一株抗人BLys单克隆抗体的制备及其生物学功能研究

研究以稳定高表达BLys的基因转染细胞L-BLys为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,L-BLys细胞和天然表达BLys的HL60细胞为抗体筛选阳性细胞株,经免疫荧光标记分析反复筛选和以有限稀释法反复克隆化培养。将获得的克隆4F7培养细胞注射入经致敏的小鼠体内,-抽取的腹水经Protein G亲和层析柱纯化。将纯化所得的抗体作用于经BLys分子刺激的扁桃体B细胞,3H-TdR检测细胞的增殖效率。结果获得1株持续、稳定分泌鼠抗人BLys单克隆抗体的杂交瘤细胞株。该细胞株所分泌的抗体能阻断膜型和可溶性BLys分子对扁桃体B细胞的促增殖作用。对这株单克隆抗体生物学功能的研究表明,这株单抗能特异性识别人BLys分子及阻断膜型和可溶性BLys发挥生物学活性。

Preparation and characterization of a functional monoclonal antibody against human tumor necrosis factor and B lymphocyte stimulator

A functional monoclonal antibody against the human tumor necrosis factor and B lymphocyte stimulator(Blys) was prepared and characterized by immunization of BALB/c mice with human Blys molecule from transfected cell line with stably expressed Blys gene as immunogen.By means of the hybridoma technique for B lymphocytes to cell fusion,repeated screening,multiple cell cloning as well as by using L-Blys cells and naturally expressed Blys-HL60 cells as the antibody screening positive cell lines, one hybridoma cell line was thus obtained and named as 4F7 clone.This clone was propagated by injection into the sensitized mice,the ascetic fluid of mice was collected and purified by affinity chromatography with protein G.The purified antibody was then added to the culture system of the tonsil B lymphocytes stimulated by the Blys molecule and ~3H-TdR incorporation assay was used to test the rate of cell proliferation.It was found that the 4F7 clone could continuously secrate the monoclonal antibody specifically recognizing Blys molecule and blocking the promoting effect on the proliferation of tonsil B lymhocytes induced by membranous and soluble types of Blys molecule.