抗人类共激活蛋白单克隆抗体的制备

目的制备抗人类共激活蛋白(CLP)单克隆抗体,并进行纯化。方法配制2×DMEM培养基,与等量的2.2%甲基纤维素溶液混合,加入HAT、胎牛血清等,配成半固体HAT选择性细胞培养基。用基因重组人CLP抗原免疫BALB/c小鼠,取免疫小鼠脾细胞,与小鼠骨髓瘤NS-2细胞融合后,直接在半固体培养基上克隆化培养,筛选可持续、稳定、高效分泌抗CLP单克隆抗体的杂交瘤细胞株。对分泌的单抗进行鉴定后,制备小鼠腹水,经DEAE离子交换柱纯化,半饱和硫酸铵沉淀,G25凝胶过滤除盐。结果共筛选出14株分泌抗CLP单抗的杂交瘤细胞,其培养上清可与相对分子质量约16000的CLP抗原结合,抗体类型为鼠IgGa1型。多数克隆分泌的抗体相对亲和力大于1∶20000,14株克隆分泌的单抗分别对应3个抗原结合位点。纯化后的单抗纯度接近95%。结论已成功制备并纯化了抗人CLP单克隆抗体,为建立CLP免疫学检测方法奠定了基础。

Preparation of Monoclonal Antibody against Human Coactosin-like Protein

Objective To prepare and purify the monoclonal antibody(McAb)against human coactosin-like protein(CLP). Methods Mix 2×DMEM with an equal volume of 2.2%methylcellulose,then add HAT and fetal calf serum et al.to prepare semisolid HAT selective medium.The splenic cells of mice immunized with recombinant human CLP were fused with murine myeloma NS-2 cells,then inoculated to the prepared medium for cloning culture.The hybridoma cell strains secreting McAb against CLP persistently,stably and effectively were screened,and the secreted McAbs were identified.BALB/c mice were injected i.p.with the screened hybridoma cells,and their ascites were purified by DEAE ion exchange chromatography and precipitated with 50%saturated ammonium sulfate,then filtrated with G25 gel for dechlorination.Results A total of 14 hybridoma cell strains secreting McAbs against CLP were screened,and their culture supernatants were bound to CLP antigen with a relative molecular mass of about 16 000. The secreted McAbs were murine IgGa1.The relative affinities(RAs)of McAbs secreted by most of the cell strains were more than 1∶20 000.The McAbs secreted by the 14 strains were corresponding to 3 antigen binding sites,and reached purities of nearly 95% after purification.Conclusion The McAbs against CLP were successfully prepared and purified,which laid a foundation of developing a method for immunological detection of CLP.