人凝血酶TM酶联免疫检测

1、人凝血酶仃M)酶联免疫检测试剂盒使用说明书使用前仔细阅读本说明书。本酶联免疫试剂盒是基于双抗体夹心技术原理, 来检测人凝血酶(TM),只能用于研究用途，不得用于医学诊断。用 途：用于人血清、血浆及相关液体样本中凝血酶 (TM)测定。工作原理本试剂盒采用的是生物素双抗体夹心酶联免疫吸附法(ELISA)测定样品中人凝血酶(TM)水平。向预先包被了人凝血酶(TM)单克隆抗体的酶标孔中加入人凝 血酶(TM)，温育；温育后，加入生物素标记的抗 TM抗体。再与链霉亲和素-HRP 结合，形成免疫复合物，再经过温育和洗涤，去除未结合的酶，然后加入底物A、 B,产生蓝色，并在酸的作用下转化成最终的黄色。颜色的深

2、浅与样品中人凝血 酶(TM)的浓度呈正相关。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份圭寸板膜2 片(48)2 片(96)密封袋1个1个酶标包被板1X 481x 9628C保存标准品1600U/L0.5ml X 1 瓶0.5ml x 1 瓶28C保存标准品稀释液3ml X 1 瓶6ml x 1 瓶2-8 C保存链霉亲和素-HRP3 mlx 1 瓶6 ml x 1 瓶2-8 C保存生物素标记的抗TM抗体0.5ml x 1 瓶1 ml x 1 瓶2-8 C保存显色剂A液3 mlx 1 瓶6 ml x 1 瓶2-8 C保存显色剂B液3 mlx 1 瓶6 ml x 1 瓶2-8 C保存

3、终止液3ml x 1 瓶6ml x 1 瓶2-8 C保存浓缩洗涤液(20ml x 20 倍)x 1 瓶(20ml x 30 倍)x 1 瓶2-8 C保存需要而未提供的试剂和器材1. 37 C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. 吸水纸注意事项1. 从2-8 C取出的试剂盒，在开启试剂盒之前要室温平衡至少 30分钟。酶标包 被板开封后如未用完，板条应装入密封袋中保存。2. 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3. 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4. 为避免交叉污染，要避免重复使用手中的吸头和

4、封板膜。5. 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。6. 底物B对光敏感，避免长时间暴露于光下。洗板方法手工洗板方法：甩掉酶标板内的液体；在实验台上铺垫几层吸水纸，酶标板朝下 用力拍几次；将稀释后的洗涤液至少 0.35ml注入孔内，浸泡1-2分钟。根据需 要，重复此过程数次。自动洗板：如果有自动洗板机，应在熟练使用后再用到正式实验过程中标本要求1不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP活性。2 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 C保存，但应避免反复冻融。操作程序1. 标准

5、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稀释。）800U/L5号标准品120卩l的原倍标准品加入120卩l标准品稀释液400U/L4号标准品120卩l的5号标准品加入120卩l标准品稀释液200U/L3号标准品120卩l的4号标准品加入120卩l标准品稀释液100U/L2号标准品120卩l的3号标准品加入120卩l标准品稀释液50U/L1号标准品120卩l的2号标准品加入120卩l标准品稀释液2. 根据代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定，能使用复孔的尽量做复孔。3. 加样：1）空白孔，空白对照孔

6、不加样品，生物素标记的抗 TM抗体，链霉亲 和素-HRP,只加显色剂 A&B和终止液，其余各步操作相同；2）标准品孔： 加入标准品50ul，链霉亲和素-HRP50ul（标准品中已事先整合好生物素抗体， 故不加）;3）代测样品孔：加入样本40ul，然后各加入抗TM抗体10ul、链 霉亲和素-HRP50ul，盖上封板膜，轻轻震荡混匀,37T温育60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. 显色：每孔先加入显色剂A50ul，再加入显色剂B50卩l，轻轻震荡混匀，37C 避光显色

7、10分钟.7. 终止：每孔加终止液50卩l，终止反应（此时蓝色立转黄色）。8. 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10分钟以内进行。9. 根据标准品的浓度及对应的 0D值计算出标准曲线的直线回归方程，再根据 样品的0D值在回归方程上计算出对应的样品浓度。也可以使用各种应用软 件来计算。操作程序总结:检测范围：30U/L800U/L 保存：2-8 C。有效期：6个月(2-8 C)。Human Thrombin (TM) ELISA KitInstructionThis kit is only for scientific research, a

8、nd shall not be used as a clinical diagnosis of use.PurposeThis kit allows for the determ in ationof TM concen trati onSn Huma n serum, cell culture super nata nt, and other biological fluids.PrincipleThe kit assay HumairM level in the sampladd HumaiTM antibody to microtiter plate wells,after Incuba

9、ting,addBiotinylatedanti -TM -antibody, then CombinedStreptavidin-HRP, become complex, after In cubat ing and wash ing Completely, Add TMB substrate solutio n,TMB substrate becomes blue color, react ion is term in ated by the additi on of a sulphuric acid soluti on and the color cha ngeis measuredsp

10、ectrophotometricallyt a wavele ngthof 450 nm. The concentration of TM in the samples is then determined by comparing the O.D. of the samples to the sta ndard curve.Materials provided with the kitMaterials provided with th kite48determ in ati ons96 determ in atio nsStorageUser manual11Closure plate m

11、embra ne 22Sealed bags11Microelisa stripplate1128CSta ndard 1600U/L0.5ml 1Xbottle0.5ml Kbottle28CSta ndard dilue nt3ml Xbottle6ml 1 bottle2-8 CStreptavidi n-HRP3ml 1 bottle6ml 1 bottle2-8 CBioti ny lated anti -TM-a ntibody0.5ml Kbottle1ml 1 bottle2-8 CChromoge n Soluti on A3ml 1 bottle6ml 1 bottle2-

12、8 CChromoge n Soluti on B3ml 1 bottle6ml 1 bottle2-8 CStop Soluti on3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml X 20 fold)x 1bottle(20ml X 30 fold)x 1 bottle2-8 CMaterials required but not supplied1.37 C incubator2. Sta ndard microplate reader(45 Onm)3. Precision pipettes and Disposable pipette

13、tips.4. dei oni zed water.5. Disposable Test tube6 Absorbe nt paperImportant notes1. The kit takes out from the refrigeration environment should be balaneed 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. add Samp

14、le with sampler Each step, And proofread its accuracy frequently, avoids the experime ntal error.3. Please accord ing to use in structi on strictly, The test result determ in ati on must take the microtiter plate reader as a sta ndard.4. Use new disposal plastic pipette tips and Closure plate membra

15、ne for each standard, in order to avoid cross con tam in atio n.5. Do not mix reagents with those from other lots.6. The substrate evade the light preservationSpecimen requirements1. extract as soon as possibleafter Specime ncollecti on,an daccord in gto the releva nt literature,a nd shouldbe experi

16、me ntas soon as possibleafter the extractio n.lf it can specime n can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.2. Can t detect the sample which coNiaNS, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Diloteinai density Standard as follow table:800U/L5 S

17、ta ndard120 d Original density Standard120 M Standard diluent400U/L4 Sta ndard120 d 5 Standard120 d Standard diluent200U/L3 Sta ndard120 d 4 Standard120 d Standard diluent100U/L2 Sta ndard120 d 3 Standarc+120 d Standard diluent50U/L1 Sta ndard120 d 2 Standarc+120 d Standard diluentt,adldtsiampied an

18、tTM2. accord ing test ing Sample nu mberdeo ne how many wells n edd, Stan dard and bla nk suggested Do holes.3. add samp：1) bla nk wells(bla nk comparis on wells don -antibody and Streptavidin-HRP ,other each step operation is same); 2) Standard wells: addStandard 0 and Streptavidin-HRP 5； 3) testin

19、g Sample wells: add sampl鶴len addanti -TM -antibody01,l Streptavidin-HRF0念 closing plate with Closure platemembra ne ,in cubate for 60 min aE374. C on figurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5. washi ng Un cover Closure plate membra ne, discard Liquid, dry by swing, add wash ing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. colo【Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservati on for 10 min a