CAJ | 학술논문

目的采用本院自行研制的ZCH-4-2E8细胞分泌的抗人CD19单克隆抗体与去甲斑蝥素(NCTD)进行连接制备免疫毒素,研究其在体外的靶向杀伤作用.方法通过凝胶过滤层析法纯化2E8单抗,SDS-PAGE电泳鉴定抗体的纯度;活泼酯法制备免疫毒素2E8-NCTD;流式细胞仪测定免疫毒素的抗体活性及Nalm-6细胞和K562细胞上CD19抗原表达情况;台盼蓝计数法比较2E8抗体、NCTD、2E8-NCTD免疫毒素对Nalm-6细胞和K562细胞的杀伤作用.结果纯化后的2E8单抗经SDS-PAGE鉴定纯度达99.00%以上;2E8培养上清在Nalm-6细胞上的阳性率为99.34%,而在K562细胞上则为0.98%,2E8-NCTD免疫毒素与Nalm-6细胞的阳性反应率为99.90%;培养96 h时,与对照组相比,2E8-NCTD(10 μg/ml)对CD19+的Nalm-6细胞的生长影响有统计学意义(96 h抑制率71.25%,P＜0.001),而纯化的2E8抗体(10 μg/ml)对Nalm-6细胞生长则无明显抑制作用(96 h抑制率为30.29%,P＞0.05);10μg/ml 2E8抗体、10μh/ml 2E8-NCTD对CD19-的K562细胞(96 h抑制率分别为4.17%和15.41%)则无明显抑制作用(P＞0.05).结论成功制备2E8-NCTD免疫毒素,该免疫毒素在体外具有明显的靶向杀伤白血病细胞Nalm-6的作用.
목적 채용본원자행연제적ZCH-4-2E8세포분비적항인CD19단극륭항체여거갑반모소(NCTD)진행련접제비면역독소,연구기재체외적파향살상작용.방법 통과응효과려층석법순화2E8단항,SDS-PAGE전영감정항체적순도;활발지법제비면역독소2E8-NCTD;류식세포의측정면역독소적항체활성급Nalm-6세포화K562세포상CD19항원표체정황;태반람계수법비교2E8항체、NCTD、2E8-NCTD면역독소대Nalm-6세포화K562세포적살상작용.결과 순화후적2E8단항경SDS-PAGE감정순도체99.00%이상;2E8배양상청재Nalm-6세포상적양성솔위99.34%,이재K562세포상칙위0.98%,2E8-NCTD면역독소여Nalm-6세포적양성반응솔위99.90%;배양96 h시,여대조조상비,2E8-NCTD(10 μg/ml)대CD19+적Nalm-6세포적생장영향유통계학의의(96 h억제솔71.25%,P＜0.001),이순화적2E8항체(10 μg/ml)대Nalm-6세포생장칙무명현억제작용(96 h억제솔위30.29%,P＞0.05);10μg/ml 2E8항체、10μh/ml 2E8-NCTD대CD19-적K562세포(96 h억제솔분별위4.17%화15.41%)칙무명현억제작용(P＞0.05).결론 성공제비2E8-NCTD면역독소,해면역독소재체외구유명현적파향살상백혈병세포Nalm-6적작용.
Objective Monoclonal antibody(mAb)conjugated with certain toxin to generate immunotoxin bears an important and promising effect as a new therapy for patients with hematopoietic malignancies.However,most toxic moieties conjugated with antibody proteins reported in the literature were toxic proteins which presented irnmunogenicitv to patients capable of inducing anti-toxin antibody.Nomantharidin(NCTD)is a small molecde toxin.It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug.In this stuay,a new clone of selfmade anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro to provide the experimental data for the further development of this new targeting agent.Methods A monodonal antibody named 2E8 wag prepared from mouse ascites and purified by gel chromatography.The purity of the antibody protein was checked by SDSPAGE assay.Immunotoxin 2E8-NCTD wag successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method.The binding activity of the immunoconjugate(2E8NCTD)against CD19 antigens on cell surface and the expression levels of CDl9 antigens on Nalm-6 and K562 cells were examined by flow cytometery.Comparisons of the inhibitory effects among PBS,purified 2E8 antibody,norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm-6 cells or K562 cells were made.Results The purity of the purified 2E8 antibody was higher than 99.00% demonstrated by SDS-PAGE assay.2E8 antibody in the supernatant reacted with 99.34%of Nalm-6 cells,while only 0.98% of K562 cells reacted with this antibody.The newly generated inununotoxin(2E8-NCTD)had a positive rate of 99.90% on Nalm-6 cells with little reduction of binding activity.From the in vitro study.both 2E8-NCTD and norcantharidin were shown to have significant inhibitory effects on the srowth of CD19+ Nalm-6 cells(P＜0.001),while the purified 2E8 antibody did not show any significant influences on the growth of Nalm-6 cells.Since no significant inhibitory effects were identified among immunotoxin 2E8-NCTD,2E8 antibody and control groups on CD19-K562 cells.a significant targeting effect of the 2E8-NCTD against Nalm-6 cells was confirmed.Conclusions The immunotoxin 2E8-NCTD Was successfully synthesized by activated ester method with an excellent targeting killing effect on CD19+ Nalm-6 leukemia cells in vitro,which provides some experimental data for the further development of this new targeting agent.