Preparation and recognition mechanism study of an scFv targeting chloramphenicol for a hybridization chain reaction-CRISPR/Cas12a amplified fluoroimmunoassay

被引:5
作者
Zhang, Fuyuan [1 ]
Hao, Dongyue [1 ]
Liu, Ruobing [1 ]
Wang, Juntao [1 ]
Sang, Yaxin [1 ]
Wang, Shuo [2 ]
Wang, Xianghong [1 ]
机构
[1] Hebei Agr Univ, Coll Food Sci & Technol, Baoding 071001, Peoples R China
[2] Nankai Univ, Med Coll, Tianjin 300500, Peoples R China
关键词
Recombinant antibody; Recognition mechanism; Hybridization chain reaction; CRISPR/Cas12a; Immunoassay; ANTIBODY;
D O I
10.1016/j.aca.2024.342283
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Recombinant antibody-based immunoassays have emerged as crucial techniques for detecting antibiotic residues in food samples. Developing a stable recombinant antibody production system and enhancing detection sensitivity are crucial for their biosensing applications. Here, we bioengineered a single-chain fragment variable (scFv) antibody to target chloramphenicol (CAP) using both Bacillus subtilis and HEK 293 systems, with the HEK 293-derived scFv demonstrating superior sensitivity. Computational chemistry analyses indicated that ASP -99 and ASN-102 residues in the scFv play key roles in antibody recognition, and the hydroxyl group near the benzene ring of the target molecule is critical for in antibody binding. Furthermore, we enhanced the scFv's biosensing sensitivity using an HCR-CRISPR/Cas12a amplification strategy in a streptavidin-based immunoassay. In the dual-step amplification process, detection limits for CAP in the HCR and HCR-CRISPR/Cas12a stages were significantly reduced to 55.23 pg/mL and 3.31 pg/mL, respectively. These findings introduce an effective method for developing CAP -specific scFv antibodies and also propose a multi -amplification strategy to increase immunoassay sensitivity. Additionally, theoretical studies also offer valuable guidance in CAP hapten design and genetic engineering for antibody modification.
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页数:10
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