人Leptin Elisa试剂盒

www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 人 Leptin 定量 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 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 Leptin 浓度。使用前请仔细阅 读说明书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。 Leptin简介： 人的瘦素蛋白是一个 16KDa 的多肽激素，主要由脂肪细胞释放，其主要作用是调控食欲的降低和能量消耗。 但是，人们还发现瘦素蛋白在血管再生、骨代谢、造血及免疫调节上有重要的作用。 瘦素可与丘脑下部的中央脊腹部的饱觉中枢结合，从而使得大脑得到食物充足的信号，这样瘦素的 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 达水平的 高低就可达到调控能量摄入和机体的能量消耗作用。 瘦素功能的实现主要跟瘦素受体结合后实现的，瘦素受体有两类，一类是固定细胞膜上的受体，主要通过 JAK/STAT信号通路起作用，另一类是游离的受体，这类受体一般是丢失了受体的细胞膜内的部分，游离出来的， 根据失去的结构不同，游离受体的种类较多。人体内游离的受体的产生主要是由金属蛋白酶介导的细胞膜外功能域 的脱落后形成的。游离的受体在晚期肾病、慢性心脏病和厌食症的病人中发现有所增高。 检测原理: 本试剂盒采用双抗体夹心ELISA法检测 样本 保单样本pdf木马病毒样本下载上虞风机样本下载直线导轨样本下载电脑病毒样本下载 中Leptin 的浓度。Leptin 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Leptin 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Leptin 抗体后，抗人Leptin 抗体与Leptin 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Leptin 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，Leptin 浓度与OD450值之间呈正比，通过参考品绘制标准曲线，对照未知样本中OD值，即可算出标本中Leptin 浓 度。 人定量分析酶联免疫检测试剂盒组成: 组分 规格 视频线规格配置磁共振要求常用水泵型号参数扭矩规格钢结构技术规格书 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 10ml/5ml 标准品 4/2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进行操作： A.细胞上清标本离心去除悬浮物后即可； 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 快递客服问题件处理详细方法山木方法pdf计算方法pdf华与华方法下载八字理论方法下载 收集； www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com D.标本收集后请分装，若待测样本不能及时检测，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:加入去离子水 0.25ml 至冻干标准品瓶中使 leptin 终浓度达到 2000pg/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 2000 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： 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50 ul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 0.0621 0.1087 0.0854 62.5 0.1488 0.1642 0.1565 125 0.2217 0.2609 0.2413 250 0.3469 0.3983 0.3726 500 0.6494 0.7724 0.7109 1000 1.3671 1.4515 1.4093 2000 1.9359 2.1219 2.0289 人Leptin参考标准曲线 人leptin标准曲线 0 0.5 1 1.5 2 2.5 0 62.5 125 250 500 1000 2000 人leptin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6.4pg/ml。 2.特异性：与鼠Leptin和大鼠Leptin等没有交叉反应。 www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 3.重复性：板内，板间变异系数均<10%. 参考文献: Myers, M.G., Jr. (2004) Recent Prog. Horm. Res. 59595959:287. Sandoval, D.A. and S.N. Davis (2003) J. Diabetes Complications 17171717:108. Sierra-Honigmann, M.R. et al. (1998) Science 281281281281:1683. La Cava, A. and G. Matarese (2004) Nat. Rev. Immunol 4444:371. ELISAELISAELISAELISA KitKitKitKit forforforfor thethethethe QuantitativeQuantitativeQuantitativeQuantitative AnalysisAnalysisAnalysisAnalysis ofofofof HumanHumanHumanHuman LeptinLeptinLeptinLeptin The human leptin ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human leptin in cell culture supernatants,human serum and plasma.THETHETHETHE ELISAELISAELISAELISA KITKITKITKIT 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 Leptin, known for its key roles in he regulation of food intake and energy metabolism, is a 16 kDa protein hormone released primarily by adipose tissues. In addition, leptin has several other important functions involved in angiogenesis, bone metabolism, hematopoiesis, and immunization. Leptin binds to the satiety center which lies in the Ventral Medial nucleus of the hypothalamus. Binding of leptin to this nucleus signals in the brain leads the body to have enough to eat. Thus, circulating leptin levels give the brain a reading of energy storage for the purposes of regulating appetite and metabolism. Leptin activities are mediated via the leptin receptor including leptin R and OB R. Leptin R is a member of the type I cytokine receptor family, which can perform function through JAK/STAT signaling pathway. OB R was truncated cytoplasmic domains with different isoforms. Soluble forms found in humans may be generated post-translationally via metalloprotease-mediated ectodomain shedding. Soluble Leptin R is also found upregulated in patients with chronic heart failure, end-stage renal disease, and anorexia. PrinciplesPrinciplesPrinciplesPrinciples ofofofof thethethethe TestTestTestTest The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human Leptin. An anti-human Leptin monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human Leptin in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human Leptin biotin-conjugated antibody were added and binds to human Leptin 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 Leptin in the original specimen. MaterialsMaterialsMaterialsMaterials providedprovidedprovidedprovided withwithwithwith thethethethe kits:kits:kits:kits: reagent 96/48Test Kit Assay Buffer 5ml/3 ml Leptin Antibody-Coated Wells 12 strips/6 strips Standard Diluent 10ml/5ml Leptin Standard 4/2vial(s) Detetion Antibody 10ml/5ml Streptavidin-HRP 10ml/5ml Wash Buffer Concentrate 20× 30ml www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 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 mixure 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 0.25ml deionized or distilled water to bottle wait15 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 into 300ul 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 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 www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com 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 data2 Average 0 0.0621 0.1087 0.0854 62.5 0.1488 0.1642 0.1565 125 0.2217 0.2609 0.2413 250 0.3469 0.3983 0.3726 500 0.6494 0.7724 0.7109 1000 1.3671 1.4515 1.4093 2000 1.9359 2.1219 2.0289 www.blkwsw.com Beijing BLKW Biotechnology Co., Ltd. Tel:010-57158602/52872342 blkwbio@blkwbio.com HHHHumanumanumanuman LeptinLeptinLeptinLeptin standardstandardstandardstandard curvecurvecurvecurve Human leptin standard Curve 0 0.5 1 1.5 2 2.5 0 62.5 125 250 500 1000 2000 Human Leptin 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 6.4pg/ml. SSSSpecificitypecificitypecificitypecificity :::: No significant cross-reactivity or interference with Mouse Leptin and Rat Leptin. Repeatability:Repeatability:Repeatability:Repeatability: The coefficient of variation between wells or plates is less than 10 per cent. REFERENCES:REFERENCES:REFERENCES:REFERENCES: Myers, M.G., Jr. (2004) Recent Prog. Horm. Res. 59:287. Sandoval, D.A. and S.N. Davis (2003) J. Diabetes Complications 17:108. Sierra-Honigmann, M.R. et al. (1998) Science 281:1683. La Cava, A. and G. Matarese (2004) Nat. Rev. Immunol 4:371.