NT-proBNP特异性单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibody Specifically Against Human NT-proBNP

旨在制备并鉴定抗人NT-pro BNP特异性单克隆抗体。采用重组NT-pro BNP蛋白免疫Balb/c小鼠,取血清效价高的脾细胞与SP2/0细胞融合,经HAT筛选和有限稀释法亚克隆,获得阳性杂交瘤细胞株,用动物体内诱生法制备腹水,用正辛酸-硫酸铵沉淀法将抗体纯化,并测定抗体的亚类、效价、特异性及相对亲和力。结果表明,获得4株能稳定分泌抗人NT-pro BNP单克隆抗体的阳性杂交瘤细胞株,分别命名为1G12、1B2、3D5和2D11。抗体Ig亚类,1G12和1B2为Ig G2a型,2D11为Ig G1型,3D5为Ig G2b型;1G12,1B2和3D5的抗体效价达到106,而2D11的效价仅达104。特异性好,1G12、1B2、3D5和2D11四株抗体的亲和常数依次为9.32×1011、1.07×1012、2.31×1012和6.33×1010。成功制备了特异性抗人NT-pro BNP单克隆抗体。

This study aims to prepare and identify monoclonal antibodies specifically against human NT-proBNP. Balb/c mice was immunized with labeled recombinant protein NT-proBNP,then the spleen cells with high titer of the mice’s serum were fused with SP2/0 myeloma cells. Further,the positive hybridoma cell lines were established after HAT selection and subcloning by limiting dilution,ascites were prepared by injecting cells to mice’s abdomen,and monoclonal antibodies were purified by caprylic acid-ammonium sulfate precipitation method. Finally,basic functions of these monoclonal antibodies such as subclasses,titers,specificity,affinity constants were analyzed. Four positive hybridoma cell lines stably secreting human monoclonal antibodies of anti-NT-proBNP were screened out,and named as 1G12,1B2, 3D5,and 2D11;1G12 and 1B2 belonged to IgG2a,2D11 to IgG1,and 3D5 to IgG2b. The titers of 1G12,1B2 and 3D5 reached 106,while the titer of 2D11 only was 104 . The affinity constants of 1G12,1B2,3D5 and 2D11 were 9.32×1011,1.07×1012,2.31×1012,and 6.33×1010, respectively. In conclusion,the monoclonal antibody of specifically against human NT-proBNP was prepared successfully.