两株CD45类单克隆抗体的产生及其特性

CD45即白细胞共同抗原,它是由同一结构基因经不同拼接后形成分子量不同的一组蛋白产物,至少包括220、205、190和180kDa的四种肽链,因而CD45尚有CD45RO、CD45RA和CD45RB的亚美。CD45具有受体蛋白的结构特征,其细胞内部分为酪氨酸磷酸酯酶,参与白细胞的激活、分化和生长调控。本实验旨在研制CD45类的单克隆抗体,为相应的基础和应用研究提供技术手段。应用杂交瘤技术共获得两个均为小鼠IgG1亚类的单克隆抗体,分别命名为NN26-2和LN2-2,它们与正常人外周血、骨髓单个核细胞、胸腺细胞和多种人白血病细胞系的反应性与CD45的分布相一致。免疫沉淀的结果表明NN26—2识别分子量为180～220kDa的蛋白成分,计算机软件分析也证明NN26-2和LN2-2同属CD45类抗体。本文报道的两个单克隆抗体已被第五届国际人白细胞分化抗原研讨会所接受,可以用于肿瘤组织免疫病理鉴别诊断、恶性淋巴瘤免疫分型、淋巴细胞亚群和造血干细胞分离。

GENERATION AND CHARACTERIZATION OF TWO MONOCLONAL ANTIBODIES REACTING WITH CD45 ANTIGEN

CD45(known as the leucocyte common antigen,LCA)is expressed on all leucocytes but is absent from non-hemopoietic tissues. There are at least four discrete chains with molecular weights of 180, 190, 205 and 220 kDa which are multiple products of a single structural gene produced by alternative splicing of the mRNA, sothat CD45 has some isoforms such as CD45RO, CD45RA and CD45RB. The sequence of CD45 showed that it had the characteristic structure of a receptor with a large intracellular segment of about 700 amino acids, which was confirmed to have tyrosine phosphatase activity and play an important role in leucocyte activation, differentiation and growth, and only the bone morrow cells expressed CD45 highly had significant numbers of self-renewing hematopoietic progenitors.The purpose of this study was the production of monoclonal antibodies(McAbs) that react with CD45. McAbs were generated by fusing NS-1 myeloma cells to spleen cells from BALE / C mice immunized with human leukemia cell line HPB-ALL intraperitoneally. Hybridomas were screened on the basis of their binding in an indirect immunofluorescence assay to various human leukemia cell lines. Two stable McAbs producing hybridoma lines were established by cloning and designated NN26-2 and LN2-2, respectively. Determination of immunoglobulin(Ig) class and subclass showed that both hybridoma cultures synthesized IgGl isotype. These hybridomas were injected into pristane-primed BALB / C mice intraperitoneally for large-scale antibody production and the liter of ascitic fluids was more than 10-4 tested with indirect immunofluorescence. Normal human peripheral blood cells, bone marrow mononuclear cells, thymocytes and a panel of human leukemia cell lines were tested and the reactivity of McAb NN26-2 and LN2-2 with the cells mentioned above was similar and equal to CD45, which had been confirmed by computer matching analysis with dataset WS4FCD. Immuno-precipitation of the antigen from lysates of 125I-labelled CEM cells indicated that McAb NN26-2 recognized a cell membrane protein with molecular weight of 180 to 220 kDa. NN26-2 and LN2-2 have been submitted to the Fifth International Workshop and Conference on Human Leucocyte Differentiation Antigens and accepted for detailed analysis. These McAbs against CD45 canbe used for immunohistochemical differential diagnosis of tumors, immunokhenotyping of malignant lymklumas, and separafim of and hemafopoiekc progenitors.