Fortebio-分子相互作用仪讲座.ppt

Click to edit Master title style,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Octet,生物分子相互作用系统,ForteBio Inc.,2004,年由华人物理学家创立,2009,年开始在中国销售,专利的生物膜干涉,Bio-Layer,Interferometry,(BL,I),技术,总部在美国硅谷,全球超过,350,个用户，中国,14,个用户,全球第二的专业非标记技术厂商,SDi,2010 Market Report survey,成长最快速的非标记技术公司,公司发展的远景：成为每个实验室都能拥有的分子互作系统,专业的服务,在中国有,2,名硬件工程师，,3,名应用工程师,除了提供批量生产的生物传感器以外，,还可以为用户定制传感器,提供实验方法开发和优化服务,国内用户名单,军事医学科学院,2,台,Novo Nordisc,中国研发中心,中国生物制品药品检定所,神州细胞工程有限公司,中科院武汉病毒所,厦门大学,天津商业大学,上海药明康德新药开发有限公司,GSK,中国研发中心,Novartis,中国研发中心,上海旭华生物科技有限公司,江苏太平洋美诺克生物药业有限公司,江苏泰康生物医药有限公司,实时检测，非标记的生物分子互作检测平台,Octet 可分析多种生物分子间的相互作用,Cells,Bacteria,Virus,IgM,IgA,IgG,IgD,IgE,Antibody Fragments,Proteins,Peptides,Nucleotides(DNA),Small Molecules,Atoms,200 nm,1000 nm,0.1 nm,1,1,000,100,000,1,000,000,75 nm,150,MW,Size,7 nm,1 nm,Bacteria,Virus,Antibody-Antigen,Receptor-Ligand,DNA-DNA,DNA-Protein,Antibody Fragment-Antigen,Antigen Fusion Protein,Antibody-Peptide,Multiple Antibody Pairings,Antibody-Small Molecule,Protein-Small Molecule,Current Octet assays within blue area,亲和力测定,Binding K,D,On and off rates,Rank order,结合特异性,Yes/No kinetic screening,Epitope,binning/mapping,蛋白质浓度定量（混合物）,Picogram,to milligram dynamic range,Contaminant testing,Octet,系统可提供的数据,Octet Solution for Drug Development,Wide Fit Within Discovery and Development Process,Lead Characterization,Screening,Preclinical and Clinical,Manufacturing,and QC,Assay development,Expression,monitoring,Clone selection and,ranking,Epitope mapping,Small molecule or ligand screening,Kinetic screening of hybridomas,Secondary hit confirmation,Affinity measurement,On and off-rates,Binding affinity ranking,Kinetic profiling of immunotherapeutics,Rapid concentration,measurements,Immunogenicity,21CFR CompliantProduction monitoring,Process optimization,Protein and small molecule therapeutics,Fast and accurate concentration measurements,Research,Octet 系统和特点,Octet 系统的优点,更高的通量,更快的实验流程,8-16,个样品同时检测，是非标记技术中通量最高的,15-30,分钟可以完成,96,个样品的分析,没有溶液控制系统，不需要清洗和维护流路，直接开始读取数据,操作简单,适应多用户的情况，经过半天的培训就可以操作和使用,不需要特别优化实验条件，传感器是抛弃式的,更宽的应用范围,直接检测粗制的样品，甚至是样品中存在不溶解的成分,耐受各种溶液环境，只有结合到传感器表面的分子才会被检测,病毒颗粒等大分子样品也能得到动力学结果,检测共价键结合的样品,更低使用和维护的成本,相对其他的非标记检测系统，耗材的成本低,系统稳定，未来的使用和维护成本也低,传感器种类,12,种,Application,Sensor Type,Regeneration,Quantitation,Anti-Human IgG Fc,Anti-Murine IgG(Fab)2,Protein A/G/L,Streptavidin(SA),Anti-Penta-HIS(HIS),Yes,Yes,Yes,Yes,Kinetics,Streptavidin(SA),Super Streptavidin(SSA),Amine Reactive(AR),Aminopropylsilane(APS),Anti-hIgG Fc Capture Surface(AHC),Anti-mouse Capture Surface,Yes,Yes,Yes,Yes,Yes,Kits,Dip and Read Immunogenicity Kit,Dip and Read Residual Protein A Detection Kit,No,No,可以根据用户的应用设计传感器,为目前种类最多的供应商,传感器可再生使用,ADA ID,Cycle#,nm Shift,Mab#2,1,0.7049,Mab#2,2,0.7435,Mab#2,3,0.7431,Mab#2,4,0.7822,Mab#2,5,0.7702,Mab#2,6,0.6712,Mab#2,7,0.7221,Mab#2,8,0.7654,Mab#2,9,0.7515,Mab#2,10,0.7729,Streptavidin(SA)sensor,表面固定了生物素化的蛋白质药物,10%,的猴血清样品，含有抗该药物的抗体,可同时平行优化,8,种传感器再生条件,每个传感器表面结合抗体以后进行再生，如此反复,10,次,由于,8,个传感器上的数据可以同时检测，因此,3,分钟内就可以得到,8,个数据,最终每个样品的检测成本仅为,4,元人民币,Avg.nm Shift,0.7427,StDev,0.0346,%CV,4.7%,Regeneration solution=1mM HCl,Octet,产品简介,Octet Instruments,5,种型号,-Octet QK,Octet,Qke,Octet RED,Octet QK384,Octet RED 384,使用通用的样品板,(96 and 384 well),没有流路系统,样品经过检测后，仍然留在原处，可以回收再使用,Biosensors,浸入样品即检测，,1,至,16个样品可以同时检测,10,种传感器可供选择，分析不同种类的样品,传感器为抛弃式设计，既可以检测共价键结合的样品,也可以再生使用,Software and computer,常用实验方法已经预制在模板中,不需要单独购买软件,Validation options,经过验证,提供,IQOQ,服务,符合美国,FDA,CFR Part 11,标准,Octet System Specifications,Octet QK,Octet QKe,Octet QK384,Octet RED96,Octet RED384,Molecular Weight Range,10,000 D,5000 D,5000 D150 D,150 D,Data Acquisition Rate,0.6 Hz,0.3 Hz or 0.6 Hz,0.3 Hz or 0.6 Hz,2,5,or 10 Hz,2,5,or 10 Hz,#Spectrometers,1,1,2,8,16,#Channels per Read,1-8,1-8,1-16,1-8,1-16,Microplate Positions,1,1,2,1,2,Biosensor Reracking,No,Yes,Yes,Yes,Yes,Robot Compatible,No,No,Yes,No,Yes,Microplate Formats,96,96 HA96,96 HA,96/96HA,384/384TW,96,96/96HA,384/384TW,Minimum Sample Volume,80 L per well,80 L per well,40 L per well,180 L per well,40 L per well,Affinity range(approximate),0.1 mM to,0.1 nM,0.1 mM to,0.1 nM,0.1 mM to,0.1 nM,1 mM to,10 pM,1 mM to,10 pM,Sample Usage,Non-destructive and recoverable,Temperature Control,4C above ambient to 40C,21 CFR Part 11 Compliance,Available as option for all systems,96 HA96 well microplate with half-area well size,384 TWForteBio 384 tilted-well microplate,Octet 384 系统的优点,兼容,96,孔板和,384,孔板,更高的通量,更少的样品消耗,最小体积只要,40ul,16,通道同时检测，让所需 实验时间更短,两个样品板位置,更多的实验方案,一次连续分析更多样品,全自动化解决方案,可与多种液体工作站连接,Octet 全自动功能,Automated door and plate stage,Works with articulated arm and track-style robotic systems,Plate stage presents the biosensor tray,sample and reagent plates for robot,Octet software has automation hooks enabled to interact with robot software,Octet“96”,系统的内部,自动化的传感器移动,样品一直保留在微孔板内，是不破坏样品的检测,“浸入”式检测，检测快,8,通道同时检测，一次可检测多个样品,Octet 384,系统的内部,两个微孔板的位置，一次可连续分析更多的样品,每个微孔板独立使用，即可以是,96,孔板，也可以是,384,孔板。即可以同时使用一块,96,孔板,+384,孔板,根据实验步骤设定将传感器在样品板的不同位置移动,当生物分子结合到传感器表面就形成了一层生物膜,生物膜对透过传感器的光的波形造成干涉现象,干涉现象以相位移动的方式被检测，从而可以检测结合到传感器分子数量的变化,生物膜光干涉技术,Bio-Layer Interferometry(BLI),Y=Y,0,+Ae,k,d,*t,Y=Y,0,+A(1-e-,kobs*t,),k,obs,k,d,Conc Ag(M),k,a,=,Ab+Ag Complex,k,a,k,d,K,D,=,k,d,k,a,For simple 1:1 binding:,k,a,=rate of association or“on-rate”,k,d,=rate of disassociation or“off-rate”,Non-linear curve fit of data,生物分子结合动力学,Buffer,Ligand-Biotin,Protein of Interest,Binding(nm),Time,Baseline,Loading,Baseline,Association,Dissociation,8-16,个样品可以被同时平行检测,检测数据被事实记录,实验方法可根据用户的要求来设计,动力学和亲和力由软件分析得到,Octet Biosensors,Octet,系统测定动力学的工作流程,Octet RED,进行蛋白质浓度测定的工作流程,Standards,TestSamples,Binding(nm),Time(sec),Binding Rate,Concentration,利用标准品在不同浓度时结合速率的差异来定量样品的浓度,15-30,分钟可以分析各样品,标准曲线可以重复使用,病毒颗粒等其他样品也可定量,Octet Biosensors,120,Octet,软件,可灵活输出各种格式，,Excel,表，,JEPG,格式等,动力学分析直接得到,ka,kd and KD,结果,浓度分析直接得到，,ug/ml,ng/ml,等浓度计算结果,快速增长的文献发表数量,从,2005,年底有第一个用户，到,2010,年有超过,100,多篇文献的发表,Multiple Systems at Top Pharma and Biotech,Octet Academic and Government Customers,Center for Disease Control,Emory University,Erasmus Medical Center,Fachhochschule Nordwestschweiz Basel,Florida Gulf Coast University,Institute of Basic Medical Sciences Academy,International AIDS Vaccine Institute,Jeonnam Institute,Kagoshima University,Kennesaw State University,Korea University,NIH,Penn State University,Scripps Florida,St.Jude University,Stanford University,Texas A&M University,TU Darmstadt,Universitt Wrzburg,University of Cambridge,University of Florida,University of South Florida,University of Michigan,University of Pittsburgh Cancer Institute,Upstate Medical College,MD Anderson Cancer Center,Van Andel Institute,Vanderbilt University,Vienna Institute of Biotechnology,Xiamen University,Study Liposomes and Virus Like Particales,Mutant 2,Full Analysis,With 3 Conc,Screening,(20nM only in duplicate),ka(1/Ms),2.56E+05,2.73E+05,kd(1/M),2.41E-05,2.59E-05,Kd(M),9.41E-11,9.49E-11,Mutant 1,Full Analysis,With 3 Conc,Screening20nM only in duplicate),ka(1/Ms),2.84E+05,3.21E+05,kd(1/M),8.59E-05,9.46E-05,Kd(M),3.02E-10,2.95E-10,Easily Determine Full Kinetics Characterization,Data provided by Genitope,Matrix Buffer,Ka,Kd,KD,50%Plasma,5.20E,6,7.91E,-6,1.52E,-12,PBS,4.39E,6,6.63E,-6,1.51E,-12,Octet QK Association,1500 ng/mL Ag,1130 ng/mL Ag,760 ng/mL Ag,390 ng/mL Ag,205 ng/mL Ag,113 ng/mL Ag,20 ng/mL Ag,Test Kinetics of Ab/Ag interaction Directly in Plasma,What are Liposomes/VLPs?,Liposomes(virus like particles-VLPs)are tiny“bubbles”(vesicles)made out of the same material as the cell membrane.They are not naturally occurring.,They are made of phospholipids,with a hydrophilic(water-soluble)head,and hydrophobic(lipid-soluble)tail,Size varies,typically from 10-150 nm in diameter,Example Workflow to study VLPs on the Octet,Data analysis flexible to accommodate inverted and non-inverted data,Donkey anti-mouse antibody binding Mouse Anti-CXCR4 antibody captured on lipoparticle-displayed CXCR4,(data flipped for analysis),Donkey anti-mouse antibody binding Mouse Anti-CXCR4 antibody captured Fab on the sensor,小分子化合物与药靶的结合,Furosemide(MW 330D),Acetazolimide(MW 222D),Sulpiride(MW 341D),Each compound run in a titration series of 5 concentrations in duplicate.,All data from one walk away run,IgG expressed in E.coli,monitored by Octet,Octet,直接分析混合样品，完全没有干扰,上清液与细胞裂解液的检测结果是一致的！,Octet,Centrifuged,Octet,Crude,0 g/ml,0 g/ml,280 g/ml,281 g/ml,42 g/ml,47 g/ml,323 g/ml,327 g/ml,132 g/ml,130 g/ml,耗材成本,实验成本,定量实验每个数据点,4-5,元人民币动力学实验，测定一组亲和力和结合速率常数,80-120,元人民币,耗材主要是传感器和,96,孔板,维护成本,接近于零,总结:Octet 系统的优点,更高的通量,更快的实验流程,8-16,个样品同时检测，是非标记技术中通量最高的,15-30,分钟可以完成,96,个样品的分析,没有溶液控制系统，不需要清洗和维护流路，直接开始读取数据,操作简单,适应多用户的情况，经过半天的培训就可以操作和使用,不需要特别优化实验条件，传感器是抛弃式的,更宽的应用范围,直接检测粗制的样品，甚至是样品中存在不溶解的成分,耐受各种溶液环境，只有结合到传感器表面的分子才会被检测,病毒颗粒等大分子样品也能得到动力学结果,检测共价键结合的样品,更低使用和维护的成本,相对其他的非标记检测系统，耗材的成本低,系统稳定，未来的使用和维护成本也低,Thanks,！,