ELISA试剂盒说明书,人骨髓抑制因子1elisa试剂盒

elisa试剂盒原理
Elisa试剂盒是一种常用的生化实验工具，用于检测特定物质
的存在和浓度。

其原理基于酶联免疫吸附实验（Enzyme-Linked Immunosorbent Assay，简称Elisa）。

Elisa试剂盒通常由多个试剂组成，包括包被抗原、检测抗体、酶标记的二抗和底物等。

首先，将要检测的被测物（如蛋白质、抗原等）固定在试板或试管的表面。

随后，向试样中加入检测抗体，该抗体与被测物具有高度的特异性结合。

接下来，加入酶标记的二抗，这个二抗能够与检测抗体结合。

二抗中的酶（如辣根过氧化物酶或碱性磷酸酶）使得酶标记的二抗能够与检测抗体结合在一起。

然后，通过洗涤，将非特异性结合的物质去除，以减少背景干扰。

最后，加入适当的底物，使酶被检测到并产生染色反应。

底物分解后产生的光学信号可以被读数仪器测量，信号的强度与被测物的浓度成正比。

Elisa试剂盒不仅可以用于科学研究，还广泛应用于医学诊断、药物检测和食品安全监测等领域。

通过改变试剂盒中的抗体和底物，Elisa可以灵活地检测各种不同的物质。

其简单、快速、灵敏和可靠的特点使其成为生命科学研究中的重要工具。

人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒供应商：上海乔羽生物有限公司上海乔羽有限公司，有elisa试剂盒，抗体，培养基人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒，猪促生长激素释放激素(GHRH)ELISA试剂盒，促生长激素释放激素elisakit，猪elisa试剂盒，GHRHelisakit上海乔羽生物供应！人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒Kit performance1 sensitivity: the minimum detection concentration is less than 1 standard. Linearity of dilution. Sample linear regression and the expected concentration correlation coefficient R value is 0.990.2 specificity: no reaction with other cytokines.3 repeatability: the variation coefficient of the plate and the plate are all less than 10%.Result judgment and analysis1, the instrument value: Yu Bo 450nm ELISA od read the hole on the instrument2, to the OD value for the longitudinal axis (y), corresponding HLA-B27 standard concentration as the abscissa (x), do the corresponding curve, HLA-B27 content in the sample can be according to its OD value by standard curve conversion out corresponding concentration.3, the detection value range: 0-8.0IU/ml4, sensitivity: 0.01 IU/ml人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒Elisa kit test rules:1, to ensure the accuracy of the gun, the error can not be more than 2%. Available water and electronic balances are determined. But it's better to have professional personnel to correct it.2, to be equipped with 20ul, 50ul, 100ul, 1000ul and a volley. Draw different liquids, to replace the gun head. Even when the standard is drawn.1, 3 hours before the experiment to remove the kit from the refrigerator, so that a variety of reagents are restored to room temperature, in order to make the results more stable.4, the experiment, to make the substrate to avoid light storage.5, with the gun to draw the liquid speed can not be too fast, so as not to produce bubbles and to absorb the amount is not accurate.6, when the liquid, to use the range and the need to close the gun to suck, reduce the error.7, when the liquid is added to the enzyme standard hole, the liquid contact of the liquid drop and the hole wall of the gun head can be avoided by avoiding the contact of the liquid drop and the hole wall in the gun head.8, after all the liquid added, the enzyme labeled plate on the table in parallel to gently shake 30 seconds, mixed with liquid. Can also use the shaking function of the enzyme standard instrument.9, should try to do two experiments, so as to ensure the accuracy of the data. 10, the results have questions about the sample to be confirmed by other methods.人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒Operation steps1 before use, all reagents fully mixed. Do not allow liquid to produce a large number of bubbles, so as to avoid adding a large number of bubbles, resulting in the addition of the error.2 according to the number of samples to be measured and the number of standard products to determine the number of required. Each standard and blank hole is recommended to do the hole. Each sample can be made according to its own quantity, and can be used as a hole in the hole.3 to join the dilution of the standard 50ul in the reaction hole, to add the sample to be measured in the reaction hole 50ul. The biotin labeled antibody was immediately added to the 50ul. Cover the membrane plate, gently oscillating mixing, 37 degrees Celsius incubation 1 hours.4 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If the washing machine with washing, washing times increased once.5 each hole to join 80ul affinity chain enzyme -HRP, gently oscillating mixing, 37 degrees Celsius incubation 30 minutes.6 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If the washing machine with washing, washing times increased once.7 each hole to join the substrate A, B each 50ul, gently oscillating mixing, 37 degrees Celsius incubation 10 minutes. Avoid light.8 remove the enzyme labeled plate, quickly join the 50ul terminator, add the end of the liquid should be measured immediately after the results.9 OD value at the wavelength of 450nm was measured by hole.limitThe result of the above 6 standard is nonlinear, and the result can not be obtained accurately according to the standard curve.人(Amylase)ELISA试剂盒说明书，淀粉酶ELISA试剂盒注意事项：1．试剂盒从冷藏环境中取出应在室温平衡15-30分钟后方可使用，酶标包被板开封后如未用完，板条应装入密封袋中保存。

产品信息和操作指南Human IgE预包被 ELISA kit Cat# : DKW12-1820-048 / DKW12-1820-096本试剂盒专用于科研，而非用于诊断Human IgEDKW12-1820目录产品简介 (1)知识背景 (1)试剂盒提供的试剂 (2)需要实验者自行准备的试剂与仪器 (2)注意事项 (3)试剂的配制 (5)操作过程 (7)结果分析 (9)试剂盒的保存 (9)操作步骤一览表 (10)参考文献 (11)ELISA测定中可能会出现的问题及解决方法 (12)预包被ELISA 试剂盒系列产品 (15)1、产品简介：达优®人IgE ELISA试剂盒是通过酶联免疫吸附技术，体外定量检测人血清、血浆、缓冲液或细胞培养液中的IgE，可同时检测天然的和重组的IgE。

本试剂盒为预包被板，整个过程孵育时间不超过4小时，洗涤9次。

本试剂盒专用于科研，而非用于诊断。

使用前请仔细阅读说明书并检查试剂盒组分，若有任何疑问请与达科为生物工程有限公司联系，E-mail：*************.检测范围：32-0.5 ng/mL灵敏度：0.1ng/mL重复性：板内、板间变异系数均＜10％。

2、知识背景：抗体根据抗原性和抗体重链分为IgG、IgA、IgM、IgE和IgD 五种。

IgE是介导I型变态反应的重要抗体，在支气管哮喘、鼻炎、食物过敏、药物、病原体等特应性疾病的发病过程中发挥着关键作用。

而抗IgE单克隆抗体可以与血液中IgE结合，降低血清总IgE水平，并下调肥大细胞、嗜碱性粒细胞表面的IgE高亲和力受体FcεRI的表达，从而抑制了IgE介导的免疫反应（1-6）。

3、试剂盒提供的试剂：试剂规格配制Cytokine standard 2/1瓶* 干粉状，按瓶上说明操作Biotinylated antibody 2/1瓶* 1：100用Dilution buffer R（1×）稀释Streptavidin-HRP 2/1瓶* 1：100用Dilution buffer R（1×）稀释Dilution buffer R（1×） 3/2瓶* 即用型Washing buffer（50×）1瓶 150∶用蒸馏水稀释TMB 1瓶即用型Stop solution 1瓶即用型Precoated ELISA plate 8×12或8×6*即用型封板膜 2/1张* 即用型说明书1份*：96/48 Tests4、需要实验者自行准备的试剂与仪器：1．酶标仪（建议参考仪器使用说明提前预热）2．微量加液器及吸头：P10，P50，P100，P200，P1000 3．蒸馏水或去离子水4．全新滤纸5．旋涡振荡器和磁力搅拌器5、注意事项：1．试剂应按瓶上标签说明储存，使用前室温平衡20-30分钟。

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。

PRRSV N蛋白抗体检测间接ELISA方法的建立1 材料：1.1 蛋白、血清、酶标二抗原核表达PRRSV N蛋白；标准阳性血清和阴性血清，待检血清；自制HRP标记兔抗猪IgG；1.2 主要试剂和溶液包被液（50 mmol/L pH9.6的碳酸盐缓冲液）Na2CO3 1.59 gNaHCO3 2.93 g准确称取后，溶于950 mL的蒸馏水中，调pH值为9.6，定容到1 L；PBS-T洗涤液1000 mL 10 mmol/L pH7.4的PBS中加入0.5 mL Tween-20；封闭液和血清稀释液含10%小牛血清的PBS-T缓冲液，10%马血清的PBS-T缓冲液，含5%BSA的PBS-T缓冲液，含10%BSA的PBS-T缓冲液，含5%脱脂奶的PBS-T缓冲液；底物溶液100 mmol/L的柠檬酸溶液（21 g柠檬酸C6H6O7•H2O溶于去离子水，定容至1 L）24.3 mL，200 mmol/L Na2HPO4•12H2O（71.6 g Na2HPO4•12H2O溶于去离子水，定容至1 L）25.7 mL混匀，加入50 mg的四甲基联苯胺（TMB），临用前加入50 μL的30% H2O2；终止液（2 mol/L H2SO4）每200 mL终止液，由蒸馏水177.8 mL和浓硫酸22.2 mL 混和而成。

1.3 主要仪器设备电热恒温培养箱；4℃冰箱；酶标仪。

2 步骤：2.1抗原包被浓度和二抗稀释度的确定将原核表达的N蛋白分别作2.0 μg/mL，1.0 μg/mL，0.5 μg/mL，0.25 μg/mL，0.1μg/mL，0.05 μg/mL连续稀释6个稀释度，每个稀释度重复两孔，100 μL/孔，4℃包被酶标反应板过夜。

将自制HRP标记的兔抗猪二抗分别做1:50、1:100、1:200和1:400一系列倍比稀释，南京金益柏生物技术有限公司Tel:025-********Fax*************每个稀释度重复两孔，100 μL/孔，组成方阵确定重组蛋白的最佳包被浓度和二抗稀释度。

人蛋白C抑制剂 (PCI)酶联免疫吸附测定试剂盒使用说明书产品编号：E-EL-H1201（本试剂盒仅供体外研究使用、不用于临床诊断!）声明：尊敬的客户，感谢您选用本公司的产品。

本产品选用世界著名生产厂家的原料，采用专业ELISA kit生产技术制造。

适用于体外定量检测人血清、血浆、组织匀浆或细胞培养上清液中天然和重组PCI浓度。

使用前请仔细阅读说明书并检查试剂组分！如有疑问，请及时联系伊莱瑞特生物科技有限公司。

*: [96T/48T]（打开包装后请及时检查所有物品是否齐全完整）检测原理:本试剂盒采用双抗体夹心ELISA法。

用抗人PCI抗体包被于酶标板上，实验时标本或标准品中的PCI会与包被抗体结合，游离的成分被洗去。

依次加入生物素化的抗人PCI抗体和辣根过氧化物酶标记的亲和素。

抗人PCI抗体与结合在包被抗体上的人PCI结合、生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去。

加入显色底物(TMB)，TMB在辣根过氧化物酶的催化下现蓝色，加终止液后变黄。

用酶标仪在450nm波长处测OD值，PCI浓度与OD450值之间呈正比，通过绘制标准曲线求出标本中PCI的浓度。

标本收集:1.血清：全血标本于室温放置2小时或4℃过夜后于1000×g离心20分钟，取上清即可检测，收集血液的试管应为一次性的无热原，无内毒素试管。

2.血浆：抗凝剂推荐使用EDTA.Na2，标本采集后30分钟内于1000×g离心15分钟，取上清即可检测。

避免使用溶血，高血脂标本。

3.组织匀浆：用预冷的PBS (0.01M, pH=7.4)冲洗组织，以去除残留血液（匀浆中裂解的红细胞会影响测量结果），称重后将组织剪碎。

将剪碎的组织与对应体积的PBS（一般按1:9的重量体积比，比如1g的组织样本对应9mL的PBS，具体体积可根据实验需要适当调整，并做好记录。

推荐在PBS中加入蛋白酶抑制剂）加入玻璃匀浆器中，于冰上充分研磨。

ELISA检测试剂盒使用指南ELISA（Enzyme-Linked Immunosorbent Assay）是一种常用的免疫学实验技术，用于检测体内特定抗原或抗体的存在和浓度。

ELISA检测试剂盒是用于进行ELISA实验的组合套装，包括抗体或抗原、酶标记物、底物、缓冲液等。

本文将介绍ELISA检测试剂盒的使用指南，以帮助用户顺利进行ELISA实验。

1.准备实验材料：-ELISA检测试剂盒：根据实验需要选择适当的ELISA检测试剂盒，确保其在储存和运输过程中无损坏。

-样本：准备需要检测的样本，如血清、尿液、组织提取物等。

确保样本的质量和浓度满足实验要求。

-微孔板：根据试剂盒的要求选择合适的微孔板，同时注意其质量有无污染。

-基本实验设备：如计量器、洗板机、显微镜等。

2.实验步骤：-取出储存在4℃的试剂，确保其达到室温（一般为20-25℃），再根据实验步骤进行下一步操作。

-预处理样本：根据试剂盒的说明书，进行样本的预处理，包括稀释、纯化、稳定化等步骤。

-加样：将样本按照试剂盒要求的体积加入微孔板中，通常每个孔需要加入100-200μL的样品。

-洗板：使用洗板机或手工操作，将孔中的杂质洗净。

洗板时应注意洗涤缓冲液的使用浓度和洗涤次数。

-加入特异性抗体：根据试剂盒的说明书，将稀释好的特异性抗体加入各孔中，确保操作的准确性和每孔的稀释倍数。

-洗板：重复第4步的操作，将未结合的抗体洗净。

-加入酶标记物：根据试剂盒的说明书，将稀释好的酶标记物加入各孔中，确保加入的量准确无误。

-洗板：重复第4步的操作，将未结合的酶标记物洗净。

-加入底物：将稀释好的底物加入各孔中，使其与酶标记物反应，生成可视化的颜色。

-终止反应：根据试剂盒的要求，在一定的反应时间后，加入终止剂停止反应。

终止剂的选择需符合试剂盒的要求。

-检测结果：使用酶标仪或目视观察检测孔中的颜色变化，通过测量吸光度或颜色强度来确定样本中目标物的浓度。

3.数据分析：-计算样本的结果：根据试剂盒的说明书，根据吸光度或颜色强度测量结果，计算样本中目标物的浓度。

本试剂盒只能用于科学研究，不得用于医学诊断。

人P21蛋白（P21）定量检测试剂盒（ELISA）使用说明书【试剂盒名称】人P21蛋白（P21）定量检测试剂盒（ELISA）【试剂盒用途】定量检测人血清、血浆及相关液体样本中P21蛋白（P21）的含量。

【检测原理】本试剂盒采用双抗体两步夹心酶联免疫吸附法（ELISA）。

将标准品、待测样本加入到预先包被人P21蛋白（P21）单克隆抗体透明酶标包被板中，温育足够时间后，洗涤除去未结合的成分，再加入酶标工作液，温育足够时间后，洗涤除去未结合的成分。

依次加入底物A、B，底物（TMB）在辣根过氧化物酶（HRP）催化下转化为蓝色产物，在酸的作用下变成黄色，颜色的深浅与样品中人P21蛋白（P21）浓度呈正相关，450nm波长下测定OD值，根据标准品和样品的OD值，计算样本中人P21蛋白（P21）含量。

【试剂盒组成】1酶标包被板12孔×8条7显色剂A液6mL2标准品0.3mL×6管8显色剂B液6mL320倍浓缩洗涤液25mL9终止液6mL4样本稀释液6mL10说明书1份5特殊稀释液6mL11封板膜2张6酶标试剂6mL12密封袋1个备注：标准品（1号→6号）浓度依次为：48、24、12、6、3、1.5ng/L【需要而未提供的试剂和器材】1、37℃恒温箱2、标准规格酶标仪3、精密移液器及一次性吸头4、蒸馏水5、一次性试管6、吸水纸【操作步骤】1、准备：从冰箱取出试剂盒，室温复温平衡30分钟。

2、配液：用蒸馏水将20倍浓缩洗涤液稀释成原倍的洗涤液。

3、加标准品和待测样本：取足够数量的酶标包被板，固定于框架上，分别设置标准品孔、待测样本孔和空白对照孔，记录各孔位置，在标准品孔中加入标准品50μL；待测样本孔中先加入待测样本10μL，再加样本稀释液40μL（即样本稀释5倍）；空白对照孔不加。

4、温育：37℃水浴锅或恒温箱温育30min。

5、洗板：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4次（也可用洗板机按说明书操作洗板）。

仅供科研使用，不得用于临床检验。

人促红细胞生成素（EPO ）酶联免疫试剂盒（ELISA试剂盒）说明书黄石市艾恩斯生物科技有限公司【产品名称】通用名称：人促红细胞生成素（EPO ）酶联免疫试剂盒（ELISA试剂盒）英文名称：Human Erythropoietin（EPO ）ELISA KIT【包装规格】48人份/盒，96人份/盒【预期用途】仅供科研使用，定量检测血清、血浆、细胞培养上清液中人促红细胞生成素（EPO ）的浓度。

【检验原理】本试剂盒采用双抗体夹心酶联免疫吸附试验（ELISA）。

在预包被抗人促红细胞生成素（EPO ）抗体（固相抗体）的微孔酶标板中，加入人促红细胞生成素（EPO ）校准品和待测样本，再加入另一株HRP标记的抗人促红细胞生成素（EPO ）抗体（酶标抗体），经过温育与充分洗涤，去除未结合的组分，在微孔板固相表面形成固相抗体-抗原-酶标抗体的夹心复合物。

加底物A和B，底物在HRP催化下，产生蓝色产物，在终止液（2M 硫酸）作用下，最终转化为黄色，在酶标仪上测定吸光度（OD值），吸光度（OD值）与待测样品中人促红细胞生成素（EPO ）的浓度正相关。

拟合校准品曲线，可以计算出样本中人促红细胞生成素（EPO ）的浓度。

【主要组成成分】主要成分校准品检测范围：47-3000pg/ml。

校准品已经通过测试，结果表明HBs抗原阴性，HIV1、HIV2和HCV抗体阴性，由于不存在一种试验方法能够完全保证没有这些物质，本品必须按照具有潜在的感染性进行处理，处理过程应当遵循通用的安全措施。

需要但未提供的材料及耗材1、酶标仪2、精密移液器及一次性吸头3、蒸馏水4、洗瓶或者自动洗板机5、37℃水浴锅或恒温箱6、500ml量筒7、无粉一次性乳胶手套【储存条件及有效期】1、2-8℃保存，切勿冷冻，有效期6个月。

2、开封使用后，包被微孔板放入带有干燥剂的自封袋中，密闭自封袋，并将全部试剂放回2-8℃冰箱。

本试剂盒只能用于科学研究，不得用于医学诊断小鼠小鼠（（MouseMouse））肿瘤坏死因子可溶性受体肿瘤坏死因子可溶性受体ⅠⅠ（TNFsR-TNFsR-ⅠⅠ）ELISA检测试剂盒使用说明书检测原理试剂盒采用双抗体一步夹心法酶联免疫吸附试验（ELISA）。

往预先包被肿瘤坏死因子可溶性受体Ⅰ（TNFsR-Ⅰ）抗体的包被微孔中，依次加入标本、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肿瘤坏死因子可溶性受体Ⅰ（TNFsR-Ⅰ）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、200-1000uL3.37℃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.浓度为0的S0号标准品即可视为阴性对照或者空白；按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。

人白血病抑制因子受体(LIFR)elisa试剂盒使用说明书Elisa kit规格：48孔配置/96孔配置标准品稀释液：1.5ml×1瓶酶标试剂：3 ml×1瓶（48）/6 ml×1瓶（96）【人白血病抑制因子受体(LIFR)试剂盒】本试剂仅供研究使用计算：以标准物的浓度为横坐标，OD值为纵坐标，在坐标纸上绘出标准曲线，根据样品的OD值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。

试剂盒组成：封板膜:2片（48）/2片（96）说明书:1份密封袋:1个标准品：2700ng/L 0.5ml×1瓶0.5ml×1瓶2-8℃保存酶标包被板: 1×48 1×96 2-8℃保存样品稀释液: 3ml×1瓶 6 ml×1瓶2-8℃保存显色剂A液: 3ml×1瓶 6 ml×1瓶2-8℃保存显色剂B液: 3ml×1瓶 6 ml×1瓶2-8℃保存终止液: 3ml×1瓶6ml×1瓶2-8℃保存浓缩洗涤液:（20ml×20倍）×1瓶（20ml×30倍）×1瓶2-8℃保存实验原理：本试剂盒应用双抗体夹心法测定标本中人白血病抑制因子受体(LIFR)水平。

用纯化的人白血病抑制因子受体(LIFR)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白血病抑制因子受体(LIFR)，再与HRP标记的白血病抑制因子受体(LIFR)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白血病抑制因子受体(LIFR)呈正相关。

人抗人类免疫缺陷病毒抗体ELISA试剂盒说明书人抗人类免疫缺陷病毒抗体ELISA试剂盒说明书产品规格：96T/48T供应商：上海樊克生物有限公司本产品只用于科研实验人抗人类免疫缺陷病毒抗体ELISA试剂盒说明书，小鼠(LR/Ob-R)ELISA试剂盒，苗条素受体elisakit elisa试剂盒上海樊克生物供应！人抗人类免疫缺陷病毒抗体ELISA试剂盒说明书Liquid type specimen:Including serum, plasma, urine, pleural effusion and ascites, cerebrospinal fluid, cell culture supernatant, etc..1) serum:Room temperature blood natural coagulation 10-20 minutes after the centrifuge about 20 minutes (2000-3000 turn / min). Carefully collect the supernatant. In the process of preservation, if there is precipitation, should be re - centrifugal.2) plasma:According to the requirements of the specimens of choice of EDTA, sodium citrate or heparin as an anticoagulant, 10-20 minutes after mixing, centrifuged for 20 minutes or so (2000-3000 r.p.m.). Carefully collect the supernatant. In the process of preservation, if there is precipitation, should be re - centrifugal.3) urine:Collecting with sterile tubes. Centrifugal 20 minutes or so (2000-3000 turn / min). Carefully collect the supernatant. In the process of preservation, if there is precipitation, should be re - centrifugal. Pleural effusion, cerebrospinal fluid with reference to this practice.4) cell culture supernatant:To collect the ingredients for the detection of secretory components. Centrifugal 20 minutes or so (2000-3000 turn / min). Carefully collect the supernatant. Detection of cell components, with PBS (PH7.2-7.4) diluted cell suspension, the cell concentration reached 1 million /ml or so. Through repeated freezing and thawing, in order to make the cell damage and release of intracellular components. Centrifugal 20 minutes or so (2000-3000 turn / min). Carefully collect the supernatant. In the process of preservation, if there is precipitation, should be re - centrifugal.5) tissue specimen:After cutting the specimen, weigh the weight. Add a certain amount of PH7.4, PBS. Rapid freezing with liquid nitrogen. The specimens were melted and still maintained at 2-8. Add a certain amount of PBS (PH7.4), with a hand or a homogenate of the specimen homogenate full. Centrifugal 20 minutes or so (2000-3000 turn / min). Carefully collect the supernatant. Be detected after a repackaging, remaining frozen spare.人抗人类免疫缺陷病毒抗体ELISA试剂盒说明书国产/进口Elisa试剂盒，96T/48TElisa试剂盒， 96T/48Telisakit价格人抗人类免疫缺陷病毒抗体ELISA试剂盒说明书Elisa试剂盒操作步骤：3. 温育：用封板膜封板后置37℃温育30分钟。

ELISA检测试剂盒使用指南ELISA（酶联免疫吸附试验）是一种常用的免疫学实验方法，用于检测病原体、抗体或其他分子的存在和浓度。

它具有高灵敏度、高特异性、易操作和较低成本的优势，被广泛应用于生物医学研究、临床诊断和免疫学领域。

本篇文章将介绍ELISA检测试剂盒的使用指南，包括实验准备、试剂的使用步骤和结果解读。

一、实验准备1.阅读检测项目的说明书：在使用试剂盒前，仔细阅读说明书，了解试剂的使用方法、灵敏度和特异性等关键信息。

2.样本准备：根据实验要求，准备样本。

如果需要检测血清中的抗体水平，可以采集血液样本，离心分离血清；如果需要检测细胞培养上清中的分子，将上清收集，并使其清晰，避免细胞或杂质的污染。

3.样本预处理：根据实验需求，对样本进行必要的预处理。

例如，可以通过加热、稀释、酶解等方式来处理样本，以改变样本组分和浓度。

4.准备品质控制样品：制备阳性和阴性控制样品，用于评估试剂盒和实验操作的稳定性和准确性。

5.实验器材准备：准备所需实验器材，如酶标板、移液器、微孔板洗涤仪等。

确保器材的干净和完整，并按照说明书要求对其进行预处理。

二、试剂使用步骤1.试剂准备：根据说明书要求，将试剂从冰箱中取出，并在室温下放置一段时间使其恢复到室温。

确保试剂瓶盖紧闭，并避免暴露在直接阳光下。

2.实验操作：按照说明书的要求，将试剂加入到酶标板中，并根据实验设计进行标准曲线的设置。

标准曲线用于测量未知样品的数量，并计算出浓度。

3.孵育：根据试剂盒的要求，将酶标板放入孵育箱中进行孵育。

孵育温度和时间应根据实验要求进行调整。

4.洗涤：使用洗涤缓冲液对酶标板上的不特异性结合物进行洗涤。

洗涤过程应准确控制洗涤孔板次数和洗涤液的体积。

5.补液：在洗涤完成后，加入辣根过氧化物酶标况稀释液，促进酶标物与特异性结合物的反应。

6.孵育：根据试剂盒的要求，将酶标板放入孵育箱中进行二次孵育。

7.反应停止：根据试剂盒的要求，加入相应的停止液，停止酶反应。

人组织因子（TF）ELISA分析检测试剂盒使用说明书人组织因子（TF）ELISA分析检测试剂盒使用说明书本试剂仅供研究使用目的：本试剂盒用于测定小鼠血清，血浆及相关液体样本中转化生长因子（TF）的含量。

实验原理：本试剂盒应用双抗体夹心法测定标本中人组织因子（TF）水平。

用纯化的转化生长因子（TF）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转化生长因子（TF），再与HRP标记的羊抗鼠抗体结合，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转化生长因子（TF）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组织因子（TF）浓度。

试剂盒内容及其配制：试剂盒成份96孔配置48孔配置96/48人份酶标板1块板（96T）半块板（48T）塑料膜板盖1块半块标准品：100ng/ml 1瓶（1.0ml）1瓶（0.5ml）空白对照1瓶（1.0ml）1瓶（0.5ml）标准品稀释缓冲液1瓶（8.0ml）1瓶（4.0ml）生物素标记的抗OT抗体1瓶（8.0ml）1瓶（4.0ml）亲和链酶素-HRP 1瓶（12ml）1瓶（5ml）洗涤缓冲液1瓶（20ml）1瓶（10ml）底物A 1瓶（6.0ml）1瓶（3.0ml）底物B 1瓶（6.0ml）1瓶（3.0ml）终止液1瓶（6.0ml）1瓶（3.0ml）试剂盒组成：试剂盒组成48孔配置96孔配置保存说明书1份1份封板膜2片（48）2片（96）密封袋1个1个酶标包被板1×48 1×96 2-8℃保存标准品：45μmol/L0.5ml×1瓶0.5ml×1瓶2-8℃保存标准品稀释液1.5ml×1瓶1.5ml×1瓶2-8℃保存酶标试剂3 ml×1瓶6 ml×1瓶2-8℃保存样品稀释液3 ml×1瓶6 ml×1瓶2-8℃保存显色剂A液3 ml×1瓶6 ml×1瓶2-8℃保存显色剂B液3 ml×1瓶6 ml×1瓶2-8℃保存终止液3ml×1瓶6ml×1瓶2-8℃保存浓缩洗涤液（20ml×20倍）×1瓶（20ml×30倍）×1瓶2-8℃保存自备材料：1. 蒸馏水。

Human secretin ELISA KitFor the quantitative in vitro determination of Human secretin concentrations in serum - plasma - celiac fluid - tissue homogenate - body fluidFOR LABORA TORY RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.This package insert must be read in its entirety before using this product.ELISAENZYME LINKED IMMUNOSORBENT ASSAYINTENDED USE AND TEST PRINCIPLEThis secretin 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 secretin in the sample, this secretin 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 of Optical Density versus secretin concentration. The concentration of secretin 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 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Remove serum and assay immediately or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles Plasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.Cell culture supernates, tissue homogenate and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles.Note: The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.MATERIALS REQUIRED BUT NOT SUPPLIED1. 37 ℃ incubator2. Standard microplate reader capable of measuring absorbance at 450 nm3. Precision pipettes, disposable pipette tips and Absorbent paper4. Distilled or deionized waterREAGENTS PROVIDEDAll reagents provided are stored at 2-8°C. Refer to the expiration date on the label.Name 96 determinations 48 determinationsMICROTITER PLATE 8*12strips 8*6stripsSTANDARD（6 vial）0.3ml/vial 0.3ml/vialSAMPLE DILUENT 6.0ml 3.0mlENZYME CONJUGATE 10.0ml 5.0mlWASH SOLUTION 25ml 15mlSUBSTRA TE A 6.0ml 3.0mlSUBSTRA TE B 6.0ml 3.0mlSTOP SOLUTION 6.0ml 3.0mlClosure plate membrane 2 2User manual 1 1Sealed bags 1 1Note:1. Standard concentration was followed by: 200, 100, 50, 25, 12.5, 0 pg/mL.2. If samples generate values higher than the highest standard, please dilute thesamples with Sample Diluent and repeat the assay.PRECAUTIONS1.Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.2.Allow kit reagents and materials to reach room temperature (20-25°C) before use. Do not use water baths to thaw samples or reagents.3.Do not use kit components beyond their expiration date.e only deionized or distilled water to dilute reagents.5.Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.e fresh disposable pipette tips for each transfer to avoid contamination.7.Do not mix acid and sodium hypochlorite solutions.8.Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from Rat blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.9.All samples should be disposed of in a manner that will inactivate viruses.10.Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.11.Substrate Solution is easily contaminated. If bluish prior to use, do not use.12.Substrate B contain 20% acetone, keep this reagent away from sources of heat or flame.13.Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C).REAGENT PREPARATION AND STORAGEWash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized or distilled water. Wash Solution is stable for 1 month at 2-8°C.ASSAY PROCEDURE1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microtiter plate.2. Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.3. Add 100μl of Enzymeconjugate to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.4. Wash the Microtiter Plate 4 times.Manual Washing - Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with Wash Solution (1X),then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame.Automated Washing- Aspirate all wells, then wash plates four times using Wash Buffer (1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.5. Add Substrate A 50μl and Substrate 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.CALCULATION OF RESULTS1.This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (X) axis versus the corresponding concentration on the horizontal (Y) axis.2.First, calculate the mean O.D. value for each standard and sample. All O.D. Values are subtracted by the mean value of the balnk well before result interpretation. Construct the standard curve using graph paper or statistical software.3.To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.4.Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.5.Intra-assay CV(%) is less than 10% and Inter-assay CV(%) is less than 15%.6.Assay range: 6.25 pg/mL – 200 pg/mL.7. Sensitivity: The minimum detectable dose of Human secretin is typically less than 1.0 pg/mL.8. Cross-reactivity: This assay recognizes recombinant and natural Human secretin. No significantcross-reactivity or interference was observed.9. Storage: 2-8℃ (Use frequently); six months (-20℃)。

ELISA检测试剂盒使用指南ELISA检测试剂盒使用指南1.简介：1.1 本文档旨在提供ELISA检测试剂盒的详细使用指南，以帮助用户正确、高效地进行ELISA检测。

1.2 ELISA（酶联免疫吸附试验）是一种常用的免疫测定方法，用于检测体液中特定抗原或抗体的存在与浓度。

1.3 本文档将涵盖ELISA检测试剂盒的准备、样本处理、试剂操作、结果读取等详细步骤。

2.准备：2.1 确保实验室环境整洁，无污染。

2.2 检查所需试剂、仪器和设备是否完整，并检查有效期和储存条件。

2.3 根据实验需要，准备所需样本和质控品，并储存于适当的条件下。

3.样本处理：3.1 根据实验目的选择合适的样本类型，如血清、血浆、尿液等。

3.2 根据试剂盒说明书的要求，对样本进行适当的稀释和预处理。

3.3 严格控制操作条件，避免样本污染和交叉污染。

4.试剂操作：4.1 根据试剂盒说明书，将所需试剂从冰箱中取出，并在室温下适当恢复。

4.2 严格按照说明书的要求，对试剂进行稀释和混合。

4.3 操作过程中注意洁净实验台面，避免试剂交叉污染。

5.检测步骤：5.1 将适量的稀释后的样本、质控品和标准品分别装入试管中。

5.2 添加适量的酶标记抗体或底物，并充分混匀。

5.3 按照试剂盒说明书的要求，进行孵育和洗涤步骤。

5.4 添加底物并进行反应，控制反应时间。

5.5 停止反应，并使用酶标仪测量吸光度。

6.结果读取：6.1 根据试剂盒说明书，根据吸光度值和标准曲线，计算样本中目标物质的浓度。

6.2 将测量结果记录，并进行统计和分析。

6.3 评估实验结果的可靠性，并进行结果的解释和报告。

7.注意事项：7.1 严格按照试剂盒说明书的要求进行操作，避免任何操作步骤的误差和误操作。

7.2 注意实验室安全，避免接触有害化学物质，正确使用防护设备。

7.3 在实验过程中遵循生物安全操作规范，避免潜在的感染风险。

此外，本文档涉及附件，请在附件部分查看相关详细信息。

ELISA kitInstruction Manual5-plate formatJuly, 2006For research use only.Not for use in diagnostic or therapeutic procedures.ContentsAbbreviations 2 Introduction 3 Contents of the kit 4 Hazard information 4 Materials and reagents required but not provided 4 Working solutions 4 General procedure 5 Coating antibodies 5 Blocking 5 Test samples and standards 5 Biotinylated detector antibodies 5 SPP conjugate 5 Substrate 5 Cytokine standards 6 Storage kit reagents 6 Directions for washing 7 Trouble shooting 7 References 8AbbreviationsAPC Antigen presenting cellsBSA Bovine serum albuminCD Cluster of differentiationCSB Cytokine stabilization bufferDMSO Dimethyl sulfoxideELISA Enzyme linked immunosorbent assayGM-CSF Granulocyte-Macrophage Colony Stimulating Factor IFN InterferonIL InterleukinMHC Major histocompatibility complexOD Optical densityPB Phosphate bufferPBS Phosphate buffered salinePBST PBS containing 0.05% Tween-20PBST-B PBST containing 0.5% bovine serum albuminSPP Streptavidin-HRP polymerT h T helper subsetTMB TetramethylbenzidineTNF Tumor necrosis factorIntroductionCytokines are a group of regulatory proteins critically involved in many physiological processes such as immune recognition, cell differentiation and cell proliferation. They have been identified in many vertebrate species and are produced by a variety of different cell types. Cytokines are usually produced transiently and locally, acting in a paracrine or autocrine manner. They interact with high affinity cell surface receptors specific for each cytokine or cytokine group and are active at very low concentrations mostly in the picogram range.It is well known now that the type of an antigen-specific immune response largely depends on the selection or preferential activation of defined CD4+T cell subsets (i.e. T h1 and T h2). Activation of these subsets is characterized by the secretion of distinct patterns of cytokines. T h1, but not T h2 cells, primarily secrete IL-2 and IFN-γ while T h2, but not T h1 cells, produceIL-4, IL-5, IL-6, IL-10 and IL-13. Other cytokines, such as TNF-α and GM-CSF are produced by both T h subsets. In addition, the production of IL-12 and IL-10, produced by antigen presenting cells (APC) such as macrophages and dendritic cells, critically contributes to the preferential expansion of T h1- or T h2-type of cells. For instance, early production of IL-12 is considered essential for the development of T h1 cells. On the other hand, the absence or low concentrations of IL-12 and IFN-γ in the early phase of an immune response and concomitant production of IL-4 by cells of the mastcell/basophil lineage or T cells themselves is known to favor the development of T h2 cells. In addition to their regulatory effects on T h subset differentiation, the cytokines released by the two types of T h cells also produce distinct effector functions. For instance, IL-4 and IFN-γhave differential or antagonistic activities on immunoglobulin isotype selection or MHC class II expression. Therefore, the properties of an immune response can be best studied by determining the amounts of cytokines produced by the responding T cells and APC.Contents of the kitItemsQuantity(5-plate format)StorageconditionsCoating antibodies 1 vial 4ºC (39ºF)Cytokine standard 5 vials 4ºC (39ºF)Biotinylated detector antibodies 1 vial 4ºC (39ºF)SPP conjugate (Streptavidin-HRP polymer) 1 vial ≤ -20ºC (-4°F)TMB substrate tablets 5 4ºC (39ºF)Substrate buffer capsules 5 Rt*BSA stock solution (10%) 2 vials (24 ml) 4ºC (39ºF)Cytokine stabilization buffer (CSB)** 1 vial (5 ml) 4ºC (39ºF)Tween-20 1 vial (5 ml) Rt*ELISA plates8 Rt*Adhesive cover slips 10 Rt** Room temperature** For serum and plasma samples only; see under “Test samples and standards”Materials and reagents required but not provided•PB stock: dissolve 96.0 g Na2HPO4.2H2O plus 17.5 g KH2PO4in 1.0 L distilled water and adjust pH to 7.4•Sterile distilled water•H2SO4•Dimethyl sulfoxide (DMSO)•Pipetting devices for the accurate delivery of volume required for the assay performance •Plate washer: automated or manual (squirt bottle, manifold dispenser, etc)•Reading device for microtiter-plate set to 370, 450 and/or 655 nmWorking solutions•PBS: add 10 ml PB stock and 8.8 g NaCl to 1 L distilled water. Adjust pH to 7.4.Alternatively, use commercially available liquid PBS from Invitrogen or other suppliers.Do not use commercially available PBS tablets for the preparation of the coating solution (the filler in the tablets interferes with the coating process).•PBST: 0.5 ml Tween-20 dissolved in 1 L PBS.•PBST-B: 2 ml BSA stock solution (10%) added to 38 ml PBST.•Blocking buffer: 2 ml BSA stock solution (10%) added to 18 ml PBS (for 1 ELISA plate). •Substrate buffer: the contents of one capsule is dissolved in 100 ml distilled water (takes approximately 5 minutes). For optimal performance, the buffer solution should be used within60 minutes.•Stopping solution: 2 M H2SO4TMB (tetramethylbenzidine) and sodium perborate (in substrate buffer)General procedureCoating antibodies•Reconstitute the lyophilized antibodies by injecting 250 µl of sterile distilled water into the vial. Mix the solution gently for approximately 15 seconds and allow it to stand for 2 minutes at room temperature. Avoid vigorous shaking. To coat 96 wells of an ELISA plate 50 µl is pipetted out of the vial (or use a frozen aliquot of 50 µl; see "Storage kit reagents") and added to 5 ml PBS. Mix gently.•Add 50 µl of diluted antibody solution to each well of the ELISA plate and fill up to 100 µl with PBS.•Seal the plate to prevent evaporation.Incubate overnight at 4ºC or alternatively 1 to 2 hours at 37ºC.Blocking•Remove the coating antibody solution and wash the wells at least six times with PBST. •Add 200 µl of blocking buffer.•Seal the plate and incubate at 37ºC for 1 hour.Test samples and standards•Remove the blocking buffer but do not wash.•Add 1/20 volume of CSB to serum or plasma samples but not to other samples such as cell culture supernatants; CSB inhibits the degradation of cytokines in pure serum or plasma. •Dilute standards and test samples in an appropriate diluent (see “Cytokine standards”). •Add 100 µl to each well.•Seal the plate and incubate at 37ºC for 2 hours or overnight at 4ºC.Biotinylated detector antibodies•Remove test samples/standards and wash the wells at least six times with PBST. •Reconstitute the lyophilized antibodies by injecting 0.5 ml of sterile distilled water into the vial. Mix the solution gently for approximately 15 seconds and allow it to stand for 2 minutes at room temperature. Avoid vigorous shaking. Hundred microliter is pipetted out of the vial (or use a frozen aliquot of 100 µl; see "Storage kit reagents") and added to 10 ml PBST-B.Mix gently.•Add 100 µl of diluted antibody solution to each well.•Seal the plate and incubate at 37ºC for 1 hour.SPP conjugate•Remove detector antibody solution and wash the wells at least six times with PBST. •Reconstitute the contents of the vial by injecting 0.5 ml of sterile distilled water into the vial.Mix the solution gently for approximately 15 seconds and allow it to stand for 1 minute at room temperature. Avoid vigorous shaking. Hundred microliter is pipetted out of the vial (or use a frozen aliquot of 100 µl; see "Storage kit reagents") and added to 10 ml PBST-B. Mix gently.•Add 100 µl to each well.•Seal the plate and incubate at 37ºC for 1 hour.Substrate•Remove SPP conjugate and wash the wells at least six times with PBST.•Dissolve one TMB tablet in 1.0 ml DMSO (vortex at high speed for 5 minutes for complete dissolution)and than add 10 ml substrate buffer.•Mix thoroughly and immediately dispense 100 µl into each well. Leave the plate on the laboratory bench at room temperature (color development between 10 and 30 minutes).The substrate produces a soluble end-product that is blue in color and can be read spectrophotometrically at 370 or 655 nm. The reaction can be stopped by adding 50 µl of2 M H2SO4 (resulting in a yellow solution which can be read at 450 nm).Cytokine standardsFor maximum recovery, the vial with lyophilized cytokine standard should be reconstituted in 0.5 ml distilled water and allowed to stand for 1 minute at room temperature. Thereafter, the reconstituted cytokine standard (stock solution) is placed on melting ice and is immediately diluted as indicated below (preferentially within one hour). Use vials with cytokine standards only once.Please note that temperature of buffers and standard solution(s) should now be kept at 0-4ºC until use in the ELISA.The total amount of cytokine standard is indicated on the label of the vial (ng/vial). After reconstitution in 0.5 ml water, the concentration (ng/ml) will become twice the amount on the label [e.g. amount on label is 4.8 ng/vial; after reconstitution, the concentration becomes9.6 ng/ml = 9600 pg/ml].The standard stock solution is diluted to 320 pg/ml in PBST-B (highest concentration cytokine to be used in the standard range).The linear region of the cytokine standard curve is now obtainable in a series of two-fold dilutions in PBST-B ranging from 320 to 5 pg/ml. Always include a blank control (PBST-B only) in the standard range.Before establishing the standard curve, the OD value of the blank control (OD.bl) is subtracted from the measured OD values of the different standard solutions. The standard curve is now plotted as the standard cytokine concentration versus the corresponding (measured) OD value minus OD.bl. In addition, the actual OD values of the test samples are determined by subtracting OD.bl from the measured OD values.The concentration of the cytokine in the test sample can then be interpolated from the standard curve. It is useful to prepare a series of dilutions of the unknown test sample to assure that the OD will fall in the linear portion of the standard curve.Note 1: The OD value measured for the blank control (OD.bl) must be below 0.2.Note 2: for measuring cytokines in cell culture supernatant, samples should be diluted inPBST-B. However, when measuring cytokines in pure serum or plasma, the diluent for the standard and blank control should preferentially be control serum or plasma originating from the same species.Storage kit reagentsThe vials with lyophilized coating antibodies and biotinylated detector antibodies can be safely stored in a refrigerator for a defined length of time (expiry date indicated on the vial). After reconstitution, the antibodies remain fully active for minimal 6 months at 4ºC (39ºF) when kept sterile. However, it is strongly recommended to divide the reconstituted antibody solutions into small aliquots for single use. These aliquots should be stored at ≤-20ºC. Under these conditions the antibodies are stable for at least one year.Upon arrival, the vial with lyophilized SPP conjugate should be stored at ≤ -20°C. Storage of the vial at room temperature or at 4ºC for several months may lead to lower OD readings in the ELISA. After reconstitution, the SPP solution is stable for 2 months at 4°C but rapidly looses activity when stored at room temperature. It is strongly recommended that after reconstitution, the solution is immediately divided into small aliquots for single use and stored at ≤-20°C. Under these conditions SPP is stable for minimal 12 months.Directions for washing•Incomplete washing will adversely affect the assay. All washing must be performed with wash buffer (PBST).•Washing can be performed manually as follows: completely aspirate the liquid from all wells by gently lowering an aspiration tip (aspiration device) into each well. After aspiration, fill the wells with at least 300 µl wash buffer. Let soak for 10 to 20 seconds, then aspirate the liquid. Repeat as directed under "General procedure". After washing, the plate is inverted and tapped dry on absorbent paper.•Alternatively, the wash buffer may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, completely filling all wells. After washing, the plate is inverted and tapped dry on absorbent paper.•If using an automated washing device, the operating instructions should carefully be followed.Trouble shooting•Poor consistency of replicates can be overcome by increasing the stringency of washes particularly after the incubation step with detector antibody.•High values of the blank control (optical density > 0.2) can be overcome by shortening the incubation time with the substrate solution or is caused by improper washing procedures. •Inconsistent replicates may be due to cross-contamination of wells by improper pipetting procedures.•If no signal is observed in the wells with the standards•try a new vial with cytokine standard•check the pH of the substrate solution (between 5.0 and 5.5)•verify whether the antibody, SPP conjugate and standardpreparations were properly diluted•Avoid sodium azide in wash buffers and diluents, as this is an inhibitor of peroxidase activity.•Storage of reconstituted SPP at room temperature for several days can lead to a significant loss of SPP activity and consequently low OD readings.ReferencesBooks:•Practice and theory of enzyme immunoassays 1985In: Laboratory techniques in biochemistry and molecular biology, Vol.15 (eds R.H.Burdon and P.H. van Knippenberg)Science Publishers bv, Amsterdam, The Netherlands•ELISA and other Solid Phase Immunoassays.Theoretical and Practical Aspects 1988(eds D.M.Kemeny and S.J.Challacombe)John Wiley & Sons Ltd, Chichester, UK• A practical guide to ELISA 1991(ed D.M.Kemeny) Pergamon Press, Oxford, UKReview of U-CyTech ELISA references:Human cytokines:•Arend, S.M. et al. 2000 J. Infect. Diseases 181: 1850-1854 •Demirkiran, A. et al. 2006 Liver Transpl. 12: 277-284 •Hoogendoorn, M. et al. 2005 Clin. Cancer Res. 11: 5310-5318 •Tang, Y-M. et al. 2006 World J. Gastroenterol. 11: 4575-4578•de Waal, L. et al. 2004 J. Virol. 78: 1775-1781Monkey cytokines:•Fallon, P.G. et al. 2003 J. Infect. Dis. 187: 939-945•Hartman, G. et al. 2005 Vaccine 23: 3310-3317•Kornfeld, C. et al. 2005 J. Clin. Invest. 115: 1082-1091 •Mascarell, L. et al. 2006 Vaccine 24: 3490-3499•Polakos, N.K. et al. 2001 J. Immunol. 166: 3589-3598•de Swart, R.L. et al. 2002 J. Virol. 76: 11561-11569Mouse cytokines:•Eijkelkamp, N. et al. 2004 J. Neuroimmun. 150: 3-9•Kavelaars, A. et al. 2005 J. Neuroimmun. 161: 162-168•Vroon, A. et al. 2005 J. Immunol. 174: 4400-4406Rat cytokines:•Dieleman, J.M. et al. 2006 Life Sci. 79: 551-558•Pacheco-López, G. et al. 2005 J. Neurosci. 25: 2330-2337•Sajti, E. et al. 2004 Brain Behav. Immun. 18: 505-514•Teunis, M.A.T. et al. 2002 J. Neuroimmun. 13: 30-38。