奶牛孕酮单克隆抗体的制备及其酶免疫测定方法的初步建立

目的]制备奶牛孕酮McAb,建立孕酮酶免疫检测方法,为研制检测试剂盒奠定基础。方法]用琥珀酰亚胺法将孕酮与钥孔血蓝蛋白(KLH)和卵清白蛋白(OVA)偶联制备完全抗原,免疫BalB/c小鼠,建立杂交瘤细胞株制备McAb,对McAb特异性、亚类、交叉反应性及相对亲和力等进行鉴定,筛选最佳McAb,建立酶免疫检测方法。结果]获得3株杂交瘤1F6、2G6和2A11,腹水效价依次为1∶1×106、1∶1×105和1∶8×104,其中1F6为IgG2b亚型,2G6和2A11为IgM亚型,3株McAb都能和游离孕酮发生特异性反应,相对亲和力1F6>2G6>2A11,其中1F6对雌二醇的交叉反应率小于0.01%。选用1F6与酶标孕酮建立ELISA检测方法,制作标准曲线,检测范围为0.5~100.0ng/ml。结论]该研究制备的单克隆抗体,可用于建立孕酮酶免疫检测方法,为奶牛孕酮水平的定量监测提供了较实用的方法。

Preparation of Monoclonal Antibodies of Diary Cattle Progesterone and Esatblishment of Enzyme Immunoassay

Objective] To produce monoclonal antibodies against progesterone and to establish enzyme immunoassay.Methods] Progesterone was conjugated with KLH and OVA by NHS.BalB/c mice were immunized to produce hybridoma cell line.Anti-progesterone monoclonal antibodies were obtained from hybridoma.The optimal McAb was selected by conduct analyses of specificity,subclass,cross-reactivity and relative affinity.The immunoassay was established with the selected McAb.Result]Obtained three hybridoma cell line 1F6,2G6 and 2A11.The titer degree of ascites fluid of three McAbs were 1∶1×106、1∶1×105 and 1∶8×104 respectively.1F6 belongs to IgG2b.2G6 and 2A11 belongs to IgM.Three McAbs can reaction with dissociative progesterone.Relative affinity showed 1F6>2G6>2A11.1F6 had 0.01% cross-reaction with estradiol.Competitive ELISA was established based on 1F6 and HRP labeled progesterone.The standard cruve was obtained.The regression equation within the range of 0.5～100.0 ng/ml.Conclusion] The prepared McAb can be used for Progesterone immunoassay.This work provides a basis for quantitative analysis of diary cattle progesterone level.