Antibody-antigen complex formation with immobilized immunoglobulins.
Antibody-antigen complex formation with immobilized immunoglobulins.
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与固定化免疫球蛋白形成抗体-抗原复合物。
DOI：
10.1016/0003-2697(92)90577-t
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发表时间：
1992
影响因子：
2.9
通讯作者：
Paek,SH
中科院分区：
文献类型：
--
作者：
Schramm,W;Paek,SH
我们研究了固定化单克隆抗体和大小相差约50倍的抗原之间的复合物形成。作为一个模型系统，我们使用碘化孕酮衍生物和胆甾酮-辣根过氧化物酶结合物作为示踪剂和单克隆抗体作为结合蛋白。通过四种不同的方法固定抗体：物理吸附、化学结合和在不存在或存在保护性蛋白（明胶）的情况下通过蛋白G结合。这些研究表明，竞争性免疫测定的性能由以下因素的组合决定：（a）分析物和示踪剂的相对大小，（B）固体基质上的抗体密度，（c）抗体的固定方法，和（d）抗体-分析物和抗体-示踪剂之间的结合常数。在设计最佳免疫测定时必须考虑所有这些相互作用。较小的抗原可以形成比较大抗原高3至35倍的最大复合物密度。剂量-反应曲线受示踪剂大小的影响小于受与抗体的结合常数的影响。因此，具有相对低的结合常数的大酶示踪剂可以提供更灵敏的测定。另一方面，用较小的示踪剂实现的复合物密度的增加产生更高的信号，这反过来可以在高度灵敏的竞争性固相免疫测定中提供更好的信噪比。我们已经提出了一个模型抗体固定，占示踪剂的大小，复合物的形成，和抗体密度的相互依赖性。所述方法可用于设计和优化预定义性能特征的免疫测定。这些结果对于将放射免疫测定法转换为酶免疫测定法特别有用。
We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.
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