瓜氨酸化α-烯醇化酶特异性单克隆抗体制备及其在阿尔茨海默病动物模型中的检测应用

Development of a monoclonal antibody specific for citrullinatedα-enolase and verification in vivo in a mouse model of Alzheimer's disease

本研究利用杂交瘤技术制备瓜氨酸化α-烯醇化酶特异性单克隆抗体,并探究瓜氨酸化α-烯醇化酶抗体在检测阿尔茨海默病(AD)动物模型中的作用。利用生物信息学软件分析预测α-烯醇化酶的B细胞优势表位,选取~9R-^(25)F位氨基酸序列,将该表位多肽中2个精氨酸替换为瓜氨酸,化学合成瓜氨酸化α-烯醇化酶~9R-^(25)F多肽(citrullinatedα-enolase peptide,CEP)。采取活泼酯法将CEP多肽与BSA偶联制备CEP-BSA偶联抗原并免疫BALB/c小鼠,杂交瘤技术制备CEP单克隆抗体。免疫组织化学和Western blotting对其特异性进行验证。CEP-BSA偶联抗原免疫小鼠能诱发特异性抗体反应,经细胞融合、筛选得到一株稳定分泌CEP特异性单克隆抗体的杂交瘤细胞,命名为9B5。Western blotting分析显示该单克隆抗体能特异性结合瓜氨酸化α-烯醇化酶,免疫组织化学检测显示9B5单抗能结合AD模型大鼠脑组织细胞上瓜氨酸化的α-烯醇化酶。本研究成功制备的瓜氨酸化α-烯醇化酶特异性单克隆抗体将有助于进一步研究瓜氨酸化α-烯醇化酶在相关疾病发病中的作用。

We generated monoclonal antibody specific for citrullinated α-enolase and examined its function in animal model of Alzheimer＇s disease. Bioinformatic analysis of B cell epitope showed that the immunodominant epitope was located in 9R-25F fragment, and the two arginines in the 9R-25F region were substituted by citrullines before synthesis and the product was named eitrullinated α-enolase peptide（CEP）. Monoelonal antibody against CEP was produced by hyhridoma technique. Conjugated antigen of CEP-BSA was prepared through coupling approach along with cell fusion and selection. We screened out one strain of hybridoma secreting CEP specific monoclonal antibody constantly, namely 9B5. The result of Western blotting demonstrated that 9B5 monoelonal antibody could specifically bind citrullinated α-enolase. IHC showed that 9B5 monoclonal antibody could specifically bind with α-enolase expressed in the brain cells of the animal model of Alzheimer＇s disease. This new antibody will aid in the further study of CEP＇s role in pathogenesis of related diseases.