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用Protein A-Sepharose亲和层析法提纯McAb-IgG,皮下多点和腹腔免疫雄性Wistar大鼠,间隔两周共3次,融合前3天采用腹腔、尾静脉和脾内注射等三种加强免疫方法,以间接ELISA法和抗体竞争ELISA法筛选Anti-Id阳性克隆。结果显示:融合前大鼠血清抗小鼠IgG或抗免疫原McAb-IgG滴度,静脉组和脾内组比腹腔组高一个滴度;Anti-Id阳性克隆出现频率,腹腔组为0.9％和0.3％,静脉组为11.0％和13.3％,脾内组为16.4％和12.9％,静脉组和脾内组明显高于腹腔组(P<0.01);上述三种加强免疫方法对抗小鼠IgG阳性克隆出现频率无明显影响(P>0.05);与腹腔加强免疫组相比,静脉和脾内加强免疫组也可提高细胞融合效果(P<0.01).本实验结果提示,腹腔和皮下多点多次长程基础免疫结合静脉或脾内加强免疫是制备Anti-Id McAb比较好的免疫方案。
McAb-IgG was purified by Protein A-Sepharose affinity chromatography. Male Wistar rats were immunized subcutaneously and intraperitoneally for 3 times at intervals of two weeks. Three kinds of enhancement, ie, abdominal cavity, tail vein and spleen injection, were performed three days before fusion Immunization methods, indirect ELISA and antibody competition ELISA screening Anti-Id positive clones. The results showed that the anti-mouse IgG or anti-immunoglobulin McAb-IgG titers in serum of pre-fusion rats were higher than those of the abdominal cavity in the venous and spleen groups. The frequency of anti-Id positive clones was 0.9% in the abdominal cavity group 0.3%, 11.0% and 13.3% in the vein group, 16.4% and 12.9% in the spleen group, and significantly higher in the intravenous group and spleen group than in the abdominal cavity group (P <0.01) (P> 0.05). Compared with the group of boosting peritoneal cavity, the immunization group could enhance the effect of cell fusion (P <0.01) .The results of this experiment suggest that the intraperitoneal and subcutaneous multiple sites Secondary long-range basic immune combined with intravenous or intra-splenic boosting is the preparation of Anti-Id McAb better immunization program.