人磷脂酰肌醇抗体IgM(PIAbIgM酶联免疫分析ELISA

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1、人磷脂酰肌醇抗体IgM (PI Ab-IgM)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磷脂酰肌醇抗体IgM (PI Ab-IgM )的含量。实验原理：本试剂盒应用双抗原夹心法测定标本中人磷脂酰肌醇抗体IgM (PI Ab-IgM)水平。 用纯化的抗原包被微孔板，制成固相抗原，往包被单抗的微孔中依次加入磷脂酰肌醇抗体 IgM (PI Ab-IgM),再与HRP标记的抗原结合，形成抗原-抗体-酶标抗原复合物，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

2、中的磷脂酰肌醇抗体IgM (PI Ab-IgM)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磷脂酰肌醇抗体IgM (PI Ab-IgM)含量。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1X481X962-8 C保存标准品：72 ng/L0.5ml X 1 瓶0.5ml X 1 瓶2-8 C保存标准品稀释液1.5ml X 1 瓶1.5ml X 1 瓶2-8 C保存酶标试剂3 mlX 1 瓶6 ml X 1 瓶2-8 C保存样品稀释液3 mlX 1 瓶6 ml X 1 瓶2-8 C保

3、存显色剂A液3 ml X 1 瓶6 ml X 1 瓶2-8 C保存显色剂B液3 ml X 1 瓶6 ml X 1 瓶2-8 C保存终止液3 ml X 1 瓶6ml X 1 瓶2-8 C保存浓缩洗涤液(20ml X 20 倍)X1 瓶(20ml X 30 倍)X1 瓶2-8 C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

4、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。5. 组织标本：切割标本后，称取重量。加入一定量的PBS，PH7.

5、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-20C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100山，然后在第一、第二孔中加标准品稀释液50山，混匀

6、；然后从第一孔、第二 孔中各取100山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50卩1, 混匀；然后在第三孔和第四孔中先各取50卩1弃掉，再各取50山分别加到第五、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50山分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液50山，混 匀后从第七、第八孔中分别取50卩1加到第九、第十孔中，再在第九第十孔分别加标准 品稀释液50卩1，混匀后从第九第十孔中各取50卩1弃掉。（稀释后各孔加样量都为50卩1, 浓度分别为48 ng/L， 32 ng/L， 16ng/L， 8ng/L， 4

7、 ng/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40卩1,然后再加待测样品10山（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37C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50卩1，空白孔除外。7. 温育：操作同 3。8. 洗

8、涤：操作同 5。9. 显色：每孔先加入显色剂A50yl，再加入显色剂B50yl，轻轻震荡混匀，37C避光显色 15分钟.10. 终止：每孔加终止液50gl，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，

9、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伍倍）后再测定，计算时请最后乘以总稀释倍数（XnX5）o5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。7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

10、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。试剂盒性能：1. 样品线性回归与预期浓度相关系数R值为0.95以上。2. 批内与批间应分别小于9%和 11%检测范围：1ng/L - 65ng/L保存条件及有效期：1试剂盒保存:;2-8C。2有效期： 6 个月RDHuman PI Ab-IgMFOR RESEARCH USE ONLYDrug NamesGeneric Name： Human PI Ab-IgM ELISA Kit.PurposeThis kit allows for the

11、 determination of PI Ab-IgM concentrations in Human serum, blood plasma, and other biological fluids.Principle of the assayThe kit assay Human PI Ab-IgM level in the sample，use Purified antigen to coat microtiter plate wells, make solid-phase antigen, then add PI Ab-IgM to wells, Combined antigen wh

12、ich With HRP labeled, become antigen -antibody - enzyme- antigen complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotome

13、trically at a wavelength of 450 nm. The concentration of PI Ab-IgM in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kitMaterials provided with the kit48determinations96 determinationsStorageUser manual11Closure plate membrane22S

14、ealed bags11Microelisa stripplate112-8 CStandard： 72 ng/L0.5mlx1 bottle0.5mlx1 bottle2-8 CStandard diluent1.5mlx1 bottle1.5mlx1 bottle2-8 CHRP-Conjugate reagent3mlx1 bottle6mlx1 bottle2-8 CSample diluent3mlx1 bottle6mlx1 bottle2-8 CChromogen Solution A3mlx1 bottle6mlx1 bottle2-8 CChromogen Solution

15、B3mlx1 bottle6mlx1 bottle2-8 CStop Solution3mlx1 bottle6mlx1 bottle2-8 Cwash solution(20ml x 20 fold) xi bottle(20ml x 30 fold) x1 bottle2-8 CSpecimen requirements1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precip

16、itation appeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min

17、at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax 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

18、-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS(PH7.2-7.4), Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supe

19、rnatant, If precipitation appeared, Centrifugal again.5. Tissue sample- sAfter cutting samples, check the weight,add PBS(PH7.2-7.4), Rapidly frozen with liquid nitrogen, maintain samples at 2-8 C after melting,add PBS (PH7.4), Homogenized by hand or Grinders, centrifugation 20-min at the speed of 20

20、00-3000 r.p.m. remove supernatant.6. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it cant, specimen can be kept in -20 C to preserve, Avoid repeated freeze-thaw cycles.7. Cant det

21、ect 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 100pl to the first and the second well, then add Standard dilution 50pl to the first and the second well, mix; take o

22、ut 100pl form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50pl to the third and the forth well ,mix ; then take out 50pl from the third and the forth well discard, add 50pl to the fifth and the sixth well ,then add Standard dilutio

23、n 50pl to the fifth and the sixth well, mix ; take out 50pl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50pl to the seventh and the eighth well ,mix ; take out 50pl from the seventh and the eighth well and add to the ninth and the tenth we

24、ll, add Standard dilution 50pl to the ninth and the tenth well, mix , take out 50pl from the ninth and the tenth well discard(add Sample 50pl to each well after Diluting ,(density: 48 ng/L，32 ng/L，16ng/L，8ng/L，4 ng/L)2. add sample：Set blank wells separately (blank comparison wells dont add sample an

25、d HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40pl to testing sample well, then add testing sample 10pl (sample final dilution is 5-fold), add sample to wells , dont touch the well wall as far as possible, and Gently mix.3.Incubate: After closi

26、ng plate with Closure plate membrane ,incubate for 30 min at 37C.4. Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5. washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for

27、 30s then drain, repeat 5 times, dry by pat.6. add enzyme： Add HRP-Conjugate reagent 50pl to each well, except blank well. 7.incubate： Operation with 3.8. 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

28、 37 C10Sop the reaction : Add Stop Solution50pl to each well, Stop the reaction(the blue color change to yellow color).11.assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Important notes1. The kit takes out from the refrigeration environment shoul

29、d be balanced 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. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sampl

30、e with sampler Each step, And proofread its accuracy frequently, avoids the experimental 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 first standard well ),please

31、 dilute Sample (n-fold), Please diluente and multiplied by the dilution factor. (xnx5).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 m

32、ust take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective material process.9. Do not mix reagents with those from other lots.CalculateTake the standard density as the horizontal, the ODThis chartis for reference onlythe result is the sample actual density.Assay range1ng/L - 65ng/LStorage and validity1. Storage :2-8C.2validity： six months.