Abstract：
BACKGROUND: Due to the serious issue of ofloxacin (OFL) abuse, there is an increasingly urgent need for accurate and rapid detection of OFL. Immunoassay has become the \"golden method\" for detecting OFL in complex matrix beneficial to its applicability for a large-scale screening, rapidity, and simplicity. However, traditional antibodies used in immunoassay present challenges such as time-consuming preparation, unstable sensitivity and specificity, and difficulty in directional evolution. In this paper, we successfully developed an OFL detection method based on a shark-derived single-domain antibody (ssdAb) to address these issues.
RESULTS: Using phage display technology and a heterologous expression system, OFL-specific clones 1O11, 1O13, 1O17, 1O19, 1O21, and 2O26 were successfully isolated and expressed in soluble form. Among all OFL-specific ssdAbs, the 1O17 ssdAb exhibited the highest binding affinity to OFL in a concentration-dependence manner. The limit of detection (IC10) of 1O17 ssdAb was calculated as 0.34 ng/mL with a detection range of 3.40-1315.00 ng/mL, and its cross reactivity with other analogs was calculated to be less than 5.98 %, indicating high specificity and sensitivity. Molecular docking results revealed that 100Trp and 101Arg located in the CDR3 region of 1O17 ssdAb were crucial for OFL binding. In fish matrix performance tests, the 1O17 ssdAb did not demonstrate severe matrix interference in OFL-negative fish matrix, achieving satisfactory recovery rates ranging from 83.04 % to 108.82 % with high reproducibility.
CONCLUSIONS: This research provides a new and efficient OFL detection recognition element with significant potential in immunoassay applications, broadening the application scenarios of ssdAbs. It offers valuable insights into the structure-activity relationship between ssdAbs and small molecules, laying a theoretical foundation for the further directional modification and maturation of ssdAbs in subsequent applications.
RESULTS: Using phage display technology and a heterologous expression system, OFL-specific clones 1O11, 1O13, 1O17, 1O19, 1O21, and 2O26 were successfully isolated and expressed in soluble form. Among all OFL-specific ssdAbs, the 1O17 ssdAb exhibited the highest binding affinity to OFL in a concentration-dependence manner. The limit of detection (IC10) of 1O17 ssdAb was calculated as 0.34 ng/mL with a detection range of 3.40-1315.00 ng/mL, and its cross reactivity with other analogs was calculated to be less than 5.98 %, indicating high specificity and sensitivity. Molecular docking results revealed that 100Trp and 101Arg located in the CDR3 region of 1O17 ssdAb were crucial for OFL binding. In fish matrix performance tests, the 1O17 ssdAb did not demonstrate severe matrix interference in OFL-negative fish matrix, achieving satisfactory recovery rates ranging from 83.04 % to 108.82 % with high reproducibility.
CONCLUSIONS: This research provides a new and efficient OFL detection recognition element with significant potential in immunoassay applications, broadening the application scenarios of ssdAbs. It offers valuable insights into the structure-activity relationship between ssdAbs and small molecules, laying a theoretical foundation for the further directional modification and maturation of ssdAbs in subsequent applications.
摘要：
背景：由于氧氟沙星（OFL）滥用的严重问题，对OFL的准确、快速检测的需求日益迫切。免疫测定已成为在复杂基质中检测OFL的“黄金方法”，有利于其适用于大规模筛查，快速性,和简单。然而,免疫测定中使用的传统抗体面临挑战，如耗时的制备，不稳定的灵敏度和特异性，和方向性进化的困难。在本文中,为了解决这些问题,我们成功开发了一种基于鲨鱼源单域抗体(ssdAb)的OFL检测方法.
结果：使用噬菌体展示技术和异源表达系统，OFL特异性克隆1O11、1O13、1O17、1O19、1O21和2O26被成功地分离并以可溶性形式表达。在所有OFL特定的ssdAb中，1O17ssdAb以浓度依赖性方式表现出对OFL的最高结合亲和力。1O17ssdAb的检测限（IC10）计算为0.34ng/mL，检测范围为3.40-1315.00ng/mL，其与其他类似物的交叉反应性经计算小于5.98%，表明高特异性和敏感性。分子对接结果表明，位于1O17ssdAbCDR3区域的100Trp和101Arg对于OFL结合至关重要。在鱼类基质性能测试中，1O17ssdAb在OFL阴性鱼类基质中没有表现出严重的基质干扰，达到令人满意的回收率范围从83.04%到108.82%，重现性高。
结论：这项研究提供了一种新型高效的OFL检测识别元件，在免疫测定应用中具有显着的潜力，拓宽ssdAbs的应用场景。它为ssdAb和小分子之间的结构-活性关系提供了有价值的见解，为ssdAb在后续应用中的进一步定向修饰和成熟奠定理论基础。
结果：使用噬菌体展示技术和异源表达系统，OFL特异性克隆1O11、1O13、1O17、1O19、1O21和2O26被成功地分离并以可溶性形式表达。在所有OFL特定的ssdAb中，1O17ssdAb以浓度依赖性方式表现出对OFL的最高结合亲和力。1O17ssdAb的检测限（IC10）计算为0.34ng/mL，检测范围为3.40-1315.00ng/mL，其与其他类似物的交叉反应性经计算小于5.98%，表明高特异性和敏感性。分子对接结果表明，位于1O17ssdAbCDR3区域的100Trp和101Arg对于OFL结合至关重要。在鱼类基质性能测试中，1O17ssdAb在OFL阴性鱼类基质中没有表现出严重的基质干扰，达到令人满意的回收率范围从83.04%到108.82%，重现性高。
结论：这项研究提供了一种新型高效的OFL检测识别元件，在免疫测定应用中具有显着的潜力，拓宽ssdAbs的应用场景。它为ssdAb和小分子之间的结构-活性关系提供了有价值的见解，为ssdAb在后续应用中的进一步定向修饰和成熟奠定理论基础。
