Department of Biochemistry, University of Turku, Kiinanmyllynkatu 10, 20520, Turku, Finland.
Labmaster Oy, Fiskarsinkatu 11, 20750, Turku, Finland.
Anal Bioanal Chem. 2019 Sep;411(22):5633-5639. doi: 10.1007/s00216-019-01952-6. Epub 2019 Jun 8.
Detection of circulatory estradiol has widespread use in various clinical applications. Particularly, the use of estradiol-specific antibodies in immunoassays is routinely used, mainly due to the cost efficiency and simplicity of the sample handling process. However, the circulatory levels of estradiol can be extremely low in some conditions, and beyond the current detection limit of existing competitive immunoassays. We describe the generation of anti-immunocomplex specific antibodies derived from synthetic antibody repertoire and the development of high-performance non-competitive immunoassay for the detection of estradiol. Phage display selections were used to isolate new antibodies from synthetic antibody library with the use of existing estradiol specific Fab fragment. The found antibodies were consecutively used to set up a time-resolved fluorescence-based immunoassay (TRFIA), which can be used to detect estradiol with exceptional sensitivity and specificity. The limit of detection and EC50 were shown to be 3.0 pg mL and 32.4 pg mL respectively. Graphical abstract.
循环雌二醇的检测在各种临床应用中得到了广泛的应用。特别是，由于样本处理过程的成本效益和简单性，雌二醇特异性抗体在免疫测定中的应用已常规化。然而，在某些情况下，循环中的雌二醇水平可能极低，超出了现有竞争性免疫测定的检测极限。我们描述了从合成抗体库中产生抗免疫复合物特异性抗体的方法，以及开发用于检测雌二醇的高性能非竞争性免疫测定法。噬菌体展示选择用于从使用现有雌二醇特异性 Fab 片段的合成抗体库中分离新抗体。所发现的抗体被连续用于建立基于时间分辨荧光的免疫测定法（TRFIA），该方法可用于检测雌二醇，具有出色的灵敏度和特异性。检测限和 EC50 分别为 3.0 pg/mL 和 32.4 pg/mL。
