小鼠补体蛋白3C3酶联免疫分析ELISA

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1、小鼠补体蛋白3（ C3）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：本试剂盒用于测定小鼠血清，血浆及相关液体样本中补体蛋白3（ C3）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补体蛋白3（C3）水平。用纯化的小鼠 补体蛋白3（C3）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补体蛋白3（C3），再与HRP标记的补体蛋白3（C3）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补体蛋白3（ C3）呈正相关。用酶标仪在 450nm波长下测

2、定吸光度（OD值），通过标准曲线计算样品中小鼠补体蛋白3（ C3）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份圭寸板膜2 片(48)2 片(96)密封袋1个1个酶标包被板1X 481X 962-8 C保存标准品：900/ml0.5ml 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 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 m

3、l 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，

4、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，PH7.4 。用液氮迅速冷冻保存备用。标本融化后仍然保持2-8C的温度。加入一定量的PBS （ PH7.4）,用手工或匀浆器将标

5、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 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（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 M，然后在第一、第二孔中加标准品稀释液50 d，混匀；然后从第一孔、第二孔中各取100 d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

6、 d,混匀；然后在第三孔和第四孔中先各取50dl 弃掉，再各取 50dl 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 600 dg/ml ， 400 dg/ml ， 200 dg/ml ， 1 00 dg/ml ， 50 dg/ml ） 。2. 加样：分别设空白孔（空白对照孔不加样

7、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 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。3. 温育：用封板膜封板后置 37 C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 dl ，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 A50

8、 d，再加入显色剂 B50 d，轻轻震荡混匀，37 C避光显色 15分钟.10. 终止：每孔加终止液 50 d，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

9、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（XnX 5）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&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

10、；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：30 用/ml -700 旧/ml 保存条件及有效期：1.试剂盒保存：;2-8Co2 有效期：6个月Mouse Compleme nt 3FOR RESEARCH USE ONLYDrug NamesGen eric NamdMouse Compleme nt 3 (C3) ELISA Kit.PurposeThis

11、 kit allows for the determ in ati on of C3 concen tratio ns in Mouse serum, blood plasma, and other biological fluids.Pr in ciple of the assayThe kit assay MouseC3 level in the sample use PurifiedMouseC3 antibodyto coat microtiter plate wells, make solid-phase antibody, then add C3 to wells, Combine

12、d C3 antibody which With HRP labeled , become an tibody- an tige n - en zyme-a ntibod擊omplex,after wash ing Completely, Add TMB substrate solutio n,TMB substrate becomes blue color At HRP en zyme-catalyzed, reacti on is term in ated by the additi on of a sulphuric acid soluti on and the color change

13、 is measured spectrophotometricallqt a wavelength of 450 nm. The concentration of C3 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 the kit48determ in ati ons96 determ in atio nsStorageUser manual11

14、Closure plate membra ne22Sealed bags11Microelisa stripplate112-8 CStandard 900/ml0.5ml 1Xbottle0.5ml Kbottle2-8 CStan dard dilue nt1.5ml Xbottle1.5ml Kbottle2-8 CHRP-Co njugate reagent3ml Xbottle6ml 1 bottle2-8 CSample dilue nt3ml 1 bottle6ml 1 bottle2-8 CChromoge n Soluti on A3ml 1 bottle6ml 1 bott

15、le2-8 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 CSpecime n requireme nts1. serum- coagulation at room temperature 10-20 ,，ceiistrifugation 20-min at the speed of 2000-3000 remove

16、supernatant. If precipitation appeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation20-min at the speed of 2000-3000 remove supernatant,lf precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container,centrifuga

17、tior20-min at the speed of 2000-3000 remove supernata nt,If precipitati on appeared, Cen trifugalaga in. The Operatio n of Hydrothorax and cerebrosp inal fluid Refere nee to it.4. cell culture supernatan-detect secretorycomponentscollect sue a sterile container, centrifugation20-min at the speed of

18、2000-3000 remove supernatant,detecthe compositionof cells, Dilut cell suspensiorwith PBS( PH7.2-7.4 , Cell concentration reached 1 million / ml, repeated freeze-thawcycles, damage cells and release of intracellularcomponents,centrifugation20-min at the speed of 2000-3000 remove supernatant, If preci

19、pitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add(PPBHS7.2-7.4) , Rapidly froze n with liquid n itroge n, mai ntain samples atQ-8fter melt in g,add PBSPH7.4 , Homogenizedby hand or Grinders,centrifugation20-min at the speed of 2000-3000 remove supern

20、atant.6. extract as soon as possibleafter Specimencollection,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction.If it cant, specime n can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Cant detect the sample which coNnataNin3, beca

21、use NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100 卩 l to the first and the second well, then add Stand50didilUrtithe first and the sec ond well, mix; take out 100stantjbthetbecioind well the n add it t

22、o the thirdand the forth well separatelythen add Standarddilution 50 卩 to the third and the forth well ,mix ; then take out 50卩 l from the third and the forth well discard, add 50the sixth well ,then add Standard dilU50)p l to the fifth and the sixth well, mix ; take out 50 from the fifth and the si

23、xth well and add to the seventh and the eighth well, then add Standard dilution50 卩 l to the seventh and the eighth well ,mix ; take out 50 e seventh and the l from th eighth well and add to the ninth and the tenth well, add Sta ndardliutioin) the ninth andthe ten th well, mix , take out 50 m the ni

24、n thpahttcthe ten th wescard(add Sample 50 卩 l toeach well after Diluting ,(density: 6gmM,40Qg/ml ,200g/ml,100_g/ml,5Qg/ml)2. add sample：Set blank wells separately(blank comparisonwells donatdd sampleandHRP-Conjugate reagent, other each step operation is same). testing sample well. add Samplediluti

25、on40 ytlo test in gsamplewel, the n add testi ng sample10 y(samplefi nal diluti onis 5-fold), add sample to welldso,nt touch the well wall as far as poasnsidblGe,ently mix.3.ln cubate: After closi ng plate with Closure plate membra ne ,in cubate for 30CBnin at 374. Configurate liquid: 30-fold (or 20

26、-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 30s then drain, repeat 5 times, dry by pat.6. add enzym: Add HRP-Conjugate reag50t l to each well, ex

27、cebtank 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 the light preservati on for 15 min at3710.Stopthe reaction Add Stop Solutio50 卩1 each well, Stop the react ion (th由lue color change to yellow col

28、or).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 should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate s

29、hould be stored in Sealed bag.2. washingbufferwill Crystallizationseparation,it can be heatedthe waterhelps dissolve when dilute . Washing does not affect the result.3. add Samplewith samplerEach step, And proofreadits accuracyfrequently,avoidsthe experimentalerror. add sample within 5 mins, if the

30、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 dilute Sample (n-fold), Please diluente and multiplied by the dilution factor. ( n5).5. Closure plate membrane only limits the d

31、isposable use, to avoid cross-contamination.6. The substrate evade the light preserv.ation7. Pleaseaccordingto use instructionstrictly,The test resultdeterminationmust take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective

32、material process.9. Do not mix reagents with those from other lots.CalculateThis chartis for refere nee onlyvalue for the vertical ,draw the sta ndardcurve on graphden sity accord ing to theTakethe sta ndardde nsityas the horiz on tal,the OD san30 lg/mD-Vai0g/iml the Samplecurve, multipliedby theSUiirag|euandbvalidHteulatethe straight line regressi on equgttojaogthe gtgndard curve with the standard density and toragethe OD value ,with the sampleOD value in the equati on.e :, isity, multiplied by the diluti on factor,the result is the sample actual den sity.