摘要
目的:通过从外周血中分离B细胞并进行培养,对外周血中存在的alpha-synuclein(α-syn)自身抗体进行分析,以探索用α-syn自身抗体开发治疗帕金森病(PD)抗体药物的可行性。方法:采集健康捐赠人的外周血并分离外周血B细胞进行培养,对B细胞培养上清进行ELISA结合活性分析,并用硫黄素T(ThT)法检测B细胞培养上清中α-syn自身抗体的生物学活性。结果:细胞培养13 d后,镜检发现B细胞扩增孔数可以达到90%以上,表明B细胞在实验条件下生长良好并能维持抗体分泌能力。ELISA结果表明检测的10530个样品中有48个可以和α-syn单体结合(OD 450>0.5),其中36个可以同时结合α-syn单体和聚合体(OD 450>0.5),并且该36个样品和α-syn家族蛋白β-synuclein以及γ-synuclein无交叉反应,具有α-syn结合特异性。用硫黄素T(ThT)法对该组样品进行的抑制聚合活性检测,结果表明36个样品中有9个样品能够显著性地抑制α-syn单体聚合,并且其中两个样品25G07和55F02的抑制率可以达到50%。结论:可以通过分离培养外周血B细胞获得具有生物学活性的α-syn自身抗体,用作治疗帕金森病的抗体药物研发。
Objective:To explore the feasibility of using alpha-synuclein(α-syn)autoantibody to develop therapeutic antibody drugs for Parkinson′s disease by analyzingα-syn autoantibody secreted by B cells in peripheral blood.Methods:B cells were isolated and enriched from peripheral blood collected from a healthy donor.ELISA and ThT functional assays were performed to check the binding and biological activities ofα-syn autoantibody in B cell supernatant.Results:After 13 days incubation,B cells were found to proliferate in over 90%wells,indicating that the primary B cells could grow and passage well under the experimental conditions and maintain the antibody secretion ability.ELISA results showed that 48 in total tested 10530 samples were able to bind toα-syn monomer and 36 of which bound to bothα-syn monomer and polymer(OD 450>0.5).The 36 samples did not cross react withβ-synuclein andγ-synuclein,indicating their good specificity toα-syn.Those samples were subsequently checked using ThT assay.It was found that 9 of 36 samples could significantly inhibitα-syn aggregation and the inhibition rate of two samples 25G07 and 55F02 could reach 50%.Conclusion:α-syn autoantibody from B cells in peripheral blood has biological functions,which can be used for the development of therapeutic antibody for Parkinson′s disease.
作者
车环宇
赵凌志
倪雪
滕国生
车翀
CHE Huan-Yu;ZHAO Ling-Zhi;NI Xue;TENG Guo-Sheng;CHE Chong(School of Medicine,Tonghua Normal University,Tonghua 134002,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2019年第23期2911-2915,共5页
Chinese Journal of Immunology
