纯化和标记抗人轻链β2m单克隆抗体的方法

Efficient purification and label of anti-human beta 2-microglobulin light chain monoclonal antibody

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：The inoculation of hybridoma cell strain onto mouse abdominal cavity may obtain ascites containing mass antibody.Previous method to purify monoclonal antibody in ascites is complex and difficult to operate.OBJECTIVE：To prepare,purify and label anti-human leukocyte antigen（HLA）-I class molecule light chain monoclonal antibody,and to detect the expression of tumor cell surface HLA-I class molecules.METHODS：Hybridoma cells were inoculated onto the mouse abdominal cavity.Ascites containing anti-human light chain beta2-microglobulin antibody were obtained and purified with the modified caprylic acid-ammonium sulfate method.The purified monoclonal antibody was labeled with fluorescein isothiocyanate to detect peripheral blood mononuclear cells,T2 cells expressing blank HLA-A2 molecule and K562 cells surface HLA-I class molecules.The expression of HLA-I class molecules was determined by using flow cytometry and fluorescent microscopy.RESULTS AND CONCLUSION：The purified anti-human light chain beta2-microglobulin-fluorescein isothiocyanate monoclonal antibodies accounted for 96% purity.Flow cytometry results showed that,the HLA-I class molecules were highly expressed in peripheral blood mononuclear cells surface,lowly expressed in T2 cells,and not expressed in K562 cells surface.It is a simple and convenient method to purify ascites with the modified caprylic acid-ammonium sulfate method,and according prepare anti-human light chain beta2-microglobulin-fluorescein isothiocyanate.This method is effective to distinguish the levels of HLA-I class molecules expressed in various cells.