Research On Rapid Immunoassay Detection Of Ethyl Carbamate In Fermented Vinegar

Ethyl Carbamate(EC)is also called uratan. It naturally exists in the fermented foods, such as bread, sour milk, cheese, soy sauce and other alcoholic beverages. EC is always accompanied by the product during the fermentation. At the same time, EC is a multi-site carcinogen, which could cause a lot of disease, such as lung cancer, lympHoma, liver cancer, skin cancer and so on. At present, the urethane pollution is regarded as another severe problem after the pollution of aflatoxin. And therefore it is vital to study an easy and rapid method of EC detection. This paper focuses on the indirect competitive ELISA detection technology and colloidal gold immune chromatography.The first experiment adopted the glutaraldehyde method which coupled EC with bovine serum albumin(BSA) and ovalbumin(OVA) to prepared the artificial antigen EC-BSA and the coated antigen EC-OVA. The next, mixed the EC-BSA and the complete adjuvant(or incomplete adjuvant) by equal quantity. After emulsification, injected multi-point on both sides of the subcutaneous spine on the back to immunize the healthy New Zealand white rabbits and was prepared immune serum containing EC polyclonal antibody. Then, the valence of antiserum was 1:2000 after purification with the indirect ELISA method. Finally, the method of EC ELISA detection was established. Experiments showed that IC50 was 7.39 ng/ml and IC15 was 0.04 ng/ml in the range of the 0.001 ng/ml to 10000 ng/ml. The cross reaction rate of methyl ester(MC) was 37.16%, which showed that the prepared antibody was specific.When added EC standard solution to the fermented vinegar solution,the EC concentration in 20 days of fermentation vinegar was 23 ng/ml and the average recovery rate was 82.83% with the ELISA method. At the same time, HPLC-FLD method was also selected as the fermentation time for 20 days, the EC concentration was 27 ng/ml and the average recovery rate was 85%.The results indicated that the indirect competitive ELISA based on the experiment has achieved rapid quantitative detection of EC.The second experiment has been established the rapid detection of EC immunoassay with colloidal gold abeled antibody. Firstly, we was adopted the three sodium citrate reduction method to prepared colloidal gold solution with the diameter of 42 nm, and labeled with EC antibody, it was acquired the gold labeled antibody. Secondly,we was smeared EC-OVA over the NC membrane as the T belt, and applied the goat anti-rabbit second antibody with reasonable dilution on the NC membrane as the C belt. Thirdly, we was applied a certain proportion of mixed solution with EC standard solution and purified gold labeled antibody in the middle of T and C belt to let the coating antigen and EC standard solution competed the gold labeled antibody. On this account, the colloidal gold strip had accomplished. Finally, the lowest limit of EC detection was 5 ng/ml after optimizing the experimental conditions. This detection method is appropriate to the qualitative and semi quantitative of EC detection because of its easy process and short detection time.