1、1牛促黄体激素(LH)酶联免疫分析（ELISA）试剂盒使用说明书本试剂盒仅供研究使用。使用目的：本试剂盒用于测定牛血清、血浆及相关液体样本中促黄体激素(LH)的含量。实验原理本试剂盒应用间接法测定标本中牛促黄体激素(LH)水平。用纯化的牛促黄体激素(LH)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已知浓度的促黄体激素(LH)标准品和未知浓度的促黄体激素(LH) 待检样品，温育后，加入生物素标记的抗 IgG 抗体，再与链霉亲和素-HRP 结合，形成免疫复合物，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。颜色的深浅和样
2、品中的促黄体激素(LH)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牛促黄体激素(LH)浓度。 试剂盒组成 1 30 倍浓缩洗涤液 20ml1 瓶 标准品 S1（30ng/L） 0.5ml1瓶2 链霉亲和素-HRP 6ml1 瓶 标准品 S2（20ng/L） 0.5ml1瓶3 酶标包被板 12 孔8条标准品 S3（10ng/L） 0.5ml1瓶4 生物素标记的抗-IgG 抗体 6ml1 瓶 标准品 S4（5ng/L） 0.5ml1瓶5 显色剂 A 液 6ml1 瓶8标准品S5（2.5ng/L ）0.5ml1瓶 6 显色剂 B 液 6ml1/瓶 9
3、说明书 1 份7 终止液 6ml1 瓶 10 封板膜 2 张标本要求 1不能检测含 NaN3 的样品，因 NaN3 抑制辣根过氧化物酶的（HRP ）活性。2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，但应避免反复冻融操作步骤1. 根据待测样品数量加上标准品的数量决定所需的板条数。每个标准品和空白孔建议做复孔。每个样品根据自己的数量来定，能使用复孔的尽量做复孔。2. 加样：分别设空白孔（空白对照孔不加样品，其余各步操作相同） 、标准品孔、待测样品孔。然后在标准品孔中加入标准品 50l，在样本反应孔中加入 50 微升样本，盖上封板膜
4、，轻轻振荡混匀，37温育 45 分钟。3. 配液：将 30 倍浓缩洗涤液用蒸馏水 30 倍稀释后备用4. 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 4 次，拍干。5. 加生物素标记的抗-IgG 抗体：每孔加入生物素标记的抗-IgG 抗体 50l。37温育 302分钟6. 洗涤：操作同 4。7. 加链霉亲和素-HRP：每孔加入 50l 的链酶亲和素-HRP，轻轻振荡混匀，37温育 30分钟。8. 洗涤：操作同 4。9. 显色：每孔先加入显色剂 A 50l，再加入显色剂 B 50l，轻轻震荡混匀，37避光显色 15 分钟.10. 终止：每孔加终止液 50
5、l，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。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 值待入方程式，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
6、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 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） ，请先将样本稀释一定倍数（n 倍）后再测定，计算时请最后乘以稀释倍数（5n） 。5 封板膜只限一次性使用，以避免交叉污染。6本试剂不同批号组分不得混用。显色剂 B 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. 如与英文说明书有异，以英文说明书为准。线形范围：1ng/L -37ng/
7、L规格：96 人份/盒保存条件及有效期1试剂盒保存：；2-8。2有效期：6 个月3Bovine LHFOR RESEARCH USE ONLYPackage size: 96 determinations.Drug NamesGeneric Name： Bovine LH ELISA Kit.PurposeThis kit allows for the determination of LH concentrations in Bovine serum, blood plasma, and other biological fluids.Principle of the assayThe ki
8、t assay Bovine LH level in the sample，use Purified Bovine LH antibody to coat microtiter plate wells, make solid-phase antibody, Samples which including standards of known LH concentrations and unknowns are pipetted into coated microtiter wells, after Incubating ,add Biotinylated anti-IgG,and Combin
9、ed Streptavidin-HRP, become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, And at the effect of acid the color finally become yellow, The intensity of this coloured product is directly prop
10、ortional to the concentration of LH present in the samples. measure the optical densit (OD) at 450 nm with microtiter plate reader, calculate Bovine BHBA concentration by standard curv.4Materials provided with the kit1 wash solution 20ml1bottle 1Standard(30ng/L) 0.5ml1 bottle2 Streptavidin-HRP 6ml1
11、bottle 2 Standard(20ng/L) 0.5ml1 bottle3 Microelisa stripplate 12well8strips 3 Standard(10ng/L) 0.5ml1 bottle4 Biotinylated anti-IgG 6ml1 bottle 4 Standard(5ng/L) 0.5ml1 bottle5 Chromogen Solution A 6ml1 bottle85 Standard(2.5ng/L) 0.5ml1 bottle6 Chromogen Solution B 6ml1 bottle 9 User manual 17 Stop
12、 Solution 6ml1 bottle 10 Closure plate membrane 2Specimen requirements1. Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.2. extract as soon as possible after Specimen collection, Extracted according to the relevant literature, and should be experiment as soon as possible
13、after the extraction. If it can not be tested immediately, specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.Assay procedure1. Determine the number of microwell strips required to test the desired number of samples,. Each sample, standard and blank should be assayed in dupli
14、cate. 2. add sample：Set blank wells separately (blank comparison wells dont add sample and ELISA reagent, other each step operation is same). Add 50 L of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate, and Gently mix. Incubate for 45 min at 373. Configurate
15、liquid: 20 times of wash solution diluted 20 times with distilled water and reserve.4. washing：remove Liquid, dry by swing, add washing buffer to every well, still for 30 second then remove, repeat 4 times.5. add Biotinylated anti-IgG: Add diluted Biotinylated anti-IgG 50ul to all wells, Incubate fo
16、r 30 min at 3756. washing：Operation with 4.7. add streptavidin-HRP : Add streptavidin-HRP 50ul to all wells, Gently mix Incubate for 15 min at 378. washing：Operation with 4.9. color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well,Incubate for 15 min at 3710. Stop the reaction：Add
17、 Stop Solution50l to each well, Stop the reaction(the blue color change to yellow color Immediately).11. assay：take blank well as zero , measure the optical densit (OD) at 450 nm after Adding Stop Solution and within 15min.CalculateTake the standard density as the horizontal, the OD value for the ve
18、rtical ,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 of the standard curve with the standard density and the OD value ,with the
19、sample OD value in the equation, calculate the sample density, multiplied by the dilution multiple, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature then use, ELISA plates coated if
20、 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 does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the
21、 experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .4. Please make specification curve when you assay, had better make duplicate well, if in the sample the testing material content is excessively high (The sample OD is bigger than the 6first stand
22、ard well OD ),please use Sample dilution to dilute certain multiple (n times),then assay. Please multiply total Dilution Times when calculate（n5）.5. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard. 6. This reagent which
23、different batch number component do not mix. Chromogen Solution B evade the light preservation.7. Closure plate membrane only limits the disposable use, in order to avoid the overlapping pollution.8. All samples, washing buffer and each kind of reject should according to infective material process.Assay range1ng/L -37ng/L Storage and validity1Storage： 2-8.2validity： six months.7
