摘要
目的制备抗重组人肝再生增强因子(recombinant human augmenter of liver regeneration,rhALR)单克隆抗体,鉴定其特性,为建立一种稳定、特异及简便的临床检测方法奠定基础。方法以纯化的rhALR免疫Balb/c小鼠,通过细胞融合建立能稳定分泌抗rhALR单抗的杂交瘤细胞株,间接ELISA方法和Western blot方法鉴定McAb特异性并对杂交瘤细胞的染色体进行分析。采用快速定性试纸鉴定McAb的Ig亚类,间接ELISA方法检测McAb的效价及亲和常数。结果筛选出一株可分泌特异性McAb的杂交瘤细胞2A6;其抗体亚类重链为IgG2b,轻链为К链,腹水效价为10-6;亲和常数为3.19×107。间接ELISA法和Western bolt检测证明这株杂交瘤细胞所分泌的McAb特异性良好。结论成功制备出高效价的抗rhALR单克隆抗体,为建立一种稳定、可靠的ALR检测方法,以便深入研究ALR在肾脏疾病中的作用奠定基础。
This study aimed to prepare monoclonal antibody（McAb） against recombinant human augmenter of liver regeneration（rhALR）,which may be useful in the development of a specific and convenient method to detect hALR.Balb/c mice were immunized with rhALR,and then splenocytes from the immunized mice were collected and fused with the mouse myeloma cell line SP2/0 cells.The hybridoma cells that secreted anti-rhALR protein McAb antibodies were cloned with limited dilution method.The ascites titers and the affinity of the obtained McAbs were determined by indirect ELISA,while the specificity of the McAbs was tested by indirect ELISA and Western blot analysis.Then the immunoglobulin（Ig） subtype was test by IsoQuick strips,the chromosome of the hybridomas was analyzed.As results,one of hybridomas was screened out,designated 2A6.Chromosome analysis revealed that the obtained hybridomas were possessed universal characteristics of the monoclonal hybridoma cells which secreted McAb,the Ig subtype of 2A6 McAb was IgG2b,and the light chain was kappa.We also detected that he ascites titers of 2A6 McAb was 1：106,and the affinity constant of 2A6 McAb was 3.19×107.Indirect ELISA and Western blot identification suggested that the McAbs react specifically with rhALR protein.In our study,one of high titer,specific McAb against rhALR protein has been successfully prepared and primarily identified.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2010年第11期987-990,共4页
Immunological Journal
基金
国家自然科学基金面上项目(30971364)
重庆市卫生局医学科学技术项目重点项目(2008-1-44)
关键词
肝再生增强因子
单克隆抗体
生物学特性
Augmenter of liver regeneration
Monoclonal antibody
Characterization
