犬腺病毒ADV酶联免疫分析ELISA.DOC

2、合的抗原和其他成分后再与 HRP 标记的腺病毒 (ADV)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 450nm 波长下测定吸光度（ OD 值），与 CUTOFF值相比较，从而判定标本中犬腺病毒 (ADV)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 1 48 1 96 2-8保存阴性对照 0.5ml

3、1 瓶 0.5ml 1 瓶 2-8保存阳性对照 0.5ml 1 瓶 0.5ml 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（ 2000-300

4、0 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。 2 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

5、.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

6、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

7、 30（ 48T 的 20 倍）倍浓缩洗涤液加蒸馏水至 600ml 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 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

8、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 阴性判定：样品 OD 值临界值（ CUT OFF）者为犬腺病毒 (ADV)阴性阳性判定：样品 OD 值临界值（ CUT OFF）者为犬腺病毒 (ADV)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，

9、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。 3 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 630nm 7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 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 个月上海逸峰生物科技有限公司代理不同品牌价格档次的 ELISA试剂盒。数万种抗体产品等 , 品种多，质量好，灵敏度高，价格实惠，并且还提供免费代检测服务。本公司的更多产品，请点击公司网址： http:/ 订货电话： 021-35300935 15821579651

10、传真： 021-35300936 网站： http:/ E-mail： 4 Canine Adenovirus FOR RESEARCH USE ONLY Drug Names Generic Name： Canine Adenovirus (ADV) ELISA Kit. Purpose This kit allows for the determination of ADV concentrations in Canine serum, and other biological fluids. Principle of the assay The kit assay ADV level

11、 in the sample， use Purified ADV antibody to coat microtiter plate wells, make solid-phase antibody, then add ADV to wells, Combined With ADV, after washing and removing non-combinative antibody and other components ,then Combined ADV antibody which with HRP labeled become antibody - antigen - enzym

12、e-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. Compared with the

13、 CUTOFF value, according to this to judge Bovine ADV exist in the sample or not. 5 Materials provided with the kit Materials provided with the kit 48determinations 96 determinations Storage User manual 1 1 Closure plate membrane 2 2 Sealed bags 1 1 Microelisa stripplate 1 1 2-8 Negative control 0.5m

14、l 1 bottle 0.5ml 1 bottle 2-8 Positive control 0.5ml 1 bottle 0.5ml 1 bottle 2-8 HRP-Conjugate reagent 3ml 1 bottle 6ml 1 bottle 2-8 Sample diluent 3ml 1 bottle 6ml 1 bottle 2-8 Chromogen Solution A 3ml 1 bottle 6ml 1 bottle 2-8 Chromogen Solution B 3ml 1 bottle 6ml 1 bottle 2-8 Stop Solution 3ml 1

15、bottle 6ml 1 bottle 2-8 wash solution （ 20ml 20 fold） 1bottle （ 20ml 30 fold） 1bottle 2-8 Specimen requirements 1. 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

16、 suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed 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 supernat

17、ant, 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, 6 centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the co

18、mposition 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 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrif

19、ugal 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 2000-3000 r.p.m. remove supernatant. 6. ext

20、ract 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, becaus

21、e NaN3 inhibits HRP active. Assay procedure 1.Number: to sample correspond microtitration well and Number Sequence, 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 ea

22、ch step the operation are same). 2.add sample： separately add Positive control and Negative control 50l to the Positive 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

23、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 until 600ml,and reserve. 5.washing： Uncover Closure plate membrane, disc

24、ard 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 50lto each well, except the blank well. 7 7.incubate： Operation with 3. 8.washing： Operation with 5. 9.color： Add Chromogen Solution A 50ul and Ch

25、romogen Solution B to each well, evade the light preservation for 15 min at 37 10.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.

26、 Determine the result Test validity: the average of Positive control well1.00; the average of Negative control well 0.10. Calculate Critical(CUT OFF) : Critical= the average of Negative control well + 0.15. Negative control: sample OD Calculate Critical(CUT OFF) is ADV Negative control. Positive con

27、trol: ample OD Calculate Critical(CUT OFF) is ADV Positive control. Important notes 1.Please according to use instruction strictly, Do 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

28、, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag. 3.washing buffer will Crystallization separation, 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 or

29、der to avoid the overlapping pollution 5.The substrate please evade the light preservation. 8 6.The test result determination must take the microtiter 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 validity 1 Storage： 2-8 . 2 validity： six months.

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