大鼠M13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：本试剂盒用于测定大鼠血清，血浆及相关液体样本中M13的含量。
实验原理：
本试剂盒应用双抗体夹心法测定标本中大鼠M13水平。用纯化的大鼠M13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M13，再与HRP标记的M13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M13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大鼠M13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 |
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封板膜 | 2片（48） | 2片（96） |
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密封袋 | 1个 | 1个 |
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酶标包被板 | 1×48 | 1×96 | 2-8℃保存 |
标准品：72pg/mL | 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。
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2-8℃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48pg/mL，32 pg/mL ，16 pg/mL，8pg/mL，4 pg/mL）。
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。
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。
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： 1.8pg/mL -65pg/mL
保存条件及有效期： 1.试剂盒保存：；2-8℃。 2．有效期：6个月
RatM13
Drug Names Generic Name：Rat M13 ELISA Kit. Purpose This kitallows for the determinationof M13 concentrations in Ratserum, blood plasma,and otherbiological fluids. Principle of the assay The kit assay Rat M13 levelin the sample，use Purified Rat M13 antibodyto coat microtiter plate wells, make solid-phase antibody, then add M13 towells, Combined M13 antibodywhich With HRP labeled，become antibody-antigen- enzyme-antibody complex,after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRPenzyme-catalyzed, reaction is terminated by the addition of asulphuric acid solution and the color change is measured spectrophotometricallyat a wavelength of 450 nm.The concentration of M13 in the samples is then determined by comparing the O.D.of the samples to the standard curve.
Materials provided with the kit
Specimen requirements 1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min atthe speed of 2000-3000r.p.m. remove supernatant, If precipitation appeared, Centrifugalagain. 2. plasma-use suitedEDTA or citrate plasma as ananticoagulant,mix 10-20mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugalagain. 3. Urine-collectsue a sterile container, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugalagain.The Operation of Hydrothorax and cerebrospinal fluidReference to it. 4. cell culture supernatant-detect secretory components,collect sue a sterile container, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant,detect the composition of cells, Dilut cellsuspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeatedfreeze-thaw cycles, damage cells and release of intracellularcomponents, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugalagain. 5. Tissuesamples- After cutting samples, check theweight,add PBS（PH7.2-7.4）, Rapidly frozen with liquidnitrogen, maintainsamples at 2-8℃after melting,add PBS（PH7.4）,Homogenized by hand or Grinders, centrifugation 20-min atthe speed of 2000-3000r.p.m. remove supernatant. 6. extract as soon as possible after Specimen collection,and according to the relevant literature,and should be experimentas 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 procedure 1.Dilute and add sample toStandard: set 10 Standard wells on the ELISA plates coated, add Standard100μl to the first and the second well, then add Standard dilution 50μl to thefirst and the second well, mix; take out 100μl form the first and the secondwell then add it to the third and the forth well separately. then add Standarddilution 50μlto the third and the forth well ,mix ; then take out 50μl from the third andthe forth well discard, add 50μl to the fifth and the sixth well ,then addStandard dilution50μl to the fifth and the sixth well, mix ; takeout 50μl from the fifth and the sixth well and add to the seventh and theeighth well, then add Standard dilution 50μl to theseventh 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 theninth and the tenth well, mix , take out 50μl from the ninth and the tenth welldiscard(add Sample 50μl to each well after Diluting ,(density: 48pg/mL，32 pg/mL ，16 pg/mL，8pg/mL，4 pg/mL) 2.add sample：Setblank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent,other each step operation is same). testing sample well. add Sample dilution40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-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 at37℃. 4.Configurateliquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) withdistilled water and reserve. 5.washing：UncoverClosure platemembrane, discard Liquid, dry byswing, add washing buffer to every well, still for 30s then drain, repeat 5times, dry by pat. 6.add enzyme：Add HRP-Conjugate reagent 50μlto each well, except blank well. 7.incubate：Operationwith 3. 8.washing：Operationwith 5. 9.color：Add Chromogen Solution A50ul andChromogen Solution B to each well, evade the light preservationfor 15 min at37℃ 10.Stop thereaction：Add Stop Solution50μl to each well, Stop the reaction(theblue color change to yellow color). 11.assay：take blank well as zero ,Read absorbance at 450nm after Adding Stop Solution and within 15min. Important notes 1. The kit takes out from the refrigerationenvironment should be balanced 15-30 minutes in the room temperature, ELISA platescoated if has not use up after opened, the plate should be stored in Sealedbag. 2. washing buffer will Crystallization separation,it can be heated the water helps dissolve when dilute . Washing does not affectthe result. 3. add Sample with sampler Each step, And proofreadits accuracy frequently, avoids the experimental error. add sample within 5 mins, if the number of sample is much , recommend touse Volley . 4. if the testing material content is excessivelyhigher (The sample OD is bigger than the firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.（×n×5）. 5. Closure platemembrane onlylimits 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 the microtiterplate reader as astandard. 8. All samples, washing buffer and each kind ofreject should according to infective material process. 9. Donot mix reagents with those from other lots.
Calculate
Assay range 1.8pg/mL -65pg/mL Storage and validity 1．Storage： 2-8℃. 2．validity： six months. |
