【作者】 戴小华；

【导师】 吴国娟； 黄燕；

【作者基本信息】 新疆农业大学 ， 基础兽医学， 2005， 硕士

【摘要】 为建立雌二醇(estradiol, E2)在畜产品中残留的快速检测方法,本研究从完全抗原的制备着手,采用碳化二亚胺法制备E2免疫原,免疫BALB/c 小鼠,用间接酶联免疫吸附法(ELISA)对抗体效价进行检测,利用杂交瘤技术筛选稳定分泌E2的单克隆抗体,用过碘酸钠氧化法制备酶标雌二醇抗原(E2-BSA-HRP)和酶标雌二醇抗体(1E3-HRP),用方阵滴定法确定包被原最适工作浓度及单抗最适稀释倍数。最终对用于检测雌二醇残留的三种酶联免疫检测方法(酶标抗原直接竞争法、酶标抗体直接抑制法、双抗夹心法)进行了筛选。实验结果为:E2抗血清效价为1∶3.2×104。利用杂交瘤技术获得了两株稳定分泌E2单克隆抗体的杂交瘤细胞株1E3和1B4,经鉴定1E3细胞株分泌的单克隆抗体为IgG2b 亚型,1B4 细胞株分泌的单克隆抗体为IgG1 亚型;杂交瘤细胞染色体数目为92~102 条;间接ELISA 测定腹水效价为1∶1×105,其亲和常数为2.9×108L/mol。1E3单克隆抗体的分子量为179KD,其中轻链分子量为28KD,重链分子量为61.2KD。该单抗与雌三醇、雌酮和孕酮交叉反应率均小于0.5%。该细胞株体外传代和冻存复苏后抗体分泌稳定。用方阵滴定法确定包被原最适工作浓度为0.625 μg/ml,单抗最适稀释倍数为1∶1000,辣根过氧化物酶标记羊抗鼠IgG(酶标二抗)最适稀释倍数为l∶5000。采用间接竞争ELISA 方法建立检测E2的标准曲线,在0.01 ng/ml~1μg/ml 之间,呈良好的线性相关,线性回归方程为:y = -8.201x + 47.814,R2 = 0.9965。以抑制率为50%时对应的浓度计算,该方法对E2的检测限为0.54 ng/ml。通过比较三种ELISA 方法,从中选出了试剂盒的检测方法为酶标抗原直接竞争法。更多还原

【Abstract】 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.更多还原