人心肌肌钙蛋白Ⅰ单克隆抗体及多克隆抗体的制备

目的:以重组人心肌肌钙蛋白Ⅰ(cTnⅠ)为抗原制备鼠源单克隆抗体(McAb)及兔源多克隆抗体,并鉴定抗体的特性。方法:以纯化的重组人cTnⅠ为抗原免疫BALB/c小鼠,取鼠脾细胞同Sp2/0骨髓瘤细胞融合,利用选择培养基筛选融合的杂交瘤细胞,用有限稀释法分离获得能够稳定分泌抗cTnⅠ的McAb阳性克隆,并利用体内诱生法大规模制备McAb,用辛酸-硫酸铵沉淀法纯化抗体;兔多抗制备以cTnⅠ为抗原常规免疫后取其血清;用间接ELISA和Western印迹鉴定抗体的性质。结果:经ELISA鉴定,筛选出5株能分泌cTnⅠMcAb的杂交瘤细胞株,即C5B2、C5B3、C5B4、C5B1、B1A6,效价最高的B1A6株分泌的McAb为IgG3型,纯化后效价为1∶10000,亲和常数为1.08×10-9mol/L,Western印迹鉴定表明cTnⅠMcAb有良好的特异性;兔多抗纯化后的效价为1∶8000。结论:制备了具有良好特性的cTnⅠMcAb和多克隆抗体。

Production of Monoclonal Antibodies and Polyclonal Antibodies Anti Human Cardiac Troponin I

Objective： We used recombinant human cardiac troponinⅠ（cTnⅠ） as antigens to prepare and identify monoclonal antibodies（McAb） and polyclonal antibodies in mouse and rabbit respectively. Methods： BALB/c mouse and New Zealand rabbit were immunized with the recombinant cTnⅠ. To prepare cTnⅠ McAb, the spleen cells of immunized mouse fused with myeloma cells Sp2/0. Hybridomas were separated by culturing in HAT cultures. The stable monoclonal hybridomas were isolated by limiting dilution. The cells of secreting cTnⅠ McAb were injected BALB/c mouse to produce a lot of antibodies. The cTnⅠ McAb were purified by caprylic acid-ammonium sulfate method. The cTnⅠ polyclonal antibodies were separated from serum of rabbit. The characters of antibodies were identified by indirect ELISA and Western blotting. Results： 5 hybridomas, named C5B2, C5B3, C5B4, C5B1 and B1A6, could secret cTnⅠ McAb. B1A6 was selected to identity. It belonged to IgG3 subtypes and had high titer and specificity. Conclusion： The monoclonal antibodies and polyclonal antibodies against cTnⅠ were prepared and identified.