抗人Leptin单克隆抗体的制备、鉴定及初步应用

本研究用本室制备的重组人Leptin为抗原,以鼠伤寒沙门氏裸菌为佐剂,通过脾内、腹腔、静脉三种途径相结合免疫BALB/c鼠,以PEG为促融剂,将免疫小鼠的脾细胞和SP2/0细胞进行融合,HAT、HT选择性培养基、间接ELISA筛选阳性克隆,有限稀释法进行4次克隆化,获得三株能稳定分泌抗人Leptin单抗的杂交瘤细胞株。对所获得的细胞株及其分泌的单抗特性进行较系统的鉴定显示,获得的单抗特异性高,亲和力强,免疫球蛋白亚类均为IgG3。初步应用的研究显示,获得的单抗不仅可用于体外Leptin的免疫印迹检测,而且可通过免疫组化、免疫荧光等技术用于脂肪组织中Leptin的检测,为Leptin的基础和临床研究奠定了基础。

PREPRATION AND PRELIMINARY APPLICATION OF MONOCLONAL ANTIBODY AGAINST HUMAN LEPTIN

Recombitant human leptin(rh-Lep)was conjugated to naked bacteria of salminella typhimurium as immunogen to immunize BALB/c mice. Rh-Lep was administered by three routes of intrasplenic inoculation, intraperitoneal and intravenous injection. The spleen cells were fused with SP2/0 cells using polyethylene glycol and hybridoma cells were selected by HAT medium and screened with enzyme-linked im-munosorbent assay(ELISA). After rounds of cloning with limited dilution method, three of hybridoma cell lines were obtained which stably secret monoclonal antibodies of IgG3 against human leptin with high specificity and affinity. MCAbs were tested for their potential uses and the results showed that these McAbs can be used in Western blotting, Immunohistochemical and Immunofluorescent techniques.