Preparation Of Ethyl Carbamate Antibodies And Study On Immunoassay Methods

Ethyl carbamate?EC?can be produced in the whole process of production and storage of fermented food.In 2007,the International Agency for Research on Cancer?IARC?of the World Health Organization listed EC as a Class 2A carcinogen.Therefore,the real-time monitoring and detection of EC concentration in the whole process of production and storage of fermented products is particularly important.The traditional instrument detection method is not suitable for such large-scale detection and on-line detection.However,the application of the immune method can meet the real-time detection on the production line.This study successfully prepared EC haptens,artificial antigens and polyclonal antibodies,evaluated the effects of polyclonal antibodies,and initially established an indirect ELISA detection method.The main research contents are as follows:?1?According to the structural characteristics of EC and the design principle of haptens,three EC haptens?ECH1,ECH2 and ECH3?were designed and synthesized by introducing specific benzene ring structures and linear linking arms into EC molecules.The structures of these three haptens were characterized by ¹H NMR and ¹³C NMR.?2?According to the structural characteristics of haptens,the activated ester method was selected to couple haptens with the carrier bovine serum protein to synthetic and obtained EC artificial antigens?ECA1,ECA2 and ECA3?.Three artificial antigens were characterized by ultraviolet spectrum,Coomassie blue method and gel permeation chromatography-eighteen-angle laser light scattering system.The protein concentration and coupling ratio were calculated respectively.The results showed that three artificial antigens?ECA1,ECA2 and ECA3?were successfully prepared.The protein content of the three artificial antigen lyophilized powders are:66%,57%and 69.5%and the coupling ratios were 14:1,17:1 and 21:1.?3?The ECA3 with the highest protein concentration and coupling ratio was selected for animal immunity tests to produce polyclonal antibodies,and antibodies were identified for immunological activity by ELISA.Then an indirect ELISA for EC was established.The results showed that the antibody of mouse No.4 had the best effect,with a titer of 128000,sensitivity of 6.02 mg/L,affinity of8.29×10⁹ L/mol,and good specificity.The ELISA optimization results are as follows:the coated antigen is diluted to 0.25?g/mL and placed overnight at 4?,blocked with 5%skimmed milk powder at 37?for 1 hour,the antibody is diluted 8000 times,the enzyme-labeled secondary antibody is diluted 2000times,and the substrate coloration time is 15 minutes.Under this condition,the standard curve of the indirect ELISA method is y=0.6827-0.2675x,the correlation coefficient is R²=0.9946,the detection limit is 0.154 mg/L,and the linear range is 0.364 mg/L-63.75 mg/L.In summary,this study has prepared EC antibodies,preliminarily established EC immunoassay method and provided a good basis for future development of rapid detection methods such as ELISA kits and immunochromatographic test strips.