1、猪辛型肝炎病毒（TTV）试剂盒使用方法检测范围 ： 96T使用目的：本试剂盒用于测定猪血清、血浆及相关液体样本中辛型肝炎病毒（TTV）表达。实验原理本试剂盒应用双抗体夹心法测定标本中猪辛型肝炎病毒（TTV）表达。用纯化的猪辛型肝炎病毒（TTV）抗体包被微孔板，制成固相抗体，可与样品中辛型肝炎病毒（TTV）相结合，经洗涤除去未结合的抗原和其他成分后再与 HRP 标记的辛型肝炎病毒（TTV）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
2、 CUTOFF 值相比较，从而判定标本中猪辛型肝炎病毒（ TTV）的存在与否。试剂盒组成 1 30 倍浓缩洗涤液 20ml1 瓶 7 终止液 6ml1 瓶2 酶标试剂 6ml1 瓶 8 阳性对照 0.5ml1 瓶3 酶标包被板 12 孔8 条 9 阴性对照 0.5ml1 瓶4 样品稀释液 6ml1 瓶 10 说明书 1 份5 显色剂 A 液 6ml1 瓶 11 封板膜 2 张 6 显色剂 B 液 6ml1/瓶 12 密封袋 1 个标本要求 1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2不能检测含 NaN3 的样
3、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 倍浓缩洗涤液用蒸馏水 30 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
4、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。计算和结果判定：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
5、OFF ）者为辛型肝炎病毒（TTV）阴性阳性判定：样品 OD 值 临界值（CUT OFF）者为辛型肝炎病毒（ TTV）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 的硫酸，使用时必须注意安
6、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 HPFOR RESEARCH USE ONLY96 determinationsPurposeThis kit allows for the determination of HP concentrations in Human serum, and other biological fluids.Principle of the assayThe kit assay HP level in the sample，use Purified HP antibody to coat microtiter plate well
7、s, make solid-phase antibody, then add HP to wells, Combined With HP, after washing and removing non-combinative antibody and other components ,then Combined HP antibody which with HRP labeled become antibody antigen - enzyme- antibody complex, after washing Completely, Add TMB substrate solution, T
8、MB 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, according to this to judge HP exist in the sample or not.Mat
9、erials provided with the kit1 wash solution 20ml1bottle 7 Stopp Solution 6ml1 bottle2 HRP-Conjugate reagent 6ml1 bottle 8 Positive control 0.5ml1 bottle3 Microelisa stripplate 12well8strips 9 Negative control 0.5ml1bottle4 Sample diluent 6ml1 bottle 10 Instruction 15 Chromogen Solution A 6ml1 bottle
10、 11 Closure plate membrane 26 Chromogen Solution B 6ml1 bottle 12 Sealed bags 1Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevant RDliterature, and should be experiment as soon as possible after the extraction. If it cant, specimen can be kept
11、 in -20 to preserve, Avoid repeated freeze-thaw cycles.2. Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1.Number: to sample correspond microtitration well and Number Sequence, each plate should be set feminine comparison 2 wells, masculine comparison 2 we
12、lls, 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 Positive and Negative well . add Sample dilution 40l to testing sample well, then add
13、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 at 37. 4.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 2
14、0-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 enzyme：Add HRP-Conjugate reagent 50lto each well, except the blank well. 7.incubat
15、e：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 bla
16、nk 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 Critical(CUT OFF) : Critical= the average of Negative control well + 0.15.Negative c
17、ontrol: sample OD Calculate Critical(CUT OFF) is HP Negative control.Positive control: ample OD Calculate Critical(CUT OFF) is HP Positive control.Important notes1.Please according to use instruction strictly, Do not mix reagents with those from other lots.2.The kit takes out from the refrigeration
18、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 separation, it can be heated the water helps dissolve when dilute . Washing does not affec
19、t 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 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 validity1Storage： 2-8.2validity： six months.
