人胱抑素C单克隆抗体的制备、鉴定及初步应用

Preparation,characterization and preliminary application of monoclonal antibody against cystatin C

目的:制备人胱抑素C(Cystatin C,Cys C)的单克隆抗体(McAb),并建立检测人血清Cys C的颗粒增强透射免疫浊度分析法(PETIA)。方法:构建人Cys C的原核表达载体pET32a(+)/Cys C,纯化Cys C重组蛋白并免疫BALB/c小鼠,杂交瘤技术制备Cys C的McAb;以自制的McAb包被乳胶颗粒,建立PETIA法测定血清Cys C,并对其分析性能进行初步方法学评价。结果:建立分泌抗Cys C单克隆抗体的杂交瘤细胞株3株,其分泌的McAb为IgG1,Western blot检测证实该抗体能与目的蛋白发生特异性结合;将杂交瘤细胞注射于小鼠腹腔,腹水中Cys C McAb效价为1∶4×106;用自制的McAb建立定量测定Cys C的PETIA,方法学评价试验结果证实我们建立的PETIA法,与进口试剂有很好的可比性,具有较满意的方法学性能。结论:成功制备了抗Cys C单克隆抗体,该抗体可用于建立定量检测Cys C的PETIA法。

Objective：To prepare monoclonal antibodies （McAb） against cystatin C （Cys C） and to estabhsh the particle enhanced turbidimetric immunoassay （PETIA） for determining human serum Cys C. Methods： The prokaryotic expression vector pET32a（ ＋ ）/Cys C was constructed and Cys C expression was induced. McAbs against Cys C were prepared with the hybridoma technique after mice were immunized with the purified recombinant protein. Then the McAbs were covalently attached to uniform microparticles, PETIA method for determination of human serum Cys C was established,and primary evaluation tests of methodology were performed.Results： Three hybridoma cell lines were obtained successfully, the secreted antibodies were isotype of IgG1, and Western blot confirmed that the antibodies reacted specifically to the Cys C protein. After one of the hybridoma cell lines was injected into mice abdominal cavity, the ascites abundant for McAb was obtained. The titer of the McAb against the purified protein was 1 ： 4 × 10^6. With the self-made McAb, PETIA for human serum Cys C was established. The primary evaluation tests of methodology revealed that self-established PE33A method had a satisfactory performance, which was equal to the import kit. Conclusion： The prepared McAb against Cys C is prepared, which could be used to establish PETIA for determining human serum Cys C.