小鼠ⅰ型胶原n末端肽ntxelisa试剂盒说明书.docx

小鼠型胶原N末端肽 (NTX)酶联免疫吸附测定试剂盒使用说明书产品编号：E-EL-M0360 （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。合用于体外定量检测小鼠血清、血浆、组织匀浆或细胞培养上清液中天然和重组NTX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。试剂盒组成：名称-中文名称-英文规格保存条件ELISA酶标板Micro ELISA Plate 812 / 86*4冻干标准品Reference Standard 2/ 1支*4标准品&样品稀释液Reference Standard & Sample Diluent 1瓶 20mL/12mL*4浓缩生物素化抗体 Concentrated Biotinylated Detection Ab 1支 120L/70L*4生物素化抗体稀释液 Diluent for Biotinylated Detection Ab 1瓶 10mL/6mL*4浓缩HRP酶结合物 Concentrated HRP Conjugate 1支 120L/70L*4(避光)酶结合物稀释液 Diluent for HRP Conjugate 1瓶 10mL/6mL* 4浓缩洗涤液（25） Concentrated Wash Buffer （25）1瓶 30mL/16mL*4底物溶液（TMB） Substrate Reagent 1瓶 10mL/6mL*4(避光)反映终止液 Stop Solution 1瓶 10mL/6mL*4封板覆膜Plate Sealer5/3张*产品说明书Product Description1份*: 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。用抗小鼠NTX抗体包被于酶标板上，实验时标本或标准品中的NTX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小鼠NTX抗体和辣根过氧化物酶标记的亲和素。抗小鼠NTX抗体与结合在包被抗体上的小鼠NTX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，NTX浓度与OD450值之间呈正比，通过绘制标准曲线求出标本中NTX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1000g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1000g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。将剪碎的组织与相应体积的PBS（一般按1:9的重量体积比，比如1g的组织样本相应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中加入蛋白酶克制剂）加入玻璃匀浆器中，于冰上充足研磨。为了进一步裂解组织细胞，可以对匀浆液进行超声破碎，或反复冻融。最后将匀浆液于5000g 离心510分钟，取上清检测。4. 细胞培养上清：取细胞培养上清于1000g离心20分钟，除去杂质及细胞碎片。取上清检测。5. 其它生物标本：1000g离心20分钟，取上清即可检测（具体解决方法可参考：）6. 标本应清澈透明，悬浮物应离心去除。7. 标本收集后若不及时检测，请按一次使用量分装，冻存于-20/-80冰箱内，避免反复冻融，1-6月内检测,4保存的应在1周内进行检测。 8. 假如您的样本中检测物浓度高于标准品最高值，请根据实际情况，做适当倍数稀释(建议先做预实验，以拟定稀释倍数)。实验所需自备物品:1. 酶标仪(450nm波长滤光片)2. 高精度移液器，EP管及一次性吸头：0.5-10L, 2-20L, 20-200L, 200-1000L3. 37恒温箱, 双蒸水或去离子水4. 吸水纸检测前准备工作:1. 请提前20分钟从冰箱中取出试剂盒，平衡至室温。2. 将浓缩洗涤液用双蒸水稀释(1:25)。未用完的放回4。从冰箱中取出的浓缩洗涤液也许有结晶，属于正常现象，可用40水浴微加热使结晶完全溶解后再配制洗涤液（加热温度不要超过50，使用时洗涤液应为室温）。3. 标准品: 加入标准品&样品稀释液1.0mL至冻干标准品中，静置10分钟，待其充足溶解后，轻轻混匀(浓度为200ng/mL)。然后根据需要进行倍比稀释（注：不要直接在反映孔中进行倍比稀释）。建议配制成以下浓度： 200、100、50、25、12.5、6.25、3.13、0 ng/mL ，样品稀释液直接作为空白孔0ng/mL。如配制100ng/mL标准品：取0.5mL 200ng/mL的上述标准品加入具有0.5mL样品稀释液的EP管中，混匀即可，其余浓度依此类推。4. 生物素化抗体工作液：实验前计算当次实验所需用量（以100L/孔计），实际配制时应多配制100-200L。使用前15分钟，以生物素化抗体稀释液稀释浓缩生物素化抗体(1:100)成工作浓度。当天使用。5. 酶结合物工作液：实验前计算当次实验所需用量（以100L/孔计），实际配制时应多配制 100-200L。使用前15分钟，以酶结合物稀释液稀释浓缩HRP酶结合物(1:100)成工作浓度。当天使用。标准品稀释方法图例：(以500L/管为例，也可根据实际用量来稀释，如200L/管)200 100 50 25 12.56.253.130ng/mL洗涤方法:1. 自动洗板机：每孔加入洗涤液350L，注入与吸出间隔60秒。2. 手工洗板：甩尽孔内液体，在洁净的吸水纸上拍干，每孔加洗涤液350L，浸泡1-2分钟，吸去（不可触及板壁）或甩掉酶标板内的液体，在厚的吸水纸上拍干。操作环节:实验开始前，各试剂均应平衡至室温；试剂或样品配制时，均需充足混匀，并尽量避免起泡。1. 加样：分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液100L，余孔分别加标准品或待测样品100L，注意不要有气泡，加样时将样品加于酶标板底部，尽量不触及孔壁，轻轻晃动混匀。给酶标板覆膜，37孵育90分钟。为保证实验结果有效性，每次实验请使用新的标准品溶液。2. 弃去液体，甩干，不用洗涤。每个孔中加入生物素化抗体工作液100L（在使用前15分钟内配制），酶标板加上覆膜，37温育1小时。3. 弃去孔内液体，甩干，洗板 3次，每次浸泡1-2分钟，大约350L/每孔，甩干并在吸水纸上轻拍将孔内液体拍干。 4. 每孔加酶结合物工作液（临用前15分钟内配制）100L，加上覆膜，37温育30分钟。5. 弃去孔内液体，甩干，洗板5次，方法同环节3。6. 每孔加底物溶液(TMB)100L，酶标板加上覆膜37避光孵育15分钟左右（根据实际显色情况酌情缩短或延长，但不可超过30分钟。当标准孔出现明显梯度时，即可终止）。7. 每孔加终止液50L，终止反映，此时蓝色立转黄色。终止液的加入顺序应尽量与底物溶液的加入顺序相同。8. 立即用酶标仪在450nm波长测量各孔的光密度（OD值）。应提前打开酶标仪电源，预热仪器，设立好检测程序。9. 实验完毕后将未用完的试剂按规定的保存温度放回冰箱保存。注意事项：1. 保存：试剂盒中各试剂请按说明书提醒合理存放。在储存及温育过程中避免将试剂暴露在强光中。所有试剂瓶盖须旋紧以防止蒸发和微生物的污染，否则也许会出现错误的结果。2. 酶标板：刚启动的酶标板孔中也许会有少许水样物质，此为正常现象，不会对实验结果导致任何影响。3. 加样：加样或加试剂时，第一个孔与最后一个孔的加样时间间隔假如太大，将会导致不同的 “预温育”时间，从而明显地影响到测量值的准确性及反复性。每次的加样时间最佳控制在10分钟内。推荐设立复孔。4. 温育：为防止样品蒸发，实验时必须给酶标板覆膜；洗板后应尽快进行下步操作，避免酶标板处在干燥状态；严格遵守给定的温育时间和温度。5. 洗涤：洗涤过程中反映孔中残留的洗涤液应在吸水纸上拍干，勿将滤纸直接放入反映孔中吸水。在读数前要注意清除底部残留的液体和手指印，以免影响酶标仪读数。6. 试剂配制：Concentrated Biotinylated Detection Ab及Concentrated HRP Conjugate体积较小，运送过程会使液体沾到管壁或瓶盖，因此使用前1000转/分离心1min，以使附着管壁或瓶盖的液体沉积到管底。取用前，请用移液器小心吹打4-5次使溶液混匀。标准品、生物素化抗体工作液、酶结合物工作液请根据所需用量配制，并使用相应的稀释液配制，不能混淆。请精确配制标准品及工作液，尽量不要微量配制（如吸取Concentrated Biotinylated Detection Ab时，一次不要小于10L），以避免由于不准确稀释而导致浓度误差；请勿反复使用已稀释过的标准品、生物素化抗体工作液、酶结合物工作液。若需要分次使用标准品应按照每一次用量分装，将其放在-20-80贮存。避免反复冻融。7. 显色时间的控制：加入底物后请定期观测反映孔的颜色变化（比如每隔5分钟），如梯度已很明显，请提前加入终止液终止反映，避免颜色过深影响酶标仪读数。8. 底物：底物请避光保存，在储存和温育时避免强光直接照射。9. 混匀：充足轻微混匀对反映结果尤为重要，最佳使用微量振荡器(使用最低频率)，如无微量振荡器，可在反映前手工轻轻敲击酶标板框混匀。10. 安全：实验中请穿着实验服并带乳胶手套做好防护工作。特别是检测血液或者其他体液标本时，请按国家生物实验室安全防护条例执行。11. 不同批号的试剂盒组份不能混用（洗涤液和反映终止液除外）12. 实验中所用的EP管和吸头均为一次性使用，严禁混用，否则将影响实验结果！结果判断:1. 每个标准品的OD值减去空白孔的OD值后作图，如设立复孔，则应取其平均值计算。以标准品的浓度为横坐标，OD值为纵坐标，绘出标准曲线。亦可以OD值为横坐标，标准品的浓度为纵坐标，绘出标准曲线。 2. 推荐使用专业的曲线制作软件，如curve expert 1.3，在软件界面既可根据样品OD值，由标准曲线查出相应的浓度，乘以稀释倍数；亦可将样品的OD值代入标准曲线的拟合方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。3. 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。灵敏度、检测范围、特异性和反复性: 灵敏度：最小可测1.88ng/mL。 检测范围：3.13 200ng/mL。 特异性：可检测重组或天然的小鼠NTX，且与其它相关蛋白无交叉反映。 反复性：板内，板间变异系数均10%。问题分析问题描述也许因素相应对策标准曲线梯度差吸液或加液不准检查移液器及吸头标准品稀释不对的溶解标准品时稍微旋转瓶身，轻轻混匀使粉末完全溶解洗涤不完全保证洗涤时间和洗涤次数及每孔的加液量显色很弱或无色孵育时间太短保证充足的孵育时间实验温度不对的使用推荐的实验温度试剂体积不够或漏加检查吸液及加液过程，保证所有试剂按顺序足量添加稀释不对的读数数值低酶标仪设立不对的在酶标仪上检查波长及滤光片设立提前打开酶标仪预热曲线偏差大加液不对的检查加液情况背景值高检测抗体的工作浓度过高使用推荐的稀释倍数酶标板洗涤不完全重新阅读操作手册，保证清洗完全；假如用自动洗板机，请检查所有的出口是否有堵塞洗液有污染配制新鲜的洗液灵敏度低ELISA试剂盒保存不妥按说明书规定保存相关试剂读数前未终止OD读数前应在每孔中加入终止液 说明 1. 限于现有条件及科学技术水平，尚不能对所有原料进行全面的鉴定分析，本产品也许存在一定的质量技术风险。2. 最终的实验结果与试剂的有效性、实验者的相关操作以及当时的实验环境密切相关，请务必准备充足的待测样品。3. 只有所有使用ElabTM试剂才干保证检测效果，不能混用其他制造商的产品。只有严格遵守ElabTM试剂的实验说明才会得到最佳的检测结果。4. 有效期：6个月。5. 本操作说明同样合用于48T试剂盒。Mouse NTX (cross linked N-telopeptide of type collagen) ELISA Kit Product Description Catalog No: E-EL-M0360（FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGONOSIS !）Dear customer, Thank you for choosing our products. This product is produced using raw materials from world-renowned manufacturer, and professional manufacturing technology of ELISA kits. Please read the instructions carefully before use and check all the reagent compositions! If in doubt, please contact Elabscience Biotechnology Co., Ltd.Intended useThis immunoassay kit allows for the in vitro quantitative determination of Mouse NTX concentrations in serum, plasma and other biological fluids.Kit Components：ItemSpecificationsStorage Micro ELISA Plate 812 or 86 *4CReference Standard 2/ 1vial *4CReference Standard & Sample Diluent 1vial 20mL/12mL *4CConcentrated Biotinylated Detection Ab 1via l 120L /70L *4CDiluent for Biotinylated Detection Ab 1vial 10mL/6mL*4CConcentrated HRP Conjugate 1vial 120L /70L *4C (shading light)Diluent for HRP Conjugate 1vial 10mL/6mL * 4CConcentrated Wash Buffer （25）1vial 30mL/16mL *4CSubstrate Reagent 1vial 10mL/6mL *4C (shading light)Stop Solution 1vial 10mL/6mL *4CPlate Sealer5/3pieces *Product Description1 copy*: 96T/48TTest principleThis ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to NTX Standards or samples are then added to the appropriate micro ELISA plate wells and combined to the specific antibody. Then a biotinylated detection antibody specific for NTX and Avidin-Horseradish Peroxidase (HRP) conjugate is added to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain NTX, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color turn yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The OD value is proportional to the concentration of NTX. You can calculate the concentration of NTX in the samples by comparing the O.D. of the samples to the standard curve.Sample collection and storageSerum - Allow samples to clot for 2 hours at room temperature or overnight at 4C before centrifugation for 20 minutes at approximately 1000g. Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.Plasma - Collect plasma using EDTA.Na2 or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000g at 2 - 8C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.Tissue homogenates: For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then minced to small pieces which will be homogenized in PBS (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces.Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000g to get the supernate.Cell culture supernate Centrifuge supernate for 20 minutes to remove insoluble impurity and cell debris at 1000g at 2 - 8C. Collect the clear supernate and carry out the assay immediately.Other biological fluids Centrifuge samples for 20 minutes at 1000g at 2 - 8C. Collect the supernatant and carry out the assay immediately. (You can refer to our website for detailed processing method: )Sample preparation Samples should be clear and transparent and be centrifuged to remove suspended solids.Note: Serum and plasma to be used within 7 days when stored at 2-8C, otherwise samples must be divided and stored at -20C (1 month) or -80C (6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. If the sample concentration is higher than the maximum standard value, please dilute it with appropriate factor according to the actual situation. (A pre-test is recommended to determine the dilute factor) Other supplies requiredMicroplate reader with 450nm wavelength filter High-precision transferpettor, EP tubes and disposable pipette tips37C Incubator, Deionized or distilled water. Absorbent paperReagent preparationBring all reagents to room temperature before use. Wash Buffer - Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized or distilled water. Put unused solution back at 4C. If crystals have formed in the concentrate, you can warm it with 40C water bath (Heating temperature should not exceed 50C) and mix it gently until the crystals have completely dissolved. The solution should be cooled to room temperature before use.Standard - Reconstitute the Standard with 1.0mL of Sample Diluent, let it stand for 10minutes until it dissolved fully. This reconstitution produces a stock solution of 200ng/mL. Then make serial dilutions as needed (Making serial dilution in the wells directly is not permitted). The recommended concentrations are as follows: 200、100、50、25、12.5、6.25、3.13、0ng/mL . As if you want to make standard solution at the concentration of 100ng/mL, you can take 0.5mL the standard at 200ng/mL, add it to an EP tube with 0.5mL sample dilution, and mix it. The procedures of making the remaining concentrations are all the same. The undiluted standard serves as the highest standard (200ng/mL). The Sample Diluent serves as the zero (0ng/mL).(500L/tube，for example. Can also be diluted according to the actual amount，such as 200L/tube)200100502512.56.253.130ng/mLBiotinylated Detection Ab Calculate the required amount before experiment (100L /well). In actual preparation you should prepare 100200L more. Dilute the concentrated Biotinylated Detection Ab to the working concentration using Diluent for Biotinylated Detection Ab (1:100).Concentrated HRP Conjugate Calculate the required amount before experiment (100L/well). In actual preparation you should prepare 100200L more. Dilute the Concentrated HRP Conjugate to the working concentration using Diluent for Concentrated HRP Conjugate (1:100).Washing Procedure:1. Automated washer: add 350L wash buffer into each well, the interval between injection and suction should be set about 60s.2. Manual wash: add 350L wash buffer into each well, soak it for 12minutes, suck(no inside wall touching) or get rid of liquid within the micro ELISA plate and pat it dry on thick clean absorbent paper.Assay procedureAllow all reagents to reach room temperature All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming.1. Add Sample: Add 100L of Standard, Blank, or Sample per well. The blank well is added with sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming to the best of your ability. Mix it gently. Cover the plate with sealer we provided. Incubate for 90 minutes at 37C.2. Biotinylated Detection Ab: Remove the liquid of each well, dont wash. Immediately add 100L of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37C. 3. Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350L) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. 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 pat it against thick clean absorbent paper.4. HRP Conjugate: Add 100L of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30 minutes at 37C.5. Wash: Repeat the wash process for five times as conducted in step 3.6. Substrate: Add 100L of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for about 15 minutes at 37C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.7. Stop: Add 50L of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.8. OD Measurement: Determine the optical density (OD value) of each well at once, using a microplate reader set to 450nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.9. After experiment, put all the unused reagents back into the refrigera