人乙肝核心抗原HBcAg酶联免疫分析ELISA

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1、人乙肝核心抗原（HBcAg）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：本试剂盒用于检测人血清，血浆及相关液体样本中乙肝核心抗原（HBcAg）水平，感染人的血液学诊断。实验原理： 本试剂盒采用双抗体夹心酶联免疫法（ELISA）测定标本中人乙肝核心抗原（HBcAg）。用纯化的人乙脑病毒抗体包被微孔板，制成固相抗体，可与样品中乙肝核心抗原（HBcAg）相结合，经洗涤除去未结合的抗体和其他成分后再与HRP标记的乙脑病毒抗体结合，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。用酶标仪在450n

2、m波长下测定吸光度（OD值），与CUTOFF值相比较，从而判定标本中人乙肝核心抗原（HBcAg）的存在与否。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个酶标包被板1481962-8保存阴性对照0.5ml1瓶0.5ml1瓶2-8保存阳性对照0.5ml1瓶0.5ml1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保存终止液3ml1瓶6ml1瓶2-8保存浓缩洗涤液（20ml20倍）1瓶（20ml30倍）1瓶

3、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

4、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

5、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. 编号：将样品对应微孔按序编号，每板应设阴性对照2孔、阳性对照2孔、空白对照1孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）2. 加样：分别在阴、阳性对照孔中加入阴性对照、阳性对照50l。然后在待测样品孔先加样品稀释液40l，然后再加待测样品10l。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）倍浓缩洗涤液加蒸馏水至600ml后备用5. 洗涤：小

6、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A 50l，再加入显色剂B 50l，轻轻震荡混匀，37避光显色15分钟10. 终止：每孔加终止液50l，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。结果判定： 试验有效性：阳性对照孔平均值1.00; 阴性对照平均值0.10 临界值（CUT OFF）计算：临界值=阴性对照孔平均值+0.15 阴性判

7、定：样品OD值 临界值（CUT OFF）者为人乙肝核心抗原（HBcAg）阴性 阳性判定：样品OD值 临界值（CUT OFF）者为人乙肝核心抗原（HBcAg）阳性注意事项1操作严格按照说明书进行，本试剂不同批号组分不得混用。2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。3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。4 封板膜只限一次性使用，以避免交叉污染。5底物请避光保存。6试验结果判定必须以酶标仪读数为准，使用双波长检测时，参考波长为630nm7所有样品，洗涤液和各种废弃物都应按传染物处理。终止液为2M

8、的硫酸，使用时必须注意安全。保存条件及有效期1试剂盒保存：；2-8。2有效期：6个月 Human HBcAgFOR RESEARCH USE ONLYDrug NamesGeneric Name：Human HBcAg ELISA Kit.PurposeThis kit allows for the determination of HBcAg concentrations in Human serum, and other biological fluids.Principle of the assayThe kit assay HBcAg level in the sample，use P

9、urified JEV antibody to coat microtiter plate wells, make solid-phase antibody, then add HBcAg to wells, Combined With HBcAg, after washing and removing non-combinative antibody and other components ,then Combined JEV antibody which with HRP labeled become antibody - antigen - enzyme- antibody compl

10、ex, 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. Compared with the CUTOFF value, a

11、ccording to this to judge HBcAg exist in the sample or not.Materials provided with the kitMaterials provided with the kit48determinations96 determinationsStorageUser manual11Closure plate membrane22Sealed bags11Microelisa stripplate112-8Negative control0.5ml1 bottle0.5ml1 bottle2-8Positive control0.

12、5ml1 bottle0.5ml1 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）1bottle2-8Spec

13、imen requirements1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of 2000-3000 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 s

14、peed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal

15、fluid Reference to it.4. cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million /

16、 ml, repeated freeze-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 froze

17、n 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 2000-3000 r.p.m. remove supernatant.6. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be ex

18、periment 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.Number: to sample correspond microtitration well and Number Sequen

19、ce, each plate should be set feminine comparison 2 wells, masculine comparison 2 wells, blank comparison 1 well(dont add sample and HRP-Conjugate reagent to blank comparison well, other each step the operation are same).2.add sample：separately add Positive control and Negative control 50l to the Pos

20、itive and Negative well . add Sample dilution 40l to testing sample well, then add testing sample 10l. add sample to the bottom of ELISA plates coated well , 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 a

21、t 37. 4.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water until 600ml,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

22、 enzyme：Add HRP-Conjugate reagent 50lto each well, except the 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

23、 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.Determine the resultTest validity: the average of Positive control well1.00; the average of Negative control well 0.10.Calculate Cr

24、itical(CUT OFF) : Critical= the average of Negative control well + 0.15.Negative control: sample OD Calculate Critical(CUT OFF) is HBcAg Negative control.Positive control: ample OD Calculate Critical(CUT OFF) is HBcAg Positive control.Important notes1.Please according to use instruction strictly, Do

25、 not mix reagents with those from other lots.2.The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature then use, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.3.washing buffer will Crystallization se

26、paration, it can be heated the water helps dissolve when dilute . Washing does not affect the result.4.Closure plate membrane only limits the disposable use, in order to avoid the overlapping pollution5.The substrate please evade the light preservation.6.The test result determination must take the m

27、icrotiter plate reader as a standard, when use dual-wavelength to assay, Reference wavelength is 630nm.7.All samples, washing buffer and each kind of reject should according to infective material process. Stopp Solution is 2M sulphuric acid. You must pay attention to safe when use .Storage and validity1Storage： 2-8.2validity： six months.8 / 8文档可自由编辑打印