一对高亲和力抗人肌钙蛋白I单克隆抗体的制备及鉴定

Preparation and Identification of a Pair of High Affinity Monoclonal Antibody Against Human Cardiac Troponin I

目的获取抗人肌钙蛋白I(cTnI)的单克隆抗体(McAb)。方法以人cTnI作为抗原,免疫Balb/c小鼠,通过杂交瘤技术制备了抗人cTnI高亲和力、高特异性单克隆抗体。随后采用间接ELISA法测定抗血清效价,用protein G亲和纯化法纯化抗体,抗原竞争ELISA法鉴定抗体亲和力,SDS-PAGE法鉴定纯度,Western blotting鉴定抗cTnI单克隆抗体的特异性,竞争ELISA法分析抗原结合位点。结果筛选出9株稳定分泌抗cTnI的单抗杂交瘤细胞株,其中A3、A9两株免疫球蛋白亚类均为IgG2a,分泌的抗体纯度高,与CK-MB、cTnT无交叉反应,效价均为:1∶1024000,亲和力分别为4.21×108mol/L、1.07×108mol/L,抗原结合位点不同。结论成功制备出了一对高亲和力、高特异性抗人cTnI单克隆抗体。

Objective To prepare monoclonal antibodies against human cardiac troponin Ⅰ（cTnⅠ）. Methods Balb/c mice were immunized by human cTnⅠ as the antigen, and the high affinity and specificity monoclonal antibody against human cardiac troponin Ⅰ was prepared through hybridoma technology. The titer of the antiserum,purified by protein G affinity of antibody,was then determined by indirect ELISA. The antigen binding site and the affinity were analyzed by competitive ELISA. SDS-PAGE and Western blotting were used to identify the specificity. Results Total 9 strains of hybridoma cell lines secreting monoclonal antibody against cTnI were obtained,and the antibody secreted by A3, A9 were of high purity and its immunoglobulin subclasses was IgG2a. There was no cross reaction with CK-MB, cTnT. Both titers were 1： 1024000, and the affinity were 4.21 ×10^8mol/L 1.07× 10^8mol/L respectively. The antigen binding sites for them were different. Conclusion A pair of affinity and specificity of monoclonal antibody against human cardiac troponin Ⅰwas prepared successfully. and high