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以北京鸭Ig为抗原免疫Balb/cJ小鼠三次,末次免疫三天后取脾细胞与小鼠NS-1骨髓瘤细胞融合,隔天换入1/2体积含有2倍HAT的完全DMEM选择培养液,借助酶联免疫吸附试验检测上清液。测得阳性的一株杂交瘤细胞经十二次克隆化培养被定名为CBH-2。在体外培养历时10月余,其分泌能力仍未见衰减。体内传至第八代,腹水中检出的分泌抗体强度比体外培养的上清液高8000倍。纯化得到的单克隆抗体经免疫双扩散分析证明该单克隆抗体属鼠1gG_1亚型。以经Sephadex G-200分离得到的北京鸭IgM作为包被抗原,酶标法测得的O.D值较高,而以北京鸭IgM经巯基乙醇断链后的蛋白或以DEAE-52柱分离得到的IgG作为包被抗原测得的O.D值较低。同时,以北京鸭Ig经Protein A-Se-pharose CL4B柱五次吸附后的流出蛋白包被,测得的O.D值也较低。由此可见CBH-2细胞产生的单克隆抗体专一性是抗北京鸭IgM。CBH-2细胞分泌的单克隆抗体与北京鸭Ig的免疫扩散未见沉淀线。用Balb/cJ、C_(57)BL/6J和ICR/JCL小鼠的腹腔渗出细胞作为克隆化的饲养细胞能达到相似效果。而以Wistar大鼠的腹腔渗出细胞作为饲养细胞效果较差,使用胸腺细胞时杂交瘤细胞则几乎不能生长。
Balb / cJ mice were immunized three times with Beijing Duck Ig as antigen and spleen cells were fused with mouse NS-1 myeloma cells three days after the last immunization and replaced with 1/2 volume of complete DMEM selection medium containing 2 × HAT The supernatants were detected by ELISA. One hybridoma cell which was positive was identified as CBH-2 after twelve clonal cultures. In vitro cultured lasted more than 10 months, its secretion capacity has not been decayed. In vivo transmission to the eighth generation, ascites secretion of antibodies detected in vitro than the supernatant of 8000 times higher. The purified monoclonal antibody proved by immunodouble diffusion analysis that the monoclonal antibody belongs to the rat 1gG1 subtype. Using Beijing duck IgM isolated by Sephadex G-200 as coating antigen, the OD value measured by enzyme-labeled method was relatively high, while the protein was screened by Beijing duck IgM by mercaptoethanol or by DEAE-52 column The OD value of IgG as coating antigen was lower. In the meantime, the O.D values measured by Peking duck Ig coated with the eluted protein after five adsorptions by Protein A-Sepharose CL4B column were also lower. This shows that the specificity of monoclonal antibodies produced by CBH-2 cells is anti-Beijing duck IgM. The immunoprecipitation of monoclonal antibodies secreted by CBH-2 cells and Pekin duck Ig did not show any precipitation. A similar effect was achieved with peritoneal exudate cells from Balb / cJ, C_ (57) BL / 6J and ICR / JCL mice as cloned feeder cells. In the Wistar rat peritoneal exudative cells as a feeder cell less effective, the use of thymocytes when the hybridoma cells are almost unable to grow.