Mouse VEGF-A(Vascular Endothelial Cell Growth Factor A) ELISA Kit
小鼠血管内皮细胞生长因子A(VEGF-A)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-M1292
反应性: 小鼠
检测范围: 31.25~2000 pg/mL
灵敏度: 18.75 pg/mL
用途
该试剂盒用于体外定量检测小鼠serum, plasma and other biological fluids中VEGF-A浓度
检测原理
本试剂盒采用双抗体夹心ELISA法。用抗小鼠VEGF-A抗体包被于酶标板上，实验时样品或标准品中的小鼠VEGF-A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小鼠VEGF-A抗体和辣根过氧化物酶标记的亲和素。抗小鼠VEGF-A抗体与结合在包被抗体上的小鼠VEGF-A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，VEGF-A浓度与OD450值之间呈正比，通过绘制标准曲线计算出样品中VEGF-A的浓度。
| 反应类型 | 夹心法 |
| 规格 | 96T |
| 反应时间 | 3.5h |
| 反应性 | 小鼠 |
| 检测方法 | Colormetric |
| 检测范围 | 31.25—2000 pg/mL |
| 灵敏度 | 18.75 pg/mL |
| 样本体积 | 100μL |
| 样本类型 | serum, plasma and other biological fluids |
特异性
可检测样本中的小鼠VEGF-A，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 |
|---|---|---|---|
| 2000 | 2.464 2.416 |
2.44 | 2.38 |
| 1000 | 1.65 1.611 |
1.631 | 1.571 |
| 500 | 0.967 0.983 |
0.975 | 0.915 |
| 250 | 0.456 0.451 |
0.454 | 0.394 |
| 125 | 0.267 0.277 |
0.272 | 0.212 |
| 62.5 | 0.171 0.18 |
0.176 | 0.116 |
| 31.25 | 0.111 0.124 |
0.118 | 0.058 |
| 0 | 0.055 0.065 |
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) |
94.00 | 212.07 | 852.78 | 83.63 | 230.40 | 893.36 |
| Standard
deviation |
4.75 | 10.50 | 46.05 | 4.25 | 11.45 | 47.44 |
| C V (%) | 5.05 | 4.95 | 5.40 | 5.08 | 4.97 | 5.31 |
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) | 97-108 | 103 |
| EDTA plasma (n=5) | 98-108 | 101 |
| Cell culture media (n=5) | 99-109 | 104 |
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 (%) | 86-93 | 82-98 | 92-98 |
| Average (%) | 90 | 90 | 95 | |
| 1:4 | Range (%) | 92-101 | 87-96 | 92-101 |
| Average (%) | 98 | 90 | 97 | |
| 1:8 | Range (%) | 90-99 | 91-103 | 88-99 |
| Average (%) | 94 | 96 | 92 | |
| 1:16 | Range (%) | 91-102 | 89-98 | 91-105 |
| Average (%) | 96 | 93 | 97 |
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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