SpG亲和肽靶向抗体Fc位点的共价偶联标记研究及临床免疫学检验应用

批准号:
81971998
项目类别:
面上项目
资助金额:
55.0 万元
负责人:
杨洪鸣
依托单位:
学科分类:
免疫学检验
结题年份:
2023
批准年份:
2019
项目状态:
已结题
项目参与者:
杨洪鸣
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中文摘要
特异Fc位点标记/固定抗体能有效提高标记免疫分析的特异性、稳定性与灵敏度。前期在NSFC资助下,课题组利用ZZ亲和肽的生物特性介导实现了抗体Fc位点的亲和标记与固定,然而这种生物亲和作用易受血清内源IgG干扰难以应用于临床免疫检验。将亲和肽与抗体的可逆结合转变为共价偶联有望突破其临床应用瓶颈。在预实验基础上,本研究拟以SpG亲和肽与Avitag、AP构建重组蛋白,先以SpG靶向抗体Fc位点形成抗体-SpG生物亲和大分子,再利用双功能交联剂对抗体-SpG亲和分子内共价交联,介导标记物对抗体Fc位点的共价偶联标记。项目通过SPR、Pull-down及SEC-HPLC等技术阐明SpG对IgG亲和力、分子内交联的关联机制,建立精准Fc位点共价偶联的抗体标记技术,在此基础上构筑一种新型标记免疫分析体系;项目以检测CEA为例综合评价应用效果与性价比,以期为提高临床免疫学检验的检测效能提供新方法与思路。
英文摘要
The labeled immunoassay plays an important role in clinical immunodiagnosis, and the Fc-specific conjugation of antibody can enhances the sensitivity, specificity and stability of the labeled immunoassay. Previously funded by the national natural science foundation of China (NSFC), the oriented immobilization and Fc-specific via the affinity-interaction for antibody were established by employing the biological characteristics of the ZZ affinity peptide. And their detection performances were significantly higher than that of random chemical covalent conjugation methodologies in immunoassays. However, a critical limitation of its clinical application is that the bio-affinity interaction is reversible, the intrinsic IgGs in serum can competitive bind to IgG affinity peptide which seriously disturbing the accuracy and the reliability of clinical immunoassays. If the reversible bio-affinity interaction be converts into covalent attachment, the bottleneck in clinical immunodiagnosis application could be solved. On the basis of the preliminary experimental results, a new strategy for Fc-specific covalent conjugation of IgG will be proposed in this project: by employing the biological characteristics of SpG, the SpG moiety is firstly fixated on the Fc-site of IgG via IgG-SpG bioaffinity macromolecule, then the marker molecules (enzyme and biotin) is covalently cross-linked to the IgG under the intramolecularly cross-linking covalent conjugation using cross-linking covalent agent. In this study, we will take advantage of SPR, Pull-down and SEC-HPLC techniques to elucidate the mechanism between the affinity constants of SpG to IgGs and the intramolecular covalent cross-linking, and then the covalent cross-linking technology for Fc-specific IgGs will be further established. Based on the Fc-specific IgG covalent conjugation, a novel labeled immunoassay will be developed, and then we take CEA detection as an example to evaluate the application effect and cost-effectiveness of this new labeled immunoassay. This Fc-specific IgG covalent conjugation strategy will provides a powerful approach to further improve the specificity, the stability and the sensitivity of the clinical immunoassays.
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DOI:10.3969/j.issn.1000-484X.2021.13.014
发表时间:2021
期刊:中国免疫学杂志
影响因子:--
作者:于晓甜;张晓坤;高晓艺;杨洪鸣;唐金宝
通讯作者:唐金宝
DOI:10.1016/j.foodcont.2021.108525
发表时间:2022
期刊:Food Control
影响因子:6
作者:Xiao-Yi Gao;Chong-Mei Xu;Xiao-Kun Zhang;Meng-Ran Li;Xiao Gong;Hong-ming Yang;Jin-Bao Tang
通讯作者:Xiao-Yi Gao;Chong-Mei Xu;Xiao-Kun Zhang;Meng-Ran Li;Xiao Gong;Hong-ming Yang;Jin-Bao Tang
DOI:10.1016/j.micromeso.2020.110627
发表时间:2020-11
期刊:Microporous and Mesoporous Materials
影响因子:5.2
作者:Meng Zhang;Fang Yan;Jingkun Bai;Xiaotong Li;Xiao-tong Zheng;Jinli Dou;Xuedong Wang;Weifen Zhang;Baolong Zhou
通讯作者:Meng Zhang;Fang Yan;Jingkun Bai;Xiaotong Li;Xiao-tong Zheng;Jinli Dou;Xuedong Wang;Weifen Zhang;Baolong Zhou
DOI:10.1039/d1nr04659d
发表时间:2021
期刊:Nanoscale
影响因子:6.7
作者:Xiao-Kun Zhang;Hong-Ming Yang;Meng-Ran Li;Xiao-Yi Gao;Xiao-Wei Sun;Xi-Feng Sun;Jin-Bao Tang
通讯作者:Jin-Bao Tang
DOI:10.1016/j.aca.2021.339054
发表时间:2021-11
期刊:Analytica chimica acta
影响因子:6.2
作者:Jin-Bao Tang;Hong-ming Yang;Xiao-Yi Gao;Xianghua Zeng;Feng-Shan Wang
通讯作者:Jin-Bao Tang;Hong-ming Yang;Xiao-Yi Gao;Xianghua Zeng;Feng-Shan Wang
国内基金
海外基金
