4-HNE(4-Hydroxynonenal) ELISA Kit
4-羟基壬烯酸(4-HNE)酶联免疫吸附测定试剂盒
| 规格： | / 96T / 48T / 96T*5 |
| 价格： | / ￥3000 / ￥2100 / ￥12750 |
货号：E-EL-0128
反应性: 通用
检测范围: 0.63~40 ng/mL
灵敏度: 0.38 ng/mL
用途
该试剂盒用于体外定量检测血清、血浆或其他相关生物液体中4-HNE浓度。
检测原理
本试剂盒采用竞争ELISA法。用4-HNE抗原包被于酶标板上，实验时样品或标准品中的4-HNE与包被的4-HNE竞争生物素标记的抗4-HNE单抗上的结合位点，游离的成分被洗去。加入辣根过氧化物酶标记的亲和素，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，4-HNE浓度与OD450值之间呈反比，通过绘制标准曲线计算出样品中4-HNE的浓度。
| 反应类型 | 竞争法 |
| 规格 | 96T |
| 反应时间 | 2.0h |
| 反应性 | 通用 |
| 检测方法 | Colormetric |
| 检测范围 | 0.63-40 ng/mL |
| 灵敏度 | 0.38 ng/mL |
| 样本体积 | 50μL |
| 样本类型 | 血清、血浆或其他相关生物液体 |
特异性
可检测样本中的4-HNE，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。
| (ng/mL) | O.D | Average |
|---|---|---|
| 40 | 0.376 0.384 |
0.38 |
| 20 | 0.49 0.496 |
0.493 |
| 10 | 0.701 0.677 |
0.687 |
| 5 | 0.979 0.983 |
0.981 |
| 2.5 | 1.342 1.318 |
1.33 |
| 1.25 | 1.656 1.648 |
1.652 |
| 0.63 | 1.878 1.888 |
1.883 |
| 0 | 2.172 2.184 |
2.178 |
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 (ng/mL) |
1.90 | 5.40 | 18.00 | 1.90 | 5.80 | 19.10 |
| Standard
deviation |
0.10 | 0.20 | 0.60 | 0.10 | 0.30 | 0.60 |
| C V (%) | 5.26 | 3.70 | 3.33 | 5.26 | 5.17 | 3.14 |
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) | 89-102 | 94 |
| EDTA plasma (n=5) | 91-107 | 99 |
| Cell culture media (n=5) | 92-106 | 100 |
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 (%) | 97-115 | 93-107 | |
| Average (%) | 103 | 105 | 101 | |
| 1:4 | Range (%) | 91-103 | 95-106 | 93-107 |
| Average (%) | 97 | 100 | 99 | |
| 1:8 | Range (%) | 86-102 | 90-102 | 99-109 |
| Average (%) | 93 | 97 | 104 | |
| 1:16 | Range (%) | 83-96 | 91-106 | 97-111 |
| Average (%) | 90 | 98 | 104 | |
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50μL标准品工作液或样本后，立即每孔加入50μL生物素化抗体工作液，37℃孵育90分钟 |
| 2. 弃掉板内液体，洗板3次 | |
| 3. 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 | |
| 4. 每孔加入90μL底物溶液，37℃孵育15分钟 | |
| 5. 每孔加入50μL终止液 | |
| 6. 立即在450nm波长下读数，处理数据 |
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