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ELISA老不成功,究竟错在哪儿?

ELISA素来以灵敏度较高、特异性较好的特点在临床上得到了广泛的应用。而它作为经典的免疫检测方法,虽然看似平常但是要真正将该实验做好并不容易,酶标板的读值总是会不尽如人意,可见实操中的各个环节对该实验的检测效果影响较大,如不注意,就会产生一些糟心的实验结果。

问题1: 低信号强度

如果ELISA读数低于吸光度下限(<000),且目标样本浓度超出标准曲线范围,则进行分析的样本浓度可能太低,或者仪器所能分析的样本检测最佳条件可能未被满足。在这种情况下,样本所产生的检测信号应该需要被级联放大。

解决方案:

1.增加抗体的孵育时间。如果依照protocol在室温下孵育您的抗体2小时后的实验结果并不理想,那么可以尝试在4°C孵育过夜。如此,可允许抗原抗体最大程度上的结合,并能扩大ELISA中的反应信号。

2.增加二抗-酶结合物的浓度。ELISA中的E代表酶(通常是辣根过氧化物酶,HRP),它与底物(通常是TMB)反应后可产生鲜艳的蓝色。因而,将HRP浓度提高50%或将其加倍,则会产生更强的颜色,易于检测。

3.充分避光,以保护TMB基质不受光照影响,并确保其能最大限度地发挥其性能。考虑到TMB对光线非常敏感,因而在黑暗中进行ELISA平板的显色反应会产生更强的信号。

问题2: 高背景

ELISA板上的空白孔通常被用作为阴性对照,当该组中任意孔的读数明显大于0,这均表示实验背景有问题。

解决方案:

1.室温过高,可能会导致实验结果偏高。因而,如果发现所有的实验孔的读数均偏高,请注意控制恒温箱温度,尽可能将其稳定在37℃左右。

2.平板孔内因清洗不彻底,而导致有酶或底物残留,同样会产生高背景。此时需要按照protocol对ELISA平板进行清洗,可不任意减少洗板液的体积以及洗板次数,同时也需要按要求操作,并适当延长清洗液浸泡孔的时间。

3.加样或加酶时导致了阴性孔的污染。此时需要及时更换吸头以避免造成污染,切忌侥幸心理。

4.试剂过期或被污染。实验操作过程中不要使用过期试剂,并防止组分污染。如使用容器时,应注意容器的清洁。

问题3: 重复性不好

同一个样本间的各个复孔的读数有显著性差异。

解决方案:

1.加样量多少不一,操作时间长短不一。重复同一样本时,加样量与加样时间理应相同,同时也应注意移液器的校准。加样后可轻微晃动酶标板使反应液充分混合。

2.样本应保证一致、无污染,并应该由同一名操作人员进行操作。

3.精密度测定方法不标准,此时理应采用正确的方法进行操作。

问题4: 干扰

干扰物质如清洗剂或NADH(存在于血清中)可能会影响ELISA平板的吸光度读数并使得实验结果偏离真实结果。

解决方案:

1.严格按照protocol进行实验操作。通常protocol中会指出会对实验产生干扰的化学物质,以及会告知实验者如何避免它们。比如在进行血红蛋白干扰的血清测定中,通常会建议不要使用溶血的血清样品。

2.有时可能你也别无选择,只能使用含有干扰物质的样品。如果你感兴趣的抗原浓度较高,可以使用一个浓度较低但仍易于检测的样品稀释液,如此可以稀释样本中的干扰物质并降低其对实验的干扰。

问题5: 阳性与阴性不能达到2.1的比值

阴性颜色太深,又或者阳性颜色太浅。

解决方案:

1.当阴性颜色太深时,则阴性对照(也就是阳性对照的除目标抗原外的其它成分)中可能有某种成分与一抗作用。此时可通过纯化抗原除去干扰成分。

2.当阳性颜色太浅时,原因可能有3个:一抗效价不好,换用一抗或增加一抗用量;抗原太稀,增加抗原浓度;封闭时间过长,应缩短封闭时间,封闭时间一般37摄氏度2个小时,或者4摄氏度过夜。

问题6: 增加抗原浓度,一抗浓度不变,显色反应没有表现出应有的梯度

一抗与抗原的比例已经饱和。

解决方案:

增加一抗用量; 或在一抗用量不变的情况下减少抗原浓度做梯度。

问题7: 增加一抗浓度,抗原浓度不变,显色反应没有表现出应有的梯度

一抗与抗原的比例已经饱和。

解决方案:

增加抗原用量;或在抗原用量不变的情况下减少一抗浓度做梯度。

问题8: 加完TMB显色后颜色梯度很明显,但加了硫酸终止反应后颜色梯度没有那么明显了

硫酸可能把其它成分都炭化了

解决方案:

加少一点硫酸,参考值:50 μlTMB+35 μl硫酸;或者采用1M的HCL作为终止液,50ulTMB+50ul1M HCL。



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