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人骨碱性磷酸酶(BALP)酶联免疫分析(ELISA)

2017-11-21 10页 doc 28KB 15阅读

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人骨碱性磷酸酶(BALP)酶联免疫分析(ELISA)人骨碱性磷酸酶(BALP)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。 药品名称: 通用名:人骨碱性磷酸酶(BALP)酶联免疫分析试剂盒 使用目的: 本试剂盒用于测定人血液中骨碱性磷酸酶(BALP)含量。 实验原理 本试剂盒应用间接法测定标本中人骨碱性磷酸酶(BALP)水平。用纯化的人骨碱性磷酸酶(BALP)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骨碱性磷酸酶(BALP)标准品和未知浓度的骨碱性磷酸酶(BALP)待检样品,温育后,加入生物素标记的抗IgG抗体,再...
人骨碱性磷酸酶(BALP)酶联免疫分析(ELISA)
人骨碱性磷酸酶(BALP)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。 药品名称: 通用名:人骨碱性磷酸酶(BALP)酶联免疫分析试剂盒 使用目的: 本试剂盒用于测定人血液中骨碱性磷酸酶(BALP)含量。 实验原理 本试剂盒应用间接法测定标本中人骨碱性磷酸酶(BALP)水平。用纯化的人骨碱性磷酸酶(BALP)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骨碱性磷酸酶(BALP)品和未知浓度的骨碱性磷酸酶(BALP)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。颜色的深浅和样品中的骨碱性磷酸酶(BALP)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人骨碱性磷酸酶(BALP)浓度。 试剂盒组成 1 20倍浓缩洗涤液 50ml×1瓶 标准品S1(16μg/L) 0.5ml×1瓶 2 链霉亲和素-HRP 6ml×1瓶 标准品S2(8μg/L) 0.5ml×1瓶 8 3 酶标包被板 12孔×8条 标准品S3(4μg/L) 0.5ml×1瓶 4 生物素标记的抗-IgG抗体 6ml×1瓶 标准品S4(2μg/L) 0.5ml×1瓶 5 显色剂A液 6ml×1瓶 标准品S5(1μg/L) 0.5ml×1瓶 6 显色剂B液 6ml×1/瓶 9 说明书 1份 7 终止液 6ml×1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50μl,在样本反应孔中加入50微升样本,盖上封 板膜,轻轻振荡混匀,37?温育45分钟。 3. 配液:将20倍浓缩洗涤液用蒸馏水20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50μl。37?温育30分 钟 1 6. 洗涤:操作同4。 7. 加链霉亲和素-HRP:每孔加入50μl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 50μl,再加入显色剂B 50μl,轻轻震荡混匀,37?避光显色 15分钟. 10. 终止:每孔加终止液50μl,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(×5×n)。 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: 0.8μg/L -16μg/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 2 RB Human BALP FOR RESEARCH USE ONLY Package size: 96 determinations. Drug Names Generic Name,Human BALP ELISA Kit. Purpose This kit allows for the determination of BALP concentrations in Human blood. Principle of the assay The kit assay Human BALP level in the sample,use Purified Human BALP antibody to coat microtiter plate wells, make solid-phase antibody, Samples which including standards of known BALP concentrations and unknowns are pipetted into coated microtiter wells, after Incubating ,add Biotinylated anti-IgG,and Combined 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 proportional to the concentration of BALP present in the samples. measure the optical densit (OD) at 450 nm with microtiter plate reader, calculate BALP concentration by standard curv. 3 Materials provided with the kit 0.5ml×1 bottle 1 wash solution 50ml×1bottle 1 Standard(16μg/L) 0.5ml×1 bottle 2 Streptavidin-HRP 6ml×1 bottle 2 Standard(8μg/L) 8 0.5ml×1 bottle 3 Microelisa stripplate 12well×8strips 3 Standard(4μg/L) 0.5ml×1 bottle 4 Biotinylated anti-IgG 6ml×1 bottle 4 Standard(2μg/L) 0.5ml×1 bottle 5 Chromogen Solution A 6ml×1 bottle 5 Standard(1μg/L 6 Chromogen Solution B 6ml×1 bottle 9 User manual 1 Closure plate 7 Stopp Solution 6ml×1 bottle 10 2 membrane Specimen requirements 1. Can’t 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 after the extraction. If it can not be tested immediately, specimen can be kept in -20 ? to preserve, Avoid repeated freeze-thaw cycles. Assay procedure 1. Determine the number of microwell strips required to test the desired number of samples,. Each sample, standard and blank should be assayed in duplicate. 2. add sample,Set blank wells separately (blank comparison wells don’t add sample and ELISA reagent, other each step operation is same). Add 50 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate, and Gently mix. Incubate for 45 min at 37? 3. Configurate 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 for 30 4 min at 37? 6. washing,Operation with 4. 7. add streptavidin-HRP : Add streptavidin-HRP 50ul to all wells, Gently mix Incubate for 15 min at 37? 8. washing,Operation with 4. 9. color,Add Chromogen Solution A 50ul and Chromogen Solution B to each well,Incubate for 15 min at 37? 10. Stop the reaction,Add Stop Solution50μl 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. 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 calculate 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 multiple, the result is the sample actual density. Important notes 1. 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. 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 experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley . 5 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 first standard well OD ),please use Sample dilution to dilute certain multiple (n times),then assay. Please multiply total Dilution Times when calculate,×n×5,. 5. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard. 6. This reagent which 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 range 0.8μg/L -16μg/L Storage and validity 1,Storage, 2-8?. 2,validity, six months. 6 7 8
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