Wu Hong-ya, Wang Qin, Chen Yong-jing, Xie Fang, Mao Yi-xiang, Liu Gao-qin, Zhang Xue-guang
Institute of Medical Biotechnology, Soochow University, Suzhou 215007, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Aug;23(8):757-60.
To prepare novel anti-OX40L functional monoclonal antibodies and characterize their distinct biological functions.
Routine immunization of BALB/c mice by a tansfected cell line L929/OX40L expressing high level of OX40L as antigens. Then fuse the immunized spleen with Sp2/0, a kind of myloma, and screen the positive clones by FCS with L929/OX40L as a positive control.After acquisition of the hybidomas secreting anti-OX40L mAb, investigation of their biological activities by Western blot, rapid isotyping analysis, karyotype analysis, competitive inhibition test, indirect immunofluorescence and MTT incorporation assay were followed.
Obtain two stable hybridomas, 4D6 and 5C2, which could continuously secret specific anti-OX40L monoclonal antibodies. The following biological activity studies showed that these monoclonal antibodies could both recognize the natural OX40L expressed on the mature DC, especially the OX40L on the several leukemia cell lines, such as Jurkat, SHI-1, U937 etc. Furthermore, they could also suppress the proliferation of SHI-1 in vitro.
Two hybridomas secreting anti-OX40L monoclonal antibodies continuously and steadily have been established. These monoclonal antibodies could specifically recognize human OX40L and effect the proliferation of leukemia cell line SHI-1 through OX40/OX40L costimulatory signals.
制备新型抗OX40L功能性单克隆抗体并表征其独特的生物学功能。
用高表达OX40L的转染细胞系L929/OX40L作为抗原常规免疫BALB/c小鼠。然后将免疫后的脾脏与骨髓瘤细胞Sp2/0进行融合，以L929/OX40L作为阳性对照，通过荧光激活细胞分选术筛选阳性克隆。获得分泌抗OX40L单克隆抗体的杂交瘤细胞后，接着通过蛋白质印迹法、快速亚型分析、核型分析、竞争抑制试验、间接免疫荧光法和MTT掺入法研究其生物学活性。
获得两个稳定的杂交瘤细胞株4D6和5C2，它们能持续分泌特异性抗OX40L单克隆抗体。随后的生物学活性研究表明，这些单克隆抗体均能识别成熟树突状细胞上表达的天然OX40L，尤其是几种白血病细胞系（如Jurkat、SHI-1、U937等）上的OX40L。此外，它们还能在体外抑制SHI-1的增殖。
已成功建立了两个能持续稳定分泌抗OX40L单克隆抗体的杂交瘤细胞株。这些单克隆抗体能特异性识别人类OX40L，并通过OX40/OX40L共刺激信号影响白血病细胞系SHI-1的增殖。
