鱼elisa试剂盒,肾上腺髓质素（ADM）酶免技术 广锐生物-国内高、优Elisa试剂盒供应商1鱼肾上腺髓质素（ADM）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：本试剂盒用于测定鱼血清，血浆及相关液体样本中肾上腺髓质素（ADM）的含量。实验原理：本试剂盒应用双抗体夹心法测定标本中鱼肾上腺髓卐质素（ADM）水平。用纯化的鱼肾上腺髓质素（ADM）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肾上腺髓质素（ADM），再与HRP 标记的肾∏上腺髓质素（ADM）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肾上腺髓质素（ADM）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鱼肾上腺髓质素（ADM）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 份封板膜2 片（48） 2 片（96）密封袋1 个1 个酶标卐包被板1×48 1×96 2-8℃保存标准品：18ng/L 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℃保存样品ぷ稀释液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ω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①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ζ　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2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ㄨ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12ng/L，8ng/L ，4ng/L，2ng/L， 1ng/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Ψ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。36． 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：0.5ng/L -16ng/L保存条◥件及有效期：1.试剂盒保存：；2-8℃。2．有效期：6 个月4Fish adrenomedullinFOR RESEARCH USE ONLYDrug NamesGeneric Name：Fish adrenomedullin (ADM) ELISA Kit.PurposeThis kit allows for the determination of ADM concentrations in Fish serum,blood plasma, and other biological fluids.Principle of the assayThe kit assay Fish ADM level in the sample ， use Purified Fish ADMantibody to coat microtiter plate wells, make solid-phase antibody, then addADM to wells, Combined ADM antibody which With HRP labeled, becomeantibody - antigen - enzyme-antibody complex, after washing Completely, AddTMB substrate solution,TMB substrate becomes blue color At HRPenzyme-catalyzed, reaction is terminated by the addition of a sulphuric acidsolution and the color change is measured spectrophotometrically at awavelength of 450 nm. The concentration of ADM in the samples is thendetermined by comparing the O.D. of the samples to the standard curve.5Materials provided with the kitMaterials providedwith the kit48determinations 96 determinationsStorageUser manual 1 1Closure platemembrane2 2Sealed bags 1 1Microelisa stripplate 1 1 2-8℃Standard：18ng/L 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃HRP-Conjugatereagent3ml×1 bottle 6ml×1 bottle 2-8℃Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃Chromogen SolutionA3ml×1 bottle 6ml×1 bottle 2-8℃Chromogen SolutionB3ml×1 bottle 6ml×1 bottle 2-8℃Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃wash solution（20ml×20 fold）×1bottle（20ml×30 fold）×1bottle2-8℃Specimen requirements1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-minat the speed of 2000-3000 r.p.m. remove supernatant, If precipitationappeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. removesupernatant, If precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of2000-3000 r.p.m. remove supernatant, If precipitation appeared,Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid6Reference to it.4. cell culture supernatant-detect secretory components, collect sue asterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant,detect the composition of cells, Dilut cell suspensionwith PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeatedfreeze-thaw cycles, damage cells and release of intracellular components,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add PBS（ PH7.2-7.4 ） , Rapidly frozen with liquid nitrogen, maintain samples at2-8℃ after melting,add PBS（PH7.4）, Homogenized by hand or Grinders,centrifugation 20-min at the speed of 2000-3000 r.p.m. removesupernatant.6. extract as soon as possible after Specimen collection,and according to therelevant literature, and should be experiment as soon as possible after theextraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoidrepeated freeze-thaw cycles.7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRPactive.Assay procedure1.Dilute and add sample to Standard: set 10 Standard wells on the ELISAplates coated, add Standard 100μl to the first and the second well, then addStandard dilution 50μl to the first and the second well, mix; take out 100μlform the first and the second well then add it to the third and the forth wellseparately. then add Standard dilution 50μl to the third and the forthwell ,mix ; then take out 50μl from the third and the forth well discard, add50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifthand the sixth well, mix ; take out 50μl from the fifth and the sixth well and addto the seventh and the eighth well, then add Standard dilution 50μl to the7seventh 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 50μlto the ninth and the tenth well, mix , take out 50μl from the ninth and the tenthwell discard(add Sample 50μl to each well after Diluting ,(density: 12ng/L ，8ng/L ，4ng/L，2ng/L， 1ng/L)2.add sample：Set blank wells separately (blank comparison wells don’t addsample and HRP-Conjugate reagent, other each step operation is same).testing sample well. add Sample dilution 40μl to testing sample well, then addtesting 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 30min at 37℃.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 30s then drain, repeat 5 times, dry bypat.6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blankwell.7.incubate：Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to eachwell, evade the light preservation for 15 min at 37℃10.Stop the reaction：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 AddingStop Solution and within 15min.Important notes81. The kit takes out from the refrigeration environment should be balanced15-30 minutes in the room temperature, ELISA plates coated if has not useup after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the waterhelps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently,avoids the experimental error. add sample within 5 mins, if the number ofsample is much , recommend to use Volley .4. if the testing material content is excessively higher (The sample OD isbigger than the first standard well ),please dilute Sample (n-fold), Pleasediluente and multiplied by the dilution factor.（×n×5）.5. Closure plate membrane only limits the disposable use, to avoidcross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determinationmust take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according toinfective material process.9. Do not mix reagents with those from other lots.CalculateTake the standard density as the horizontal,the OD value for the vertical ,draw the standardcurve on graph paper, Find out the correspondingdensity according to the sample OD value by theSample curve, multiplied by the dilution multiple,or calculate the straight line regression equationof the standard curve with the standard densityand the OD value ,with the sample OD value inthe equation, calculate the sample density,This chartis for reference only9Assay range0.5ng/L -16ng/LStorage and validity1．Storage： 2-8℃.2．validity： six months.