[Preparation and characterization of a monoclonal antibody against human c-Kit].
OBJECTIVE
To prepare anti-c-Kit monoclonal antibodies and characterize their specificity of epitope recognition.


METHODS
cDNA encoding human c-Kit extracellular domain was constructed into a procaryotic expression vector pQE30 and the correctness of the reconstructed plasmid pQE30-KitD4-5 was verified by sequencing. The plasmid was transformed into E.coli M15 strain. Recombinant 6 x His pQE30-KitD4-5 was expressed after induction by IPTG for 4 h. Then SDS-PAGE results suggested that the products mainly formed inclusion bodies. The fusion protein was further purified with Ni-NTA-His affinity chromatography and then used to immunize BALB/c mice. The hybridomas were achieved by fusing the immunized spleen cells with the Sp2/0 myeloma cell line. The positive clones were screened by FCM with CHO-hKit cells. Hybidoma clones secreting anti-c-Kit antibodies were further subcloned and investigated for their biological activities by Western blot, rapid isotyping analysis and FCM.


RESULTS
Recombinant human c-Kit fusion proteins were in vitro expressed and purified to be used as immunogen. One stable hybridoma cell line, which continuously secrets specific anti-c-Kit monoclonal antibody ((SRJ1)) was established. The biological activity studies showed that the monoclonal antibody recognized the natural c-Kit expressed on the Kasumi leukemia cell line, but failed to bind to the normal human peripheral blood cells. Interestingly, this monoclonal antibody failed to recognize a subpopulation of Kasumi cells that is reactive with the commercial anti-c-Kit mAb Ab81 suggesting that the c-Kit expressed by this subpopulation contains some sequencial and/or structural aberrations that are distinguishable by mAb SRJ1.


CONCLUSIONS
With an immunization procedure using purified recombinant human c-Kit fusion proteins. a hybridoma cell line continuously and stably secreting anti-c-Kit monoclonal antibody has been established. The monoclonal antibody SRJ1 specifically recognizes human c-Kit expressed on the leukemia cells, and may provide a novel approach to analyze the possible structural variations of c-Kit expressed by different cells.
Liu
Hong-chen
HC
State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. liuhcsky@163.com
Mao
Chao-ming
CM
Su
Xiao-yu
XY
Ruan
Zheng
Z
Ding
Qiu-lan
QL
Wang
Xue-feng
XF
Wang
Hong-li
HL
Xi
Xiao-dong
XD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D019009:Proto-Oncogene Proteins c-kit / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D019009:Proto-Oncogene Proteins c-kit