Hang Ao, Wencheng Xiao, Wenrui Hu, Jie Wu, Huangxian Ju
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After the KHL peptide was recognized by the target antibody, the strand replacement hybridization could be inhibited by the bound antibody, which retained the high catalytic activity of hemin overhung on the antibody-bound affinity probe for a CL reaction, leading to a \"signal-on\" process for CL antibody detection. Using a KHL-specific antibody, anti-proprotein convertase subtilisin/kexin type 9 antibody (PCSK9-Ab), as a target model and common L012-1,2,4-triazole-H<sub>2</sub>O<sub>2</sub> CL system, the designed switch showed a detection range of 10 ng mL<sup>-1</sup> to 1 μg mL<sup>-1</sup> with a detection limit of 4.16 ng mL<sup>-1</sup> (56.2 pM) and a short analytical time of 6.5 min. The proposed quick method could simply be used for lab-on-chip CL detection of PCSK9-Ab in situ-secreted from PCSK9-6E3 hybridoma cells, which showed an accuracy of 90.2% compared with the statistical results from general fluorescence imaging, providing a potential technique for screening specific hybridoma cells.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7000,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"DNA Conformation-Regulated Hemin Switch for Lab-on-Chip Chemiluminescent Detection of an Antibody Secreted from Hybridoma Cells.\",\"authors\":\"Hang Ao, Wencheng Xiao, Wenrui Hu, Jie Wu, Huangxian Ju\",\"doi\":\"10.1021/acs.analchem.4c04122\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>This work designed a DNA conformation-regulated hemin switch for rapid chemiluminescent (CL) detection of a monoclonal antibodies. This switch was performed with an affinity probe and an inhibition probe, which were conveniently prepared by hybridizing hemin-labeled DNA1 with KHL peptide-labeled DNA2 and binding biotin-labeled DNA3 to streptavidin, respectively. In the absence of the target antibody, streptavidin-DNA3 could hybridize with hemin-DNA1/KHL-DNA2 to release KHL-DNA2, which led to the loss of hemin activity due to the affinity hindrance of streptavidin-DNA3. After the KHL peptide was recognized by the target antibody, the strand replacement hybridization could be inhibited by the bound antibody, which retained the high catalytic activity of hemin overhung on the antibody-bound affinity probe for a CL reaction, leading to a \\\"signal-on\\\" process for CL antibody detection. 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引用次数: 0
摘要
这项研究设计了一种 DNA 构象调控hemin开关,用于快速化学发光(CL)检测单克隆抗体。亲和探针和抑制探针分别通过将hemin标记的DNA1与KHL肽标记的DNA2杂交,以及将生物素标记的DNA3与链霉亲和素结合而制备。在没有目标抗体的情况下,链霉亲和素-DNA3可以与hemin-DNA1/KHL-DNA2杂交,释放出KHL-DNA2,由于链霉亲和素-DNA3的亲和性阻碍,导致hemin活性丧失。当 KHL 肽被目标抗体识别后,结合的抗体可以抑制链置换杂交,从而保留了悬浮在抗体结合的亲和探针上的 hemin 的高催化活性,以进行 CL 反应,从而实现了 CL 抗体检测的 "信号开启 "过程。该方法以KHL特异性抗体--抗前列腺素转化酶亚基酶/前列腺素9型抗体(PCSK9-Ab)为靶标模型,采用常见的L012-1,2,4-三唑-H2O2 CL体系,检测范围为10 ng mL-1 至1 μg mL-1,检测限为4.16 ng mL-1 (56.2 pM),分析时间短,仅需6.5 min。所提出的快速方法可简单地用于 PCSK9-6E3 杂交瘤细胞原位分泌 PCSK9-Ab 的实验室芯片 CL 检测,与一般荧光成像的统计结果相比,准确率达 90.2%,为筛选特定的杂交瘤细胞提供了一种潜在的技术。
DNA Conformation-Regulated Hemin Switch for Lab-on-Chip Chemiluminescent Detection of an Antibody Secreted from Hybridoma Cells.
This work designed a DNA conformation-regulated hemin switch for rapid chemiluminescent (CL) detection of a monoclonal antibodies. This switch was performed with an affinity probe and an inhibition probe, which were conveniently prepared by hybridizing hemin-labeled DNA1 with KHL peptide-labeled DNA2 and binding biotin-labeled DNA3 to streptavidin, respectively. In the absence of the target antibody, streptavidin-DNA3 could hybridize with hemin-DNA1/KHL-DNA2 to release KHL-DNA2, which led to the loss of hemin activity due to the affinity hindrance of streptavidin-DNA3. After the KHL peptide was recognized by the target antibody, the strand replacement hybridization could be inhibited by the bound antibody, which retained the high catalytic activity of hemin overhung on the antibody-bound affinity probe for a CL reaction, leading to a "signal-on" process for CL antibody detection. Using a KHL-specific antibody, anti-proprotein convertase subtilisin/kexin type 9 antibody (PCSK9-Ab), as a target model and common L012-1,2,4-triazole-H2O2 CL system, the designed switch showed a detection range of 10 ng mL-1 to 1 μg mL-1 with a detection limit of 4.16 ng mL-1 (56.2 pM) and a short analytical time of 6.5 min. The proposed quick method could simply be used for lab-on-chip CL detection of PCSK9-Ab in situ-secreted from PCSK9-6E3 hybridoma cells, which showed an accuracy of 90.2% compared with the statistical results from general fluorescence imaging, providing a potential technique for screening specific hybridoma cells.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.