抗hMIF单克隆抗体的活性鉴定及其对脓毒症小鼠的保护作用

Bioactivity identification of anti-hMIF monoclonal antibody and its protective effect on sepsis mice

目的对制备的人巨噬细胞移动抑制因子(anti-human macrophage migration inhibitory factor,hMIF)单克隆抗体进行生物学活性鉴定,并研究其对脓毒症小鼠的保护作用。方法通过ELISA、Western blot、免疫组织化学等方法鉴定抗MIF单克隆抗体F11的亚类、抗体亲和力及特异性;通过一氧化氮(nitric oxide,NO)释放抑制试验和异构酶活性抑制试验,鉴定F11抗体的生物学活性;选用C57BL/6J小鼠构建CLP、LPS诱导脓毒症模型,研究F11抗体对脓毒症小鼠生存率的保护作用及对血清细胞因子TNF-α、IL-6的影响。结果 F11抗体亚类为IgG1型,亲和力为2.5×1010,Western blot及免疫组织化学显示强阳性;F11抗体显著抑制MIF刺激RAW264.7细胞释放NO(P<0.01)及MIF异构酶活性(P<0.01);明显提高脓毒症小鼠生存率(CLP模型:P<0.01,LPS模型:P<0.05)且具有剂量依赖关系,并对CLP术后血清TNF-α和IL-6的升高具有明显抑制作用(P<0.05)。结论成功制备了高特异性、高亲和力、具有异构酶活性抑制作用的抗MIF单克隆抗体,显著提高致脓毒症模型小鼠生存率并降低小鼠血清TNF-α、IL-6水平。

Objective To identify bioaetivity of anti-human macrophage migration inhibitory factor （hMIF） monoclonal antibody （mAb） Fll, and to investigate its protective effect on sepsis mice. Methods Subclass, affinity and specificity of anti-hMIF mAb F11 were analyzed by ELISA, Western blotting and immunohistochemistry. Bioactivity of mAb Fll was identified by nitric oxide （NO） release inhibition test in MIF-stimulated RAW264.7 cells and tautomerase activity inhibition test. Protective effect of mAb Fll and its effect on serum levels of TNF-α and IL-6 were determined in C57BL/6J mice model of sepsis. Results The mAb F11 was of IgG1 subclass, and its affinity was 2.50 x 1010. Western blotting and immunocytoehemistry showed strong positive signals. In bioactivity research, mAb F11 significantly inhibited NO releasing from MIF-stimulated RAW264.7 cells as well as the activity of MIF tautomerase （P 〈0.01 ）. mAb Fll significantly improved the survival rate of sepsis mice （ CLP model ： P 〈 O. 01 ; LPS model ： P 〈 0.05 ） in a dose-dependent manner, and significantly suppressed the elevation of serum levels of TNF-α and IL-6 （P 〈 0.05 ）. Conclusion Anti-MIF mAb Fll is successfully prepared, which has high specificity and affinity. The mAb F11 can inhibit activity of MIF tautomerase, significantly improve the survival rate of sepsis mice and reduce serum levels of TNF-α and IL-6.