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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。
Objective To prepare monoclonal antibodies against human coactivator (CLP) and purify them. Methods 2 × DMEM medium was prepared, mixed with the same amount of 2.2% methyl cellulose solution, HAT, fetal calf serum and the like were added to prepare semi-solid HAT selective cell culture medium. BALB / c mice were immunized with recombinant human CLP antigen. The splenocytes of immunized mice were fused with mouse myeloma NS-2 cells and directly cloned and cultured in semi-solid medium to screen for a sustainable, stable and efficient Hybridoma cell line secreting anti-CLP monoclonal antibody. After identification of the secreted monoclonal antibody, mouse ascites was prepared, purified by DEAE ion exchange column, precipitated with half-saturated ammonium sulfate and gel-filtered desalted by G25 gel filtration. Results A total of 14 hybridoma cells secreting anti-CLP McAb were screened. The supernatant of the hybridoma cells can be combined with the CLP antigen of about 16000 molecular weight. The antibody type was mouse IgGa1. The relative affinity of the antibodies secreted by most clones was greater than 1: 20000, and the monoclonal antibodies secreted by 14 clones corresponded to 3 antigen binding sites respectively. Purified monoclonal antibody purity of nearly 95%. Conclusion The anti-human CLP monoclonal antibody has been successfully prepared and purified, which lays the foundation for the establishment of the immunological detection method of CLP.