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背景:通过杂交瘤细胞株接种小鼠腹腔可获得含大量抗体的腹水,但以往纯化腹水中单克隆抗的方法较复杂,不易操作。目的:制备、纯化和标记抗人类白细胞抗原Ⅰ类分子轻链的单克隆抗体,以检测肿瘤细胞表面的人类白细胞抗原Ⅰ类分子的表达。方法:将杂交瘤细胞接种至小鼠腹腔,获得含抗人轻链β2m抗体的腹水,用改良的辛酸-硫酸铵方法纯化腹水,将纯化后的单克隆抗体标记异硫氰酸荧光素(FITC),标记后的抗体用于检测外周血单个核细胞、表达空载人类白细胞抗原A2分子的T2细胞和白血病K562细胞表面的人类白细胞抗原Ⅰ类分子,并用流式细胞仪和荧光显微镜观察人类白细胞抗原Ⅰ类分子的表达。结果与结论:纯化后的抗人轻链β2m-FITC单克隆抗体纯度为96%。流式细胞检测结果表明,人类白细胞抗原Ⅰ类分子在外周血单个核细胞表面高表达,在表达空载人类白细胞抗原A2分子的T2细胞表面低表达,而在白血病K562细胞表面不表达。结果证实,用改良的辛酸-硫酸铵方法纯化腹水以此制备的抗人轻链β2m-FITC能有效区别不同细胞表达人类白细胞抗原Ⅰ类分子的强弱,其纯化方法简便易行。
BACKGROUND: Ascites fluid containing large amounts of antibodies can be obtained by inoculating mice intraperitoneally with hybridoma cell lines. However, in the past, monoclonal antibodies against ascites were complicated and difficult to manipulate. OBJECTIVE: To prepare, purify and label monoclonal anti-HLA class I monoclonal antibodies to detect the expression of human leukocyte antigen class I molecules on tumor cells. Methods: The hybridoma cells were inoculated into the peritoneal cavity of mice to obtain ascites containing anti-human light chain β2m antibody. The ascites was purified by modified octanoic acid-ammonium sulfate method. The purified monoclonal antibody was labeled with fluorescein isothiocyanate (FITC ). The labeled antibodies were used to detect peripheral blood mononuclear cells, T2 cells expressing empty HLA-A2 and HLA class I molecules on leukemia K562 cells. Human leukocytes were observed by flow cytometry and fluorescence microscopy Antigen class I molecule expression. RESULTS AND CONCLUSION: The purity of purified anti-human light chain β2m-FITC monoclonal antibody was 96%. Flow cytometry showed that human leukocyte antigen class I molecules were highly expressed on the surface of peripheral blood mononuclear cells, but not expressed on the surface of T2 cell of leukemia K562 cells. The results demonstrate that the purified anti-human light chain β2m-FITC prepared by modified octanoic acid-ammonium sulfate method can effectively distinguish the expression of human leukocyte antigen class I molecules in different cells, and the purification method is simple and easy.