抗人IL-17C单克隆抗体的制备和功能鉴定

Preparation and identification of monoclonal antibodies against human IL-17C

目的制备特异性识别人IL-17C的单克隆抗体并进行功能鉴定。方法使用6周龄雌性BALB/c小鼠,利用杂交瘤技术制备抗人IL-17C单克隆抗体。对所制备的单克隆抗体进行抗体效价检测、交叉反应性测定、特异性鉴定以及亚型鉴定。利用免疫荧光和免疫组化对所制备的IL-17C单克隆抗体进行初步功能鉴定。结果成功筛选出9株能稳定分泌抗人IL-17C单克隆抗体的杂交瘤细胞株,分别命名为1F11、3A3、7F10、9B3、10C6、10A7、10B10、12F1和12F7,该9株杂交瘤克隆均具有受体阻断活性。其中,抗人IL-17C单抗7F10和10A7可与小鼠IL-17C发生交叉反应;可选择性结合人IL-17C,而与IL-17家族其他成员均无交叉反应,且均属于IgG1亚类抗体,轻链为κ链。以抗人IL-17C单抗7F10为代表进行了功能实验,结果显示,其作为一抗可成功应用于免疫荧光和免疫组化实验。结论成功制备了抗人IL-17C单克隆抗体,且7F10成功应用于免疫荧光和免疫组化检测,为IL-17C单抗在相关疾病的诊断和治疗方面奠定了基础。

Objective To prepare and identify monoclonal antibodies against human IL-17C.Methods The monoclonal antibodies against human IL-17C(mAbs)with hybridoma technique using six-week-old female BALB/c mice.The titers,cross-reactivity,specificity and subclass of the mAbs were determined.The applicability of the mAb was verified by immunoassay,and the mAb 7F10 was used as primary antibody for immunofluorescence and immunohistochemistry analysis.Results Nine hybridoma clones stably secreting mAb against human IL-17C were successfully screened out and named as 1F11,3A3,7F10,9B3,10C6,10A7,10B10,12F1 and 12F7,which all possessed significant receptor-blocking activity.Further studies showed that clone 7F10 and 10A7 could cause a cross-reaction between human and mouse IL-17C;and no cross-reactivity between mAb 7F10 or 10A7 and other members of the IL-17 family was observed.Both 7F10 and 10A7 were the IgG1 subclass antibodies,and the light chain was kappa type.The mAb 7F10 as primary antibody was successfully applied in immunofluorescence and immunohistochemistry assays.Conclusion The monoclonal antibodies against human IL-17C have been successfully prepared,among which 7F10 can be successfully applied in immunofluorescence and immunohistochemical detection,indicating that 7F10 may be used for diagnosis and treatment of IL-17C-associated diseases clinically.