Zhang Dengyang, LA Wenjun, Li Liuping, Wang Yan
School of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, Shenzhen 518044, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 Nov;31(11):1549-53.
To prepare the monoclonal antibody (mAb) against recombinant human interferon-β1a (rhIFN-β1a).
BALB/c mice were immunized with purified rhIFN-β1a. The spleen lymphocyte cells from the one whose blood specific antibody titer exceeded 1:10 000 were fused with Sp2/0 cells. The fused cells went through HAT selection, serial dilution, and positive selection until stable hybridoma cell lines were obtained. The equilibrium dissociation constant (K(D)) of antibodies was calculated based on ELISA result. The cell line with the best KD value was cultured in a stirred-tank bioreactor with a fed-batch strategy to produce secreted anti-rhIFN-β1a monoclonal antibody. The mAb released into the cell culture supernatant were purified by ultrafiltration and protein-G affinity chromatography.
Thirteen hybridoma cell lines which stably produced anti-rhIFN-β1a antibody were retrieved from standard hybridoma fusion and selection procedures. One of the 13 cell lines, 1E8 with the KD value of 4.6 × 10⁻⁹ mol/L was cultured and antibody titer in the cell culture supernatant reached 1:5000. The mAb with 90% purity was recovered from the two-steps purification.
The anti-rhIFN-β1a mAb with high purity has been successfully obtained.
制备抗重组人干扰素-β1a（rhIFN-β1a）的单克隆抗体（mAb）。
用纯化的rhIFN-β1a免疫BALB/c小鼠。选取血液特异性抗体效价超过1:10 000的小鼠的脾淋巴细胞与Sp2/0细胞进行融合。融合细胞经HAT筛选、系列稀释和阳性筛选，直至获得稳定的杂交瘤细胞系。根据ELISA结果计算抗体的平衡解离常数（K(D)）。将KD值最佳的细胞系在搅拌罐式生物反应器中采用补料分批策略进行培养，以生产分泌型抗rhIFN-β1a单克隆抗体。通过超滤和蛋白G亲和层析对释放到细胞培养上清液中的mAb进行纯化。
从标准的杂交瘤融合与筛选程序中获得了13个稳定产生抗rhIFN-β1a抗体的杂交瘤细胞系。其中1个细胞系1E8的KD值为4.6×10⁻⁹ mol/L，对其进行培养，细胞培养上清液中的抗体效价达到1:5000。通过两步纯化回收了纯度为90%的mAb。
已成功获得高纯度的抗rhIFN-β1a mAb。
