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Elisa酶联检测


技术原理:

     酶联免疫吸附试验(又称酵素免疫分析法,Enzyme-linked immunosorbent assay,简称 ELISA)利用抗原抗体之间专一性键结之特性,对检体进行检测;由于结合于固体承载物(一般为塑胶孔盘)上之抗原或抗体仍可具有免疫活性,因此设计其键 结机制后,配合酵素呈色反应,即可显示特定抗原或抗体是否存在,并可利用呈色之深浅进行定量分析。根据待测样品与键结机制的不同,ELISA可设计出各种 不同类型的检测方式,主要以三明治法(sandwich)、间接法(indirect)、以及竞争法(Competitive)三种为主,以下为各种方法 之介绍。

 



三明治法


三明治法常用于检测大分子抗原,一般之操作步骤为:

  •  将具有专一性之抗体固著(coating)于塑胶孔盘上,完成后洗去多余抗体;
  •  加入待测检体,检体中若含有待测之抗原,则其会与塑胶孔盘上的抗体进行专一性键结;
  •  洗去多余待测检体,加入另一种对抗原专一之一次抗体,与待测抗原进行键结;
  •  洗去多余未键结一次抗体,加入带有酵素之二次抗体,与一次抗体键结;
  •  洗去多余未键结二次抗体,加入酵素受质使酵素呈色,以肉眼或仪器读取呈色结果。

三明治法分别以两种抗体对检体中的抗原进行两次专一性辨认,因此专一性相当高,但此待测抗原必须是多价抗原,如此才可获得两种以上的专一性抗体,以分别进行夹心;而且此法需要足够的表位空间以进行抗原抗体的夹心,所以并不适用于半抗原或小分子抗原等分子量较小之标的。


间接法

间接法常用于检测抗体,一般之操作步骤为:

  •  将已知之抗原固著于塑胶孔盘上,完成后洗去多余之抗原;
  •  加入待测检体,检体中若含有待测之一次抗体,则其会与塑胶孔盘上的抗原进行专一性键结;
  •  洗去多余待测检体,加入带有酵素之二次抗体,与待测之一次抗体键结;
  •  洗去多余未键结二次抗体,加入酵素受质使酵素呈色,藉仪器(ELISA reader)测定塑胶盘中的吸光值(OD值),以评估有色终产物的含量即可测量待测抗体的含。量。


竞争法

竞争法是一种较少用到的ELISA检测机制,一般用于检测小分子抗原,其操作步骤为:

  •  将具有专一性之抗体固著于塑胶孔盘上,完成后洗去多余抗体;
  •  加入待测检体,使检体中的待测抗原与塑胶孔盘上的抗体进行专一性键结;
  •  加入带有酵素之抗原,此抗原也可与塑胶孔盘上的抗体进行专一性键结,由于塑胶孔盘上固著的抗体数量有限,因此当检体中抗原的量越多,则带有酵素之抗原可键。结的固著抗体就越少,亦即,两种抗原皆竞相与塑胶孔盘上抗体键结,即所谓竞争法之由来;
  •  洗去检体与带有酵素之抗原,加入酵素受质使酵素呈色,当检体中抗原量越多,代表塑胶孔盘内留下之带有酵素的抗原越少,显色也就越浅;
  •  当需要侦测无法获得两种以上单一性抗体的抗原,或是不易得到足够的纯化抗体以固著于孔盘上时,一般会考虑使用竞争法ELISA。


结果判断

定性测定

  定性测定的结果判断是对受检标本中是否含有待测抗原或抗体作出”有”或”无”的简单回答,分别用”阳性”、”阴性”表示。” 阳性”表示该标本在该测定系统中有反应。”阴性”则为无反应。用定性判断法也可得到半定量结果,即用滴度来表示反应的强度,其实质仍是一个定性试验。在这 种半定量测定中,将标本作一系列稀释后进行试验,呈阳性反应的最高稀释度即为滴度。根据滴度的高低,可以判断标本反应性的强弱,这比观察不稀释标本呈色的 深浅判断为强阳性、弱阳性更具定量意义。

在间接法和夹心法ELSIA中,阳性孔呈色深于阴性孔。在竞争法ELISA中则相反,阴性孔呈色深于阳性孔。

定量测定

        ELSIA操作步骤复杂,影响反应因素较多,特别是固相载体的包被难达到各个体之间的一致,因此在定量测定中,每批测试均须用一系列不同浓度的参考 标准品在相同的条件下制作标准曲线。测定大分子量物质的夹心法ELISA,标准曲线的范围一般较宽,曲线最高点的吸光度可接近2.0,绘制时常用半对数 纸,以检测物的浓度为横坐标,以吸光度为纵坐标,将各浓度的值逐点连接,所得曲线一般呈S形,其头、尾部曲线趋于平坦,中央较呈直线的部分是最理想的检测 区域。

测定小分子量物质常用竞争法,其标准曲线中吸光度与受检物质的浓度呈负相关。标准曲线的形状因试剂盒所用模式的差别而略有不同。ELISA测定的标准曲线注意图中横坐标为对数关系,这更有利于测定系统的表达。


服务内容


您需要提供以下材料:

细胞上清和血清等,试剂盒(可代购)。

您将获得以下结果:

  • (1)实验数据;
  • (2)数据分析;
  • (3)实验报告。
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