[Preparation of three functional monoclonal antibodies against human FL molecule and analysis of their bioactivity].
OBJECTIVE
To Prepare three functional monoclonal antibodies(mAbs) against human FL molecule and analyze their bioactivity.


METHODS
The cell line L929-FL transfected with human FL gene was used as immunogen. The hybridomas secreting the antibodies against human FL were obtained by fusing splenoecytes from the immunized mice with murine myeloma cells(Sp2/0). Their subclasses were analyzed using fast-strip method. The monoclonal antibodies were produced in mouse peritoneal cavity and purified by Protein G affinity chromatography. The inhibitory effect of mAbs against FL on leukemia cell lines U937 and HL-60 was detected by MTT. The apoptosis of U937 and HL-60 cells stained by annexin-V/PI was determined by FCM.


RESULTS
Three hybridomas named 3C2, 3C6 and 8D10 were successfully obtained, which secreted monoclonal antibodies against human FL molecule stably. Their subclasses were the mouse IgG2a with kappa light chains. The three monoclonal antibodies recognized the FL molecule on U937 and HL-60 cells that also coexpressed Flt3 molecule. When U937 and HL-60 cells were cultured in presence of 3C2, 3C6 and 8D10, their proliferation was reduced as compared to that in control in MTT assay(P < 0.05). The analysis of annexin-V/PI binding to U937 and HL-60 cells by FCM showed the mAbs had the apoptotogenic activity of the monoclonal antibodies against human FL molecule.


CONCLUSIONS
3C2, 3C6 and 8D10 are three funtional monoclonal antibodies against human FL molecule. They may be of some value in the study of the roles of FL/Flt3 interaction in leukemia pathogenesis.
Zhou
Chun-gang
CG
Department of Immunology, School of Medicine, Soochow University, Suzhou 215007, China.
Tian
Xiao-jing
XJ
Ju
Song-guang
SG
Xu
Ying
Y
Yu
Ge-hua
GH
Zhang
Xue-guang
XG
Gu
Zong-jiang
ZJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D050440:Immunoglobulin Subunits / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C