Development Of Antibody And Rapid Detection Methods Against Estriol

Estriol is an important endocrine disruptors, which even at low concentrations can exert potential biological effects. It could disturb the endocrine system, cause endocrine disorder, affect the health. In recent, it has been reported estrogen was applied to livestock by illegal merchants. The harm has become the focus of attention, country has also enacted relevant laws and regulations to forbid the abuse of estrogen in livestock. But under benefit’s urge, the phenomenon of abuse of estrogen still exists. thus, it is necessary to strengthen monitoring and testing. Immunoassay methods are high sensitivity, high specificity, cost effective and easy to use, exhibiting good potential in widespread application, which has important significance for establish and improve food safety monitoring system and protect health of the people. In this study, on the basis of previous studies, new hapten against estriol were designed in attempts to obtain a specific antibody, and established ELISA method and electrochemical sensor method, to meet the needs of different detection. The main results are shown as follow:Synthesis and identification of haptens and antigens. Aiming at the problem of original estriol antibody sensitivity low and according to the chemical structural characteristics of estriol, hapten E3-A(E3-3- carboxymethyl ether) was designed and synthesized. Spacer arm containing two carbon atoms and a the carboxyl group in terminal was introduced in the position of phenolic hydroxyl, All of the haptens were confirmed by mass spectrometry and nuclear magnetic resonance techniques, and antigens were identified by UV scanning. The results indicated that haptens and antigens were synthesized successfully.A highly sensitive indirect competitive ELISA(ic ELISA) was developed based on polyclonal antibody against E3. New immunogen was used to immunize rabbits to obtain polyclonal antibody, and then ELISA was developed. The specific of ELISA was studied under the conditions of different coating antigens, the results indicated that heterology coating could directly affect sensitivity of method and specificity of antibodies. When using E2-A-OVA coating antigens, the detection test limit(IC10) of ELSIA was 0.017 ng/m L, IC50 was 0.185 ng/m L, the linear range was 0.041~0.837 ng/m L, and the antibody did not cross-reactivity with the related compounds of E3. The average recovery ranged from 85.0% to 106.5% in dairy samples.The results of ic ELSIA are very compatible with that of HPLC-MS/MS(R2=0.973).A one step electro deposition immunosensors method was developed based on monoclonal antibody against E3. To further ensure a good source of the antibody, monoclonal antibody was preparated. New immunogen was used to immunize Balb/c mice, a specific Mc Ab cell line of E3-2C2 were established through technique of cellular fusion between spleen cells with myeloma cells. Murine monoclonal antibodies of m Ab-E3-A-BSA was preparated by vivo culture. The titer of it was 64000, and inhibition was 95.2% with 1 μg/m L E3. Further, electrochemical immunosensor method was developed. Prussian blue- chitosan-gold nanoparticles(PB-CS-Au NPs) were modified onto glassy carbon electrode through cyclic voltammetry to enhance the electrode specific surface area, electrical conductivity and bioaffinity. Antigen was fixed through gold nanoparticles, competitive immune response has happened when antibody and drugs was added after blocking protein. and then second antibody was added, form antigen-antibodysecond antibody complex, which changed the electrochemical properties of the surface of surface of electrode. Hereby, the label-free immunosensor of E3 was constructed. The detection linear range of the proposed immunosensor was 0.001~100 ng/m L with a detection limit of 0.001 ng/m L, the recovery from spiked sample of this method was between 92.5% to 113.0%. The results of the immunosensor are very compatible with that of HPLC-MS/MS(R2=0.993).