Preparation Of Monoclonal Antibody Against Estradiol And Preliminary Study Of ELISA Kit

To esabolish the quick test for the residues of Estradiol(E2) in the animal products, E2 was coupled to carrier protein of bovine serum albumin(BSA) and ovalbumin(OVA) using carbodiimide method. The E2-BSA and the E2-OVA were used as immunogen and coating antigen, respectively, then BALB/c mice were immunised with E2-BSA. The titer of anti-E2 serum was tested with indirectly competitive ELISA. The constantly secreting anti-E2 monoclonal antibody was screened with hybridomas techinque. HRP-labelled estradiol antigen and antibody (E2-BSA-HRP and 1E3-HRP) were prepared by coupling the E2-BSA and 1E3 with HRP by the improved sodium periodate method. The optimised working concentrations of coated antigen and antibody were performed with phalanx titration. Consequently, the enzyme-labelled antigen directly competitive, enzyme-labelled antibody directly inhibition and double antibody sandwich enzyme linked immunosorbent assay (ELISA) methods for E2 residues testing were compared to select the optimised one. Results were that the titer of anti-E2 serum was 1:3.2×104. Two hybridomas producing anti-E2 monoclonal antibodies (McAb) were obtained by limited dilution, named 1E3 and 1B4. Aditionally, the characteristic of McAb was performed by ELISA, the subclass of the McAb was IgG2b and IgG1, the titer of ascites was 1:1×105, the affinity constant of E2-McAb for coated complete antigen was 2.9 ×108L/mol, the molecular weight was 179KD. Chromosomal numbers of the hybridoma 92-102. The cross-reaction rate of McAb to estriol, estrone and progesterone was all below 0.5%. The antibody secretion of 1E3 was stable after in vitro passage and the anabiosis from the frozened store. The optimised working concentration and dilution ratio of coated antigen and McAb were 0.625μg/ml, 1:1000, respectively. The optimised dilution ratio of HRP linked goat anti-mouse IgG antibody was 1:5000. The linear detecting range of standard curves was between 0.01ng -1μg/ml. The linear regression equation was y = -8.201x + 47.814 with R2 = 0.9965. When regarding the 50% of inhibition concentration (IC50) as the detecting limitation it was 0.54ng/ml. It is concluded that the enzyme labelled antigen direct competitive ELISA could be used in the kit-test for the E2 residues.