Rat NFKB-p65(Nuclear Factor Kappa B p65) ELISA Kit
大鼠核因子κB亚基p65(NFKB-p65)酶联免疫吸附测定试剂盒
| 规格： | / 96T / 48T / 96T*5 |
| 价格： | / ￥3200 / ￥2300 / ￥13600 |
货号：E-EL-R0674
反应性: 大鼠
检测范围: 78.13~5000 pg/mL
灵敏度: 46.88 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NFKB-p65浓度
检测原理
本试剂盒采用双抗体夹心ELISA法。用抗大鼠NFKB-p65抗体包被于酶标板上，实验时样品或标准品中的大鼠NFKB-p65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NFKB-p65抗体和辣根过氧化物酶标记的亲和素。抗大鼠NFKB-p65抗体与结合在包被抗体上的大鼠NFKB-p65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，NFKB-p65浓度与OD450值之间呈正比，通过绘制标准曲线计算出样品中NFKB-p65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 | 78.13—5000 pg/mL |
| 灵敏度 | 46.88 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NFKB-p65，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 |
|---|---|---|---|
| 5000 | 2.509 2.567 |
2.538 | 2.466 |
| 2500 | 1.729 1.761 |
1.745 | 1.673 |
| 1250 | 0.977 0.971 |
0.974 | 0.902 |
| 625 | 0.549 0.583 |
0.563 | 0.494 |
| 312.5 | 0.26 0.258 |
0.259 | 0.187 |
| 156.25 | 0.196 0.17 |
0.183 | 0.111 |
| 78.13 | 0.126 0.13 |
0.128 | 0.056 |
| 0 | 0.066 0.078 |
0.070 | -- |
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) |
247.16 | 793.15 | 2350.95 | 248.82 | 741.40 | 2123.84 |
| Standard
deviation |
13.77 | 35.06 | 123.19 | 12.69 | 32.03 | 93.02 |
| C V (%) | 5.57 | 4.42 | 5.24 | 5.10 | 4.32 | 4.38 |
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 | 98 |
| EDTA plasma (n=5) | 88-99 | 93 |
| Cell culture media (n=5) | 95-111 | 102 |
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 (%) | 97-112 | 95-112 | 95-107 |
| Average (%) | 103 | 103 | 100 | |
| 1:4 | Range (%) | 88-103 | 87-102 | 84-97 |
| Average (%) | 95 | 93 | 89 | |
| 1:8 | Range (%) | 90-103 | 85-97 | 83-96 |
| Average (%) | 97 | 90 | 89 | |
| 1:16 | Range (%) | 92-105 | 88-99 | 88-99 |
| Average (%) | 97 | 93 | 94 |
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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