| Quinolones are a class of antimicrobial drugs with broad-spectrum, which are widely used in the prevention and treatment of animal diseases. However, its residues in animal foods harm the people's health seriously. It is imperative to monitor the quinolones residues to prevent people from the harm of quinolones poisoning. Traditionally, analytical methods for quinolones mainly are high performance liquid chromatography and capillary electrophoresis methods. These methods are sensitive and accurate, however, they are time-consuming, have low-throughput, and are laborious. For high-throught and rapid screening, immunoassays based on the binding properties of an antibody to an antigen are simple, rapid, sensitive and cost-effective, which are more suitable for rapid screening. The key of broad specificity immunoassays is broad specificity antibodies. Therefore, in this work, several quinolones haptens/antigens were designed and synthesized to produce the polyclonal antibody for quinolones and developed several immunoassays. The mechanism of antibody recognition were also studied using quantitative structure-activity relationship(QSAR). The results are shown as follows.(1) Study of broad specificity immunoassays using pipemidic acid as a haptenPipemidic acid has the basic structure of quinolones, and is similar with the norfloxacin which is broad specificity. Thus, pipemidic acid was used as a hapten to produce polyclonal antibody. Based on a novel heterologous strategy, a highly sensitive indirect competitive ELISA was successfully constructed with an IC50(50% inhibitory concentration) of 6.0 ng/m L, indicating that the sensitivity was much better than that of the homologous combination and that of the reported physio-chemical method. Fine QNs showed a CR higher than 10 % while the others lower than 9.2%, which demonstrated high specificity. Comparing with the structure of norfloxacin, QSAR results showed that piperazine ring at C-7 position shaped like a Chinese word ‘one', which formed a flat cavity that could not accommodate a large group, mainly lead to high specificity of pipemidic acid antibody. In addition, it was negatively associated with the large bulky substructure at the N-1 position of quinolones. Besides, the concentration of pipemidic acid peaked on day 5 and then fell to below 100 ng/g at day 14 when hens were administrated pipemidic acid. The data indicated that the ELISA is reliable for pipemidic acid residues detection and supervision in food products, and provided a scientific basis for the possible future risk assessment of pipemidic acid residues in egg.(2) Study of broad specificity immunoassays using clinafloxacin as a haptenThe strucure of clinafloxacin is similar with the strucures of ciprofloxacin and enrofloxacin, just the C-7 position and C-8 position are different. Comparing with pipemidic acid, C-8 position of clinafloxacin is a chlorine atom, which could form a ring-like structure with cyclopropyl group at N-1 position. In this study, clinafloxacin was used as a hapten to produce polyclonal antibody. Two fluorescein-labeled tracers were synthesized and their binding response with clinafloxacin-specific antibody was evaluated. The effects of tracer concentration and matrix on FPIA performance were also studied. A rapid and specific FPIA was developed successfully for clinafloxacin residues with the IC50 value of 25.6 ng/m L. Nineteen QNs showed a CR lower than 5% and indicated that the proposed FPIA was highly specific to the clinafloxacin analyte. QSAR results showed that amino group and 5-membered ring at C-7 position could formed a small cavity and could not accommodate a large group, for example, a piperazine ring, which mainly lead to the high specificity of clinafloxacin antibody. In addition, due to the non-cyclic structure at C-8 position and N-1 position, clinafloxacin could not recognize the quinolones which have a cyclic structure at the same position. The recoveries from milk ranged from 97.9 to 112.7 % and the coefficient of variation were low than 7.1%. The FPIA results were validated by HPLC, this indiced that HPLC results were concordant with FPIA results. This study indicated that the proposed method could be used as an ideal surveillance screening method for the analysis of clinafloxacin in milk samples.Besides, based on the combination of a heterologous tracer(PAZ-FITC, synthesized with pazufloxacin and FITC) and the antibody against clinafloxacin, a highly sensitive FPIA was established for the detection of clinafloxacin residues in goat milk. The IC50 value was 29.3 ng/m L for clinafloxacin in the heterologous format–six times lower than that of the combination of the homologous tracers and the antibody. The mean recoveries was 96.6% with the mean relative standard deviation was 5.3%. In conclusion, the proposed FPIA using heterologous strategy could be considered a highly sensitive and rapid high-throughput method for monitoring clinafloxacin residues in goat milk.(3) Study of broad specificity immunoassays using pazufloxacin as a haptenPazufloxacin is a fluoroquinolone drug containing a 1-aminocyclopropyl group at the 7- position but not a piperazinyl group common in many quinolones, which may lead to broad specificity. In this study, pazulfoxacin was used as immunizing hapten to generate a polyclonal antibody. The assay exhibited an IC50 of 10.3 ng/m L for pazufloxacin. It is interesting that the resultant antibody against pazufloxacin demonstrated an extremely broad recognition spectrum up to 24 quinolones. 3D QSAR studies showed that steric field plays a major role on antibody recognition. The amino group and cyclopropyl group at C-7 position shaped like an alphabet V, which could formed a big cavity to accommodate a large group, mainly lead to broad specificity of pazufloxacin antibody. In addition, due to the cyclic structure at C-8 position and N-1 position, pazulfoxacin could recognize the quinolones which have a similar structure at the same position. While it was negatively associated with the large bulky substructure at the N-1 position of quinolones.In conclusion, in order to better understanding broadly specific recognition mechanism of quinolones, pipemidic acid, clinafloxacin and pazufloxacin were selected as haptens to produce polyclonal antibody, respectively. 3D QSAR studies showed that steric field plays a major role on antibody recognition. The cavity formed by the group at C-7 position is the major factor to affect the antibody recognition. In addition, due to the cyclic structure at C-8 position and N-1 position, antibody could recognize the quinolones which have a similar structure at the same position. While it was negatively associated with the large bulky substructure at the 1-position of quinolones. |
PDF Full Text Request