消减免疫法制备抗肝癌凋亡细胞相关抗原单克隆抗体

Preparation of mAb against apoptosis-associated antigen of hepatomacells with subtractive immunization

目的 分析肝癌细胞凋亡过程中相关分子的表达情况 ,制备抗凋亡相关抗原的 m Ab.方法 用 6 0 m L· L- 1乙醇作用 6 h诱导 HCC- 92 0 4细胞凋亡 . May- Grunwald- Giemsa(MGG)法染色观察细胞形态 .流式细胞仪分析细胞 DNA含量 .环磷酰胺消减免疫法免疫 10只小鼠 ,另取 10只小鼠用常规法免疫作为对照 .末次免疫后取鼠尾血清进行免疫细胞化学法检测 .选取尾血清与凋亡细胞和非凋亡细胞的反应差异最明显者取脾细胞 ,与骨髓瘤细胞 SP2 /0融合 . EL ISA法筛选 .计算杂交瘤细胞融合率和抗体阳性率 .有限稀释法连续克隆化 .免疫细胞化学法进一步筛选 .Western blot法分析m Ab相关抗原的分子质量 .结果 MGG染色可见大部分细胞出现凋亡 .流式细胞仪分析可见亚二倍体凋亡峰 .实验组有 2只鼠的尾血清在凋亡细胞呈强阳性 ,而与非凋亡细胞的反应很弱 .两组的细胞融合率无明显差异 ;在凋亡细胞高表达 ,而在非凋亡细胞低表达的抗体的产生率也未见明显差异 .实验组总的抗体产生率明显低于对照组 (P<0 .0 1) ,在凋亡细胞与非凋亡细胞均高表达的抗体的产生率也明显低于对照组 (P<0 .0 1) .筛选出 1株在乙醇诱导的 HCC- 92 0 4凋亡细胞核内高表达 ,而在非凋亡的 HCC- 92 0 4细胞低表达的 m Ab.此 m

AIM To analyze the expression of associated m olecules of hepatoma during apoptosis, and prepare mAb against apoptosis-associ ated antigens. METHODS Apoptosis of HCC-9204 cells was induce d with 60 mL·L -1 ethanol for 6 h. The morphological changes in the cells were observed with May-Grunwald-Giemsa (MGG) staining. Cell DNA contents were analyzed with flow cytometer. Ten mice were immunized with cyclophosphamide sub tractive immunization method, and the other 10 mice with normal method as the c ontrol. The tail serum after last immunization, were detected with immunocyto chemistry. The spleen cells were taken from the mice whose serum reacted posit ively with apoptotic cells but poorly with unapoptotic cells, and were fused wit h myeloma cell line SP2/0. The molecular mass of the mAb-associated antigen wa s analyzed with Western blot. RESULTS Most cells showed apoptot ic changes. Sub-G1 apoptotic peak was seen. There were two mice whose tail ser um reacted strongly with apoptotic cells, but very weakly with unapoptotic cells . Between the experimental and control groups, the fusion rates weren't signific antly different; the production rates of antibodies which reacted strongly with apoptotic cells, but weakly with unapoptotic cells, weren't significantly differ ent, either. The total production rate of antibodies in the experimental group w as significantly lower than that in the control group (P<0.01). The producti on rates of antibodies which reacted with both apoptotic and unapoptotic cells s trongly in the experimental group were markedly lower than those in the control group (P<0.01). An mAb was screened out after cloning, and it reacted strong ly with the ethanol-induced apoptotic HCC-9204 cells, but weakly with unapopto tic HCC-9204 cells. The relative molecular mass of this mAb-associated antigen was about 75×103. CONCLUSION Subtractive immunization is he lpful to prepare mAb against apoptosis-associated antigen.