1、植物甘薯羽狀斑駁病毒（SPFMV）酶聯免疫分析（ELISA）試劑盒使用說明書本試劑僅供研究使用 目的：本試劑盒用于檢測植物組織，細胞上清及相關液體樣本中甘薯羽狀斑駁病毒（SPFMV）水平。實驗原理： 本試劑盒采用雙抗體夾心酶聯免疫法（ELISA）測定標本中甘薯羽狀斑駁病毒（SPFMV）。用純化的甘薯羽狀斑駁病毒（SPFMV）抗體包被微孔板，制成固相抗體，可與樣品中甘薯羽狀斑駁病毒（SPFMV）相結合，經洗滌除去未結合的抗體和其他成分后再與HRP標記的甘薯羽狀斑駁病毒（SPFMV）抗體結合，形成抗體-抗原-酶標抗體復合物，經過徹底洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉化成藍色，并在酸

2、的作用下轉化成最終的黃色。用酶標儀在450nm波長下測定吸光度（OD值），與CUTOFF值相比較，從而判定標本中甘薯羽狀斑駁病毒（SPFMV）的存在與否。試劑盒組成：試劑盒組成48孔配置96孔配置保存說明書1份1份封板膜2片（48）2片（96）密封袋1個1個酶標包被板1×481×962-8保存陰性對照0.5ml×1瓶0.5ml×1瓶2-8保存陽性對照0.5ml×1瓶0.5ml×1瓶2-8保存酶標試劑3 ml×1瓶6 ml×1瓶2-8保存樣品稀釋液3 ml×1瓶6 ml×1瓶2-8保存顯色劑A液3

3、 ml×1瓶6 ml×1瓶2-8保存顯色劑B液3 ml×1瓶6 ml×1瓶2-8保存終止液3ml×1瓶6ml×1瓶2-8保存濃縮洗滌液（20ml×20倍）×1瓶（20ml×30倍）×1瓶2-8保存樣本處理及要求：1. 血清：室溫血液自然凝固10-20分鐘，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如出現沉淀，應再次離心。2. 血漿：應根據標本的要求選擇EDTA或檸檬酸鈉作為抗凝劑，混合10-20分鐘后，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過

4、程中如有沉淀形成，應該再次離心。3. 尿液：用無菌管收集，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如有沉淀形成，應再次離心。胸腹水、腦脊液參照實行。4. 細胞培養上清：檢測分泌性的成份時，用無菌管收集。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。檢測細胞內的成份時，用PBS（PH7.2-7.4）稀釋細胞懸液，細胞濃度達到100萬/ml左右。通過反復凍融，以使細胞破壞并放出細胞內成份。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。保存過程中如有沉淀形成，應再次離心。5. 組織標本：切割標本后，稱取重量。加入一定量的PBS，PH7.4。用

5、液氮迅速冷凍保存備用。標本融化后仍然保持2-8的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標本勻漿充分。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。分裝后一份待檢測，其余冷凍備用。6. 標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上進行試驗，可將標本放于-20保存，但應避免反復凍融.7. 不能檢測含NaN3的樣品，因NaN3抑制辣根過氧化物酶的（HRP）活性。操作步驟：1. 編號：將樣品對應微孔按序編號，每板應設陰性對照2孔、陽性對照2孔、空白對照1孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）2. 加樣：分別在陰、陽性對照孔中

6、加入陰性對照、陽性對照50l。然后在待測樣品孔先加樣品稀釋液40l，然后再加待測樣品10l。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻，3. 溫育：用封板膜封板后置37溫育30分鐘。 4. 配液：將30（48T的20倍）倍濃縮洗滌液加蒸餾水至600ml后備用5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復5次，拍干。6. 加酶：每孔加入酶標試劑50l，空白孔除外。 7. 溫育：操作同3。8. 洗滌：操作同5。9. 顯色：每孔先加入顯色劑A 50l，再加入顯色劑B 50l，輕輕震蕩混勻，37避光顯色15分鐘10. 終止：每孔加終止液50l，終止

7、反應（此時藍色立轉黃色）。11. 測定：以空白空調零，450nm波長依序測量各孔的吸光度（OD值）。 測定應在加終止液后15分鐘以內進行。結果判定： 試驗有效性：陽性對照孔平均值1.00; 陰性對照平均值0.10 臨界值（CUT OFF）計算：臨界值=陰性對照孔平均值+0.15 陰性判定：樣品OD值< 臨界值（CUT OFF）者為甘薯羽狀斑駁病毒（SPFMV）陰性 陽性判定：樣品OD值 臨界值（CUT OFF）者為甘薯羽狀斑駁病毒（SPFMV）陽性注意事項1操作嚴格按照說明書進行，本試劑不同批號組分不得混用。2試劑盒從冷藏環境中取出應在室溫平衡15-30分鐘后方可使用，酶標包被板開封后如

8、未用完，板條應裝入密封袋中保存。3濃洗滌液可能會有結晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。4 封板膜只限一次性使用，以避免交叉污染。5底物請避光保存。6試驗結果判定必須以酶標儀讀數為準，使用雙波長檢測時，參考波長為630nm7所有樣品，洗滌液和各種廢棄物都應按傳染物處理。終止液為2M的硫酸，使用時必須注意安全。保存條件及有效期1試劑盒保存：；2-8。2有效期：6個月 Plant SPFMV FOR RESEARCH USE ONLYDrug NamesGeneric Name：Plant SPFMV ELISA Kit.PurposeThis kit allows for the

9、 determination of SPFMV concentrations in Plant serum, blood plasma, and other biological fluids.Principle of the assayThe kit assay SPFMV level in the sample，use Purified SPFMV antibody to coat microtiter plate wells, make solid-phase antibody, then add SPFMV to wells, Combined With SPFMV, after wa

10、shing and removing non-combinative antibody and other components ,then Combined SPFMV which with HRP labeled become antigen - antibody - enzyme- antigen complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by

11、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 SPFMV exist in the sample or not.Materials provided with the kitMaterials provided with the kit48determinations96 de

12、terminationsStorageUser manual11Closure plate membrane22Sealed bags11Microelisa stripplate112-8Negative control0.5ml×1 bottle0.5ml×1 bottle2-8Positive control0.5ml×1 bottle0.5ml×1 bottle2-8HRP-Conjugate reagent3ml×1 bottle6ml×1 bottle2-8Sample diluent3ml×1 bottle6m

13、l×1 bottle2-8Chromogen Solution A3ml×1 bottle6ml×1 bottle2-8Chromogen Solution B3ml×1 bottle6ml×1 bottle2-8Stop Solution3ml×1 bottle6ml×1 bottle2-8wash solution（20ml×20 fold）×1bottle（20ml×30 fold）×1bottle2-8Specimen requirements1. serum- coagula

14、tion 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 speed of 2000-3000 r.p.m. remove su

15、pernatant, 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 fluid Reference to it.4. cell cult

16、ure 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 / ml, repeated freeze-thaw cycles,

17、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 frozen with liquid nitrogen, maintain s

18、amples 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 experiment as soon as possible after

19、 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 Sequence, each plate should be set femin

20、ine 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 Positive and Negative well . add Samp

21、le 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 at 37. 4.Configurate liquid: 30-fol

22、d (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 enzyme：Add HRP-Conjugate reagent

23、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 well, Stop the reaction(the blue

24、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 Critical(CUT OFF) : Critical= the av

25、erage of Negative control well + 0.15.Negative control: sample OD< Calculate Critical(CUT OFF) is SPFMV Negative control.Positive control: ample OD Calculate Critical(CUT OFF) is SPFMV 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 environment