ENZYME-IMMUNOASSAY TECHNIQUES - AN OVERVIEW
ENZYME-IMMUNOASSAY TECHNIQUES - AN OVERVIEW
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DOI：
10.1016/0022-1759(92)90061-w
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发表时间：
1992-06-24
影响因子：
2.2
通讯作者：
KIESSIG, ST
中科院分区：
文献类型：
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作者：
PORSTMANN, T;KIESSIG, ST
尽管酶免疫分析（EIA）的种类繁多，但根据其反应原理，可将其分为“分析物观察”和“试剂观察”两类。后者通过使用单克隆抗体而受到青睐，其特征在于更高的灵敏度，更大的测量范围，对干扰影响的敏感性较低。它们只能用于检测大分子。对于在实验室规模上使用的异质EIA，抗原和抗体的简单吸附仍然是可消除的，尽管亲和常数降低至少一个数量级，并且由于空间位阻增加，固相处的抗体密度和分析物结合能力不平行。因此，具有较高亲和常数的抗体应始终用作固相抗体。微粒作为固相用于多相分析，由于其对分析物的结合能力非常高，扩散距离极短，保证了“一步”分析仅需几分钟。在有限数量的适合作为免疫分析标记的酶中，辣根过氧化物酶是首选酶，其次是碱性磷酸酶。虽然酶和酶标记试剂可通过荧光产物检测，其灵敏度比使用显色底物高10-1000倍，但测定的灵敏度仅可提高2- 10倍。标记酶不仅与抗体共价结合，而且还通过抗酶抗体结合。讨论了酶/抗酶复合物与含分析物的免疫复合物偶联的不同方法的优缺点，综述了检测特异性抗体的不同EIA变体。其中，只有捕获EIA允许不同独特型抗体的精确同种型分析。均质EIA广泛应用于半抗原测定，但即使是基于近端连接的变体也不能替代异质EIA用于测定大分子。定义了不同的参数，这些参数允许评估免疫测定的质量，并应在实验室的常规测定中用作内部对照。
In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant' should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee 'one step' assays of only a few minutes.Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10-1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2-10.Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme / anti-enzyme complex to analyte-containing immune complexes are discussed.Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules.Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory.