Chang Liu,Hong Lin,Limin Cao,Kaiqiang Wang,Jianxin Sui
The third generation of genetic engineering antibodies, single-domain antibodies, have been widely reported as potential biomaterials in recognizing small molecular hazards. In this study, a shark-derived single-domain antibody was used as the recognition element for the first time to detect enrofloxacin (ENR), one of the most representative hazards in aquaculture. An ENR-specific clone named 2E6 was isolated by phage display technology. Experimental results proved that 2E6 ssdAb showed high affinity to ENR-PEI complete antigen, with the highest OD 450 value of 1.348 in binding ELISA . Through icELISA, it was determined that the IC 50 of 2E6 ssdAb to ENR was 19.230 ng/mL, while the IC 10 was 0.975 ng/mL, with rare recognition to other fluoroquinolones, which showed high sensitivity and specificity to ENR. The 2E6 ssdAb also performed excellently in fish matrix immunoassay . Results showed that the ENR-negative fish matrix did not seriously interfere with the recognition of 2E6 ssdAb to ENR-OVA, with the matrix index between 4.85% and 11.75%, while the results of icELISA in ENR-spiked fish matrix showed that 2E6 ssdAb could recognize the target ENR in different ENR-spiked concentrations of the fish matrix (10–1000 ng/mL), with the recovery between 89.30% and 126.38% and the RSD between 1.95% and 9.83%. This study broadens the application scenario of shark-derived single-domain antibodies as small molecule recognition biomaterials, providing a new recognition element on ENR detection for immunoassay.
Chang Liu,Yuan Chen,Hong Lin,Limin Cao,Kaiqiang Wang,Xiudan Wang,Jianxin Sui
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 (IC 10 ) 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. Significance 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.
危险品化学品经营许可证（带存储）