番茄黄化曲叶病毒TYLCV酶联免疫分析ELISA.DOC

1、1番茄黄化曲叶病毒(TYLCV)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：本试剂盒用于测定组织,细胞及相关液体样本中番茄黄化曲叶病毒(TYLCV)含量。实验原理：本试剂盒应用双抗体夹心法测定标本中番茄黄化曲叶病毒(TYLCV)水平。用纯化的番茄黄化曲叶病毒(TYLCV) 抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入番茄黄化曲叶病毒(TYLCV) ，再与 HRP 标记的番茄黄化曲叶病毒 (TYLCV)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。颜色的深

2、浅和样品中的番茄黄化曲叶病毒(TYLCV)呈正相关。用酶标仪在 450nm 波长下测定吸光度（ OD 值） ，通过标准曲线计算样品中番茄黄化曲叶病毒(TYLCV)浓度。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 148 196 2-8 保存标准品：135ng/ml 0.5ml1 瓶 0.5ml1 瓶 2-8 保存标准品稀释液 1.5ml1 瓶 1.5ml1 瓶 2-8 保存酶标试剂 3 ml1 瓶 6 ml1 瓶 2-8 保存样品稀释液 3 ml1 瓶 6 ml1 瓶 2-8 保存显色剂

3、A 液 3 ml1 瓶 6 ml1 瓶 2-8 保存显色剂 B 液 3 ml1 瓶 6 ml1 瓶 2-8 保存终止液 3ml1 瓶 6ml1 瓶 2-8 保存浓缩洗涤液 （20ml20 倍）1 瓶 （20ml30 倍）1 瓶 2-8 保存样本处理及要求：1. 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 转/分） 。仔细收集上清。保存过程

4、中如有沉淀形成，应再次离心。2. 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 2-8的温度。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。23. 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.4. 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. 标准品的稀释与加样：在酶标包被板上设

5、标准品孔 10 孔，在第一、第二孔中分别加标准品 100l，然后在第一、第二孔中加标准品稀释液 50l，混匀；然后从第一孔、第二孔中各取 100l 分别加到第三孔和第四孔，再在第三、第四孔分别加标准品稀释液50l，混匀；然后在第三孔和第四孔中先各取 50l 弃掉，再各取 50l 分别加到第五、第六孔中，再在第五、第六孔中分别加标准品稀释液 50ul，混匀；混匀后从第五、第六孔中各取 50l 分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液50l，混匀后从第七、第八孔中分别取 50l 加到第九、第十孔中，再在第九第十孔分别加标准品稀释液 50l，混匀后从第九第十孔中各取 50l 弃掉

6、。 （稀释后各孔加样量都为 50l，浓度分别为 90ng/ml，60ng/ml ，30ng/ml，15ng/ml，7.5ng/ml） 。2. 加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同） 、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液 40l，然后再加待测样品10l（样品最终稀释度为 5 倍） 。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温育 30 分钟。4. 配液：将 30（48T 的 20 倍）倍浓缩洗涤液用蒸馏水 30（48T 的 20 倍）倍稀释后备用。5. 洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满

7、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 50l，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 A50l，再加入显色剂 B50l，轻轻震荡混匀，37避光显色15 分钟. 10. 终止：每孔加终止液 50l，终止反应（此时蓝色立转黄色） 。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。注意事项：1 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。2 浓洗涤液可能会有结晶析

8、出，稀释时可在水浴中加温助溶，洗涤时不影响结果。3 各步加样均应使用加样器，并经常校对其准确性，以避免试验误差。一次加样时间最好控制在 5 分钟内，如标本数量多，推荐使用排枪加样。4 请每次测定的同时做标准曲线，最好做复孔。如标本中待测物质含量过高（样本 OD值大于标准品孔第一孔的 OD 值） ，请先用样品稀释液稀释一定倍数（ n 倍）后再测定，计算时请最后乘以总稀释倍数（n5） 。5 封板膜只限一次性使用，以避免交叉污染。6 底物请避光保存。7 严格按照说明书的操作进行，试验结果判定必须以酶标仪读数为准.8 所有样品，洗涤液和各种废弃物都应按传染物处理。9 本试剂不同批号组分不得混用。310

9、. 如与英文说明书有异，以英文说明书为准。计算：以标准物的浓度为横坐标，OD 值为纵坐标， 在坐标纸上绘出标准曲线，根据样品的 OD 值由标准曲线查出相应的浓度；再乘以稀释 倍数；或用标准物的浓度与 OD 值计算出标 准曲线的直线回归方程式，将样品的 OD 值 代入方程式，计算出样品浓度，再乘以稀释 倍数，即为样品的实际浓度。 （此图仅供参考）试剂盒性能：1.样品线性回归与预期浓度相关系数 R 值为 0.92 以上。2.批内与批间应分别小于 9%和 15%检测范围： 5ng/ml - 100ng/ml 保存条件及有效期：1.试剂盒保存：；2-8。2有效期：6 个月4FOR RESEARCH U

10、SE ONLYTomato yellow leaf curl virus Drug NamesGeneric Name： Tomato yellow leaf curl virus (TYLCV) ELISA Kit.PurposeThis kit allows for the determination of TYLCV concentrations in Tissue, cell culture supernatant and other biological fluids.Principle of the assayThe kit assay TYLCV level in the sam

11、ple，use Purified TYLCV antibody to coat microtiter plate wells, make solid-phase antibody, then add TYLCV to wells, Combined TYLCV which With HRP labeled , become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HR

12、P 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. The concentration of TYLCV in the samples is then determined by comparing the O.D. of the samples to the standard curve.5Material

13、s provided with the kitMaterials provided with the kit 48determinations 96 determinations StorageUser manual 1 1Closure plate membrane 2 2Sealed bags 1 1Microelisa stripplate 1 1 2-8Standard：135ng/ml 0.5ml1 bottle 0.5ml1 bottle 2-8Standard diluent 1.5ml1 bottle 1.5ml1 bottle 2-8HRP-Conjugate reagent

14、 3ml1 bottle 6ml1 bottle 2-8Sample diluent 3ml1 bottle 6ml1 bottle 2-8Chromogen Solution A 3ml1 bottle 6ml1 bottle 2-8Chromogen Solution B 3ml1 bottle 6ml1 bottle 2-8Stop Solution 3ml1 bottle 6ml1 bottle 2-8wash solution （20ml20 fold）1bottle （20ml30 fold）1bottle 2-8Specimen requirements1. cell cultu

15、re 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, d

16、amage 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.2. Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain sa

17、mples 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.3. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after

18、 the extraction. If it cant, specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.4. Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure61.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 1

19、00l to the first and the second well, then add Standard dilution 50l to the first and the second well, mix; take out 100l form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50l to the third and the forth well ,mix ; then take out 50l

20、 from the third and the forth well discard, add 50l to the fifth and the sixth well ,then add Standard dilution 50l to the fifth and the sixth well, mix ; take out 50l from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50l to the seventh and the

21、eighth well ,mix ; take out 50l from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50l to the ninth and the tenth well, mix , take out 50l from the ninth and the tenth well discard(add Sample 50l to each well after Diluting ,(density: 90ng/ml，60ng/ml

22、，30ng/ml ，15ng/ml ，7.5ng/ml)2.add sample： Set blank wells separately (blank comparison wells dont add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40l to testing sample well, then add testing sample 10l (sample final dilution is 5-fol

23、d), add sample to wells , 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-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5.washing

24、：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 50l to each well, except blank well. 7.incubate：Operation with 3.8.washing：Operation with 5.9.color： Add Chromo

25、gen 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 color change to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solut

26、ion and within 15min.7Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it ca

27、n be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 mins, if the number of sample is much , recommend to use Volley .4. if the testing mat

28、erial content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.（ n5）.5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the lig

29、ht preservation.7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective material process.9. Do not mix reagents with those from other lots.C

30、alculateTake the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation

31、 of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.8Assay range5ng/ml - 100ng/mlStorage and validity1Storage： 2-8.2validity： six months.This chart for reference only