Chang Liu,Yuan Chen,Hong Lin,Limin Cao,Kaiqiang Wang,Xiudan Wang,Jianxin Sui
ABSTRACT Low specificity and sensitivity often restrict the fluoroquinolones (FQs) detection when traditional IgG antibodies are used as recognition elements in immunoassay. Herein, shark-derived single-domain antibodies (ssdAbs) were used as new recognition elements to enhance the sensitivity and specificity for norfloxacin detection in immunoassay. Firstly, NOR-specific clones were isolated by phage display technology. Finally, five clones were successfully isolated from a NOR-immunized library. Secondly, a soluble expression system was constructed to produce ssdAbs. The 1N9 ssdAb exhibits the best affinity to the NOR complete antigen, with a limit of detection of 0.392 ng/mL and the cross-reaction rates of less than 8.240%, indicating its high sensitivity and specificity as a new recognition element in NOR detection. More importantly, the ELISA result proved that in NOR-negative fish matrix, no serious interference was observed, the 1N9 ssdAb could still recognize complete antigen NOR-OVA effectively. While in icELISA, 1N9 ssdAb could recognize NOR in different NOR-spiked concentrations between 10 to 1000 ng/mL, with the recovery rates between 93.66 - 116.49% with high reproducibility. Our study provides a new and effective recognition element with great potential for NOR detection.
Chang Liu,Qing Yang,Hong Lin,Limin Cao,Kaiqiang Wang,Jianxin Sui
Nowadays, obesity severely impacts human health and is the fifth leading risk factor that leads to death globally. Pancreatic lipase (PL) inhibitors have attracted extensive attention owing to their role in effective prevention and treatment of obesity. Here, a shark-derived single-domain antibody fusion protein was used to inhibit PL for the first time. After biopanning, the heterologous expression system pET28a-SUMO-D2 was constructed using the method of double restriction enzyme digestion and T4 ligase to achieve the soluble expression of the PL-specific antibody gene D2. According to the calculation of protein concentration, the final expression of fusion protein PL-D2S was 1.183 mg per liter of Luria Bertani broth. The binding ability of the soluble fusion protein PL-D2S to PL was identified. Enzyme-linked immunosorbent assay results showed that the fusion protein PL-D2S exhibited a strong binding affinity to PL. The experimental results of PL inhibition of PL-D2S in vitro showed that the fusion protein could significantly inhibit the activity of PL, with an IC50 of 404 μg/mL. Our study shows that the fusion protein PL-D2S is a potential PL inhibitor to prevent and treat obesity.
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.
危险品化学品经营许可证（带存储）