Aim: 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.
Conclusion: 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.