Preparation Of Monoclonal Antibodies Against DBP And The Establishment And Preliminary Application Of An Indirect Competition ELISA Method

Dibutyl phthalate (d-in-butyl phthalate, DBP) is one of phthalates (PAEs),which is an environmental endocrine disruptor, been called environmental hormones.DBP has a good ability to dissolve more of the resins, and when it was used for PVC, it can make the products excellent in softness; when it was used for cellulose nitrate, it can be enhanced the gel capacity; when it was used in nitrocellulose coating, It can make products having a good softening effect, excellent stability, adhesion, flex resistance and water resistance, so it was often widely used as a plasticizer.But because during the processing, DBP are not polymerized with the polymer carbon chain, as time goes, it will be gradually released and therefrom out into the environment and organisms,then causes kinds of potential harm. Currently, the United States, European Union and China, have been Made provision about plasticizers prohibited or restricted in plastics and other products by regulations. However, affected by various factors, current illegal use or abuse of phthalate esters remains pervasive.Many physical and chemical methods to detect residue of DBP have been established at home and abroad. Such as HPLC, GS, Lc-Ms, GC-MS, although chromatography technology are precise and efficient to detect the residue of DBP, it is not suitable for large batch sample screening. Because of their complication, long time consuming, big cost and high technical request, AS a method that has been widely used in drug residue detection,elisa can solve these shortcomings, it is simple,accurate,efficient,cheap and unique test methods, suitable for rapid screening of large samples.In this research, we synthesized the antigen and prepared anti-clenbuterol monoclonal antibody by using monoclonal antibody and ELISA technique.Finally, the CiELISA method to detect residue of Clenbuterol was established.1. DBP-conjugated antigens were synthesized by coupling DBP with BSA and OVA.The synthesized antigens were demonstrated to be successful by UV-scanning and immunological methods.2. BALB/c female mice of6-8weeks immunized with the complete antigen by routine method. Immunological methods, dose, carrier proteins and times having effecs on immune were considered. The results showed that gradually strengthen the immune by back subcutaneous injection was better than the same dose of immune, the first immunization dose of100ug per one was better than50ug,as carrier protein BSA was better than OVA,and five immunity had a good immune response.3. The DBAP-BSA was used as an immune antigen in mice while the DBAP-OVA is was used for detection, the titer of blood serum collected after the fifth immunization reached1:12000,then a hybridoma cell line named2B3which could excrete anti-diethylstilbestrol monoclonal antibody steadily by hybridoma’s technique was received and then ascetic fluid was prepared.The protein concentration of ascetic fluid was22.4mg/mL,the titer of the McAb was1:16000.4. The CiELISA’s methed was established by the monoclonal antibody in the abdominal dropsy which was got by injecting hybridoma cellas2B3into BABL/c mouse. The optimum conditions were established through the matrix test. The coating antigen was attenuated by1:4000, the monoclonal antibody in the abdominal dropsy was attenuated by1:16000. Under optimized conditions, the standard ELISA inhibition curve of DBP to antibody-antigen reaction was developed and the regression equation was Y=-0.1574x+0.6603(R2=0.9889). The half-maximal inhibition (IC50) was5.73ng/mL. The cross reaction experiment showed that the very high specificity of McAb for DBP was expressed with endogenous estrogen and other compounds and drugs.5. The addition and reclamation test of DBP.The CiELISA’s method was initially applied in the addition and reclamation test of the liver tissue and muscle tissue of swine.The sample was determined after pre-processing.The intervention disappeared when the extraction of liver and muscle sample was diluted4times.The standard curve equation was: Y=-0.3015x+0.6461(R2=0.9867) between0-50ng/mL, IC50was4.39ng/g, LOD was0.5ng/g. When adding0.5,1.5and2.5ng/mL DBP to the liver tissue of swine, the recovery ratio was between85%and100%.The intraassay coefficient of variation was between4.12%and6.71%,and the interassay coefficient of variation was between5.98%and7.13%.