Establishment and application of multiple immunoassays for environmental estrogens based on recombinant Japanese flounder (Paralichthys olivaceus) choriogenin protein
Establishment and application of multiple immunoassays for environmental estrogens based on recombinant Japanese flounder (Paralichthys olivaceus) choriogenin protein
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DOI：
10.1016/j.talanta.2022.124135
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发表时间：
2022-12-02
期刊：
影响因子：
6.1
通讯作者：
Wang，Jun
中科院分区：
文献类型：
--
作者：
Li，Yuejiao;Zhang，Yabin;Wang，Jun
环境雌激素因其对水生生物的潜在威胁而受到广泛关注，但目前常用的卵黄蛋白原（Vtg）生物标志物检测方法不能检测10 ng/L以下17β-雌二醇的雌激素活性。本研究首次克隆了牙鲆Chg基因（ChgLandChgH），并利用原核表达系统制备了分子量约为52 kDa的ChgL重组蛋白，利用Ni亲和层析柱纯化ChgL蛋白，建立了以Chg为抗原的多种免疫检测方法。随后，针对ChgL的特异性单克隆抗体的制备和用于开发夹心酶联免疫吸附测定（ELISA），其具有检测范围为3.9-250 ng/mL和检测限为1.9 ng/mL。还建立了免疫荧光方法，并用于目视检测组织中的ChgL诱导。此外，侧流免疫测定ChgL，可以在10分钟内检测雌激素活性的发展。最后，通过测定暴露于0、2、10和50 ng/L 17α-乙炔基甜菜碱（EE 2）的牙鲆血浆和组织中ChgL的诱导，检验了免疫测定法的可靠性。结果表明，2 ng/LEE 2显著增加血浆中ChgL水平，表明ChgL对环境雌激素的敏感性高于Vtg; 50 ng/LEE 2诱导肝窦血管中Chg的明显诱导。总之，本研究提供了可靠的方法，敏感和快速检测的雌激素活性的水环境。
Environmental estrogens have generated great concern because of their potential threat to aquatic organisms; however, the commonly used vitellogenin (Vtg) biomarker detection methods are not capable of detecting estrogenic activity below 10 ng/L 17β-estradiol. In this study, we developed multiple immunoassays based on Japanese flounder (Paralichthys olivaceus) choriogenin (Chg), a highly sensitive biomarker of environmental estrogens.Chggenes (ChgLandChgH) of Japanese flounder were cloned for the first time, and a recombinant ChgL protein with a molecular weight of approximately 52 kDa was prepared using a prokaryotic expression system and purified using Ni-affinity column chromatography. Subsequently, specific monoclonal antibodies against ChgL were prepared and used to develop sandwich enzyme-linked immunosorbent assays (ELISAs), which had a detection range of 3.9–250 ng/mL and detection limit of 1.9 ng/mL. An immunofluorescence method was also established and used to visually detect ChgL induction in the tissues. In addition, a lateral flow immunoassay for ChgL that could detect estrogen activity within 10 min was developed. Finally, the reliability of the immunoassays was examined by measuring ChgL induction in the plasma and tissues of Japanese flounder exposed to 0, 2, 10, and 50 ng/L 17α-ethinylestradiol (EE2). The results showed that 2 ng/L EE2notably increased ChgL levels in the plasma, demonstrating that ChgL is more sensitive than Vtg to environmental estrogens; 50 ng/L EE2induced obvious Chg induction in the sinusoidal vessels of the liver. Conclusions taken together, this study provides reliable methods for sensitive and rapid detection of estrogenic activity in aquatic environments.