抗腺病毒载体单克隆抗体的制备及其初步鉴定

Preparation and Identification of Monoclonal Antibodies against Adenovirus Vector

目的研制抗腺病毒载体（Adv）表面蛋白的单克隆抗体（McAb）。方法将Adv以Al（OH）3佐剂化，常规免疫BALB／c小鼠。采用细胞融合技术制备抗Adv McAb；采用ELISA、免疫细胞化学及Western blot法对McAb进行筛选与鉴定。结果共获得6株可稳定分泌特异性McAb的细胞（A4H11、A8C7、F1H5、G1D2、G4E3、H2G8），其染色体数目为102～106条，所分泌McAb均属IgG1亚类，持续3个月适应培养仍能保持其稳定的抗体分泌能力。腹水抗体的ELISA效价在1：10^6～1：10^8之间；Western blot结果显示6株McAb均与来自腺病毒载体及野生3、5、7型腺病毒抗原提取物中分子量约为114kD的多肽反应，据此推测它们均是抗ADV-TK六邻体蛋白的McAb。结论成功制备出6株抗腺病毒载体六邻体单克隆抗体，为腺病毒载体应用于肿瘤基因治疗的临床前研究提供物质基础。

Objective To prepare and identify monoclonal antibodies （McAb） against the capsid proteins of adenovirus vector. Methods BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and Al（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of McAb were identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blot. Results Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After subculture and low concentration of serum adapting culture for 3 months, 6 strains retained their stability to secrete McAb. The chromosomal number of the 6 cell lines ranged from 102 to 106 and all McAbs types were IgG1. The ascites titers were between 1 ： 10^6 and 1 ： 10^8. Western blot analysis revealed that all the McAbs reacted with one protein （about 114 kD） which was present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion Successfully prepared 6 strains of hybridoma cells secreted McAbs against the hexon proteins of adenovirus vector, which provided the substantial foundation of preclinical research of adenovirus vectors.