State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Zhejiang-Malaysia Joint Research Laboratory for Agricultural Product Processing and Nutrition and College of Food Science and Engineering, Ningbo University, Ningbo 315800, China.
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Zhejiang-Malaysia Joint Research Laboratory for Agricultural Product Processing and Nutrition and College of Food Science and Engineering, Ningbo University, Ningbo 315800, China.
Enzyme Microb Technol. 2024 Dec;181:110507. doi: 10.1016/j.enzmictec.2024.110507. Epub 2024 Sep 4.
An immunoassay method based on penicillin-binding protein (PBP) was developed for the quantitative determination of 10 kinds of beta-lactam antibiotics (BLAs). First, two kinds of PBPs, which are named PBP1a and PBP2x, were expressed and purified, and they were characterized by SDS-PAGE and western blotting analysis. Then, the binding activity of PBP1a and PBP2x to template BLAs, cefquinome (CEFQ) and ampicillin (AMP), was determined. The effect of the buffer solution system, e.g., pH, ion concentration, and organic solvent, on the immune interaction efficiency between PBPs and BLAs was also evaluated. In the end, the PBP-based immunoassay method was developed and validated for the detection of 10 kinds of BLAs. Under optimal conditions, PBPs exhibited high binding affinity to BLAs. In addition, this method showed a high sensitivity for the detection of 10 kinds of BLAs with the limits of detection from 0.21 to 9.12 ng/mL, which are much lower than their corresponding maximum residual limit of European Union (4-100 ng/mL). Moreover, the developed PBP-immunoassay was employed for BLA detection from milk samples, and satisfactory recoveries (68.9-101.3 %) were obtained.
建立了一种基于青霉素结合蛋白（PBP）的免疫分析方法，用于定量测定 10 种β-内酰胺类抗生素（BLAs）。首先，表达并纯化了两种 PBP，分别命名为 PBP1a 和 PBP2x，并通过 SDS-PAGE 和 Western blot 分析进行了表征。然后，测定了 PBP1a 和 PBP2x 与模板 BLAs（头孢喹肟（CEFQ）和氨苄西林（AMP））的结合活性。还评估了缓冲溶液体系（例如 pH 值、离子浓度和有机溶剂）对 PBP 与 BLAs 之间免疫相互作用效率的影响。最后，开发并验证了基于 PBP 的免疫分析方法用于检测 10 种 BLAs。在最佳条件下，PBP 对 BLAs 表现出高结合亲和力。此外，该方法对 10 种 BLAs 的检测具有很高的灵敏度，检出限为 0.21-9.12ng/mL，远低于欧盟的最大残留限量（4-100ng/mL）。此外，该方法还用于检测牛奶样品中的 BLA，获得了令人满意的回收率（68.9-101.3%）。
