抗Aβ和AL双抗原的单克隆抗体亲和力与活性检测方法的建立

Establishment of Affinity and Bioactivity Analysis Methods for Monoclonal Antibody Targeting Aβ and AL

随着靶向β-淀粉样蛋白(β-amyloid,Aβ)和淀粉样蛋白轻链(amyloid light chain,AL)单克隆抗体的开发,建立一种有效的抗Aβ和AL单克隆抗体亲和力及其生物活性检测方法十分必要。Crenezumab是基于Aβ靶点研制的IgG4型全人源化抗Aβ单克隆抗体,对Aβ寡聚体具有较强的亲和力。前期计算机方法模拟抗体抗原分子结合试验证实,可能设计1种新型的抗体对2种抗原均有结合力和生物活性。本文基于研发同时具备抗Aβ和AL 2种抗原新型抗体的需求出发,以Crenezumab为例,建立间接ELISA方法,并用该方法分别检测Crenezumab抗体对Aβ寡聚体和AL纤维的亲和力。进一步建立SH-SY5Y细胞活性实验方法和ThT荧光分析方法,分别检测Crenezumab对Aβ和AL抗原的生物活性。本研究建立的检测方法可为同时靶向Aβ和AL的抗体的药学初步研究提供检测方法。

With the development of monoclonal antibodies targetingβ-amyloid(Aβ)and amyloid light chain(AL),it is necessary to establish effective analytical methods for the anti-Aβand AL antibody affinity and bioactivity assay.In this report we have used Crenezumab,a fully humanized IgG4 anti-Aβmonoclonal antibody(mAb)with relatively high affinity to Aβoligomers,as a model to establish ELISA and cell based activity assays.Our previous research of antigen-antibody binding simulation using computational method indicated that it was possible to mutate the existing mAb to generate novel antibody mutants with modified affinity and bioactivity to the Aβand AL,which may create novel mAbs targeting both antigens.To facilitate screening the novel antibodies targeting both Aβand AL,we established indirect ELISA method for quick affinity measurement of the mAb to Aβoligomers and AL fibrils.The results demonstrated assay reproducibility and reliability.Furthermore,cytotoxicity assay in SH-SY5Y neuroblastoma cell line and ThT fluorescence test were established for the antibody bioactivity detection to Aβand AL to show cell toxicity of Crenezumab to Aβ42 oligomers and inhibitory effect of AL fibrillogenesis by Crenezumab.So,both critical methods were successfully established and be applied to the screening and preliminary bioactivity assay of novel monoclonal antibodies targeting Aβand AL.