抗人淋巴细胞再循环受体单克隆抗体的制备与鉴定

Preparation and characterization of monoclonal antibody to lymphocyte homing receptor in human

用人外周血高纯度的淋巴细胞做抗原，通过杂交瘤技术，获得一鼠抗人单克隆抗体（ＭｃＡｂ）－ＱＭ５（ＩｇＧ＿１，Ｋ轻链）。ＭｃＡｂＱＭ５能特异地与９８％的淋巴细胞，５２％的单核细胞和１０％左右的胸腺细胞反应，而不与红细胞、血小板和多核白细胞反应，与ＯＫＴ＿３和ＯＫＴ＿８抗原亦不相关。ＭｃＡｂＱＭ５尤其能特异地阻断人淋巴细胞与周围淋巴结内毛细血管后静脉内皮细胞结合，阻断作用与剂量相关，３０μｇ的ＭｃＡｂＱＭ５可完全抑制其结合。定量分析ＭｃＡｂＱＭ５的结合特性，证明每一淋巴细胞表面能结合大约１．２×１０￣５个ＭｃＡｂＱＭ５分子。用Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析，ＭｃＡｂＱＭ５能与淋巴细胞蛋白溶解产物中一种多肽成分结合，其分子量为１３５±３Ｋｄ（还原条件下为１４８±３Ｋｄ）。出此，ＭｃＡｂＱＭ５可用于鉴定新的淋巴细胞再循环受体。

High purity(98%)peripheral blood lymphocytes from healthy humandonors was used as immunogen,and defined by means of stringent selection mousemonoclonal antibody(McAb)QM5(IgGl,κ light chain). McAbQM5 specifically bindto 98%of lymphocytes, and 10%of thymocytes,neither to erythrocytes, platelets norto granulocytes,and did not recognize OKT3 and OKT8 antigen. McAbQM5 blocks lymph- ocytes adhesion to the high endothelial venules(HEV) of peripheral and mensentericlymph nodes in the Stamper-Woodruff assay,and the inhibition was dose dependent withmaximal effect at 0.03mg/ml.Quantitation with labeled ￣125I-McAbQM5 revealed 1.2×10_5 binding sites/lymphocyte.By Western blot, McAbQM5 reacted to a single polypep- tide(QM5 antigen)from lymphocyte cytoplasmic membrane,with a MW of 135 ± 3Kdwhich increased to 148±3Kd by reduction,indicating presence of intrachain disulfidebond(s).These results strongly suggested that McAbQM5 recognizes a new homingreceptor of human lymphocyte,and would be a useful tool for detectinghoming receptor.