牛細(xì)小病毒I型(PV-I)酶聯(lián)免疫分析(ELISA)

1、牛細(xì)小病毒I型 （PV-I）酶聯(lián)免疫分析（ELISA）試劑盒使用說明書本試劑僅供研究使用 目的：本試劑盒用于檢測牛血清，血漿及相關(guān)液體樣本中細(xì)小病毒I型 （PV-I）水平，感染人的血液學(xué)診斷。實(shí)驗(yàn)原理： 本試劑盒采用雙抗體夾心酶聯(lián)免疫法（ELISA）測定標(biāo)本中牛細(xì)小病毒I型 （PV-I）。用純化的牛細(xì)小病毒I型 （PV-I）抗體包被微孔板，制成固相抗體，可與樣品中細(xì)小病毒I型 （PV-I）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)記的細(xì)小病毒I型 （PV-I）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過徹底洗滌后加底物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

2、轉(zhuǎn)化成最終的黃色。用酶標(biāo)儀在450nm波長下測定吸光度（OD值），與CUTOFF值相比較，從而判定標(biāo)本中牛細(xì)小病毒I型 （PV-I）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(gè)1個(gè)酶標(biāo)包被板1×481×962-8保存陰性對(duì)照0.5ml×1瓶0.5ml×1瓶2-8保存陽性對(duì)照0.5ml×1瓶0.5ml×1瓶2-8保存酶標(biāo)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 ml&#

3、215;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(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如出現(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，保存過程中如有沉

4、淀形成，應(yīng)該再次離心。3. 尿液：用無菌管收集，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：檢測分泌性的成份時(shí)，用無菌管收集。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。檢測細(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/ml左右。通過反復(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

5、凍保存?zhèn)溆谩?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，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(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、陽性對(duì)照孔中加入陰性對(duì)

6、照、陽性對(duì)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(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)

7、藍(lán)色立轉(zhuǎn)黃色）。11. 測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。 測定應(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+0.15 陰性判定：樣品OD值< 臨界值（CUT OFF）者為副流感病毒III型 （PIV-III）陰性 陽性判定：樣品OD值 臨界值（CUT OFF）者為副流感病毒III型 （PIV-III）陽性注意事項(xiàng)1操作嚴(yán)格按照說明書進(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

8、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)，使用雙波長檢測時(shí)，參考波長為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 FOR RESEARCH USE ONLY Bovine PV-II Drug NamesGeneric Name：Bovine PV-II ELISA Kit.PurposeThis kit allows f

9、or the determination of PV-II concentrations in Bovine serum, and other biological fluids.Principle of the assayThe kit assay PV-II level in the sample，use Purified PV-II antibody to coat microtiter plate wells, make solid-phase antibody, then add PV-II to wells, Combined With PV-II, after washing a

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

11、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 PV-II exist in the sample or not.Materials provided with the kitMaterials provided with the kit48determinations96

12、 determinationsStorageUser 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 bottl

13、e6ml×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- coag

14、ulation 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

15、 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 cerebrospinal fluid Reference to it.4. cell c

16、ulture 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 cycle

17、s, 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, maintai

18、n 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, and should be experiment as soon as possible af

19、ter 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 fe

20、minine 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 S

21、ample 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-

22、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, dry by pat.6.add enzyme：Add HRP-Conjugate reage

23、nt 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 bl

24、ue 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

25、 average of Negative control well + 0.15.Negative control: sample OD< Calculate Critical(CUT OFF) is PV-II Negative control.Positive control: ample OD Calculate Critical(CUT OFF) is PV-II 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 environmen