1、馬鈴薯卷葉病毒(plrv)酶聯(lián)免疫分析（elisa）試劑盒使用說(shuō)明書(shū)本試劑僅供研究使用 目的：本試劑盒用于檢測(cè)植物組織，細(xì)胞上清及相關(guān)液體樣本中馬鈴薯卷葉病毒(plrv)水平。實(shí)驗(yàn)原理： 本試劑盒采用雙抗體夾心酶聯(lián)免疫法（elisa）測(cè)定標(biāo)本中馬鈴薯卷葉病毒(plrv)。用純化的馬鈴薯卷葉病毒(plrv)抗體包被微孔板，制成固相抗體，可與樣品中馬鈴薯卷葉病毒(plrv)相結(jié)合，經(jīng)洗滌除去未結(jié)合的抗體和其他成分后再與hrp標(biāo)記的馬鈴薯卷葉病毒(plrv)抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過(guò)徹底洗滌后加底物tmb顯色。tmb在hrp酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成最終的黃色。用

2、酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（od值），與cutoff值相比較，從而判定標(biāo)本中馬鈴薯卷葉病毒(plrv)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(shuō)明書(shū)1份1份封板膜2片（48）2片（96）密封袋1個(gè)1個(gè)酶標(biāo)包被板1481962-8保存陰性對(duì)照0.5ml1瓶0.5ml1瓶2-8保存陽(yáng)性對(duì)照0.5ml1瓶0.5ml1瓶2-8保存酶標(biāo)試劑3 ml1瓶6 ml1瓶2-8保存樣品稀釋液3 ml1瓶6 ml1瓶2-8保存顯色劑a液3 ml1瓶6 ml1瓶2-8保存顯色劑b液3 ml1瓶6 ml1瓶2-8保存終止液3ml1瓶6ml1瓶2-8保存濃縮洗滌液（20ml20倍）1瓶（20m

3、l30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(zhuǎn)/分）。仔細(xì)收集上清，保存過(guò)程中如出現(xiàn)沉淀，應(yīng)再次離心。2. 血漿：應(yīng)根據(jù)標(biāo)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(zhuǎn)/分）。仔細(xì)收集上清，保存過(guò)程中如有沉淀形成，應(yīng)該再次離心。3. 尿液：用無(wú)菌管收集，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過(guò)程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實(shí)行。4. 細(xì)胞培養(yǎng)上清：檢測(cè)分泌性的成份時(shí)，用無(wú)菌管收集。離心20分鐘左右（2000-3000轉(zhuǎn)/

4、分）。仔細(xì)收集上清。檢測(cè)細(xì)胞內(nèi)的成份時(shí)，用pbs（ph7.2-7.4）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到100萬(wàn)/ml左右。通過(guò)反復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。保存過(guò)程中如有沉淀形成，應(yīng)再次離心。5. 組織標(biāo)本：切割標(biāo)本后，稱取重量。加入一定量的pbs，ph7.4。用液氮迅速冷凍保存?zhèn)溆?。?biāo)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(biāo)本勻漿充分。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。分裝后一份待檢測(cè)，其余冷凍備用。6. 標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快

5、進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20保存，但應(yīng)避免反復(fù)凍融.7. 不能檢測(cè)含nan3的樣品，因nan3抑制辣根過(guò)氧化物酶的（hrp）活性。操作步驟：1. 編號(hào)：將樣品對(duì)應(yīng)微孔按序編號(hào)，每板應(yīng)設(shè)陰性對(duì)照2孔、陽(yáng)性對(duì)照2孔、空白對(duì)照1孔（空白對(duì)照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）2. 加樣：分別在陰、陽(yáng)性對(duì)照孔中加入陰性對(duì)照、陽(yáng)性對(duì)照50l。然后在待測(cè)樣品孔先加樣品稀釋液40l，然后再加待測(cè)樣品10l。加樣將樣品加于酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動(dòng)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

6、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(fù)5次，拍干。6. 加酶：每孔加入酶標(biāo)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，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。11. 測(cè)定：以空白空調(diào)零，450nm波長(zhǎng)依序測(cè)量各孔的吸光度（od值）。 測(cè)定應(yīng)在加終止液后15分鐘以內(nèi)進(jìn)行。結(jié)果判定： 試驗(yàn)有效性：陽(yáng)性對(duì)照孔平均值1.00; 陰性對(duì)照平均值0.10 臨界值（cut off）計(jì)算：臨界值=陰性對(duì)照孔平均值+0

7、.15 陰性判定：樣品od值 臨界值（cut off）者為馬鈴薯卷葉病毒(plrv)陰性 陽(yáng)性判定：樣品od值 臨界值（cut off）者為馬鈴薯卷葉病毒(plrv)陽(yáng)性注意事項(xiàng)1操作嚴(yán)格按照說(shuō)明書(shū)進(jìn)行，本試劑不同批號(hào)組分不得混用。2試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30分鐘后方可使用，酶標(biāo)包被板開(kāi)封后如未用完，板條應(yīng)裝入密封袋中保存。3濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。4 封板膜只限一次性使用，以避免交叉污染。5底物請(qǐng)避光保存。6試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn)，使用雙波長(zhǎng)檢測(cè)時(shí)，參考波長(zhǎng)為630nm7所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。終

8、止液為2m的硫酸，使用時(shí)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(gè)月 plant plrv for research use onlydrug namesgeneric name：plant plrv elisa kit.purposethis kit allows for the determination of plrv concentrations in plant serum, blood plasma, and other biological fluids.principle of the assaythe kit assay plrv level in

9、the sample，use purified plrv antibody to coat microtiter plate wells, make solid-phase antibody, then add plrv to wells, combined with plrv, after washing and removing non-combinative antibody and other components ,then combined plrv antibody which with hrp labeled become antibody - antigen - enzyme

10、- 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

11、 cutoff value, according to this to judge plrv 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 bottle2-8pos

12、itive control0.5ml1 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

13、）1bottle2-8specimen 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

14、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 supernatant, if precipitation appeared, centrifugal again. the operation of hydrothorax and

15、 cerebrospinal 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 reac

16、hed 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, centrifugal again.5. tissue samples- after cutting samples, check the weight,add pbs（ph7.2-7.4）

17、, 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. extract as soon as possible after specimen collection,and according to the relevant literature, a

18、nd 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, because nan3 inhibits hrp active.assay procedure1.number: to sample correspond microtitration well an

19、d 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 each step the operation are same).2.add sample：separately add positive control and negative control

20、 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 far as possible, and gently mix.3.incubate: after closing plate with closure plate membrane ,incuba

21、te 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, discard liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, d

22、ry by pat.6.add 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 solu

23、tion50l 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.determine the resulttest validity: the average of positive control well1.00; the average of negative control well 0.

24、10.calculate critical(cut off) : critical= the average of negative control well + 0.15.negative control: sample od calculate critical(cut off) is plrv negative control.positive control: ample od calculate critical(cut off) is plrv 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 should be balanced 15-30