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免疫原设计:双功能螯合剂对螯合铜免疫反应的影响。

Design of immunogens: The effect of bifunctional chelator on immunological response to chelated copper.

机构信息

School of Public Health, Nantong University, No.9 Seyuan Road Nantong, Jiangsu, 226019, China.

School of Public Health, Nantong University, No.9 Seyuan Road Nantong, Jiangsu, 226019, China.

出版信息

J Pharm Biomed Anal. 2019 Sep 10;174:263-269. doi: 10.1016/j.jpba.2019.06.001. Epub 2019 Jun 3.

Abstract

To produce specific antibodies for the detection and quantification of copper ions, bifunctional chelators (BFCs) are commonly applied in the preparation of copper conjugates. However, some copper-chelator complexes exhibit limited stability under in vivo conditions. In this study, Cu was coupled with carrier proteins via three different macrocyclic BFCs: p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, and p-SCN-Bn-TETA. The stability in plasma and the immunogenicity of three copper immunoconjugates were compared. The chelators other than p-SCN-Bn-DOTA were very stable in plasma, with <9% dissociation of Cu over 96 h. The immune response varied depending on the choice of chelator; notably, antisera from the Cu-NOTA-KLH conjugate demonstrated the best reactivity toward chelated Cu. p-SCN-Bn-NOTA, which showed significant advantages over the other chelators, was used for antibody production. The efficiency of immune-positive hybridoma production was satisfactory, and the resultant monoclonal antibodies (McAbs) 4B7 showed sensitivity (half-maximal inhibitory concentration (IC) of 8.9 ng/mL) to chelated Cu, with a working range from 1.21 to 48.9 ng/mL. The recovery of Cu from water samples was 85.7-108%, and the intra- and inter-assay coefficients of variation were 4.0-10.1% and 7.1-11.4%, respectively. Compared with previously reported McAb specific to Cu, DF4, the sensitivity of the newly developed assay was improved 100-fold. The results of this study indicate the utility of NOTA for the efficient generation of highly sensitive McAbs against Cu.

摘要

为了制备用于检测和定量铜离子的特异性抗体,通常在制备铜缀合物时应用双功能螯合剂(BFCs)。然而,一些铜-螯合剂复合物在体内条件下表现出有限的稳定性。在这项研究中,通过三种不同的大环 BFC(p-SCN-Bn-DOTA、p-SCN-Bn-NOTA 和 p-SCN-Bn-TETA)将 Cu 与载体蛋白偶联。比较了三种铜免疫缀合物在血浆中的稳定性和免疫原性。除了 p-SCN-Bn-DOTA 以外的螯合剂在血浆中非常稳定,在 96 小时内 Cu 的解离率<9%。免疫反应取决于螯合剂的选择;值得注意的是,Cu-NOTA-KLH 缀合物的抗血清表现出对螯合 Cu 的最佳反应性。p-SCN-Bn-NOTA 比其他螯合剂具有显著优势,因此用于抗体生产。免疫阳性杂交瘤的产生效率令人满意,所得单克隆抗体(McAb)4B7 对螯合 Cu 具有敏感性(半抑制浓度(IC)为 8.9ng/mL),工作范围为 1.21-48.9ng/mL。从水样中回收 Cu 的回收率为 85.7-108%,批内和批间变异系数分别为 4.0-10.1%和 7.1-11.4%。与之前报道的针对 Cu 的特异性 McAb DF4 相比,新开发的测定方法的灵敏度提高了 100 倍。本研究结果表明,NOTA 可有效用于生成针对 Cu 的高灵敏度 McAb。

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