| Diethylstilbestrol is an artificially synthesized estrogen. Due to the low price and high efficiency, diethylstilbestrol is widely used in clinical medicine and livestock breeding industry. A wide range of domestic and foreign studies prove the certain toxicity of diethylstilbestrol on human, animal’s reproductive system and immune system. Diethylstilbestrol also has carcinogenicity on reproductive system and is strongly harmful to the ecological environment. Due to the toxicity and side effects of diethylstilbestrol on human and their living environment, many countries have published laws to prohibit the use of diethylstilbestrol.Detection methods of diethylstilbestrol are experiencing a rapid development all over the world, GC-MS and LC-MS are common standard methods for diethylstilbestrol residue detection due to the high detection accuracy, whereas accompanied by a series of disadvantages: expensive instruments,complex pretreatment, professional operator and not easy to popularize. Based on immunoreactions and its advantages of rapidity, sentitivity, simple operation and low cost, fluorescence immunochromatographic method shows a wide development prospects. This study aims to establish a diethylstilbestrol competitive fluorescence-immunochromatographic assay and develop a fast, efficient and compact diethylstilbestrol detection kit.Two diethylstilbestrol hapten(DES-HS and DES-MCME) were synthesized and identified by elemental analysis and mass spectrum analysis. Carbodiimide method together with mixed anhydride method were used to couple synthesized haptens with BSA. Ultraviolet analysis proved that the successful synthesis of the two complete antigens( DES-HS-BSA and DES-MCME-BSA). Selecting the most appropriate pair of antigen and antibody among the synthesized and purchasing antigens and antibodies on ELISA platform and fluorescenceimmunochromatographic platform, a pair of coating antigen B2 and antibody A1 were eventually got, which were suitable for fluorescence quantitative immunochromatographic detection.A series of experiments were carried out to optimize the conditions of the fluorescence quantitative immunochromatographic platform. The best process conditions for the strips were found that 1 mL fluorescent latex added with 200 μg antibody, antigen concentration of the test line is 2.0 mg/m L, a 100-fold dilution for fluorescent latex labeled antibody, a 1:1 ratio for adding proportion of fluorescent latex labeled antibody solution and the samples, 36 h for drying time of nitrocellulose membrane and 15 min for reaction time, the standard curve built at the five DES concentrations of 0 μg /L、2 μg /L、4 μg /L、8 μg /L、16 μg /L showed good linear with the R2=0.9998. The experiment also did performance evaluation on the kit. The LOD and LOQ of the kit for pig urine samples are 0.4684 μg /L and 1.4316 μg /L. The average recoveries for immunochromatographic strips are in the range of 95.1%-97.2%. The within-run and between-run precision meet the requirements. The specificity and stability of the strip are good.This study discussed the different pretreatment methods on different diethylstilbestrol residue samples(pork, fish, feed and milk samples) which were tested by DES kit. Results showed that adding recovery rate for the four kind of samples are 91.50%,93.25%,91.07% and 90.39% respectively which can meet the requirements of the test. The adding recovery rates proved that pretreatment methods on the samples are effective and can be used in samples which have similar matrix. |
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