人-鼠杂交瘤制备人单克隆抗体技术的研究

Study on Preparation of Human Monoclonal Antibodies by Human-mouse Heterohybridomas

本文采用小鼠骨髓瘤细胞系(Sp2/0)与破伤风类毒素(TT)免疫的人外周血淋巴细胞(PBL)进行融合,对人—鼠杂交瘤细胞系制备人单克隆抗体的技术条件进行了探讨。按免疫时间(d)及体外刺激与否分为4组。结果表明:(1)PWM+TT体外刺激可导致B细胞总数增加,且融合率高,容易建成稳定分泌人单抗的细胞株。(2)经TT免疫的人PBL,6d就有抗TT抗体(IgG)的前体细胞出现。15d及21d时,抗TT抗体前体细胞的频率更高。(3)体内免疫7d及14d并分别经体外刺激的两组共建立5株阳性株。经双扩碓定IgG2株,IgM3株。冻存10个月后复苏、传代及适当的克隆化后仍为阳性。上清液中人Ig的分泌量为0.42～1.15μg/ml。经染色体核型鉴定,碓认为人-鼠杂种细胞。

The techniques for preparing human monoclonal antibodies by human-mouse heterohybridomas were studied. The murine myeloma cells ( Sp2/0 ) were fused with human peripheral blood lymphocytes ( PBL ) immunized with tetanus toxoid(TT). The results indicated: ( 1 ) The total amount of B cells in PBL was increased by in vitro activation with PWM + TT and the fusion ratio was higher. The cell lines stably secr-eting the antibodies were obtained easily. (2 ) The precursor B cells capable of secreting specific anti-TT antibodies appeared on the 6th day after immunization with TT and the frequency of precursor B cells was much higher on the 15th and 21th day. ( 3 ) 5 positive cell lines in two groups were established on the 7th and 14th day after immunization and by in vitro activation, respectively. Two of the cell lines secreting IgG, and the other three IgM by the double diffusion assay were also established. The hybrid cells still produced anti-TT antibodies 10 month's freezing after and the human Ig concentration in the culture supernatants was 0.42-1.15μg/ml, Karyotype analysis confirmed that they were human-mouse hybridomas.