The Development Of Rapid Detection Of Method For Phenobarbital In Food

Phenobarbital is psychotropic drugs of category I and most used for sedation and hypnosis. It was often illegality added to the health food of Improvement Sleep by lawbreaker, because of interest. Long-term consumption of the health food with Phenobarbital, may cause great threat to human health. Therefore, handy, rapid, and highly sensitive screening methods are urgently needed to develop. In view of this,this dissertation carried out the following studies:To synthesize artificially and identify the complete antigen of Phenobarbital.Artificial Phenobarbital hapten with the active amino and carboxyl groups were designed and synthesized by chemical modification and linked to BSA and OVA through diazotization and carbodiimide method to form artificial Phenobarbital antigen. Phenobarbital hapten is identified by TLC、MS、NMR, and its structure was identified by ultraviolet scanning. The biological identification of artificial Phenobarbital antigen was done by mice immunization, and polyclonal antiserum was obtained, identified and analyzed by ELISA. The result showed that the synthesis of Phenobarbital hapten was successful. The Phenobarbital was successfully coupled with BSA and OVA, and the conjugation ratio of PAPB-BSA was about 23.20:1, the conjugation ratio of PCPB-BSA was about 15.44:1, the conjugation ratio of PAPB-OVA was about 10.91:1. The immunization results showed that titer against Phenobarbital were above 103 in mice, and IC50 of NO.5 mice was 95.49 ng/m L.Artificial Phenobarbital antigens were synthesized successfully, in the study, and make it possible to establish a base of the generation specific antibody and development of immunoassay of Phenobarbital.Two immunogens were applied to immunize BALB/C mice to produce anti-Phenobarbital monoclonal antibody. The titer of blood serum was collected after the thrice immunization reached 104. And four monoclonal cell lines which could steadily secrete anti-Phenobarbital monoclonal antibody were obtained successfully by PEG-induced cells fusion. The ascites were purified by n-Caprylic acid-ammonium sulfate precipitation and determined with the BCA kit for antibodyconcentration. The antibody affinity constant was calculated to be 4.13×108 L/mol,IC50 was 10.76 ng/m L, and the titer of the antibody was about 105. The most important property of the prepared antibody was that there was no cross-reaction with other Phenobarbital analogues except with Phenobarbital Sodium.Colloidal gold chromatography technique was convenient, accurate, and high specificity and sensitivity combinated with monoclonal antibody. This experiment developed colloidal gold chromatography strip with anti-Phenobarbital monoclonal antibody marked by colloidal gold, which had good bioactivity and specificity to Phenobarbital. Colloidal gold with about 33 nm particle diameter were prepared by reducing the gold chloride with sodium cit rate. The optimum p H was about 7 and the optimum labeling rate of anti-Phenobarbital monoclonal antibody was 18 μg/m L. The combination was optimized with the condition of the check line and the contrast line was sprinkled with 1.5 mg/m L and 0.75 mg/m L when making the strip, meanwhile 2lines was sprinkled with 0.75 μL/cm. The detection limit of the strip was 30 ng/m L for detecting the Phenobarbital standard solution. The detection limit of the strip was30 ng/m L for detecting the aqueous extracts of health food raw materials with Phenobarbital. The result of specificity test proved that colloidal gold test strip had no significant cross-reaction with barbital, pentobarbital, and amobarbital. Colloidal gold test trips can be stable in 3 months at room temperature and dry environment. There were no difference between same batch and different batches.The test strip prepared in this experiment was convenient and efficient, the detection result would be intuitive judgment in 15 min, and the strip was suitable for on-site and real time rapid test.