大鼠组胺HIS说明指导书.doc

大鼠组胺（HIS）酶联免疫分析试剂盒使用阐明书本试剂仅供研究使用 目：本试剂盒用于测定大鼠血清，血浆及有关液体样本中组胺（HIS）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组胺（HIS）水平。用纯化大鼠组胺（HIS）抗体包被微孔板，制成固相抗体，往包被单抗微孔中依次加入组胺（HIS），再与HRP标记HIS抗体结合，形成抗体-抗原-酶标抗体复合物，通过彻底洗涤后加底物TMB显色。TMB在HRP酶催化下转化成蓝色，并在酸作用下转化成最后黄色。颜色深浅和样品中组胺（HIS）呈正有关。用酶标仪在450nm波长下测定吸光度（OD值），通过原则曲线计算样品中大鼠组胺（HIS）浓度。试剂盒构成：试剂盒构成48孔配备96孔配备保存阐明书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1481962-8保存原则品：18g/L0.5ml1瓶0.5ml1瓶2-8保存原则品稀释液1.5ml1瓶1.5ml1瓶2-8保存酶标试剂3 ml1瓶6 ml1瓶2-8保存样品稀释液3 ml1瓶6 ml1瓶2-8保存显色剂A液3 ml1瓶6 ml1瓶2-8保存显色剂B液3 ml1瓶6 ml1瓶2-8保存终结液3ml1瓶6ml1瓶2-8保存浓缩洗涤液（20ml20倍）1瓶（20ml30倍）1瓶2-8保存样本解决及规定：1. 血清：室温血液自然凝固10-20分钟，离心20分钟左右（-3000转/分）。仔细收集上清，保存过程中如浮现沉淀，应再次离心。2. 血浆：应依照标本规定选取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（-3000转/分）。仔细收集上清，保存过程中如有沉淀形成，应当再次离心。3. 尿液：用无菌管收集，离心20分钟左右（-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：检测分泌性成分时，用无菌管收集。离心20分钟左右（-3000转/分）。仔细收集上清。检测细胞内成分时，用PBS（PH7.2-7.4）稀释细胞悬液，细胞浓度达到100万/ml左右。通过重复冻融，以使细胞破坏并放出细胞内成分。离心20分钟左右（-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PBS，PH7.4。用液氮迅速冷冻保存备用。标本融化后依然保持2-8温度。加入一定量PBS（PH7.4），用手工或匀浆器将标本匀浆充分。离心20分钟左右（-3000转/分）。仔细收集上清。分装后一份待检测，别的冷冻备用。6. 标本采集后尽早进行提取，提取按有关文献进行，提取后应尽快进行实验。若不能立即进行实验，可将标本放于-20保存，但应避免重复冻融.7. 不能检测含NaN3样品，因NaN3抑制辣根过氧化物酶（HRP）活性。操作环节：1. 原则品稀释与加样：在酶标包被板上设原则品孔10孔，在第一、第二孔中分别加原则品100l，然后在第一、第二孔中加原则品稀释液50l，混匀；然后从第一孔、第二孔中各取100l分别加到第三孔和第四孔，再在第三、第四孔分别加原则品稀释液50l，混匀；然后在第三孔和第四孔中先各取50l弃掉，再各取50l分别加到第五、第六孔中，再在第五、第六孔中分别加原则品稀释液50ul，混匀；混匀后从第五、第六孔中各取50l分别加到第七、第八孔中，再在第七、第八孔中分别加原则品稀释液50l，混匀后从第七、第八孔中分别取50l加到第九、第十孔中，再在第九第十孔分别加原则品稀释液50l，混匀后从第九第十孔中各取50l弃掉。（稀释后各孔加样量都为50l，浓度分别为12g/L，8g/L ，4g/L，2g/L， 1g/L）。2. 加样：分别设空白孔（空白对照孔不加样品及酶标试剂，别的各步操作相似）、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40l，然后再加待测样品10l（样品最后稀释度为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（48T20倍）倍浓缩洗涤液用蒸馏水30（48T20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50l，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A50l，再加入显色剂B50l，轻轻震荡混匀，37避光显色15分钟. 10. 终结：每孔加终结液50l，终结反映（此时蓝色立转黄色）。11. 测定：以空白空调零，450nm波长依序测量各孔吸光度（OD值）。 测定应在加终结液后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 浓洗涤液也许会有结晶析出，稀释时可在水浴中加温助溶，洗涤时不影响成果。3 各步加样均应使用加样器，并经常校对其精确性，以避免实验误差。一次加样时间最佳控制在5分钟内，如标本数量多，推荐使用排枪加样。4 请每次测定同步做原则曲线，最佳做复孔。如标本中待测物质含量过高（样本OD值不不大于原则品孔第一孔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（n5）。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 严格按照阐明书操作进行，实验成果鉴定必要以酶标仪读数为准.8 所有样品，洗涤液和各种废弃物都应按传染物解决。9 本试剂不同批号组分不得混用。10. 如与英文阐明书有异，以英文阐明书为准。计算：以原则物浓度为横坐标，OD值为纵坐标， 在坐标纸上绘出原则曲线，依照样品OD 值由原则曲线查出相应浓度；再乘以稀释 倍数；或用原则物浓度与OD值计算出标 准曲线直线回归方程式，将样品OD值 代入方程式，计算出样品浓度，再乘以稀释 倍数，即为样品实际浓度。 （此图仅供参照）试剂盒性能：1.样品线性回归与预期浓度有关系数R值为0.990以上。2.批内与批见应分别不大于9%和11%检测范畴： 0.75g/L -15g/L 保存条件及有效期：1.试剂盒保存：；2-8。2有效期：6个月RB Rat Histamine(HIS)FOR RESEARCH USE ONLYDrug NamesGeneric Name：Rat Histamine (HIS) ELISA Kit.PurposeThis kit allows for the determination of HIS concentrations in Rat serum，blood plasma，and other biological fluids.Principle of the assayThe kit assay Rat HIS level in the sample，use Purified Rat HIS antibody to coat microtiter plate wells，make solid-phase antibody，then add HIS to wells，Combined HIS antibody which With HRP labeled,become antibody - antigen - enzyme-antibody 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 spectrophotometrically at a wavelength of 450 nm. The concentration of HIS 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 membrane22Sealed bags11Microelisa stripplate112-8Standard：18g/L0.5ml1 bottle0.5ml1 bottle2-8Standard diluent1.5ml1 bottle1.5ml1 bottle2-8HRP-Conjugate reagent3ml1 bottle6ml1 bottle2-8Sample diluent3ml1 bottle6ml1 bottle2-8Chromogen Solution A3ml1 bottle6ml1 bottle2-8Chromogen Solution B3ml1 bottle6ml1 bottle2-8Stop Solution3ml1 bottle6ml1 bottle2-8wash solution（20ml20 fold）1bottle（20ml30 fold）1bottle2-8Specimen requirements1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of -3000 r.p.m. remove supernatant，If precipitation 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 -3000 r.p.m. remove supernatant，If precipitation appeared，Centrifugal again.3. Urine-collect sue a sterile container，centrifugation 20-min at the speed of -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 -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 -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 at 2-8 after melting,add PBS（PH7.4），Homogenized by hand or Grinders，centrifugation 20-min at the speed of -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 to preserve，Avoid repeated freeze-thaw cycles.7. Cant detect 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，add Standard 100l to the first and the second well，then add Standard dilution 50l to the first and the second well，mix；take out 100l form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50l to the third and the forth well ,mix ；then take out 50l from the third and the forth well discard，add 50l to the fifth and the sixth well ,then add Standard dilution 50l to the fifth and the sixth well，mix ；take out 50l from the fifth and the sixth well and add to the seventh and the eighth well，then add Standard dilution 50l to the seventh and the eighth well ,mix ；take out 50l from the seventh and the eighth well and add to the ninth and the tenth well，add Standard dilution 50l to the ninth and the tenth well，mix ，take out 50l from the ninth and the tenth well discard(add Sample 50l to each well after Diluting ,(density：12g/L,8g/L ,4g/L,2g/L，1g/L )2.add sample：Set blank wells separately (blank comparison wells dont add sample and HRP-Conjugate reagent，other each step operation is same). testing sample well. add Sample dilution 40l to testing sample well，then add testing sample 10l (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 closing plate with Closure plate membrane ,incubate for 30 min 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 by pat.6.add enzyme：Add HRP-Conjugate reagent 50l 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 the light preservation for 15 min at 3710.Stop the reaction：Add Stop Solution50l 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 should 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 Sample 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 dilute Sample (n-fold)，Please diluente and multiplied by the dilution factor.（n5）.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 must 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.Take the standard density as the horizontal，the OD value for the vertical ,draw the standard curve on graph paper，Find out the corresponding density according to the sample OD value by the Sample curve，multiplied by the dilution multiple，or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation，calculate the sample density，multiplied by the dilution factor，the result is the sample actual density.CalculateThis chartis for reference only Assay range0.75g/L -15g/LStorage and validity1Storage： 2-8.2validity： six months.