个氨基酸的成熟蛋白。BDNF有六个保守的半胱氨酸残基，在蛋白内部形成三个二硫键。与 NGF 相似， 活性 BDNF被预测为一个非共价结合的同源二聚体。BDNF 在中枢神经系统中广泛以自分泌和旁分泌的形式分泌出 来，具有刺激中枢神经的营养和分化的作用，已证明在记忆形成的过程中和染色体调控方面具有重要的作用。此外， 在脑垂体、脊髓，心肌和肺及平滑肌中也有低水平的 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 达。BDNF 可与其高亲和力的受体 TrkB酪氨酸激酶受体结合 特异性的激活下游信号通路。 BDNF在血浆中以pg/ml水平存在，但在血清中则以ng/ml水平存，该差异明显是由于血小板中的BDNF的释放 造成差异。BDNF不同种属间在结构上的高度的保守性，使得人的BDNF ELISA可以用于其它种属的BDNF的检测。 检测原理:::: 本试剂盒采用双抗体夹心ELISA法检测样本中BDNF的浓度。BDNF捕获抗体已预包被于酶标板上，当加入标本 或参考品时，其中的BDNF会与捕获抗体结合，其它游离的成分通过洗涤的过程被除去。当加入生物素化的抗人BDNF 抗体后，抗人BDNF抗体与BDNF接合，形成夹心的免疫复合物，其它游离的成分通过洗涤的过程被除去。随后加入 辣根过氧化物酶标记的亲合素。生物素与亲合素特异性结合，亲合素连接的酶就会与夹心的免疫复合物连接起来； 其它游离的成分通过洗涤的过程被除去。最后加入显色剂，若样本中存在BDNF将会形成免疫复合物，辣根过氧化物 酶会催化无色的显色剂氧化成蓝色物质，在加入终止液后呈黄色。通过酶标仪检测，读其450nm处的OD值，BDNF 浓度与OD450值之间呈正比，通过参考品绘制 标准 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 曲线，对照未知样本中OD值，即可算出标本中BDNF浓度。 人定量分析酶联免疫检测试剂盒组成:::: 组分 规格 预包被板 12条 或 6条 样本分析缓冲液 1瓶5ml/3 ml 标准品稀释液 10 ml/5ml 标准品 2支/1支(冻干) 生物素化抗体 1瓶10ml/5ml HRP连接的酶结合物 1瓶10ml/5ml 浓缩洗涤液 20× 30ml/瓶 TMB底物 1瓶10ml/5 ml 终止液 1瓶5ml/3 ml 封板胶纸 3/2张 说明书 1份 标本收集:::: 1.标本的收集请按下列 流程 快递问题件怎么处理流程河南自建厂房流程下载关于规范招聘需求审批流程制作流程表下载邮件下载流程设计 进行操作： A.细胞上清标本离心去除悬浮物后即可； www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com B.血清标本应是自然凝固后，取上清，避免在冰箱中凝固血液； C.血浆标本，推荐用EDTA的方法收集若待测样本不能及时检测； D.标本收集后请分装，冻存于－20℃，避免反复冻融。 2.血清标本不应添加任何防腐剂或抗凝剂； 3.标本应清澈透明，检测前样本中如有悬浮物应通过离心去除； 4.请勿使用溶血，高血脂或污染的标本检测，否则结果将不准确。 注：正常人血清或血浆样本请用标本缓冲液做倍比稀释后再检测。 注意事项:::: 1.试剂盒请保存在2～8℃。 2.浓缩洗涤液因在低温下可能有结晶，请水浴加热使结晶完全溶解后再配制工作液。 3.若标准品复溶后，请在三天内用完。 4.底物请勿接触氧化剂和金属。 5.加样时，请及时更换枪头，避免交叉污染。 6.严禁混用不同批号的试剂盒组份。 7.充分混匀对保证反应结果的准性很重要，在加液后请轻轻叩击边缘以保证混匀。 8.室温反应，请严格控制在25～28℃。 9.洗涤过程是至关重要的，洗涤不充分会使精确度下降并导致结果误差较大。 10.试验中标准品和样本检测时建议作双复孔。 11.加样过程中避免气泡的产生。 12.血清和血浆标本的检测时，检测抗体的孵育时间应适当延长。 检测前准备工作:::: 1.试剂盒自冰箱中取出后应置室温平衡20分钟；每次检测后剩余试剂请及时于2～8℃保存。 2.将浓缩洗涤液用双蒸水或去离子水稀释(1份加19份水)。 3.标准品:加入标准品稀释液1ml至冻干标准品瓶中使BDNF终浓度达到1500pg/ml，静置15分钟后轻轻混悬待彻底溶 解，用标准品稀释液倍比梯度稀释后依次加入检测孔中。（标准曲线取七个点，最高浓度为1500 pg/ml,标准品稀释液 直接加入作为0浓度.） 洗涤方法:::: 自动洗板机或人工洗板：每孔洗涤液为300ul，注入与吸出间隔15-30秒。洗板5次。最后一次洗板完成后将板倒扣 着在厚吸水纸上用力拍干。 实验过程需自备的 材料 关于××同志的政审材料调查表环保先进个人材料国家普通话测试材料农民专业合作社注销四查四问剖析材料 ： 1.不同规格的加样枪及相应的枪头； 2.酶标仪； 3．自动洗板机； 4．去离子水或双蒸水； 操作步骤:::: 1.通过计算并确定一次性实验所需的板条数，取出所需板条放置在框架内，暂时用不到板条请放回铝箔袋密封，保存 于4℃。 2.建议设置本底较正孔，即空白孔,设置方法为该孔只加TMB显色液和中止液。每次实验均需做标准品对照并画出标 准曲线。 www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 3.分别将标本或不同浓度标准品(100ul/孔)加入相应孔中，用封板胶纸封住反应孔，室温孵育120分钟。对于血清或血 浆标本，请加入50ul样本分析缓冲液后加50ul标本，如稀释量大，请将样本与样本分析缓冲液等量加入，不足部分用 标准品稀释液补充至100ul。 4.洗板5次，且最后一次置厚吸水纸上拍干。 5.加入生物素化抗体工作液(100ul/孔)。用封板胶纸封住反应孔，室温孵育60分钟。 6.洗板5次，且最后一次置厚吸水纸上拍干。 7.加入酶结合物工作液(100ul/孔)。用封板胶纸封住反应孔，避光室温孵育20分钟。 8.洗板5次，且最后一次置厚吸水纸上拍干。 9.加入显色剂TMB100ul/孔，避光室温孵育20分钟。 10.加入终止液50ul/孔,混匀后即刻测量OD450值。 结果判断:::: 1.复孔的值在20%的差异范围内结果才有效，复孔的值平均后可作为测量值。 2.每个标准品或标本的OD值应减去本底校正孔的OD值。 3.手工绘制标准曲线。以标准品浓度作横坐标，OD值作纵坐标，以平滑线连接各标准品的坐标点。通过标本的OD值 可在标准曲线上查出其浓度。 4.若标本 OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。 典型数值和参考曲线 浓度pg/ml 典型OD值1 典型OD值2 OD平均值 0 0.0992 0.079 0.0891 46.875 0.1881 0.1349 0.1615 93.75 0.3081 0.2149 0.2615 187.5 0.5087 0.4395 0.4741 375 0.8142 0.7248 0.7695 750 1.4832 1.4108 1.447 1500 2.2141 2.0289 2.1215 人 BDNFBDNFBDNFBDNF参考标准曲线 人BDNF参考标准曲线 0 0.5 1 1.5 2 2.5 0 46.88 93.75 187.5 375 750 1500 人BDNF浓度（pg/ml） O D 值 注意：本图仅供参考，应以同次试验标准品所绘标准曲线计算标本含量。 灵敏度，特异性和重复性:::: 1.灵敏度：多次重复结果表明，最小检出量为21pg/ml。 2.特异性：与人EGF，Epo，FGF acidic，FGF basic，G-CSF，GDNF，GM-CSF，HGF，IGF-I，IGF-II，IL-13，KGF， PDGF-AB等没有交叉反应。 3.重复性：板内，板间变异系数均<10%. www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 参考文献:::: 1. Leibrock, J. et al. (1989) Nature 341:149. 2. Barakat-Walter, I. (1996) J. Neurosci. Methods 68:281. 3. Green, L.A. and D.R. Kaplan (1995) Curr. Opin. Neurobiol. 5:579. 4. Carter, B.D. et al. (1996) Science 272:542. 5. Eide, F.F. et al. (1996) J. Neurosci. 16:3123. 6. Kishino, A. et al. (1997) Exp. Neurol. 144:273. ELISA Kit for the Quantitative Analysis of Human BDNF The human BDNF ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human BDNF in cell culture supernatants,human serum and plasma.THETHETHETHE ELISAELISAELISAELISAKITKITKITKIT ISISISIS FORFORFORFOR RESEARCHRESEARCHRESEARCHRESEARCH USEUSEUSEUSE ONLYONLYONLYONLY. Please read this instruction manual carefully and check out the material provided before use, and you can contact with our company, if any questions. You can enter our website or call us for other aim. IntroductionIntroductionIntroductionIntroduction Brainderived neurotrophic factor (BDNF) is a member of the NGF family of neurotrophic factors (also named neurotrophins) that are required for the differentiation and survival of specific neuronal subpopulations in both the central as well as the peripheral nervous system.BDNF is synthesized as pre-proBDNF, with a signal peptide and a proprotein that are cleaved to yield the 119 amino acid residue mature BDNF. BDNF have six conserved cysteine residues that are involved in the formation of three disulfide bonds. Similarly to NGF, bioactive BDNF is predicted to be a noncovalently linked homodimer. BDNF is widely expressed in the central nervous system, and acts in an autocrine and paracrine manner on several classes of neurons. BDNF promotes neuronal survival and differentiation, and has been shown to play a critical role in memory formation and synaptic regulation. . In addition, low levels of BDNF expression are also found in the pituitary gland, spinal cord, heart, lung and skeletal muscle. BDNF binds with high affinity and specifically activates the TrkB tyrosine kinase receptor.BDNF in plasma is detected in the pg/mL range, while BDNF in serum is measured in the ng/mL range, the difference apparently attributable to platelet degranulation and BDNF release during clotting . The conservation of BDNF structure potentially allows a human BDNF ELISA to be widely applied across species. PrinciplesPrinciplesPrinciplesPrinciples ofofofof thethethethe TestTestTestTest The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human BDNF. An anti-human BDNF monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human BDNF in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human BDNF biotin-conjugated antibody were added and binds to human BDNF captured by the first antibody, which formed a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be removed during a wash step. After this, subtrate solution would be added and catalyzed by the HRP, and a coloured product is formed. The intensity of the colored product is used to calculate in proportion to the amount of human BDNF in the original specimen. MaterialsMaterialsMaterialsMaterials providedprovidedprovidedprovided withwithwithwith thethethethe kits:kits:kits:kits: reagent 96/48Test Kit Assay Buffer 5ml/3 ml BDNF Antibody-Coated Wells 12 strips/6 strips Standard Diluent 10 ml/5ml BDNF Standard 2/1vial(s) www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com Detetion Antibody 10ml/5ml Streptavidin-HRP 10ml/5ml Wash Buffer Concentrate 20× 30ml TMB 10ml/5 ml Stop Solution 5ml/3 ml Plate Covers 3/2 Complete Instruction Manual 1 SpecimenSpecimenSpecimenSpecimen CollectionCollectionCollectionCollection 1.Collecting specimen as following: A.The particulate of the cell culture supernatants should be removed before use. B.Serum was obtained from clot at room temperature. C.Please collect plasma with EDTA. D.Assay immediately or store samples at －20℃. Avoid free-thaw cycles. 2.Antiseptic and anticoagulant should not appear in Serum samples. 3.Any particulate should be removed from samples before use. 4. Do not use grossly hemolyzed or lipemic samples. Note: Srongly recommend that the serum and plasma samples should be diluent as doubling dilution before use. PrecautionsPrecautionsPrecautionsPrecautions forforforfor use:use:use:use: 1.Please storage the Kit at 2～8℃。 2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use. 3. Please discard the dissolved standard after 3 days for use. 4. Avoid contact of substrate solution with oxidizing agents and metal. 5. Usage of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens. 6. Do not mix or substitute reagents with those from other lots or other sources. 7. To ensure the adequate mixture of added reagents, please tap gently the plate after the wells were filled with liquid. 8. Incubation temperature should be 25～28℃. 9. Wash step was crucial for whole assay process. 10. Duplicate wells of the same sample were recommended in assay process. 11. Avoid the foam while pour the liquid into wells. 12. For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes. ReagentReagentReagentReagent PreparationPreparationPreparationPreparation 1.The reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refrigeratory again as soon as possible. 2.Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work. 3. Add 0.6ml deionized or distilled water to bottle wait 15 minutes for complete dissolution. And in turn add the half concentration diluent by standard diluent . WashWashWashWash step:step:step:step: Automated microplate washer or operating by pipette: Each well should be pour into300ul wash buffer and soak 15 or 30 seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by aspirating.Invert the plate and blot it against clean paper towels. MaterialsMaterialsMaterialsMaterials RequiredRequiredRequiredRequired ButButButBut NotNotNotNot ProvidedProvidedProvidedProvided 1. pipettes and pipette tips www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 2. Microwell strip reader capable of reading at 450 nm (540 nm as optional reference wave length) 3. automated microplate washer 4.Glass-distilled or deionized water AssayAssayAssayAssay procedureprocedureprocedureprocedure 1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed. 2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient density of standard for standard curve. 3.Add 100ul of standard or sample.Cover with the Plate Covers provided.Incubate for 2 hours at room temperature, If assay the serum sample,you should add 50μl assay buffer with 50μl sample into the wells,if the protein concentration is higher than the range of the Kit, add the same quantitys assay buffer with the sample, the deficiency should be complemented with sample diluent to 100μl per well. 4.Five times wash process were repeated. 5.Add 100ul of detetion antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature. 6.Five times wash process were repeated. 7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature. 8.Five times wash process were repeated. 9.Add 100ul of TMB，Lucifugal incubation for 20 minutes at room temperature. 10.Add 50ul of stop solution to each well, determine the optical density of each well within 10 minutes. CalculationCalculationCalculationCalculation ofofofof ResultsResultsResultsResults 1.Duplicates should be within 20 per cent of the mean. Average absorbance values for each set of duplicate samples were used as detection results. 2.The blank absorbance values of subtract should be deducted. 3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD value (on the Y axis) vs. concentration (on the X axis). The sample concentration was obtained based on its OD value founding in the standard concentration curve. 4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again. TTTTypicalypicalypicalypical DataDataDataData andandandand SSSStandardtandardtandardtandard CCCCurveurveurveurve concentration (pg/ml) Typical data 1 Typical data 2 Average 0 0.0992 0.079 0.0891 46.875 0.1881 0.1349 0.1615 93.75 0.3081 0.2149 0.2615 187.5 0.5087 0.4395 0.4741 375 0.8142 0.7248 0.7695 750 1.4832 1.4108 1.447 1500 2.2141 2.0289 2.1215 www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com HHHHumanumanumanuman BDNFBDNFBDNFBDNFstandardstandardstandardstandard curvecurvecurvecurve Human BDNF standard curve 0 0.5 1 1.5 2 2.5 0 46.88 93.75 187.5 375 750 1500 Human BDNF concentration(pg/ml) O p t i c a l d e n s i t y Sensitivity,Sensitivity,Sensitivity,Sensitivity, SSSSpecificity,pecificity,pecificity,pecificity, RepeatabilityRepeatabilityRepeatabilityRepeatability Sensitivity:Sensitivity:Sensitivity:Sensitivity: repeated assays were evaluated and the minimum detectable dose was 21pg/ml. SSSSpecificitypecificitypecificitypecificity :::: No significant cross-reactivity or interference with human EGF，Epo，FGF acidic，FGF basic，G-CSF，GDNF， GM-CSF，HGF，IGF-I，IGF-II，IL-13，KGF，PDGF-AB Repeatability:Repeatability:Repeatability:Repeatability: The coefficient of variation between wells or plates is less than 10 per cent. REFERENCESREFERENCESREFERENCESREFERENCES:::: 1. Leibrock, J. et al. (1989) Nature 341:149. 2. Barakat-Walter, I. (1996) J. Neurosci. Methods 68:281. 3. Green, L.A. and D.R. Kaplan (1995) Curr. Opin. Neurobiol. 5:579. 4. Carter, B.D. et al. (1996) Science 272:542. 5. Eide, F.F. et al. (1996) J. Neurosci. 16:3123. 6. Kishino, A. et al. (1997) Exp. Neurol. 144:273.