牛布氏杆菌抗体(Brucella-Ab)酶联免疫分析试剂盒使用说明书
本试剂盒仅供研究使用。
96T
使用目的：
本试剂盒用于测定牛血清、血浆及相关液体样本中布氏杆菌抗体(Brucella-Ab)表达。
实验原理
本试剂盒应用双抗原夹心法测定标本中牛布氏杆菌抗体(Brucella-Ab)表达。用纯化的抗原包被微孔板，制成固相抗原，可与样品中布氏杆菌抗体(Brucella-Ab)相结合，经洗涤除去未结合的抗原和其他成分后再与HRP标记的抗原结合，形成抗原-抗体-抗原复合物，经过洗涤后加底物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，从而判定标本中牛布氏杆菌抗体(Brucella-Ab)的存在与否。
试剂盒组成
| 1 | 30倍浓缩洗涤液 | 20ml×1瓶 | 7 | 终止液 | 6ml×1瓶 |
| 2 | 酶标试剂 | 6ml×1瓶 | 8 | 阳性对照 | 0.5ml×1瓶 |
| 3 | 酶标包被板 | 12孔×8条 | 9 | 阴性对照 | 0.5ml×1瓶 |
| 4 | 样品稀释液 | 6ml×1瓶 | 10 | 说明书 | 1份 |
| 5 | 显色剂A液 | 6ml×1瓶 | 11 | 封板膜 | 2张 |
| 6 | 显色剂B液 | 6ml×1/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的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。
操作步骤
操作程序结：
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 OFF）者为布氏杆菌抗体(Brucella-Ab)阴性
阳性判定：样品OD值≥ 临界值（CUT OFF）者为布氏杆菌抗体(Brucella-Ab)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。
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
| RD |
Bovine Brucella-Ab
FOR RESEARCH USE ONLY
96determinations
Purpose
This kit allows for the determination ofBrucella-Abconcentrationsin Bovine serum and otherbiological fluids.
Principle of the assay
The kitassayBrucella-Ablevel in the sample，use PurifiedBrucellato coat microtiter plate wells make solid-phase antigen then addBrucella-Ab to wellsCombined WithBrucella-Abafter washing and removing non-combinative antibody and other components thenCombinedBrucella which with HRP labeledbecome antigen – antibody - antigen complex after washing CompletelyAdd TMB substrate solution TMB substrate becomes blue color At HRP enzyme-catalyzedreaction 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 Brucella-Abexist in the sample or not.
Materials provided with the kit
| 1 | wash solution | 20ml×1bottle | 7 | Stopp Solution | 6ml×1 bottle |
| 2 | HRP-Conjugate reagent | 6ml×1 bottle | 8 | Positivecontrol | 0.5ml×1 bottle |
| 3 | Microelisa stripplate | 12well×8strips | 9 | Negative control | 0.5ml×1bottle |
| 4 | Sample diluent | 6ml×1 bottle | 10 | Instruction | 1 |
| 5 | Chromogen Solution A | 6ml×1 bottle | 11 | Closure plate membrane | 2 |
| 6 | Chromogen Solution B | 6ml×1 bottle | 12 | Sealed bags | 1 |
Specimen requirements
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(don’t add sample and HRP-Conjugate reagent to blank comparison well other each step the operation aresame).
2.add sample：separately add PositivecontrolandNegative control 50μl to the Positiveand Negative well . add Sample dilution 40μl to testing sample well then add testing sample 10μl. add sample to the bottom of ELISA plates coated well don’t 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 solutiondiluted 30-fold (or 20-fold) with distilled wateruntil 600mland reserve.
5.washing：Uncover Closure plate membrane discardLiquid 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 reagent50μlto each well except the blank well.
7.incubate：Operation with 3.
8.washing：Operation with 5.
9.color：AddChromogen Solution A50ul andChromogen Solution B to each wellevade the light preservationfor 15 min at 37℃
10.Stop the reaction：Add Stop Solution50μlto 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.
Determine the result
Test validity: the average of Positive control well≥1.00;the average ofNegative controlwell ≤0.10.
Calculate Critical(CUT OFF) : Critical= the average of Negative controlwell + 0.15.
Negativecontrol: sample OD< Calculate Critical(CUT OFF) isBrucella-AbNegative control.
Positivecontrol: ample OD≥ Calculate Critical(CUT OFF) isBrucella-Ab Positivecontrol.
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 useELISA 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 membraneonly limits the disposable use in order to avoid the overlapping pollution
5.The substrate please evade the light preservation.
6.The test result determination must take themicrotiter plate readeras 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.
牛布氏杆菌抗体(Brucella-Ab)酶联免疫分析试剂盒使用说明书
