小鼠色素上皮衍生因子PEDF酶联免疫分析ELISA

1、1小鼠色素上皮衍生因子（PEDF酶联免疫分析（ELISAELISA）试剂盒使用说明书关液体样本中色素上皮衍生因子（PEDF的含量。实验原理：本试剂盒应用双抗体夹心法测定标本中小鼠色素上皮衍生因子（PEDF）水平。用纯化的小鼠色素上皮衍生因子（PEDF）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依 次加入色素上皮衍生因子（PEDF），再与HRP标记的色素上皮衍生因子 （PEDF）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色素上皮衍生因子（PEDF）呈正相关。用酶标仪在45

2、0nm波长下测定吸光度（OD值），通过标准曲线计 算样品中小鼠色素上皮衍生因子（PEDF）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：180 jjg/L0.5ml X 1瓶0.5ml X 1瓶28C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 ml X 1瓶6 ml X 1瓶2-8 C保存样品稀释液3 ml X 1瓶6 ml X 1瓶2-8 C保存显色剂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

3、X 1瓶2-8 C保存终止液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转份）。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转份）。仔细收集上清，保存过程中如有沉淀形成

4、，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20分钟左右（2000-3000转/2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 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,PH7.4。用液氮迅速冷冻保存备2-8 C的温度。加入一定量的PBS（PH7.4）,用手工或匀浆器20

5、分钟左右（2000-3000转/分） 。仔细收集上清。分装后一份待20分用。标本融化后仍然保持 将标本匀浆充分。离心 检测，其余冷冻备用。6.标本采集后尽早进行提取，进行试验，可将标本放于提取按相关文献进行，提取后应尽快进行实验。若不能马上-20 C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（ 操作步骤1.HRP）活性。2.3.4.5.6.7.8.9.10.11.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 M，然后在第一、第二孔中加标准品稀释液50也混匀；然后从第一孔、第二孔中各取100 d分别加到第三孔和

6、第四孔， 再在第三、第四孔分别加标准品稀释液50 M, 混匀；然后在第三孔和第四孔中先各取50 d弃掉，再各取50 d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 d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 d，混匀后从第七、第八孔中分别取50dl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 d，混匀后从第九第十孔中各取50 d弃掉。（稀释后各孔加样量都为50 d,浓度分别为120dg/L，80 dg/L，40 dg/L，20 dg/L，10dg/L）。 加样：分别设空白孔（空白对照孔不加样品及

7、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 d,然后再加待测样品10 d（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。温育：用封板膜封板后置37 C温育30分钟。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。 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。加酶：每孔加入酶标试剂50 dl，空白孔除外。 温育：操作同3。 洗涤：操作同5。显色：每孔先加入显色剂A50 d，再加入显色剂B50 d，轻轻震荡混匀，15分钟.终止：每孔

8、加终止液50 d，终止反应（此时蓝色立转黄色）。测定：以空白空调零，450nm波长依序测量各孔的吸光度（0D值）。 测定应在加终止 液后15分钟以内进行。37 C避光显色注意事项：1234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。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。 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。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0D值

9、），请先用样品稀释液稀释一定倍数（n倍）后再测定，计34算时请最后乘以总稀释倍数（X n X 5）o封板膜只限一次性使用，以避免交叉污染。 底物请避光保存。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 所有样品，洗涤液和各种废弃物都应按传染物处理。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，0D值为纵坐标，在坐标纸上绘出标准曲线，根据样品的0D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0D值计算出标 准曲线的直线回归方程式，将样品的0D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

10、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批内与批见应分别小于9%和11%检测范围:5.0用/L -150 /L保存条件及有效期：1试剂盒保存：；2-8Co2.有效期：6个月Mouse p igme nt ep ithelium-derived factorFOR RESEARCH USE ONLY（此图仅供参考）5.6.7.&9.5DrugDrug NamesNamesGen eric Nam:Mouse p igme nt epi thelium-derived factor (PEDF) ELISA Kit.PurposePurposeThis ki

11、t allows for the determ in ationof P EDF concen trati onSn Mouse serum, bloodpl asma, and other biological fluids.P P rincrinc ipleiple ofof thethe assayassayThe kit assay Mouse P EDF level in the sample, use Purified Mouse P EDF an tibody tocoat microtiterplate wells, make solid-p hasea ntibody,the

12、 n add P EDF to wells, Comb inedP EDF an tibody which With HRP labeled, become an tibody- an tige n - en zyme-a ntibody complex, after wash ing Comp letely, Add TMB substrate solutio n,TMB substrate becomes blue colorAt HRP en zyme-catalyzecheacti onis term in atedby the additi onof a sulp huricacid

13、 solution and the color cha nge is measured spectrop hotometrically at a wavele ngth of 450 nm.The concen trati on of P EDF in the samp les is the n determ ined by comparing the O.D. ofthe samp les to the sta ndard curve.6Materials pro videdwith the kit48determ in ations96 determ in atio nsStorageUs

14、er manual11Closure pl ate membra ne22Sealed bags11Microelisa stri pplate112-8 CSta ndard 180冯/L0.5ml 1Xbottle0.5ml Kbottle2-8 CStan dard dilue nt1.5ml Xbottle1.5ml Kbottle2-8 CHRP-Co njugate reagen t3ml Xbottle6ml 1 bottle2-8 CSample dilue nt3ml 1 bottle6ml 1 bottle2-8 CChromoge n Soluti on A3ml 1 b

15、ottle6ml 1 bottle2-8 CChromoge n Soluti on B3ml 1 bottle6ml 1 bottle2-8 CStop Soluti on3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml X 20fold)X 1bottle(20ml X 30fold)X 1bottle2-8 CMaterialsMaterials p p rovidedrovided withwith thethe kitkitSpSp ecimenecimen requirementsrequirements1.serum- coagula

16、tio n at room temp erature 10-20 ,raeintrifugati on 20-min at the speed of2000-3000 r.p .m. remove supern ata nt. If precip itati on app eared, Cen trifugalaga in.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20mins ,ce ntrifugatior20-min at the sp eed of 2000-3000r. p.m. re

17、move supern atant,Ifprecip itati on app eared, Cen trifugalaga in.3. Urine collect sue a sterile container, cen trifugati on 20-min at the sp eed of 2000-3000r.p.m.remove supern ata nt,lf precip itati onapp eared, Cen trifugalaga in. The Operatio n ofHydrothorax and cerebros pinal fluid Refere nee t

18、oit.4. cell culture supern ata n-detect secretoryco mponen tscollect sue a sterilecontainer,cen trifugatior2 0-min at the sp eed of 2000-3000r. p.m. removes upern atant,detechecompositionof cells, Dilut cell suspensionwith PBS (PH7.2-7.4 , Cellconcentrationreached 1 million / ml, repeated freeze-tha

19、wcycles, damage cells and release ofin tracellular componen ts, cen trifugati on 20-min at the sp eed of 2000-3000 r.p.m.removesupern ata nt, If precip itati on app eared, Cen trifugalaga in.785. Tissue samples- After cutting samples, check the weight,add(PPBHS7.2-7.4),Rapidlyfroze n with liquid n i

20、troge n, mai ntain samp les af(2-8fter melt in g,add PBSPH7.4 ,Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant.6. extract as soon as possibleafter Specimencollection,andaccordingto the relevantliterature,and shouldbe experimentas soon as poss

21、ibleafter the extraction.If itcant,sp ecime n can be kept in -20 to p reserve, Avoid rep eated freeze-thaw cycles.7.Cant detect the sample which coNnataNin3, because NaN3 inhibits HRP active.AssayAssay procedureprocedure1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates c

22、oated, addStandard 100卩l to the first and the seconewadd Standard diluti50i卩l to the firstandthe second well, mix; take out 100卩l form the first and the second well then add it tothe thiand the forth well separatelythen add Standarddilution 50卩to the third and theforthwell ,mix ; thetake out 50卩l fr

23、om the third and the forth well discard, add50the sixth well ,then add Standard dilL50)p l to the fifth and the sixth well, mix ; takeout 50from the fifth and the sixth well and add to the seventh and the eighth well, then add Standarddilution50卩l to the seventh and the eighth well ,mix ; takeout 50

24、卩l from theseveneighth well and add to the ninth and the tenth well, add Sta ndard50liutionD the ninthandthe tenth well, mix , take out 50 m the nin thpahdcihe ten th wescard(add Samp le 50卩ltoeach well after Diluting ,(density: jg20,8O旧/L ,4Qg/L,20fg/L,10旧/L)2.add sample：Set blank wells separately(

25、blank comparisonwells donatdd sampleandHRP-Conjugate reagent, other each step operation is same). testing sample well. add Samplediluti on40 ytlo test in gsa mp lewell the n add testi ng sampi e10卩(sa mplefinaldiluti onis5-fold), add sample to welldso,nt touch the well wall as far aspoasnsidblGe,ent

26、ly mix.3.ln cubate: After closi ng p late with Closure plate membra ne ,in cubate for 30(minat 374.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) withdistilledwater and reserve.5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buf

27、ferto every well, still for 30s then drain, repeat 5 times, dryby pat.9106.add enzym: Add HRP-Conjugate reagent l to each well, excebtank well.7.incubate： Operation with3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight p reservat

28、i on for 15 minat371O.Stopthe reaction Add Stop Solutio50卩1 each well, Stop the react ion (th由lue colorchange to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.ImportantImportant notesnotes1.The kit takes out from the refrigeratio

29、n environment should be balanced 15-30 minutesinthe room temperature, ELISA plates coated if has not use up after opened, the plate shouldbe stored in Sealed bag.2. washingbufferwill Crystallizationseparation,it can be heatedthe water helpsdissolvewhen dilute . Washing does not affect the result.3.add Samplewith samplerEach st