Penicillin binding proteins-based immunoassay for the selective and quantitative determination of beta-lactam antibiotics

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Enzyme and Microbial Technology Pub Date : 2024-09-04 DOI:10.1016/j.enzmictec.2024.110507

Rilong Liu , Hangzhen Lan , Song Yan , Lu Huang , Daodong Pan , Yichun Wu

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Abstract

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.

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基于青霉素结合蛋白的免疫测定法，用于选择性定量测定β-内酰胺类抗生素。

建立了一种基于青霉素结合蛋白（PBP）的免疫测定方法，用于定量检测10种β-内酰胺类抗生素（BLAs）。首先，表达并纯化了两种 PBPs，分别命名为 PBP1a 和 PBP2x。然后，测定了 PBP1a 和 PBP2x 与模板 BLAs（头孢喹诺（CEFQ）和氨苄青霉素（AMP））的结合活性。此外，还评估了缓冲溶液体系（如 pH 值、离子浓度和有机溶剂）对 PBPs 和 BLAs 免疫相互作用效率的影响。最后，建立了基于 PBP 的免疫测定方法，并对 10 种 BLAs 的检测进行了验证。在最佳条件下，PBPs 与 BLAs 具有很高的结合亲和力。此外，该方法对10种BLAs的检测灵敏度高，检出限为0.21-9.12 ng/mL，远低于欧盟规定的相应最大残留限量（4-100 ng/mL）。此外，所开发的 PBP 免疫分析法还被用于牛奶样品中 BLA 的检测，并获得了令人满意的回收率（68.9%-101.3%）。

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.