人试剂盒说明书人血清一氧化氮NOELISA检测试剂盒
一氧化氮(Nitric oxide ,NO)含量测定试剂盒说明书
货号：QS1804 规格：50管/24样一氧化氮（Nitric oxide，NO）含量测定试剂盒说明书可见分光光度法注意：正式测定之前选择2-3个预期差异大的样本做预测定。
测定意义：NO（Nitric Oxide，NO）广泛分布于生物体内神经、循环、呼吸、消化、泌尿生殖等系统中，特别是神经组织中较丰富。
它作为细胞间及细胞内的信息物质，发挥信号传递的作用，是一种新型的生物信使分子，在机体的生理、病理过程中起着重要的作用。
测定原理：NO在体内或水溶液中极易氧化生成NO2—，在酸性条件下，NO2—与重氮盐磺酸胺生成重氮化合物，进一步与萘基乙烯基二胺偶合，产物在550nm处有特征吸收峰，测定其吸光值，可以计算NO含量。
为了排除样品色素等的影响，每个样品需做对照管。
自备实验用品及仪器：天平、研钵或匀浆器、可见分光光度计、1mL玻璃比色皿、蒸馏水。
试剂组成和配制：提取液：液体50mL×1瓶，4℃保存。
试剂一：液体6mL×1瓶，4℃避光保存。
试剂二：液体6mL×1瓶，4℃保存。
试剂三：液体12mL×1瓶，4℃避光保存。
样品处理：1.组织：按照组织质量（g）：提取液体积(mL)为1：5~10的比例（建议称取约0.1g组织，加入1mL提取液）进行冰浴匀浆。
10000g，4℃离心15min，取上清，置冰上待测。
2.细菌、真菌：按照细胞数量（104个）：提取液体积（mL）为500~1000：1的比例（建议500万细胞加入1mL提取液），冰浴超声波破碎细胞（功率300w，超声3秒，间隔7秒，总时间3min）；然后10000g，4℃，离心15min，取上清置于冰上待测。
3.体液和培养液等其它液态样品：直接测定。
NO含量计算：标准曲线回归方程为：y= 0.016x -0.0103，R2= 0. 99861、组织样品：NO含量定义：25℃时，每克样品或每毫克蛋白15min生成1μmoL NO2—，相当于1μmoL NO。
Elabscience 一氧化氮（NO）比色法测试盒说明书
（本试剂盒仅供体外研究使用，不用于临床诊断！）Elabscience®一氧化氮（NO）比色法测试盒Nitric Oxide (NO) Colorimetric Assay Kit产品货号：E-BC-K035-S产品规格：50 assays(48 samples)/100 assays(96 samples)检测仪器：紫外-可见光分光光度计（550 nm）使用前请仔细阅读说明书。
如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话131****6790具体保质期请见试剂盒外包装标签。
请在保质期内使用试剂盒。
联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。
用途本试剂盒适用于检测血清、血浆、及动植物组织样本中的NO含量。
检测原理NO在体内或水溶液中极易氧化生成NO2-，与显色剂生成淡红色偶氮化合物（如下图），生成偶氮化合物的浓度与NO的浓度具有线性关系，通过比色可以间接计算NO的浓度。
试剂一和试剂二的作用，为除去样本有色物质的干扰。
本试剂盒检测组织样本时，需测定总蛋白浓度，推荐使用本公司BCA试剂盒(货号E-BC-K318-M)进行测定。
提供试剂和物品说明：试剂严格按上表中的保存条件保存，不同测试盒中的试剂不能混用。
对于体积较少的试剂，使用前请先离心，以免量取不到足够量的试剂。
所需自备物品仪器：紫外-可见光分光光度计（550nm）、涡旋混匀仪、磁力搅拌器、分析天平、微量移液器（1000 μL，200 μL，100 μL，10 μL）、离心机、烧杯（50 mL，25 mL）。
耗材：枪头（1000 μL，200 μL，10 μL）、EP管（5 mL，2 mL）、吸水纸、擦镜纸、磁力搅拌子。
试剂：双蒸水、生理盐水（0.9% NaCl）或PBS（0.01 M，pH 7.4）。
试剂准备①试剂盒中的试剂平衡至室温。
②试剂四工作液：将试剂四加入37.5 mL双蒸水溶解，混匀即可，2-8℃避光保存2个月。
一氧化氮检测试剂盒(化学法)
一氧化氮检测试剂盒（化学法）说明书修订日期：2016.06.22 Cat number：KGT008Store at4℃for for6months,避光For Research Use Only（科研专用）一、实验原理一氧化氮NO(即血管内皮舒张因子)，在生物体内作为一种反应性极强的自由基，兼有第二信使和神经递质作用，同时又是一种效应分子，在体内具有广泛的生理作用，如松弛血管平滑肌，抑制血小板聚集，调节血流，介导细胞毒效应和免疫调节，参与学习和记忆、动脉粥样硬化等作用。
NO本身半衰期极短，血液中的NO主要由血管内皮细胞、血管平滑肌细胞、血小板、巨噬细胞等产生以硝酸盐的形式存在，通过其浓度可以间接测知NO浓度。
NO遇氧及水生成硝酸盐及亚硝酸盐，后二者遇硝酸盐显色剂可生成淡红色偶氮化合物，通过比色可间接测知NO浓度。
二、试剂盒组份组份KGT008（100assays）保存条件Buffer A100mL4℃Buffer B50mL室温或4℃试剂C粉剂一支4℃避光试剂D粉剂一支4℃避光Buffer E12mL室温亚硝酸钠标准液（2mmol/L）1mL4℃注意事项1．试剂C工作液配制：用时加入双蒸水至30mL，4℃避光保存。
（难溶，可以60℃隔水加热至溶解）2．试剂D工作液配制：用时加入双蒸水至12mL，4℃避光保存。
3．显色剂配制：试剂C工作液︰试剂D工作液︰Buffer E＝2.5︰1︰1的体积比配制（现配现用），配好的显色剂放冰箱保存，如颜色变得很深则不可再用。
4.亚硝酸钠标准液（20umol/L）配制：将亚硝酸钠标准液（2mmol/L）用双蒸水100倍稀释待用，现用现配。
三、操作步骤1．操作方法：于一次性塑料试管中按下表加入试剂试剂空白管标准管测定管双蒸水（mL）a﹡20μmoL/L亚硝酸钠标准液（mL）a﹡样本（mL）a﹡Buffer A（mL）0.80.80.8Buffer B（mL）0.40.40.4混匀后室温放置10分钟，3500～4000转/分，离心10分钟，取澄清的上清液上清液（mL）0.80.80.8显色剂（mL）0.40.40.4混匀,15分钟后,550nm比色，0.5cm光径比色杯，水调零，测各管OD值注意事项a﹡为取样量=标准品量=蒸馏水量)/()/20()mol/gprot (L gprot L mol OD OD OD OD NO 待测样本蛋白浓度标准管浓度值管－空白管标准管值空白管管测定管含量÷⨯-=μμ样品测试前稀释倍数标准管浓度值管－空白管标准管值空白管管测定管含量⨯⨯-=)/20()mol/L (L mol OD OD OD OD NO μμ四、计算1．计算公式血清：组织：2．计算举例(1)兔血清测定管OD 值为0.018，空白管OD 值为0.004，标准管OD 值为0.024。
碧云天生物技术一氧化氮检测试剂盒说明书
碧云天生物技术/Beyotime Biotechnology 订货热线：400-1683301或800-8283301 订货e-mail ：******************技术咨询：*****************网址：碧云天网站 微信公众号一氧化氮检测试剂盒产品编号 产品名称包装 S0021S 一氧化氮检测试剂盒 500次 S0021M一氧化氮检测试剂盒2500次产品简介：碧云天生产的一氧化氮检测试剂盒采用了经典的Griess Reagent ，并对其测定的溶液体系进行了优化，使检测下限达到1µM ，在1-100µM 范围内有非常完美的线性关系。
检测速度极快，完成一条标准曲线或5-10个样品的测定只需3分钟。
样品范围广，可以检测细胞或组织及其培养液中的一氧化氮的含量，酚红和10%血清均对测定无明显干扰，也可以检测血清、血浆和尿液中一氧化氮的含量。
包装清单：产品编号 产品名称 包装 S0021S-1 1M NaNO 2 1ml S0021S-2 Griess Reagent I 25ml S0021S-3 Griess Reagent II25ml —说明书1份产品编号 产品名称 包装 S0021M-1 1M NaNO 2 1ml S0021M-2 Griess Reagent I 125ml S0021M-3Griess Reagent II125ml —说明书1份保存条件：-20ºC 避光保存，一年有效。
4ºC 避光保存，半年有效。
注意事项：本产品对人体有害，操作时请小心，并注意有效防护以避免直接接触人体或吸入体内。
如保存不当导致溶液变色或沉淀，则说明该溶液已经失效，请购买新的试剂盒。
不建议使用RIPA 裂解液对细胞或者组织进行裂解，使用RIPA 裂解液可能在后续反应中产生沉淀，影响测试。
推荐使用碧云天的细胞与组织裂解液(一氧化氮检测用)(S3090)或Western 及IP 细胞裂解液(P0013)。
人一氧化氮NOELISA试剂盒使用方法
人一氧化氮(NO)ELISA试剂盒使用方法本试剂盒仅供研究使用。
检测范围：48T6μmol/L -200μmol/L使用目的：本试剂盒用于测定人血清、血浆及相关液体样本中一氧化氮(NO)含量。
实验原理本试剂盒应用双抗体夹心法测定标本中人一氧化氮(NO)水平。
用纯化的人一氧化氮(NO)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入一氧化氮(NO)，再与HRP 标记的一氧化氮(NO)抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。
TMB在HRP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一氧化氮(NO)呈正相关。
用酶标仪在450nm波长下测定吸光度（OD 值），通过标准曲线计算样品中人一氧化氮(NO)浓度。
1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。
若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。
操作步骤1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。
在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。
加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。
3.温育：用封板膜封板后置37℃温育30分钟。
4.配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。
6.加酶：每孔加入酶标试剂50μl，空白孔除外。
7.温育：操作同3。
8.洗涤：操作同5。
9.显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色15分钟.10.终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。
碧云天生物技术 Human IL-1β ELISA Kit 说明书
碧云天生物技术/Beyotime Biotechnology 订货热线：400-168-3301或800-8283301 订货e-mail ：******************技术咨询：*****************网址：碧云天网站 微信公众号Human IL-1β ELISA Kit产品编号 产品名称包装 PI305Human IL-1β ELISA Kit96次产品简介：碧云天的Human IL-1β ELISA Kit (Human Interleukin-1β Enzyme-Linked ImmunoSorbent Assay Kit)，即人白细胞介素1β酶联免疫吸附检测试剂盒，是一种用于特异性地高灵敏地定量检测人血清、血浆、细胞或组织裂解液、或细胞培养上清液中的IL-1β的ELISA 试剂盒。
本产品检测灵敏度高，特异性强，重复性好。
多次重复检测结果表明，最小检出量为2.2pg/ml ，与人IL-1r α、IL-1sRII 、IL-1sRI 、小鼠IL-1α、小鼠IL-1β等均没有交叉反应，板内、板间变异系数均小于10%。
IL-1即白细胞介素-1(简称白介1)，其家族由IL-1α(也称IL-1F1)、IL-1β(也称IL-1F2)、IL-1受体拮抗剂(IL-1 receptor antagonist, 简称IL-1RA, 或IL-1F3)及IL-18、IL-33和IL-1F5~F10组成。
IL-1主要由巨噬细胞产生，此外几乎所有的有核细胞，如B 细胞、NK 细胞、体外培养的T 细胞、角质细胞、树突状细胞、星形细胞、成纤维细胞、中性粒细胞、内皮细胞以及平滑肌细胞均可产生IL-1。
正常情况下只有皮肤、汗液及尿液中含有一定量的IL-1，绝大多数细胞在受到外来抗原或细菌内毒素刺激后才能合成和分泌IL-1。
IL-1β在免疫和炎症反应、骨重建(bone remodeling)、发烧、碳水化合物代谢等生理、病理过程中都有重要作用。
一氧化氮检测试剂盒(化学法)
一氧化氮检测试剂盒（化学法）说明书修订日期：2016.06.22 Cat number：KGT008Store at4℃for for6months,避光For Research Use Only（科研专用）一、实验原理一氧化氮NO(即血管内皮舒张因子)，在生物体内作为一种反应性极强的自由基，兼有第二信使和神经递质作用，同时又是一种效应分子，在体内具有广泛的生理作用，如松弛血管平滑肌，抑制血小板聚集，调节血流，介导细胞毒效应和免疫调节，参与学习和记忆、动脉粥样硬化等作用。
NO本身半衰期极短，血液中的NO主要由血管内皮细胞、血管平滑肌细胞、血小板、巨噬细胞等产生以硝酸盐的形式存在，通过其浓度可以间接测知NO浓度。
NO遇氧及水生成硝酸盐及亚硝酸盐，后二者遇硝酸盐显色剂可生成淡红色偶氮化合物，通过比色可间接测知NO浓度。
二、试剂盒组份组份KGT008（100assays）保存条件Buffer A100mL4℃Buffer B50mL室温或4℃试剂C粉剂一支4℃避光试剂D粉剂一支4℃避光Buffer E12mL室温亚硝酸钠标准液（2mmol/L）1mL4℃注意事项1．试剂C工作液配制：用时加入双蒸水至30mL，4℃避光保存。
（难溶，可以60℃隔水加热至溶解）2．试剂D工作液配制：用时加入双蒸水至12mL，4℃避光保存。
3．显色剂配制：试剂C工作液︰试剂D工作液︰Buffer E＝2.5︰1︰1的体积比配制（现配现用），配好的显色剂放冰箱保存，如颜色变得很深则不可再用。
4.亚硝酸钠标准液（20umol/L）配制：将亚硝酸钠标准液（2mmol/L）用双蒸水100倍稀释待用，现用现配。
三、操作步骤1．操作方法：于一次性塑料试管中按下表加入试剂试剂空白管标准管测定管双蒸水（mL）a﹡20μmoL/L亚硝酸钠标准液（mL）a﹡样本（mL）a﹡Buffer A（mL）0.80.80.8Buffer B（mL）0.40.40.4混匀后室温放置10分钟，3500～4000转/分，离心10分钟，取澄清的上清液上清液（mL）0.80.80.8显色剂（mL）0.40.40.4混匀,15分钟后,550nm比色，0.5cm光径比色杯，水调零，测各管OD值注意事项a﹡为取样量=标准品量=蒸馏水量)/()/20()mol/gprot (L gprot L mol OD OD OD OD NO 待测样本蛋白浓度标准管浓度值管－空白管标准管值空白管管测定管含量÷⨯-=μμ样品测试前稀释倍数标准管浓度值管－空白管标准管值空白管管测定管含量⨯⨯-=)/20()mol/L (L mol OD OD OD OD NO μμ四、计算1．计算公式血清：组织：2．计算举例(1)兔血清测定管OD 值为0.018，空白管OD 值为0.004，标准管OD 值为0.024。
Human kynurenine (KYN) ELISA Kit说明书
Human kynurenine(KYN)ELISA KitCatalog Number.CSB-E13659hFor the quantitative determination of endogenic human kynurenine(KYN) concentrations in serum,urine,tissue homogenates.This package insert must be read in its entirety before using this product.If You Have ProblemsTechnical Service Contact informationPhone:86-27-87582341Fax:86-27-87196150Email:****************Web:In order to obtain higher efficiency service,please ready to supply the lot number of the kit to us(found on the outside of the box).PRINCIPLE OF THE ASSAYThis assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antigen. Standards or samples are added to the appropriate microtiter plate wells with antibody specific for KYN and Horseradish Peroxidase(HRP)conjugated goat-anti-rabbit antibody.The competitive inhibition reaction is launched between with pre-coated KYN and KYN in samples.A substrate solution is added to the wells and the color develops in opposite to the amount of KYN in the sample.The color development is stopped and the intensity of the color is measured.DETECTION RANGE7.8pmol/ml-500pmol/ml.SENSITIVITYThe minimum detectable dose of human KYN is typically less than3.9pmol/ml. The sensitivity of this assay,or Lower Limit of Detection(LLD)was defined as the lowest human KYN concentration that could be differentiated from zero.SPECIFICITYThis assay has high sensitivity and excellent specificity for detection of human KYN.No significant cross-reactivity or interference between human KYN and analogues was observed.Note:Limited by current skills and knowledge,it is impossible for us to complete the cross-reactivity detection between human KYN and all the analogues, therefore,cross reaction may still exist.PRECISIONIntra-assay Precision(Precision within an assay):CV%<8%Three samples of known concentration were tested twenty times on one plate to assess.Inter-assay Precision(Precision between assays):CV%<10%Three samples of known concentration were tested in twenty assays to assess.LIMITATIONS OF THE PROCEDURE●FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTICPROCEDURES.●The kit should not be used beyond the expiration date on the kit label.●Do not mix or substitute reagents with those from other lots or sources.●If samples generate values higher than the highest standard,dilute thesamples with Sample Diluent and repeat the assay.●Any variation in Sample Diluent,operator,pipetting technique,washingtechnique,incubation time or temperature,and kit age can cause variation in binding.●This assay is designed to eliminate interference by soluble receptors,binding proteins,and other factors present in biological samples.Until all factors have been tested in the Immunoassay,the possibility of interference cannot be excluded.MATERIALS PROVIDEDReagents QuantityAssay plate1(96wells) Standard(Freeze dried)2Antibody(100x concentrate)1x60μl Antibody Diluent1x10mlHRP-conjugate(100x concentrate)1x120μlHRP-conjugate Diluent1x20ml Sample Diluent2x20mlWash Buffer(25x concentrate)1x20mlTMB Substrate1x10mlStop Solution1x10ml Adhesive Strip(For96wells)4Instruction manual1STORAGEUnopened kit Store at2-8°C.Do not use the kit beyond the expiration date.Opened kitCoated assayplateMay be stored for up to1month at2-8°C.Try to keep it in a sealed aluminum foil bag,and avoid the damp.Standard May be stored for up to1month at2-8°C.Ifdon’t make recent use,better keep it store at-20°C.AntibodyHRP-conjugateAntibodyDiluentMay be stored for up to1month at2-8°C. HRP-conjugateDiluentSample DiluentWash BufferTMB SubstrateStop Solution*Provided this is within the expiration date of the kit.OTHER SUPPLIES REQUIRED●Microplate reader capable of measuring absorbance at450nm,with thecorrection wavelength set at540nm or570nm.●An incubator which can provide stable incubation conditions up to37°C±0.5°C.●Squirt bottle,manifold dispenser,or automated microplate washer.●Absorbent paper for blotting the microtiter plate.●100ml and500ml graduated cylinders.●Deionized or distilled water.●Pipettes and pipette tips.●Test tubes for dilution.PRECAUTIONSThe Stop Solution provided with this kit is an acid solution.Wear eye,hand,face, and clothing protection when using this material.SAMPLE COLLECTION AND STORAGE●Serum Use a serum separator tube(SST)and allow samples to clot fortwo hours at room temperature or overnight at4°C before centrifugation for15minutes at1000×g.Remove serum and assay immediately or aliquot and store samples at-20°C or-80°C.Avoid repeated freeze-thaw cycles.●Urine Use a sterile container to collect urine samples.Remove anyparticulates by centrifugation for15minutes at1000xg,2-8°C and assay immediately or aliquot and store samples at-20°C or-80°C.Avoid repeated freeze-thaw cycles.Centrifuge again before assaying to remove any additional precipitates that may appear after storage.●Tissue Homogenates100mg tissue was rinsed with1X PBS,homogenized in1ml of1X PBS and stored overnight at-20°C.After two freeze-thaw cycles were performed to break the cell membranes,the homogenates were centrifuged for5minutes at5000x g,2-8°C.The supernate was removed and assayed immediately.Alternatively,aliquot and store samples at-20°C or-80°C.Centrifuge the sample again after thawing before the assay.Avoid repeated freeze-thaw cycles.Note:1.CUSABIO is only responsible for the kit itself,but not for the samplesconsumed during the assay.The user should calculate the possible amount of the samples used in the whole test.Please reserve sufficient samples in advance.2.Samples to be used within5days may be stored at2-8°C,otherwisesamples must be stored at-20°C(≤1month)or-80°C(≤2month)to avoid loss of bioactivity and contamination.3.Grossly hemolyzed samples are not suitable for use in this assay.4.If the samples are not indicated in the manual,a preliminary experiment todetermine the validity of the kit is necessary.5.Please predict the concentration before assaying.If values for these arenot within the range of the standard curve,users must determine the optimal sample dilutions for their particular experiments.6.Tissue or cell extraction samples prepared by chemical lysis buffer maycause unexpected ELISA results due to the impacts of certain chemicals.7.Owing to the possibility of mismatching between antigen from otherresource and antibody used in our kits(e.g.,antibody targets conformational epitope rather than linear epitope),some native or recombinant proteins from other manufacturers may not be recognized by our products.8.Influenced by the factors including cell viability,cell number and alsosampling time,samples from cell culture supernatant may not be detected by the kit.9.Fresh samples without long time storage are recommended for the test.Otherwise,protein degradation and denaturalization may occur in those samples and finally lead to wrong results.REAGENT PREPARATIONNote:●Kindly use graduated containers to prepare the reagent.Please don'tprepare the reagent directly in the Diluent vials provided in the kit.●Bring all reagents to room temperature(18-25°C)before use for30min.●Prepare fresh standard for each e within4hours and discardafter use.●Making serial dilution in the wells directly is not permitted.●Please carefully reconstitute Standards according to the instruction,andavoid foaming and mix gently until the crystals have completely dissolved.To minimize imprecision caused by pipetting,use small volumes and ensure that pipettors are calibrated.It is recommended to suck more than 10μl for once pipetting.●Distilled water is recommended to be used to make the preparation forreagents.Contaminated water or container for reagent preparation will influence the detection result.1.Antibody(1x)-Centrifuge the vial before opening.Antibody requires a100-fold dilution.A suggested100-fold dilution is10μl of Antibody+990μl of Antibody Diluent.2.HRP-conjugate(1x)-Centrifuge the vial before opening.HRP-conjugate requires a100-fold dilution.A suggested100-fold dilution is10μl of HRP-conjugate+990μl of HRP-conjugate Diluent.3.Wash Buffer(1x)-If crystals have formed in the concentrate,warm up toroom temperature and mix gently until the crystals have completely dissolved.Dilute20ml of Wash Buffer Concentrate(25x)into deionized or distilled water to prepare500ml of Wash Buffer(1x).4.StandardCentrifuge the standard vial at6000-10000rpm for30s before opening.Reconstitute the Standard with 1.0ml of Sample Diluent.Do not substitute other diluents.This reconstitution produces a stock solution of 500pmol/ml.Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of15minutes with gentle agitation prior to making dilutions.Pipette150μl of Sample Diluent into each tube(S0-S6).Use the stock solution to produce a2-fold dilution series(below).Mix each tube thoroughly before the next transfer.The undiluted Standard serves as the high standard(500pmol/ml).Sample Diluent serves as the zero standard (0pmol/ml).Tube S7S6S5S4S3S2S1S0pmol/ml50025012562.531.215.67.80ASSAY PROCEDUREBring all reagents and samples to room temperature before use.Centrifuge the sample again after thawing before the assay.It is recommended that all samples and standards be assayed in duplicate.1.Prepare all reagents,working standards,and samples as directed in theprevious sections.2.Refer to the Assay Layout Sheet to determine the number of wells to beused and put any remaining wells and the desiccant back into the pouch and seal the ziploc,store unused wells at4°C.3.Set a Blank well without any solution.4.Add50μl of standard and sample per well.Add50μl Antibody(1x)toeach well immediately(not to Blank well).Mix well with the pipette or shake the plate gently for60seconds.A plate layout is provided to record standards and samples assayed.5.Cover with the adhesive strip provided.Incubate for40minutes at37°C.6.Aspirate each well and wash,repeating the process two times for a total ofthree washes.Wash by filling each well with Wash Buffer(200μl)using a squirt bottle,multi-channel pipette,manifold dispenser,or autowasher, and let it stand for2minutes,complete removal of liquid at each step is essential to good performance.After the last wash,remove any remaining Wash Buffer by aspirating or decanting.Invert the plate and blot it against clean paper towels.7.Add100μl HRP-conjugate(1x)to each well immediately(not to Blankwell).Cover with the adhesive strip provided.Incubate for30minutes at 37°C.8.Repeat the aspiration/wash process for five times as in step6.9.Add90μl of TMB Substrate to each well.Incubate for20minutes at37°C.Protect from light.10.Add50μl of Stop Solution to each well,gently tap the plate to ensurethorough mixing.11.Determine the optical density of each well within5minutes,using amicroplate reader set to450nm.If wavelength correction is available,set to540nm or570nm.Subtract readings at540nm or570nm from the readings at450nm.This subtraction will correct for optical imperfections in the plate.Readings made directly at450nm without correction may be higher and less accurate.Note:1.The final experimental results will be closely related to validity of theproducts,operation skills of the end users and the experimental environments.2.Samples or reagents addition:Please use the freshly prepared Standard.Please carefully add samples to wells and mix gently to avoid foaming.Do not touch the well wall as possible.For each step in the procedure,total dispensing time for addition of reagents or samples to the assay plate should not exceed10minutes.This will ensure equal elapsed time for each pipetting step,without interruption.Duplication of all standards and specimens,although not required,is recommended.To avoid cross-contamination,change pipette tips between additions of each standard level,between sample additions,and between reagent additions.Also,use separate reservoirs for each reagent.3.Incubation:To ensure accurate results,proper adhesion of plate sealersduring incubation steps is necessary.Do not allow wells to sit uncovered for extended periods between incubation steps.Once reagents have been added to the well strips,DO NOT let the strips DRY at any time during the assay.Incubation time and temperature must be observed.4.Washing:The wash procedure is plete removal of liquid at eachstep is essential to good performance.After the last wash,remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate.Insufficient washing will result in poor precision and falsely elevated absorbance reading.When using an automated plate washer,adding a30second soak period following the addition of wash buffer,and/or rotating the plate180degrees between wash steps may improve assay precision.5.Controlling of reaction time:Observe the change of color after adding TMBSubstrate(e.g.observation once every10minutes),TMB Substrate should change from colorless or light blue to gradations of blue.If the color is too deep,add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.6.TMB Substrate is easily contaminated.TMB Substrate should remaincolorless or light blue until added to the plate.Please protect it from light. 7.Stop Solution should be added to the plate in the same order as the TMBSubstrate.The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution.Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the TMB Substrate.ASSAY PROCEDURE SUMMARY*Please determine whether the sample needs to be diluted or the optimal dilution factor based on preliminary experiment result.CALCULATION OF RESULTSUsing the professional soft"Curve Expert"to make a standard curve is recommended,which can be downloaded from our web.Average the duplicate readings for each standard and sample and subtract the average optical density of Blank.Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic(4-PL)curve-fit.As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph.The data may be linearized by plotting the log of the KYN concentrations versus the log of the O.D.and the best fit line can be determined by regression analysis.This procedure will produce an adequate but less precise fit of the data.If samples have been diluted,the concentration read from the standard curve must be multiplied by the dilution factor.人犬尿氨酸（KYN）酶联免疫试剂盒使用说明书【产品编号】CSB-E13659h【预期应用】ELISA法定量测定人血清、尿液、组织裂解液中KYN含量。
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人试剂盒说明书人血清一氧化氮NOELISA检测试剂
盒
人试剂盒说明书,人血清一氧化氮（NO）ELISA检测试剂盒樊克生物专业供应：
使用目的：本试剂盒用于测定人血清、血浆及相关液体样本血清
试验原理：
NO试剂盒是固相夹心法酶联免疫吸附实验（ELISA）.已知NO浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。
先将NO和生物素标记的抗体同时温育。
洗涤后，加入亲和素标记过的HRP。
再经过温育和洗涤，去除未结合的酶结合物，然后加入底物A、B，和酶结合物同时作用。
产生颜色。
颜色的深浅和样品中NO的浓度呈比例关系。
试剂盒内容及其配制
自备材料
1．蒸馏水。
2．加样器：5ul、10ul、50ul、100ul、200ul、500ul、1000ul。
3．振荡器及磁力搅拌器等。
安全性
1．避免直接接触终止液和底物A、B。
一旦接触到这些液体，请尽快用水冲洗。
2．实验中不要吃喝、抽烟或使用化妆品。
3．不要用嘴吸取试剂盒里的任何成份。
操作注意事项
1．试剂应按标签说明书储存，使用前恢复到室温。
稀稀过后的标准品应丢弃，不可保存。
2．实验中不用的板条应立即放回包装袋中，密封保存，以免变质。
3．不用的其它试剂应包装好或盖好。
不同批号的试剂不要混用。
保质前使用。
4．使用一次性的吸头以免交叉污染，吸取终止液和底物A、B液时，避免使用带金属部分的加样器。
5．使用干净的塑料容器配置洗涤液。
使用前充分混匀试剂盒里的各种成份及样品。
6．洗涤酶标板时应充分拍干，不要将吸水纸直接放入酶标反应孔中吸水。
7．底物A应挥发，避免长时间打开盖子。
底物B对光敏感，避免长时间暴露于光下。
避免用手接触，有毒。
实验完成后应立即读取OD值。
8．加入试剂的顺序应一致，以保证所有反应板孔温育的时间一样。
9．按照说明书中标明的时间、加液的量及顺序进行温育操作。
样品收集、处理及保存方法
1、血清－－－－-操作过程中避免任何细胞刺激。
使用不含热原
和内毒素的试管。
收集血液后，1000×g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、肝素血浆可用于检测。
1000
×g离心30分钟去除颗粒。
3、细胞上清液－－-1000×g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。
1000×g
离心10分钟，取上清液
5、保存－－－－－－如果样品不立即使用，应将其分成小部分-
70 ℃保存，避免重复冷冻。
尽可能的不要使用溶血或高血脂
血。
如果血清中大量颗粒，检测前先离心或过滤。
不要在37℃
或更高的温度加热解冻。
应在室温下解冻并确保样品均匀地充分解冻。
试剂的准备
1．标准品：标准品的系列稀释应在实验时准备，不能储存。
稀释前将标准品振荡混匀。
稀释比例按下表中进行：
2．洗涤缓冲液（50×）的稀释：蒸馏水50倍稀释。
操作步骤
1．使用前，将所有试剂充分混匀。
不要使液体产生大量的泡沫，以免加样时加入大量的气泡，产生加样上的误差。
2．根据待测样品数量加上标准品的数量决定所需的板条数。
每个标准品和空白孔建议做复孔。
每个样品根据自己的数量来。