人纤溶酶原激活物抑制因子1(PAI

人纤溶酶原激活物抑制因子1 （PAI-1） ELISA试剂盒分析检测说明书本试剂盒仅供研究使用。检测范围：48T25 ng/L -800 ng/L使用目的：本试剂盒用于测定人血清、血浆及相关液体样本纤溶酶原激活物抑制因子1 （PAI-1）含量。实验原理本试剂盒应用双抗体夹心法测定标本中人纤溶酶原激活物抑制因子1（PAI-1） 水平。用纯化的人纤溶酶原激活物抑制因子1（PAI-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纤溶酶原激活物抑制因子1（PAI-1）， 再与HRP标记的纤溶酶原激活物抑制因子1（PAI-1 ）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。颜色的深浅和样品中的纤溶酶原 激活物抑制因子1（PAI-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人纤溶酶原激活物抑制因子1（PAI-1）浓度。试剂盒组成120倍浓缩洗涤液20ml X1瓶7终止液3ml X 1 瓶2酶标试剂3ml X1 瓶 8标准品(1600ng/L)0.5ml X 1 瓶3酶标包被板12孔X4条9标准品稀释液1.5ml X 1 瓶4样品稀释液3ml X1 瓶 10说明书1份5显色剂A液3ml X1 瓶 11封板膜2张6显色剂B液3ml X1/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-20C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。150p l的原倍标准品加入150l标800 ng/L 5号标准品准品稀释液400 ng/L 4号标准品150l的5号标准品加入150l标准品稀释液200 ng/L1501的4号标准品加入1501标13号标准品准品稀释液100 ng/L1501的3号标准品加入1501标2号标准品准品稀释液50 ng/L1501的2号标准品加入1501标1号标准品准品稀释液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50卩1,待测样品 孔中先加样 品稀释液40p 1,然后再加待测样品10p 1 （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37C温育30分钟。4. 配液：将20倍浓缩洗涤液用蒸馏水20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50p 1，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A50p 1，再加入显色剂B50p 1，轻轻震荡混匀, 37C避光显色15分钟.10. 终止：每孔加终止液50p 1,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，做复孔。如标本中待测物质含量过高（样 本OD值大于标准品孔孔的0D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（XnX5）o5. 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. 试剂盒保存：；2-8Co2. 有效期：6个月The performa nee of kit:1 sen si tivity: min imum detee tion concen tra tion is less tha n 1 sta ndard. Lin earity of dilu tion. Sample lin ear regressi on and the expected concen tra tion correla tion coefficie nt R value is 0.990.2: no specific reac tion with other cytok in es.3 repeatability: plate, plate betwee n the coefficie nts of varia tion were less tha n 10%.Huma n type III procollage n amino term inal pep tide ELISA kit steps:1 before use, all reage nts and mixing. Do not allow liquid to produce a large n umber of bubbles, so as to avoid add ing a large n umber of bubbles, result ing in the additi on of the error.2 accord ing to determ ine the n umber of sample n umber plus sta ndard strip n umber. Each sta ndard and bla nk hole is recomme nded to do the hole. Each sample can be made accord ing to its own qua ntity, and can be used as a hole in the hole.3 diluted after sta ndard 50ul in reac tion hole, added to the sample 50 UL in reac tion hole to be measured. Immediately joined the 50 UL anti body bio tin. Cover the membra ne plate, gen tly oscilla ting mixing, 37 degrees Celsius for 45 minu tes.4 left hole liquid, each hole filled with wash ing liquid, oscilla ting 30 sec onds off the wash ing liquid, pat dry with absorbe nt paper. Repeat this opera tion 4 廿 mes. If the wash ing mach ine with wash ing, wash ing times in creased on ce.5 per hole add ing cha in affin ity en zyme -HRP 100ul, gen tly oscilla ting mixing, 37 degrees 30 minu tes in cuba tion.6 left hole liquid, each hole filled with wash ing liquid, oscilla ting 30 sec onds off the wash ing liquid, pat dry with absorbe nt paper. Repeat this opera tion 4 廿 mes. If the wash ing mach ine with wash ing, wash ing times in creased on ce.7 per hole add ing substrate A, B 50ul, gen tly oscilla ting mixing, 37 degrees 5 minu tes in cuba tion. Avoid light.8 remove ELISA plate, quickly add 50ul term in ated liquid, add ing the stop solution immediately after the determ ination results.9 od determ ination of each hole at the wavele ngth of 450nm.The result of judgme nt and an alysis:1, in strume nt value: Yu Bo 450nm ELISA od read the hole on the in strume nt2, to the OD value as a ver tical coord in ate (y), corresp onding ot sta ndardconcen tra tions as a horiz on tal coord in ate (x), do have corresp onding curve, sample ot content can be accord ing to the OD value by sta ndard curve con versi on out corresp onding concen tra tion, multiplied by the dilu tion multiple; or with the sta ndard concen tra tion and the OD value calculated the regressi on equa tion of the sta ndard curve, the sample OD value in the equa tion to calculate the sample concen tra tion, multiplied by the dilu tion factor is the actual concen tra tion of the sample.3, detec tion ran ge: 0-10 On g/ml4, sen sitivity: 0.39 ng/ml