Rat ET-1(Endothelin 1) ELISA Kit
大鼠内皮素1(ET-1)酶联免疫吸附测定试剂盒
| 规格： | / 96T / 48T / 96T*5 |
| 价格： | / ￥2400 / ￥1650 / ￥10200 |
货号：E-EL-R1458
反应性: 大鼠
检测范围: 0.78~50 pg/mL
灵敏度: 0.47 pg/mL
用途
该试剂盒用于体外定量检测大鼠serum, plasma and other biological fluids中ET-1浓度
检测原理
本试剂盒采用双抗体夹心ELISA法。用抗大鼠ET-1抗体包被于酶标板上，实验时样品或标准品中的大鼠ET-1会与包被抗体结合，游离的成分被洗去。依次加入生物素化的抗大鼠ET-1抗体和辣根过氧化物酶标记的亲和素。抗大鼠ET-1抗体与结合在包被抗体上的大鼠ET-1结合、生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去。加入显色底物(TMB)，TMB在辣根过氧化物酶的催化下呈现蓝色，加终止液后变成黄色。用酶标仪在450nm波长处测OD值，ET-1浓度与OD450值之间呈正比，通过绘制标准曲线计算出样品中ET-1的浓度。
| 反应类型 | 夹心法 |
| 规格 | 96T |
| 反应时间 | 3.5h |
| 反应性 | 大鼠 |
| 检测方法 | Colormetric |
| 检测范围 | 0.78—50 pg/mL |
| 灵敏度 | 0.47 pg/mL |
| 样本体积 | 100μL |
| 样本类型 | serum, plasma and other biological fluids |
特异性
可检测样本中的大鼠ET-1，且与其它相关蛋白无明显交叉反应。
重复性
板内，板间变异系数均<10%。
典型数据
由于实验操作条件的不同（如操作者、移液技术、洗板技术和稳定条件等），标准曲线的OD值会有所差异。以下数据和曲线仅供参考，实验者需根据自己的实验建立标准曲线。
| (pg/mL) | O.D | Average | Corrected |
|---|---|---|---|
| 50 | 2.509 2.416 |
2.463 | 2.403 |
| 25 | 1.609 1.663 |
1.636 | 1.576 |
| 12.5 | 0.95 0.919 |
0.935 | 0.875 |
| 6.25 | 0.513 0.465 |
0.489 | 0.429 |
| 3.13 | 0.265 0.26 |
0.263 | 0.203 |
| 1.57 | 0.185 0.175 |
0.18 | 0.12 |
| 0.78 | 0.129 0.125 |
0.127 | 0.067 |
| 0 | 0.058 0.061 |
0.06 | -- |
精密度
板内精密度:低浓度样本,中浓度样本和高浓度样本分别在1块板子上检测20次。
板间精密度:低浓度样本,中浓度样本和高浓度样本分别在3块板子上检测20次。
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) |
2.14 | 5.93 | 24.08 | 2.31 | 5.77 | 23.01 |
| Standard
deviation |
0.11 | 0.27 | 1.08 | 0.14 | 0.29 | 1.14 |
| C V (%) | 5.14 | 4.55 | 4.49 | 6.06 | 5.03 | 4.95 |
回收率
分别往5个不同样本中添加已知浓度的目标蛋白，做回收实验，得出回收率范围和平均回收率。
| Sample Type | Range (%) | Average Recovery (%) |
|---|---|---|
| Serum (n=5) | 93-105 | 101 |
| EDTA plasma (n=5) | 97-104 | 101 |
| Cell culture media (n=5) | 97-109 | 103 |
线性
分别往5个样本中添加已知浓度的目标蛋白，做回收实验，得出回收率范围及平均回收率。将5个样本分别稀释2倍，4倍，8倍，16倍做回收实验，得出回收率范围及平均回收率。
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 98-106 | 85-96 | 87-98 |
| Average (%) | 101 | 93 | 91 | |
| 1:4 | Range (%) | 100-110 | 90-101 | 98-107 |
| Average (%) | 103 | 97 | 103 | |
| 1:8 | Range (%) | 96-109 | 98-108 | 86-96 |
| Average (%) | 104 | 105 | 92 | |
| 1:16 | Range (%) | 100-108 | 93-103 | 99-111 |
| Average (%) | 104 | 99 | 103 |
试剂盒组成及保存
未拆封的试剂盒可在4℃保存一周；如果一周以后才使用试剂盒，请拆开试剂盒按照下表中的条件分别保存各组分。
| 中文名称 | 英文名称 | 规格 | 保存条件 |
|---|---|---|---|
| ELISA酶标板(可拆卸) | Micro ELISA Plate(Dismountable) | 8 孔×12 条 | -20℃，可存放6个月 |
| 冻干标准品 | Reference Standard | 2 支 | |
| 浓缩生物素化抗体(100×) | Concentrated Biotinylated Detection Ab | 1支 120 μL | |
| 浓缩HRP酶结合物(100×) | Concentrated HRP Conjugate | 1支 120 μL | -20℃(避光)，可存放6个月 |
| 标准品&样品稀释液 | Reference Standard & Sample Diluent | 1瓶 20 mL | 4℃.可存放6个月 |
| 生物素化抗体稀释液 | Biotinylated Detection Ab Diluent | 1瓶 14 mL | |
| 酶结合物稀释液 | HRP Conjugate Diluent | 1瓶 14 mL | |
| 浓缩洗涤液(25×) | Concentrated Wash Buffer (25×) | 1瓶 30 mL | |
| 底物溶液(TMB) | Substrate Reagent | 1瓶 10 mL | 4℃(避光) |
| 反应终止液 | Stop Solution | 1瓶 10 mL | 4℃ |
| 封板覆膜 | Plate Sealer | 5 张 | |
| 产品说明书 | Product Description | 1 份 | |
| 质检报告 | Certificate of Analysis | 1 份 |
说明：所有试剂瓶盖须旋紧以防止蒸发和微生物的污染。
试剂体积以实际发货版说明书为准。相关试剂在分装时会比标签上标明的体积稍多一些，请在使用时量取而非直接倒出。
试验所需自备物品
- 1. 酶标仪(450nm波长滤光片)
- 2. 高精度移液器，EP管及一次性吸头：0.5-10 μL, 2-20 μL, 20-200 μL, 200-1000 μL
- 3. 37℃恒温箱，双蒸水或去离子水
- 4. 吸水纸
操作步骤
|
|
1.对应板孔中加入100μL标准品工作液或样本，37℃孵育90分钟 |
| 2.弃掉板内液体后，立即加入100μL生物素化抗体工作液，37℃孵育60分钟 | |
| 3.弃掉板内液体，洗板3次 | |
| 4.每孔加入100μL HRP酶结合物工作液，37℃孵育30分钟，弃掉板内液体，洗板5次 | |
| 5.每孔加入90μL底物溶液，37℃孵育15分钟 | |
| 6.每孔加入50μL终止液 | |
| 7.立即在450nm波长下读数，处理数据 |
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