Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product

作     者：Guoxin Wu Sethu Sankaranarayanan Donna L.Montgomery Adam J.Simon Zhiqiang An Mary J.Savage 

作者机构：Department of NeurologyMerck Research LaboratoryWest PointPA 19486USA Department of Biologics ResearchMerck Research LaboratoryWest PointPA 19486USA 

出 版 物：《Protein & Cell》 (蛋白质与细胞（英文版）)

年 卷 期：2011年第2卷第7期

页      面：573-584页

核心收录：

学科分类：1002[医学-临床医学] 10[医学] 

主　　题：scFv antibody BACE1 amyloid-βprecursor protein(APP) immunoassay 

摘      要：We have previously described a novel artificial NFEVβ-secretase(BACE1)cleavage site,which when introduced into the amyloid-βprecursor protein(APP),significantly enhances APP cleavage by BACE1 in in vitro and cellular *** this study,we describe the identification and characterization of a single chain fragment of variable region(scFv),specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide,and its conversion to *** the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage ***-IgG1 can detect as little as 0.3 nmol/L of the EV ***-IgG1 antibody was purified,conjugated with alkaline phosphatase and utilized in various biological *** the BACE1 enzymatic assay using NFEV substrate,a BACE1 inhibitor MRK-3 inhibited cleavage with an IC50 of 2.4 nmol/L with excellent *** an APP_NFEV stable SH-SY5Y cellular assay,the EC_(50) for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L,consistent with values derived using an EV polyclonal *** an APP_NFEV knock-in mouse model,both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage *** conclusion,the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro,cellular screening assays and in vivo biochemical *** methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.