基于重组抗体的免疫测定已成为检测食品样品中抗生素残留的关键技术。开发稳定的重组抗体生产系统和提高检测灵敏度对于其生物传感应用至关重要。这里,我们使用枯草芽孢杆菌和HEK293系统将单链片段可变（scFv）抗体生物工程用于靶向氯霉素（CAP），HEK293衍生的scFv表现出优异的敏感性。计算化学分析表明scFv中的ASP-99和ASN-102残基在抗体识别中起关键作用,目标分子苯环附近的羟基对于抗体结合至关重要。此外，我们在基于链霉亲和素的免疫测定中使用HCR-CRISPR/Cas12a扩增策略增强了scFv的生物传感敏感性。在双步扩增过程中，CAP在HCR和HCR-CRISPR/Cas12a阶段的检测限显着降低至55.23pg/mL和3.31pg/mL，分别。这些发现引入了用于开发CAP特异性scFv抗体的有效方法,并且还提出了增加免疫测定灵敏度的多扩增策略。此外,理论研究也为CAP半抗原设计和基因工程抗体修饰提供了有价值的指导。
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.
