所需设备及试剂
•450 nm滤光片酶标仪,含540 nm或570nm校正波长更佳
•单道或多道微量移液器,移液器枪头
•去离子水
• 500 mL量筒
•样品稀释管或不同规格EP管
•吸水纸
一、试剂准备
使用前将所有试剂和样本置于室温平衡。
20×浓缩清洗液: 如果浓缩液中有晶体析出,加热至室温并轻轻混合,直到晶体完全溶解,25mL浓缩清洗液使用去离子水定容至500mL,得到清洗工作液。
标准品:用1mL样本稀释液溶解RBD蛋白标准品冻干粉,溶解后标准品母液浓度为8000pg/mL。震荡混匀后静置10分钟,然后进行标准品的稀释,将标准品母液使用样本稀释液10倍稀释得到第一个标准点800pg/mL,依次进行倍比稀释操作得到共计7个标准点800 pg/mL、 400 pg/mL、200 pg/mL、100 pg/mL、50 pg/mL、25 pg/mL、12.5pg/mL、最后再以100ul样本稀释液为零标准(0 pg/mL),共计8个标准点,建议每个标准点至少做2个平行孔。
检测溶液A(生物素标记):震荡混匀后用掌上离心机瞬时离心,使液体集中于管底,临用前取合适体积用检测溶液稀释液1:2000稀释至工作浓度。
检测溶液B(HRP标记):震荡混匀后用掌上离心机瞬时离心,使液体集中于管底,临用前取合适体积用检测溶液稀释液1:2000稀释至工作浓度。
显色液: 避光保存,显色时100μL /孔。
终止液:显色完毕后50μL /孔。
二、实验步骤
使用前将所有试剂和样本置于室温,建议对所有标准品和样本进行一式两份的分析
1. 按照前面章节的指示准备所有试剂和准备工作。
2. 从板架上取下多余的微孔板条,将其放回装有干燥剂包的箔袋中,然后重新密封保存于-20℃。
3. 每孔加入100μL 稀释好的标准品(共计8点)和待测样本,用封板膜封住,37℃孵育60分钟。
4. 弃去孔内液体,每孔加入 300μL的清洗工作液,轻微震荡后弃去孔内液体,将酶标板倒扣在吸水纸上轻拍,使残留在孔内的液体全部去除,重复洗板3次,此过程也可采用尖嘴喷射瓶,多道移液器或自动洗板机来完成。
5. 每孔加入100μL检测溶液A工作液(生物素标记),用新的封板膜封住,37℃孵育60分钟。
6. 重复步骤4中的洗板程序。
7. 每孔加入100μL检测溶液B工作液(HRP标记),用新的封板膜封住,37℃孵育30分钟。
8. 重复步骤4中的洗板程序。
9. 每孔加入100μL显色液,室温避光显色10分钟。
10. 每孔加入50μL终止液,孔里的颜色应该由蓝色变至黄色,如果孔内颜色为绿色或颜色变化不均匀,轻轻震动酶标板板使颜色均一。
11. 擦干酶标板底部水气,立刻用酶标仪在 450nm 波长测量各孔的光密度值(O.D.值),如果波长校正可用,则设置为540 nm或570 nm,如果波长校正不可用,则从450 nm的读数中减去540 nm或570 nm的读数,这种修正可去除酶标板读数中的光学偏差,如果没有校正波长,可直接读取450 nm的读数,但读值有可能会更高、更不准确。
三、标准曲线制作
取每个标准品和样本的复孔平均值O.D.,减去零标准孔平均值O.D.后,以阳性对照的浓度为横坐标,O.D.值为纵坐标,绘出标准曲线(R2值越趋近于1曲线越精确),然后再将样本的平均值O.D.带入标准曲线的回归方程式,计算出样本中目标蛋白浓度。
如果样本已经稀释,从标准曲线上算出的浓度须乘以稀释系数,即为样本中目标蛋白的实际浓度。
四、标曲案例
标准曲线仅用于演示,每次实验均需生成单独的标准曲线。
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)
五、检测范围
12.5pg/mL—800pg/mL
六、最低检测限(MDD)
10pg/mL
MDD是测试20个零标准(稀释液)的平均O.D.值上加两倍标准差后计算相应的浓度来确定的。
七、精密度
批内差: CV<12%
批间差: CV<15%
八、稳定性
试剂盒按推荐温度保存6个月,信号强度降低小于 10%。
九、实验细节说明
1. 在试剂盒标签上的有效期内使用,不要与其他厂家的试剂盒混合使用。
2. 如果样本检测值超出标准曲线范围,可适当调整稀释倍数并重新测定,或预估样本中蛋白浓度,在实验之前预先稀释数个梯度同时进行测定。
3. 为避免交叉污染,不同标准品、样本、试剂的加样需更换移液枪头,每管试剂使用单独的容器储存。
4. 在孵育过程中贴紧封板膜。
5. 显色液避光保存,临用前15分钟从冰箱取出在室温平衡。
6. 浓缩清洗液(20×)需使用去离子水1:20稀释至工作浓度,去离子水不包含在试剂盒中,需实验人员自备。
7. 终止液为酸性溶液,具有轻微腐蚀性,使用时避免接触皮肤、眼、耳、口等暴露部位,如不慎接触,使用大量清水冲洗后观察接触部位反应,如情节严重请及时就医。
8. 试剂盒酶标板条可按需拆卸使用,已开封的试剂盒建议在1个月内使用,未开封的试剂盒所有试剂均按试剂瓶标签上所示保存,收到试剂盒后请将标准品、检测溶液 A、检测溶液 B、以及预包板保存于-20℃,其余试剂请置于 4 ℃保存备用。
十、常见问题解决指南
问题 | 原因 | 解决方案 |
标准曲线差 | 移液不精确 | 检查和校正移液器 |
标准品准备不正确 | 进行正确的标准品梯度稀释 |
O.D 值低 | 孵育时间太短 | 保证充足的孵育时间 |
温度不正确 | 试剂平衡至室温并保证孵育温度正确 |
试剂体积不正确 | 检查移液器并确保加样体积与说明书一致 |
精密度低 | 移液不精确 | 检查和校正移液器 |
混匀不充分 | 充分混匀和吸取试剂 |
洗涤不充分 | 按说明书要求充分洗涤和浸泡 |
背景值高 | 洗涤不充分 | 按说明书要求充分洗涤和浸泡 |
清洗液污染 | 重新配置清洗液 |
灵敏度低 | 试剂盒储存不当 | 试剂盒短期整体保存于4℃,长期保存根据说明书分开保存各组分 |