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1、如有帮助，欢迎支持。马铃薯M病毒（PVM）酶联免疫分析（ELISA）试剂盒使用说明书本试剂仅供研究使用目的：本试剂盒用于检测马铃薯组织，细胞上清及相关液体样本中M病毒（PVM）水平。实验原理：本试剂盒采用双抗体夹心酶联免疫法（ ELISA）测定标本中马铃薯 M病毒（PVM）。用纯 化的马铃薯M病毒（PVM）抗体包被微孔板，制成固相抗体，可与样品中马铃薯 M病毒（PVM） 相结合，经洗涤除去未结合的抗体和其他成分后再与 HRP标记的马铃薯 M病毒（PVM）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
2、。用酶标仪在450nm波长下测定吸光度（OD值），与CUTOFF值相比较，从而判定标本中马铃薯 M病毒（PVM）的存在与 否。试剂盒组成试剂盒组成48孔配置96孔配置保存说明书1份1份封板膜2 片(48)2 片(96)密封袋1个1个酶标包被板1X481X 962-8 C保存阴性对照0.5ml X 1 瓶0.5ml X 1 瓶2-8 C保存阳性对照0.5ml X 1 瓶0.5ml X 1 瓶2-8 C保存酶标试剂3 ml x 1 瓶6 ml x 1 瓶2-8 C保存样品稀释液3 ml x 1 瓶6 ml x 1 瓶2-8 C保存显色剂A液3 ml x 1 瓶6 ml x 1 瓶2-8 C保存显色
3、剂B液3 ml x 1 瓶6 ml x 1 瓶2-8 C保存终止液3ml x 1 瓶6ml x 1 瓶2-8 C保存浓缩洗涤液(20ml X 20 倍)X 1 瓶(20ml x 30 倍)x 1 瓶2-8 C保存样本处理及要求：1 .血清：室温血液自然凝固 10-20分钟，离心20分钟左右（2000-3000转/分）。仔细收集上 清，保存过程中如出现沉淀，应再次离心。2 .血浆：应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂，混合 10-20分钟后，离心20分钟左右（2000-3000转/分）。仔细收集上清，保存过程中如有沉淀形成，应该再次离，3 .尿液：用无菌管收集，离心 20分钟左右（2
4、000-3000转/分）。仔细收集上清，保存过程 中如有沉淀形成，应再次离心。胸腹水、脑脊液参照实行。4 .细胞培养上清：检测分泌性的成份时，用无菌管收集。离心 20分钟左右（2000-3000转/ 分）。仔细收集上清。检测细胞内的成份时，用 PBS （PH7.2-7.4）稀释细胞悬液，细胞 浓度达到100万/ml左右。通过反复冻融，以使细胞破坏并放出细胞内成份。离心 20分 钟左右（2000-3000车t/分）。仔细收集上清。保存过程中如有沉淀形成，应再次离心。5 .组织标本：切割标本后，称取重量。加入一定量的 PBS, PH7.4。用液氮迅速冷冻保存备 用。标本融化后仍然保持2-8C的温度
5、。加入一定量的PBS （PH7.4）,用手工或匀浆器将标本匀浆充分。离心 20分钟左右（2000-3000转/分）。仔细收集上清。分装后一份待 检测，其余冷冻备用。6 .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 C保存，但应避免反复冻融.7 .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：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50晨 然后在待
6、测样品孔先加样品稀释液 40屋然后再加待测样品 103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 c温育30分钟。4 .配液：将30 （48T的20倍）倍浓缩洗涤?加蒸储水至600ml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 重复5次，拍干。6 .加酶：每孔加入酶标试剂 50J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 A 50再加入显色剂 B 50科J轻轻震荡混匀，37 c避光显色15分钟10 .终止：每孔加终止液 50终止反应（此时蓝色立转黄色）
7、。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。结果判定：试验有效性：阳性对照孔平均值*.00;阴性对照平均值 旬.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）者为马铃薯 M病毒（PVM）阴性阳性判定：样品 OD值R临界值（CUT OFF）者为马铃薯 M病毒（PVM）阳性注意事项12 操作严格按照说明书进行，本试剂不同批号组分不得混用。13 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。14
8、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。15 封板膜只限一次性使用，以避免交叉污染。16 底物请避光保存。17 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630nm18 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 C。2 .有效期：6个月7FOR RESEARCH USE ONLYPVMDrug NamesGeneric Name PVM ELISA Kit.PurposeThis kit allows for the determinationVMf con
9、centrations in Plant serum, blood plasma, and other biological fluids.Principle of the assayThe kit assayVM level in the sampleuse PurifiecPVM antibody to coat microtiter plate wells, make solid-phase antibody, thenfOdd to wells, Combined WithVM , after washing and removing non-combinative antibody
10、and other components ,then CPVbinedibody 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 by the addition of a sulphuric acid solution and
11、 the color change is measured spectrophotometrically at a wavelength of 450 nm. Compared with the CUTOFF value, according to this to jPVge exist in the sample or not.Materials provided with the kitMaterials provided with the kit48determinations96 determinationsStorageUser manual11Closure plate membr
12、ane22Sealed bags11Microelisa stripplate112-8 CNegative control0.5ml 1Xbottle0.5ml 1Xbottle2-8 CPositive control0.5ml 1Xbottle0.5ml 1Xbottle2-8 CHRP-Conjugate reagent 3ml 1 bottle6ml 1 bottle2-8 CSample diluent3ml 1 bottle6ml 1 bottle2-8 CChromogen Solution A3ml 1 bottle6ml 1 bottle2-8 CChromogen Sol
13、ution B3ml 1 bottle6ml 1 bottle2-8 CStop Solution3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml x 20 folcD x 1bottle(20mlx 30 folcD x 1 bottle2-8 CSpecimen requirements1. serum- coagulation at room temperature 10-20 geistrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If prec
14、ipitation appeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugatior20-min at the speed of 2000-3000r.p.m. remove supernatant,If precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min a
15、t the speed of 2000-3000 r.p.m. remove supernatant,If precipitationappeared, Centrifugalagain. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.4. cell culture supernatan-detect secretorycomponentscollect sue a sterile container, centrifugatior20-min at the speed of 2000-3000r.p.
16、m. removesupernatant,detedttie compositionof cells, Dilut cell suspensionwith PBS (PH7.2-7.4 , Cell concentration reached 1 million / ml, repeated freeze-thawcycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If pr
17、ecipitation appeared, Centrifugal again.5. Tissue samples After cutting samples, check the weight,add PPHS7.2-7.4 , Rapidly frozen with liquid nitrogen, maintain samples aC2-after melting,add PBSPH7.4 ,Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove sup
18、ernatant.6. extract as soon as possibleafter Specimencollection,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction If it cant, specimen can be kept in - 20 to preserve, Avoid repeated freeze-thaw cycles.7. Can t detect the sample which conaN3, becau
19、se NaN3 inhibits HRP active.Assay procedure1 .Number: to sample correspond microtitration well and Number Sequence, each plate should be set femininecomparison2 wells, masculinecomparison2 wells, blank comparisonl well(don t add sampHRPndConjugate reagent to blank comparison well, other each step th
20、e operation are same).2 .add sample separately add Positive control and Negacivetrol 50 仙 l to the Positive Negativewell . add Sample dilution 40 仙 l to testing samddlteweig thample 10 仙 l. add sample to the bottom ofLISA plates coated welldon t touch the weall as far as possible, and Gently mix.1 .
21、Incubate: After closing plate with Closure plate membrane ,incubate for 30Cmin at 374.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 b
22、uffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6 .add enzyme Add HRP-Conjugate reagent仙 lto 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
23、preservation for 15 min a t23710.Stopthe reaction Add Stop Solutio50 仙 to each well, Stop the reaction(thdolue colorchange to yellow color).11. assa y take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Determine the resultTest validity: the average of Posi
24、tive control well the average of Negative control well 010.Calculate Critical(CUT OFF) : Critical= the average of Negative control well + 0.15.Negative control: sample OD Calculate Critical(CUT OFF) isositive control.Important notes1 .Please according to use instruction strictly, Do not mix reagents
25、 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 use, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.3 .washingbuffer will Crystallizationseparation it can b
26、e heatedthe water helps dissolve when dilute . Washing does not affect 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
27、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 validity1. Storage2-8C .2. validity six months.