Preparation of mouse anti-human B7-2 monoclonal antibody and analysis of its role in inducing Fas and FasL on the surface of 8266 cells
10.3760/cma.j.issn.0254-5101.2019.01.009
- VernacularTitle:小鼠抗人B7-2单克隆抗体的制备及对8266细胞表面Fas及FasL的诱导作用
- Author:
Yuyu WANG
1
;
Tianming YAN
;
Zhiyao WANG
;
Lijun SHEN
;
Yuhua QIU
Author Information
1. 苏州大学医学部免疫学系 215123
- Keywords:
B7-2 molecule;
Hybridoma cell;
Monoclonal antibody;
FasL;
Fas
- From:
Chinese Journal of Microbiology and Immunology
2019;39(1):50-56
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare a mouse anti-human B7-2 monoclonal antibody ( McAb) and to study its effect on the induction of death-related molecules on the surface of tumor cells. -ethods Trans-genic cells, L929-B7-2, were used as the immunogen to immunize BALB/c mice. Through cell fusion, mul-tiple screening by immunofluorescence labeling and continuous subcloning, the hybridoma secreting B7-2 McAb was obtained. Biological characteristics of the McAb were analyzed using Ig subclass identification test strip, antigenic site competition inhibition assay and specific cell membrane molecules binding test. McAb was prepared through inducing ascites in vivo and then purified by protein G affinity chromatography. The purified McAb was co-cultured with 8266 cells, naturally expressing B7-2 molecules, to observe the expres-sion of Fas and FasL on cell surface by flow cytometry ( FCM) . Results The prepared B7-2 McAb labeled as 12G4 was successfully obtained with a titer of 0. 1 μg/5×105 cells. Its heavy and light chains were IgG2b and κ, respectively. The concentration of the purified ascites-derived antibody was 1. 61 mg/ml. FCM re-sults showed that the 12G4 McAb recognized cell membrane molecules well with a positive binding rate of 89. 6％ to 8266 cells. The mean value of the Fas molecule on the cell surface increased after incubating with 20 μg/ml of 12G4 McAb for 12 h and reached the peak of 62575. 8 at 48 h, which was significantly higher than the maximum value of 57135. 4 in the IgG control group (P<0. 05). After culturing the cells with 20μg/ml of 12G4 McAb for 12 h, the expression of FasL on the cell surface also increased and reached the maximum of 7. 98％ at 48 h, which was significantly higher than the 1. 10％ in the IgG control group ( P<0. 05). Conclusions B7-2 McAb was successfully prepared. It could be used to induce the expression of some death-related molecules on the surface of tumor cells.
