摘要
免疫分析技术在食品安全快速检测领域发挥着重要作用,其中抗体是免疫分析的核心元件。然而,目前食品安全领域所使用的抗体主要基于多克隆抗体或通过杂交瘤细胞体内培养而获得的单克隆抗体,不仅涉及动物福利及病毒污染问题,还可能面临非靶向抗体污染和细胞抗体基因丢失等挑战。以食品中常见的真菌毒素玉米赤霉烯酮(zearalenone,ZEN)为研究对象,基于作者所在实验室自制的抗ZEN单克隆杂交瘤细胞株(Z14),采用亚型特异性引物分别扩增抗ZEN抗体的重、轻链全长基因序列,构建了ZEN重组全长抗体的表达载体,并使用PEI介导实现了ZEN重组全长抗体在Expi 293F细胞内的瞬时转染表达。间接竞争酶联免疫吸附检测(indirect competitive enzyme-linked immunosorbent assay,IC-ELISA)结果显示,基于重组全长抗体建立的ZEN抑制标准曲线,其半数抑制质量浓度(IC_(50))为2.43 ng/mL,线性范围(IC_(20)~IC_(80))的质量浓度为0.66~8.88 ng/mL,亲和常数Ka为3.87×10^(10)L/moL,性能均优于传统腹水制备的ZEN单克隆抗体,为未来采用体外细胞培养技术大规模生产抗ZEN的重组全长抗体奠定了基础。
Immunoassay technology plays an important role in the field of rapid detection of food safety,with antibodies being the core component of immunoassay.However,the current detection of antibodies used in foods is mainly based on polyclonal antibodies or monoclonal antibodies obtained in large quantities through the in vivo cultivation of hybridoma cells,which not only face animal welfare and viral contamination,but also face problems,such as non-targeted antibody contamination and loss of cellular antibody genes.This study focused on the common fungal toxin zearalenone(ZEN)in foods.Based on the self-made anti-ZEN monoclonal hybridoma cell line(Z14)in our laboratory,sub-type specific primers were used to amplify the full-length gene sequences of the heavy and light chains of the anti-ZEN antibody.The expression vector of the ZEN recombinant full-length antibody was constructed and the PEI mediated transient transfection expression of the ZEN recombinant fulllength antibody in Expi 293F cells was achieved.The results of the indirect competitive enzyme-linked immunosorbent assay(IC-ELISA)showed that the half inhibitory mass concentration(IC_(50))of the ZEN inhibition standard curve established was based on recombinant full-length antibodies,which was 2.43 ng/mL.Moreover,the mass concentration in the linear range(IC_(20)~IC_(80))was 0.66~8.88 ng/mL,while the affinity constant(Ka)was 3.87×10^(10) L/mol.The performance of the ZEN monoclonal antibodies prepared from ascites was superior to that of traditional ascites,providing a basis for a large-scale production of recombinant full-length antibodies against ZEN based on in vitro cell culture in the later stage.
作者
王何瑶
梁赟
钟于红
郭原臻
黄斌辉
何庆华
WANG Heyao;LIANG Yun;ZHONG Yuhong;GUO Yuanzhen;HUANG Binhui;HE Qinghua(College of Food Science&Technology,Nanchang University,Nanchang 330000,China;State Key Laboratory of Food Science and Resources,Nanchang University,Nanchang 330000,China;Sino German Joint Research Institute,Nanchang University,Nanchang 330000,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2024年第7期67-76,共10页
Journal of Food Science and Biotechnology
基金
江西省“双千计划”人才培养项目(JXSQ2023201029)
江西省自然科学基金重点项目(20232ACB205027)。
关键词
玉米赤霉烯酮
重组抗体
真核表达
zearalenone
recombinant antibodies
eukaryotic expression
