Study On Methodology For Detection Of Diethylstibetrol Residues In Aquatic Products

Diethylstibetrol is a kind of artificial synthesis estrogen analog with stronger function, which is a forbidden medicine in aquaculture and is one of the critical residue controlled medicines in aquatic products in China. At present, there is not a effective and fast method to detect Diethylstibetrol residues in aquatic products. So, it is significant to set up a faster and accurate detection method of Diethylstibetrol residues in aquatic products. We studied Diethylstibetrol ELISA competition method and gas chromatogram- mass spectrum method to detect Diethylstibetrol residues in aquatic products in this paper.一,Study on the ELISA detection technique of DiethylstibetrolA carboxyl group was introduced into the molecule of DES under reflux with ethyl chloroacetate. The reaction products of the monocarboxymethyl-DES were determined in quality and quantitative analysis. The monocarboxymethyl-DES purity was more than 95%. The monocarboxymethyl-DES was then conjugated with BSA by CDI method. The DES-BSA conjugation was characterized by UV spectroscopy and gave a DES/BSA molar ratio of 14:1. Five New Zealand big ear rabbits were immunized with subcutaneous injections in this antigen for five times. Purification antibody was collected after the antiserum was precipitated by (NH4 ) 2SO4 and purified by Sephedex-G-200 . The IgG effective price ratio is about 1:30000 with ELISA test. The monocarboxymethyl-DES was then conjugated with HRP in carbodiimide(EDC) catalytic action and was synthesized as DES-HRP. DES-HRP conjugation had good enzyme activation and immune activation through spectral analysis. The molar ratio of monocarboxymethyl-DES and HRP was 10.92:1. The best working concentration of the enzyme was the dilution of 1/6400. The antisera was diluted at the ratio of 1:500 with coated solution. Every well on 96 microtier well was added 50μL antisera dilution, 20μL of a serial of standard solution and enzyme conjugate DES-HRP solution sequentially, and 50μL substrate and 50μL chrommgen respectively. Finally 100μL stop reagent was added to each well and measured the absorbance to set up the standard curve of DES.We have set up the competition DES ELISA detection method through the optimization of the experimental condition. The linear range of this method is 50-400ppt; the coefficient of variation is 15.68%; and it has better sensitivity and stability.二,The study on the Analysis of Residues of Diethylstibetrol in aquatic...