人端粒酶(TE)elisa酶联试剂盒说明书 广锐生物-国内高、优Elisa试剂盒供应商 1人端粒酶(TE)酶联免疫分析（ELISA）试剂盒使用说明书本试剂仅供研究使用 目的：本试剂盒用于测定人血清，血浆及相关液体样本中端粒酶(TE)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端粒酶(TE)水平。用纯化的人端粒酶(TE)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端粒酶(TE)，再与HRP 标记的端粒酶(TE)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人端粒酶(TE)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端粒酶(TE)浓度。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：36U/L 0.5ml×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℃保存样本处理及要求：1. 血清：室温血液自然凝固10-20 分钟，离心20 分钟左右（2000-3000 转/分）。仔细♀收集上清，保存过程中如出现沉淀，应再◤次离心。2. 血浆：应根据标本的要求选择EDTA 或柠檬酸钠作为抗凝剂，混合10-20 分钟后，离心20 分钟左右（2000-3000 转/分）。仔细收集上清〗，保存过程中如有沉淀形成，应该再次离心。3. 尿液：用无↓菌管收集，离心20 分钟左右（2000-3000 转/分）。仔细收集上清，保存过程中如有沉淀形成，应再次离心。胸腹水、脑脊液参照实行。4. 细胞培养上清：检测分泌性的成份时，用无菌管收集。离心20 分钟左右（2000-3000 转/分）。仔细收集上清。检测细胞内的成份时，用PBS（PH7.2-7.4）稀释细胞悬液，细胞浓度达到100 万/ml 左右。通过反复冻融，以使细胞破坏并放出细胞内成份。离心20 分钟左右（2000-3000 转/分）。仔细收集上清。保存过程中如有沉淀形成，应再次离心。25. 组织标本：切割标本后，称取重量。加入一定量的PBS，PH7.4。用液氮迅速冷冻保存备用。标本融化后仍然保持2-8℃的温度。加入一定量的PBS（PH7.4），用手工或匀浆器将标本匀浆充分。离心20 分钟左右（2000-3000 转/分）。仔细收集上清。分装后一份⌒　待检测，其余冷冻备用。6. 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.7. 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▽与加样：在酶标包被板上设标准品孔10 孔，在第一、第二孔中分别加标准品100μl，然后在第一、第二孔中加标准品稀释液50μl，混匀；然后从第一孔、第二孔中各取100μl 分别加〗到第三孔和第四孔，再在第三、第四孔分别加标准品稀释液50μl，混匀；然后在第三孔和第四孔中先各取50μl 弃掉，再各取50μl 分别加到第五、第六孔中，再在第五、第六孔中分别加标准品稀释液50ul，混匀；混匀后从第五、第六孔中各取50μl 分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液50μl，混匀后从第七、第八孔中分别取50μl 加到第九、第十孔中，再在第九第十孔分别加标准品稀释液50μl，混匀后从第九第十孔中各取50μl 弃掉。（稀释后各孔加样量都为50μl，浓度分别为24U/L，16U/L ，8U/L，4U/L， 2U/L）。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）、待测样品孔。在酶标包被板上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（48T 的20 倍）倍浓缩洗涤液用蒸∩馏水30（48T 的20 倍）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）。11. 测定：以空白空调零，450nm 波长依序测量各孔的吸光度（OD 值）。 测定♀应在加终止液后15 分钟以内进行。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。2． 浓洗涤液可能会有结晶析出，稀释时可在水浴中加温助溶，洗涤时不影响结果。3． 各步加样均应使用加样器，并经常校对其准确性，以避免试验误差。一次加样时间最好控制在5 分钟内，如标本数量多，推荐使用排枪加样。4． 请每次测定的同时做标准曲线，最好做复孔。如标本中待测物质含量过高（样本OD 值大于标准品孔第一孔的OD 值），请先用样品稀释液稀释一定倍数（n 倍）后再测定，计算时请最后乘以总稀释倍数（×n×5）。5． 封板膜只限一次性使用，以避免交叉污染。6． 底物请避光保存。37． 严格按照说明书的操作进行，试验结果判定必须以酶标仪读数为准.8． 所有样品，洗涤液和各种废弃物都应按传染物处理。9． 本试剂不同批号组分不得混用。10. 如与英文说明书有异，以英文说明书为准。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。（此图仅供参考）试剂盒性能：1.样品线性回归与预期浓度相关系数R 值为0.990 以上。2.批内与批见应分别小于9%和11%检测范围：1U/L -30U/L保存条件及有效期：1.试剂盒保存：；2-8℃。2．有效期：6 个月4Human telomeraseFOR RESEARCH USE ONLYDrug NamesGeneric Name：Human telomerase (TE)ELISA Kit.PurposeThis kit allows for the determination of TE concentrations in Human serum, blood plasma,and other biological fluids.Principle of the assayThe kit assay Human TE level in the sample，use Purified Human TE antibody to coatmicrotiter plate wells, make solid-phase antibody, then add TE to wells, Combined TE antibodywhich With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washingCompletely, Add TMB substrate solution,TMB substrate becomes blue color At HRPenzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and thecolor change is measured spectrophotometrically at a wavelength of 450 nm. Theconcentration of TE in the samples is then determined by comparing the O.D. of the samples tothe standard curve.5Materials provided with the kitMaterials provided withthe kit48determinations 96 determinations StorageUser manual 1 1Closure plate membrane 2 2Sealed bags 1 1Microelisa stripplate 1 1 2-8℃Standard：36U/L 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃HRP-Conjugate reagent 3ml×1 bottle 6ml×1 bottle 2-8℃Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃Chromogen Solution A 3ml×1 bottle 6ml×1 bottle 2-8℃Chromogen Solution B 3ml×1 bottle 6ml×1 bottle 2-8℃Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃wash solution（20ml×20 fold）×1bottle（20ml×30 fold）×1bottle2-8℃Specimen requirements1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, Ifprecipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugal again. The Operation ofHydrothorax and cerebrospinal fluid Reference to it.4. cell culture supernatant-detect secretory components, collect sue a sterile container,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect thecomposition of cells, Dilut cell suspension with PBS（PH7.2-7.4）, Cell concentrationreached 1 million / ml, repeated freeze-thaw cycles, damage cells and release ofintracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. removesupernatant, If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidlyfrozen with liquid nitrogen, maintain samples at 2-8℃ after melting,add PBS（PH7.4）,6Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant.6. extract as soon as possible after Specimen collection,and according to the relevantliterature, and should be experiment as soon as possible after the extraction. If it can’t,specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, addStandard 100μl to the first and the second well, then add Standard dilution 50μl to the first andthe second well, mix; take out 100μl form the first and the second well then add it to the thirdand the forth well separately. then add Standard dilution 50μl to the third and the forthwell ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth andthe sixth well ,then add Standard dilution 50μl to the fifth and the sixth well, mix ; take out 50μlfrom the fifth and the sixth well and add to the seventh and the eighth well, then add Standarddilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and theeighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth andthe tenth well, mix , take out 50μl from the ninth and the tenth well discard(add Sample 50μl toeach well after Diluting ,(density: 24U/L，16U/L ，8U/L，4U/L， 2U/L)2.add sample：Set blank wells separately (blank comparison wells don’t add sample andHRP-Conjugate reagent, other each step operation is same). testing sample well. add Sampledilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.4.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilledwater and reserve.5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing bufferto every well, still for 30s then drain, repeat 5 times, dry by pat.6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.7.incubate：Operation with 3.78.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight preservation for 15 min at 37℃10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(the blue colorchange to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes inthe room temperature, ELISA plates coated if has not use up after opened, the plate shouldbe stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids theexperimental error. add sample within 5 mins, if the number of sample is much ,recommend to use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilutionfactor.（×n×5）.5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take themicrotiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective materialprocess.9. Do not mix reagents with those from other lots.8CalculateAssay range1U/L -30U/LStorage and validity1．Storage： 2-8℃.2．validity： six months.Take the standard density as the horizontal, the ODvalue for the vertical ,draw the standard curve on graphpaper, Find out the corresponding density according to thesample OD value by the Sample curve, multiplied by thedilution multiple, or calculate the straight line regressionequation of the standard curve with the standard density andthe OD value ,with the sample OD value in the equation,calculate the sample density, multiplied by the dilution factor,the result is the sample actual density.This chartis for reference only