小鼠16α羟基雌酮11αOHE-1试剂盒使用方法.DOC

1、小鼠 16羟基雌酮 1(16- OHE-1)试剂 盒使用 方法 检测范围： 96T 20ng/L - 700ng/L 使用目的： 本试剂盒用于测定 小鼠血清、血浆及相关液体样本中 16羟基雌酮 1(16- OHE-1)含量 。 实验原理 本试剂盒应用双抗体夹心法测定 标本 中 小鼠 16羟基雌酮 1(16- OHE-1)水平。用纯化的 小鼠 16羟基雌酮 1(16- OHE-1)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 16羟基雌酮 1(16- OHE-1)， 再与 HRP 标记的 16羟基雌酮 1(16- OHE-1)抗体结合，形成抗体 -抗原 -酶标抗体复合物 ，经过彻底

2、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。颜色的深浅和样品中的 16羟基雌酮 1(16- OHE-1)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 小鼠 16羟基雌酮 1(16- OHE-1)浓度。 试剂盒组成 1 30 倍浓缩洗涤液 20ml 1 瓶 7 终止液 6ml 1 瓶 2 酶标试剂 6ml 1 瓶 8 标准 品（ 1200 ng/L） 0.5ml 1 瓶 3 酶 标包被 板 12 孔 8 条 9 标准品 稀释液 1.5ml 1 瓶 4 样品 稀释液 6ml 1 瓶 10

3、说明书 1 份 5 显色剂 A 液 6ml 1 瓶 11 封板膜 2 张 6 显色剂 B 液 6ml 1/瓶 12 密封袋 1 个 标本要求 1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 2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：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。 600ng/L 5 号标准品 150l 的原倍标准品加入 150l 标准品稀释液 300ng/L 4 号标准品 150l 的 5 号标

4、准品加入 150l 标准品稀释液 150ng/L 3 号标准品 150l 的 4 号标准品加入 150l 标准品稀释液 75ng/L 2 号标准品 150l 的 3 号标准品加入 150l 标准品稀释液 37.5ng/L 1 号标准品 150l 的 2 号标准品加入 150l 标准品稀释液 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）、标准孔、待测样品孔 。在酶标包被板上标准品准确加样 50l， 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

5、：用封板膜封板后置 37温育 30 分钟 。 4. 配液：将 30 倍浓缩洗涤液用蒸馏水 30 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） 。

6、 测定应在加终止液后 15 分钟以内进行。 操作程序总结： 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。 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

7、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（ n 5）。 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。 10. 如与英文说明书有异，以英文说明书为准。 保存条件及有效期 1 试剂盒保

8、存： ； 2-8 。 2有效期： 6 个月 Mouse 16- OHE-1 FOR RESEARCH USE ONLY Assay range： 20ng/L - 700ng/L 96 determinations Purpose For the quantitative in vitro determination of 16- OHE-1 concentrations in Mouse serum, cell culture supernates and other biological fluids Principle of the assay The kit assay Mouse 1

9、6- OHE-1 level in the sample， use Purified Mouse 16- OHE-1 antibody to coat microtiter plate wells, make solid-phase antibody, then add 16- OHE-1 to wells, Combined 16- OHE-1 antibody which With HRP labeled goat anti-Human become antibody - antigen - enzyme-antibody complex, after washing Completely

10、, 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. The concentration of Mouse 16- OHE-1 in the samples is then de

11、termined by comparing the O.D. of the samples to the standard curve . Materials provided with the kit 1 wash solution 20ml 1bottle 7 Stopp Solution 6ml 1 bottle 2 HRP-Conjugate reagent 6ml 1 bottle 8 Standard（ 1200ng/L） 0.5ml 1 bottle 3 Microelisa stripplate 12well 8strips 9 Standard diluent 1.5ml 1

12、bottle 4 Sample diluent 6ml 1 bottle 10 Instruction 1 5 Chromogen Solution A 6ml 1 bottle 11 Closure plate membrane 2 RD 6 Chromogen Solution B 6ml 1 bottle 12 Sealed bags 1 Specimen requirements 1. extract as soon as possible after Specimen collection,and according to the relevant literature, and s

13、hould 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. 2. Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active. Assay procedure 1. Dilute and add sample:Dilute Original density Stand

14、ard as follow table: 600ng/L 5 Standard 150l Original density Standard+150l Standard diluent 300ng/L 4 Standard 150l 5 Standard+150l Standard diluent 150ng/L 3 Standard 150l 4 Standard+150l Standard diluent 75ng/L 2 Standard 150l 3 Standard +150l Standard diluent 37.5ng/L 1 Standard 150l 2 Standard

15、+150l Standard diluent 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-fold), a

16、dd 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

17、： 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

18、 Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37 10.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

19、 Stop Solution and within 15min. Steps description Standard, Sample diluent Add Standard, Sample diluent, incubate for 30 min at 37 . Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37 . Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37 . Add Stopp Solution Read ab

20、sorbance at 450nm within 15 min calculate Calculate Take 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 cal

21、culate the straight line regression equation 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. Important notes 1. The kit takes out from

22、 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 can be heated the water helps dissolve when dilute . Washing do

23、es not affect the result. 3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley . 4. if the testing material content is excessively higher (The sample OD is bigger

24、 than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.（ n 5） . 5. Closure plate membrane only limits the disposable use, to avoid cross-contamination. 6. The substrate evade the light preservation. 7. Please according to use instruction

25、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. Storage and validity 1 Storage： 2-8 . 2 validity： six months