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文章编号:100028020(2007)0620759204・综述・多重快速鉴别病原微生物的新技术:xM AP 液态芯片胡瑞 综述 王景林1 审校西北农林科技大学动物科技学院,杨凌 712100摘要:多指标同步分析(Flexible Multi 2Analyte Profiling ,xM AP )液态芯片技术是美国Luminex 公司近年来开发的一种新型生物芯片技术。
这种基于微球的芯片技术能够对单孔内多达100种不同的反应同时进行检测,与固相芯片或片膜芯片相比,具有多重、快速、灵敏度高(可达0101pg )、重复性好(C V <5%)以及检测动态范围宽(可达012～32000pg Πml )等优点。
目前,该技术已被广泛应用于各研究领域,尤其在核酸、蛋白质和其他生物分子的大规模分析中。
关键词:多指标同步分析 液态芯片 微球 病原微生物 检测中图分类号:R15114 文献标识码:AA rapid ,multiplexed ne w technology xMAP liquid chip fordetection and identification of pathogensHU Rui ,WANG JinglinC ollege of Animal Science and T echnology ,N orthwest A &F University ,Y angling 712100,ChinaAbstract :xM AP liquid chip is a new biochip technology developed by the Luminex corporation in recent years.This chip technology based on microsphere is capable of simultaneously detecting up to 100different analytes in a single reaction vessel.In com parition with the s olid chip or membrane chip ,it possesses the features of multi 2analytes ,high sensitivity(0101pg ),g ood reproducibility (C V <5%),wide channel (012-32000pg Πml ),and s o on.F or these advantages ,the xM AP liquid chip technology has been used in many fields ,especially in the analysis and identification of nucleic acid ,protein or other biological m olecules.K ey w ords :xM AP liquid chip ,microsphere ,pathogens ,detection基金项目:国家863计划资助项目(N o.2006AA06Z 414);国家自然科学基金资助项目(N o.30671824)作者简介:胡瑞,男,硕士研究生,研究方向:分子病原学与免疫学,E 2mail :hurui 2a @1通讯作者:王景林,军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,E 2mail :wangjlin @bm ,wangjl6481@ 目前,基于微生物学、免疫学、生物化学和分子生物学发展起来的病原微生物检测方法大致有以下几种:培养法、最大可能数法(Maximum Probable Number ,MPN )、免疫学方法、聚合酶链反应(PCR )和多重PCR (multiplex PCR )、实时定量PCR (real 2time PCR )、寡核苷酸芯片检测法等。
Electric heating cable 加热电缆field development 油田开发flexible riser 挠性隔水管heating line 加热管线…host‟ platform “主”平台…surface‟ conductor “表层”导管“dual bore” type tree “双筒”型采油树“temporary” guide base “临时”导向基座“two stack” approach “双防喷器组”法Abandonment and Recovery 弃置与回收acceptance criteria 验收标准Acceptance Test 验收测试accumulator bottle 蓄能器瓶alignment 找正alternating current 交流电ambient seawater temperature 周围海水温度ambient temperature 环境温度ancillary equipment 辅助设备annular space 环形空间annulus bore 环空孔annulus master valve 环空主阀annulus seal assembly 环空密封总成anti-rotation pin 防旋转销armour wire 铠装线armours layer 铠装层artificial lift 人工举升artificial lift method 人工举升法Asgard project Asgard项目Atmospheric Diving Suit 常压潜水服atmospheric pressure 大气压力attachement point 固着点Austenitic Stainless Steel 奥氏体不锈钢austenitic-ferric steel 奥氏体-铁素体钢auto depth 自动定深automatic connector 自动连接器average unevenness height 平均不均匀高度axial tension 轴向拉力backing steel 背钢backup function 备份功能barrier coating 防护涂层barrier protection 隔层保护basic oxygen furnace 碱性氧气转炉basket storage carousel 篮式存管盘bend limiter 弯曲限幅器bend radius 弯曲半径bend restrictor 弯曲限制器bend stiffener 弯曲加强杆bending loads 弯曲载荷bending moment 弯曲力矩bending reaction ring 弯曲止推环bending stiffness 抗弯刚度bending stress 弯曲应力Billet piercing process 坯板料穿孔工艺bleed valve 泄放阀blind cap 盲帽block valves 断流阀bypass valves 旁通阀Blowout Preventer 防喷器bollard pull 系缆拉力bolt flange connection 螺栓法兰连接bolted flange connection 螺栓法兰连接BOP connector 防喷器连接器BOP pipe ram 防喷器管闸板BOP Stack 防喷器组Bore Protector 井筒保护器bottom crawling vehicle 海底爬行车Buckle propagation 弯曲传播buckle propagation 弯曲传播Bull Nose 圆头管堵bundle 管束buoyancy module 浮力块Campos basin Campos盆地carbon steel 碳钢Carbon steel tube 碳钢管Carbon-Manganese steel pipe 碳锰钢管carrier pipe 输送管Casing Hanger 套管悬挂器casing pup joint 套管短节catenary curve 悬链曲线catenary shape 悬链线型cathodic protection 阴极保护detection system 检测系统Centrifugation Process 离心工艺Check Valve 止回阀chemical fluids 化学流体chemical injection 化学剂注入choke actuator 油嘴促动器choke insert 油嘴嵌件choke position 油嘴位置circuit breaker 断路器circuit length 电路长度circulate hot water 循环热水clad corrosion resistant alloy 复合防腐合金Clad Metal 复合金属clad pipe structure 复合管结构Clad pipe system 复合管系统clamp connector 紧固连接器clamp hub 卡箍Class I 第一类Class II 第二类Class III 第三类Class IV 第四类C-Mn steel pipe 碳锰钢管coating defect 涂层缺陷Coil tubing 连续油管coiled skelp 挠性管材collet connection 套管连接collet connector 套管连接器Completion / Workover Riser System 完井/修井隔水管系统completion equipment 完井设备completion guide base 完井导向基座Completion Guidebase 完井导向基座completion riser system 完井隔水管系统Component Change-Out tool 部件更换工具composite armour 复合铠装composite armour layer 复合铠装层composite material 复合材料compression force 压缩力compressive load 压缩载荷concentric tubing hanger 同心油管悬挂器Conductor Housing 导管头conductor resistance 导体电阻conductor string 导管柱Connection/reaction loads 连接/反作用载荷Connector profile 连接器剖面Continuous lamination 连续分层continuous length 连续长度continuous slab caster 板坯连铸机continuous strand caster 流坯连铸机control cabin 控制舱control fluid 压井液Control Function 控制功能Control junction plate 控制连接板control module 控制模块control pod 控制盒control room 控制室Control Station 控制站control umbilical 控制脐带管control valve 控制阀conventional style trees 常规型采油树Conventional Tree 常规采油树correct orientation 正确方向corrosion allowance 腐蚀裕量corrosion control 腐蚀控制corrosion inhibition 腐蚀抑制corrosion inhibitor 缓蚀剂CORROSION MONITORING 腐蚀监控corrosion protection 腐蚀防护corrosion protection system 腐蚀防护系统corrosion rate 腐蚀速度corrosion resistance 耐腐蚀corrosion resistant alloy 耐腐蚀合金corrosion resistant materials 耐腐蚀材料corrosive agent 腐蚀剂corrosive environment 腐蚀性环境coupler hydraulic 接头液压CROSS SECTION 截面cross sectional area 截面面积Cross Wound Polyethylene 交叉卷绕聚乙烯cross-flow oscillations 横流振动cross-section 截面crushing load 压碎载荷residual strain 残余应变Cumulative strain deformation 累积应变变形cumulative strain 累积应变current drag 海流阻力Current Limiter 限流器current loads 海流载荷current saturation 当前饱和current velocity 海流速度cylindrical billet 圆柱坯料data collection 数据采集data sheets 数据表data transmission 数据传输DC conductor resistance 直流导体电阻Dead Weight 船舶载重量debris cap 岩屑帽deck level 甲板水平面deck load 甲板载荷Deck space 甲板空间deep water 深水deepwater developments 深水开发deepwater field development 深水油气田开发DEEPWATER FIELD DEVELOPMENT REFERENCE BOOK 深水油气田开发参考手册deepwater flowline 深水出油管线deepwater pipeline 深水管道DEEPWATER REFERENCE BOOK 深水参考手册deepwater riser 深水隔水管Destructive Examination 破坏性检验development option 开发方案Differential Global Positioning System 差分全球定位系统Differential Pressure 压力差Digging chain 开挖链direct current 直流Direct Heating System 直接加热系统Directional Control Valve 方向控制阀Distributed Control System 分布式控制系统distribution piping systems 分配管道系统distribution system 配电系统Distribution Unit 分配装置Diving Support Vessel 潜水供应船Dolly base 转管滚座downhole gauges 井下压力计Downhole Pressure 井下压力Downhole Safety Valve 井下安全阀drag loads 拉力载荷drill pipe string 钻杆柱drilling operation 钻井作业drilling riser 钻井隔水管Driving wheel 驱动轮Dual Bore Split Body Tree 双筒拼合本体采油树dual bore subsea tree 双筒水下采油树dual bore tree 双筒采油树dual redundant 双冗余dual SEMs 双SEMDual Systems 双机系统Duplex stainless pipe 双相不锈钢管Duplex stainless steel 双相不锈钢Dynamic analysis 动态分析Dynamic positioning 动力定位dynamic positioning (DP) system 动力定位系统dynamic positioning system 动力定位系统dynamic response 动态响应dynamic riser 动力隔水管dynamically positioned lay vessel 动力定位铺管船Earth continuity test 接地连续性试验Edge trimming 切边edge-crimping 卷边Effective tension 有效张力electric cable 电缆electric current 电流electric heating 电加热electric power 电力Electric Power Unit 电力装置electric pump 电动泵electric resistance welded 电阻焊Electric resistance welded pipe 电阻焊管electric submersible pump 电动潜油泵electrical cable 电缆electrical component 电气部件electrical conductor 电气导线Electrical connection 电气连接electrical connector 电气连接件electrical current 电流Electrical Distribution Unit 配电装置Electrical electrode 电极Electrical Flying Lead 电引线Electrical isolation 电气隔离Electrical Jumper 电气跨接线Electrical junction box 电气接线盒electrical motor 电动机electrical noise 电噪音Electrical penetrators 电动穿孔器Electrical Power Unit 电力装置ELECTRICAL POWER UNIT 电力装置（EPU）electrical pump 电动泵Electrical signal 电信号Electrical Umbilical 电气脐带管electric-arc furnace 电弧炉electric-welded pipe 电焊接管electro-hydraulic control systems 电动液压控制系统electro-hydraulic multiplexed subsea control system 电动液压多路复用水下控制系统Electro-hydraulic umbilical 电动液压脐带管electronic components 电子元件Emergency Shut Down 应急关断empirical curve 经验曲线empirical uplift coefficient 经验上提系数end fitting 端部配件end riser termination 端部隔水管终端environment loads 环境载荷environmental condition 环境条件environmental loads 环境载荷ERW line pipe 电阻焊管线ESD sequence 应急关断顺序ESD signal 应急关断信号export line 输送管线external corrosion 外部腐蚀external diameter 外径external hydrostatic pressure 外部静水压力external pressure 外部压力External Welding 外部焊接Extrusion Process 挤压工艺Extrusion throat 挤压式喉管fabrication 制造fabrication times 制造时间Factory Acceptance Test 工厂验收试验fatigue damage 疲劳损坏fatigue life 疲劳寿命female connector 母连接器female couplers 母接头female couplings 母接头fibre optic 光纤fibre optic cables 光纤电缆fibre optic technology 光纤技术field development 油气田开发field joint 现场连接接头field life 油田寿命Field Signature Method (FSM) stations FSM技术检测点（备注：FSM是挪威CorrOcean公司开发的一种新型无损检测技术）final position 最终位置Firing line 流水作业线first-line barrier protection 一级隔层保护Flex joint 挠性接头flexible flowline 挠性出油管线flexible jumper 挠性跨接管flexible laying system 挠性管铺设系统flexible line 挠性管线flexible pipe flowlines 挠性出油管线flexible pipe structure 挠性管结构Flexible pipe system 挠性管系统flexible riser 挠性隔水管Flexible storage carousel 挠性管存管盘flexible structure 挠性结构flexible subsea pipe 挠性水下管道Flexible technology 挠性技术floating production 浮式采油floating production platform 浮式采油平台floating production system 浮式采油系统Flow Line 出油管flow meter 流量计Flowbase 出油基座fluid barrier 流体隔层fluid characteristics 流体特性fluid compatibility 流体相容性fluid level 流体液位fluid samples 流体样本fluid temperature 流体温度FPS motion FPS运动Free Hanging 自由悬挂free standing 自由竖立Frequency Shift 频移friction coefficient 摩擦系数full bore annulus circulation 全井筒环空循环Functional requirements 功能要求fusion-bonded epoxy 热熔结环氧Gas compressor 气体压缩机gas export line 天然气输出线路gas export line 天然气输出线路gas lift 气举Gas lift line 气举管线Gas Metal Arc Welding 气体保护金属极电弧焊gas pipeline 天然气管道Gas pipeline system 天然气管道系统gas trunkline 天然气干线Gas Tungsten Arc Welding 钨极气体保护电弧焊general arrangement 总体布局general arrangement 总体布置general corrosion 全面腐蚀Gimbal 常平架Girth welds 管道环形焊缝Global Positioning System 全球定位系统gravel pack 砾石充填green canyon Green峡谷grit blasting 喷钢砂guide frame 导向架guide funnel 漏斗形导向承口guide pin 导向销guide pin 导向销guide pin 导向销guide post 导向柱guide sleeve 导向套guide wires 导向绳Guideline Drilling 导向绳钻井Guiding roller 导向滚轮hammer unions 锤击由任hand valve 手动阀Hazardous Area 危险区Heat Affected Zone 热影响区Heat soak tests 热浸试验Heat tracing 伴热heat tracing 伴热heat transfer 热传导heat transfer 热传导heat transfer 热传导heat treatment 热处理heating lines 加热管线heating system 加热系统heating technique 加热技术helical strake 螺旋箍条High Collapse Resistance 高抗挤毁（性）high collapse resistance hose 高抗挤毁（HCR）软管high cost 高成本High Density 高密度High Density Polyethylene 高密度聚乙烯high frequency alternative current 高频交流电流high lay tensions 高敷设张力high pressure 高压high pressure jets 高压喷射装置high procurement cost 高采购成本HIGH TEMPERATURE 高温high-density 高密度hollow glass micospheres 空心玻璃微球hollow glass microsphere 空心玻璃微球hoop strength layer 环向强度层horizontal 水平horizontal connection 水平连接Horizontal Connector 水平连接器horizontal plane 水平面horizontal position 水平位置Horizontal powered reel 水平动力卷轴horizontal reel 水平卷轴horizontal subsea Christmas tree 水平水下采油树horizontal subsea tree 水平水下采油树horizontal tree 水平采油树Horizontal Tree stinger 水平采油树插入管hose liner 软管内衬hose liner 软管内衬Host' platform “主”平台Hot fluid 热流体Hot rolling clad pipe 热轧复合管Hot rolling process 热轧工艺hot stab 热入扣hot-rolling mill 热轧机hot-stab 热入扣hybrid riser 复合隔水管hybrid riser system 复合隔水管系统hybrid riser tower 复合隔水管塔hybrid riser tower 复合隔水管塔hydrate formation 水合物形成hydrate formation temperature 水合物形成温度hydraulic 液压Hydraulic Analysis 液压分析hydraulic connection 液压连接部分hydraulic connectors 液压连接器Hydraulic coupler 液压接头Hydraulic Couplers 液压接头hydraulic couplings 液压接头hydraulic couplings 液压接头Hydraulic cylinder 液压缸Hydraulic Distribution 液压分配Hydraulic Distribution Module 液压分配模块hydraulic expansion method 液压膨胀法hydraulic fluid 液压油hydraulic fluid 液压液Hydraulic fluid cleanliness 液压液清洁度hydraulic flying leads 液压引线hydraulic function 液压功能hydraulic galleries 液压坑道Hydraulic Hoses 液压软管hydraulic interface 液压接口hydraulic jet pump 水力喷射泵hydraulic line 液压管线hydraulic line 液压管线Hydraulic lock 液压锁hydraulic motor 液压马达Hydraulic oil 液压油Hydraulic output 液压输出Hydraulic Power Unit 液压动力装置hydraulic press 液压机hydraulic pressure 水压hydraulic pressure 水压hydraulic supplies 液压供应hydraulic supply 液压供应Hydraulic Supply 液压供应Hydraulic System 液压系统Hydraulic tubes 液压管hydraulic umbilicals 液压脐带管hydrodynamic load 水动力载荷hydrogen embrittlement 氢脆变hydrogen embrittlement 氢脆变Hydrogen Induced Cracking 氢致开裂hydrostatic collapse 静水压挤毁hydrostatic pressure 静水压力I.D. punch 内径穿孔Indicator rod 指示杆Induction cable 感应电缆Induction current 感应电流induction heating 感应加热Induction Welded pipe 感应焊管Induction Welding 感应焊接In-house experience 内部经验In-house technical database 内部技术数据库injection fluids 注入流体injection line 注入管线injection of chemicals 化学剂注入injection point 注入点injection point 注入点injection point 注入点Injection Valves 注入阀injection well 注入井in-line oscillations 平行振动inner pipe 内管Insert choke 嵌入式油嘴installation 安装installation 设备（一般大型设备，注意文中是否有出现）installation method 安装方法installation methods / sequences 安装方法/安装顺序installation parameters 安装参数installation phase 安装阶段installation procedure 安装程序installation process 安装过程installation processes 安装过程installation technique 安装技术installation tolerances 安装公差Installation tolerances 安装公差Installation Vessel 安装船Installation vessels 安装船instrument unit 测量装置instruments 仪器insulating properties 绝热性质Insulation foam 隔热泡沫Insulation Foam 保温泡沫insulation layer 隔热层Insulation resistance 绝缘电阻Integral Valves 集成阀Integration Test 完整性试验interaction model 相互作用模型interface area 接口区interface flange 接口法兰INTERFACE REQUIREMENT 接口要求interface with Xmas tree flanges 与采油树法兰的接口interfaces 接口interfaces 接口internal carcass 内部架构internal corrosion 内部腐蚀internal diameter 内径internal pressure 内压Internal pressure containment 内压限制Internal Profile 内剖面Internal Profile Datum 内剖面基准internal sealing profile 内部密封剖面internal service pressure 内部工作压力internal temperature 内部温度internal tree cap 内部采油树帽Internal welding 内部焊接International Electrotechnical Commission 国际电工委员会Intervention 干预intervention 干预INTERVENTION PHILOSOPHY 干预原理Intervention System 干预系统intervention task 干预任务isolation test tool 绝缘试验工具isolation valve 隔离阀J lay ramp J型敷管滑道J Lay semi-submersible J型铺管半潜式平台J-Lay J型铺管J-Lay method J型铺管法junction 连接junction box 接线盒junction box 接线盒junction plate 连接板keel joint 龙骨接头Key issues 关键问题Kvaerner integrated service umbilical Kvaerner综合功能脐带管Kvaerner umbilical Kvaerner脐带管landing shoulder 坐放台肩landing string system 坐放式管柱系统large diameter 大直径large diameter 大直径Latch Mechanism 锁紧机构Latching mechanism 锁紧机构lateral loads 侧向荷载Lay ramp 敷管滑道laying parameters 铺管参数Lazy S 低弯度S型Lazy Wave 低弯度波形Lazy Wave 低弯度波形Lazy wave 低弯度波形Leading Tug 首拖船leak path 渗漏通道level indicator 液面指示器life cycle cost 寿命周期成本Life Cycle Costing 寿命周期成本Life Cycle Costing 寿命周期成本（LCC）Line Insulation Monitor 管道保温监控line pipe 输送管Linear pullin winch 直线式引入绞车Linear Variable Differential Transformers (LVDT) 线性差动变压器（LVDT）linear winch 直线状绞车liner material 内衬材料liner tube 衬管liner tube 衬管Load ring 承载环load transfer 载荷转移load transfer 载荷转移local buckling criteria 局部折皱标准local control 本地控制器local panel control 本地面板控制盘Lock ring 锁环locking mechanism 锁定机构long beam 长臂long term 长期longitudinal seam 纵向缝longitudinal seam welding 纵向缝焊接low-alloy carbon-steel 低合金碳钢Lower Marine Riser Package 下部海洋隔水管插件（LMRP）Lower tool joint 下部工具接头major component 主要组件major subsea components 主要海底组件male hub 公卡箍Man Machine Interface 人机接口management system 管理系统Management System 管理系统Mandrel 工作筒manipulator 机械手manipulator arm 操作手臂manipulator arm 机械手臂manipulator arms 机械手臂manipulators 机械手manipulators 机械手manual valves 手动阀manufacturer 制造商marginal field 边际油田Marine Drilling Riser 海洋钻井隔水管marine environment 海洋环境marine growth 海生物聚集marine riser 海洋隔水管Master Control Station 主控台mechanical properties 机械性能Mechanised Pulsed Gas Metal Arc Welding 机械脉冲气体保护金属极电弧焊Medium size flowline 中等尺寸出油管线Melting steel 熔融钢水messenger line 引缆metal to metal seals 金属对金属密封metal tube 金属管Metallic Materials 金属材料metallic sealing materials 金属密封材料Module Replacement Tool (MRT) 模块更换工具（MRT）monitoring system 监控系统Mono Bore Tree 单筒采油树moon pool 月池mooring line 系泊绳Mounting Base 安装基座Mudline Conversion System 泥线转换系统mudline suspension system 泥线悬挂系统Mudline Tree 泥线采油树Mudline Wellhead 泥线井口Multi Mode Running Tool (MMRT) 多模式下送工具（MMRT）multi-phase flow meters 多相流量计multiphase flows 多相流体Multiple load shoulder profile 多载荷台肩剖面National Aerospace Standard 国家宇航标准natural period 固有周期navigation system 导航系统neutrally buoyant 中和浮力normal operating 正常操作normal operation 正常操作North Sea 北海offshore hydrocarbon pipeline 海上油气管道offshore installation 海上安装Offshore Installations 海上设备offshore pipeline 海洋管道offshore production platform 海上生产平台offshore production platform piping system 海上生产平台管道系统Offshore Technology Conference 海洋技术会议Oil pipeline transportation systems 石油管道运输系统oil/gas production rate 油/气生产速度open-hearth steelmaking 平炉炼钢operating conditions 作业条件operating cost 运营成本operating cost 运营成本operating pressure 操作压力operating temperature 操作温度operation 作业operational temperatures 操作温度operator skill 操作者技能operators 操作者optical fibre 光纤optical fibres 光纤orbital welding 轨道焊接Orientation device 定向仪orientation system 定向系统O-Ring seals O环密封圈O-rings seals O环密封圈outboard hub 外侧轮毂outer pipe 外管outer sheath 外部护套outer sheath 外部护套outer sheath 外部护套outer surface 外表面outer thermoplastic sheath 热塑外部护套overall length 总长overall System 整个系统Overboard chute 船外下降溜槽overboarding chute 船外下降溜槽override functions 优先功能pack off retrieval tool 封隔收回工具Packoff 封隔Pack-Off 封隔Pack-Off Assembly 封隔总成paraffin deposits 结蜡parallelepipedic billets 立方坯板料parking position 停放位置partial pressure 分压performance verification 性能验证permanent guide base 永久导向基座Piercing of billets 坯板料穿孔Pig Detection 清管器检测Pig Detector 清管器检测器pig launcher 清管器发送筒pig receiver 清管器接收筒pilot valves 先导阀pin connection 销连接pin connector 销连接器PIPE DIAMETER 管道直径Pipe dimensions 管道尺寸Pipe expansion calculations 管道膨胀计算pipe in pipe system 管中管系统Pipe inside diameter 管道内径pipe joints 管接头pipe joints 管接头pipe layer dimensions 管层尺寸pipe material 管道材料pipe outside diameter 管道外径pipe section 管道截面Pipe storage Reel 存管卷筒pipe strings 管柱pipe structure 管道结构pipe structure 管道结构pipe system 管道系统pipe wall 管壁Pipe wall thickness 管道壁厚Pipe-in-pipe system 管中管系统pipelay vessel 铺管船pipeline 管道pipeline 管道Pipeline bundle system 管束系统pipeline burial 管道埋设pipeline design 管道设计Pipeline Design Method 管道设计方法Pipeline End Manifold 管道末端管汇Pipeline end manifold (PLEM) 管道末端管汇（PLEM）Pipeline end terminations 管道端部终端设备pipeline systems 管道系统Pipeline Valve 管道阀piping system 管道系统plastic deformation 塑性变形plastic deformation 塑性变形Poisson coefficient 泊松系数Polyurethane Foam 聚氨酯泡沫Polyvinylidene Fluoride 聚偏氟乙烯position indicators 位置指示器Position Reference System 定位参考系统positioning system 定位系统Positive mechanical lockdown mechanism 正向机械锁定机构post-weld heat treatment 焊后热处理power cable 电缆power cables 电源电缆power channel 电力通道power management 电源管理power source 电源power source 电源power supplies 电力供应Power Supply 电源power supply 电源Power Supply 电源Power System 动力系统pressure balance 压力平衡pressure balanced 压力平衡pressure drop 压降Pressure Drop 压降pressure drops 压降pressure gauges 压力表pressure integrity 压力完整性pressure monitoring 压力监测Pressure Sensor 压力传感器pressure test 压力测试Pressure Vessel 压力容器pressure-drop 压降primary pressure 原始压力Product Flow 产品流动product leaflets 产品说明书production and annulus master valve 生产和环空总阀production bore 生产井筒production casing 生产套管production control system 生产控制系统Production Deck 生产甲板production facilities 生产设施production flow rate 生产流量production flowline 生产出油管线production flowline 生产出油管线production flowline system 生产出油管线系统production fluids 生产流体production gathering 生产集输Production line 生产管线production lines 生产管线Production Master Valve 采油主阀production mode 生产模式production phase 生产阶段production platform 生产平台production rate 采油速度production rates 采油速度Production Riser 生产隔水管production riser 生产隔水管production riser 生产隔水管production risers 生产隔水管Production String 生产管柱Production System 生产系统production system 生产系统Production Template 采油基盘production tree 采油树production tubing 生产油管Project Management 项目管理Project Manager 项目经理project specification 项目规范propagation buckling 延伸折皱propagation pressure 延伸压力propulsion system 推进系统protection cap 保护帽protective structure 防护结构pull force 拉力pull in cable 牵引线缆Pull in winch 牵引绞车pull-in and connection tool 牵引和连接工具pull-in cable 牵引线缆pull-in head 拉入头pull-in operation 引入作业pull-in winch 牵引绞车pull-in/connection method 牵引/连接法Pump Down System 泵送系统pump systems 泵系统qualification 资质Qualification 资质qualification test 合格性测试Quality Assurance 质量保证quality control 质量控制Quality Plan 质量计划Quality System 质量体系Quenching 淬火Quick Connect 快速连接radial forces 径向力reaction loads 反作用载荷real time 实时Reel-Lay 卷管式铺管法Reference Book 参考手册reliability analysis 可靠性分析relief valves 安全阀removable guide posts 活动导向柱reservoir 油藏reservoir 油藏reservoir 油藏reservoir 油藏reservoir pressure 储集层压力residual axial tension 残余轴向拉力residual strains 残余应变resin binder 树脂粘合剂resistance factor 阻力系数Response time 响应时间Retrieval Tool 收回工具return line 回油管线return line 回油管线return lines 回油管线return tank 回油罐rigid line 刚性管道rigid pipe 刚性管道Rigid Pipe 刚性管rigid pipeline 刚性管道rigid production riser 刚性生产隔水管rigid reeled gas line 刚性卷筒式天然气管道rigid steel pipe 刚性钢管rigid steel riser 刚性钢质隔水管ring grooves 环形槽riser 隔水管riser base 隔水管基座riser connector 隔水管连接器riser joint 隔水管接头Riser joints 隔水管单根Riser Pressure 隔水管压力riser system 隔水管系统riser tension 隔水管张力riser tensioner 隔水管张紧器rolling process 轧制工艺rotary table 转盘ROV 水下机器人（ROV）ROV 水下机器人ROV 水下机器人ROV AND TOOLS 水下机器人和潜水工具ROV control cabin ROV控制舱ROV deck equipment ROV甲板设备ROV interface ROV接口ROV intervention ROV修井ROV locking interface ROV锁紧接口ROV manufacturers and subsea contractor ROV制造商和水下承包商ROV panels ROV面板ROV system ROV系统ROV vehicles ROV装置ROVs and Tools 水下机器人和潜水工具running sequence 送入顺序running tool 下入工具running tool 下入工具running tools 下入工具Safe Area 安全区safety factor 安全系数Safety Valve 安全阀safety valve control 安全阀控制Sand Detector 测砂器sand monitor 测砂器sand monitoring 防砂监控Sand Monitors 测砂器Satellite tree 卫星采油树satellite well 卫星井saturation diving 饱和潜水scan cycle time 扫描周期scan time 扫描时间sea bed 海床sea bed 海床sea bottom 海底sea bottom 海底sea level 海平面Sea level 海平面sea state 海况sea states 海况sea surface level 海面sea water 海水sea-bed 海底sea-bed level 海底水位seafloor wellhead 海底井口Seal area 密封区seal assembly 密封总成Seal Elements 密封元件SEAL Engineering SEAL工程seal material 密封材料Seal plate 密封板seal pocket 密封腔seal surfaces 密封表面Sealines 海下管线sealing system 密封系统seam welding process 管缝焊接工艺seamless pipe 无缝管seamless tube 无缝钢管sea-states 海况seawater tolerance 耐海水Seaway Condor Seaway Condor selector valve 选择阀semiconductor device 半导体装置sensing element 传感元件sensing pins 感应针sensor systems 传感器系统serial number 序列号service life 使用寿命service line 维修管线Service line 维修管线seven-function 七功能seven-function 七功能seven-function 七功能severe weather 恶劣天气shallow water 浅水shear pins 剪切销钉sheave package 滑轮组件side valve tree 旁阀采油树Signal Pair 信号线对signal transmission line 信号传输线silicate microspheres 硅酸盐微球体Single Bore Tree 单筒采油树Skin Effect Current Tracing 集肤电伴热sled piping 滑车管道sleeve pipe 套管soft soil 软土soil conditions 土壤条件solenoid valve 电磁阀Span calculations 跨度计算spare parts 备件special tools 专用工具specific project 指定项目Spirally-oriented seam welded pipe 螺旋焊缝管splash zone 飞溅区splash zones 飞溅区Split lock ring 开口锁环Spool Piece 短管Spool tree 四通采油树spooling 缠绕spooling system 缠卷系统spray cooling 喷淋冷却法stab plate 插板stainless steel 不锈钢stainless steel strips 不锈钢带static analysis method 静态分析法steel armoured pipe 钢质铠装管道steel catenary riser 钢质悬链线隔水管Steel Flying Lead 钢引线steel jacket pipe 钢质夹套管steel pipe 钢管steel riser 钢质隔水管steel tube umbilical 钢管脐带管steel tube umbilical 钢管脐带管steel tube umbilical 钢管脐带管Steelmaking process 炼钢工艺Steep S-Lay 陡S型铺管法Steep S-Lay vessel 陡S型铺管船Steep Wave 高弯度波形Steep Wave 高弯度波形Steep wave 高弯度波形Steep wave 高弯度波状Stepwise Cracking 阶式断裂storage reel 存储盘架storage reel 存储盘架straight line 直线strength members 强度元件Stress Corrosion Cracking 应力腐蚀开裂strong currents 强电流strong seawater currents 海水强流Submarine pipeline code 海底管道规范submarine pipeline system 海底管道系统Submerged Arc Weld 埋弧焊Submerged arc welded pipe 埋弧焊管submerged weight of the pipeline 管道浮重submerged weight of the soil 土壤浮重submerged-arc welded 埋弧焊submerged-arc welded seam 埋弧焊缝subsea blowout preventer 水下防喷器subsea christmas tree 水下采油树Subsea Christmas Tree System 水下采油树系统Subsea cluster manifold 水下丛式管汇Subsea cluster well system 水下丛式井系统subsea contractor 水下承包商Subsea Control Module 海底控制模块subsea control module mounting base 海底控制模块安装基座（SCMMB）Subsea Control Module Running Tool 海底控制模块下入工具Subsea Control Systems 水下控制系统subsea drilling system 水下钻井系统Subsea Electronics Module 水下电子装置模块subsea field 海底油气田subsea intervention tree 水下修井采油树subsea manifold 水下管汇Subsea Production Control System 海底生产控制系统SUBSEA PRODUCTION EQUIPMENT 海底采油设备Subsea Production Manifold 海底采油管汇Subsea Production System 海底生产系统Subsea Production Template 海底生产基盘subsea system 水下系统Subsea tree single-piece spool body construction 水下采油树单件四通本体结构Subsea wellhead 海底井口装置Subsurface Safety Valve 水下安全阀Suction Pile 吸力桩Suction Piles 吸力桩Sulphide Stress Corrosion Cracking 硫化物应力腐蚀开裂super duplex 超双相super-duplex 超双相Supply Tank 供应罐supply voltage 电源电压supply voltage 电源电压support frame 支撑框架surface area 表面积surface BOP 地面防喷器surface casing string 表层套管柱Surface Conductor 地面导管Surface Controlled Sub-Surface Safety Valve 地面控制水下安全阀surface production processing facilities 地面生产处理设施Surface Safety Valve 地面安全阀surface support vessel 水面供应船surface tree 地面采油树Surface Tree 地面采油树survey vessel 测量船swab valve 抽汲阀syntactic foam 复合泡沫syntactic foam 复合泡沫Tag Number 标号target area 目标区域temperature sensor 温度传感器Temporary Guidebase 临时导向基座tensile armours 抗拉铠装tensile strength 拉伸强度tension capacity 张力大小Tension Leg Platform 张力脚平台tension leg platforms 张力腿平台tension load 张力载荷tension loads 拉伸载荷terminal head 终端头termination 终端termination 终端termination point 终端点termination unit 终端装置test cap 测试帽Test Electrical Power Unit 试验电源装置test method 测试方法test pressures 测试压力Test Tool 试验工具Teta profile T形剖面Teta spiral T形螺旋层thermal expansion 热膨胀thermal expansion coefficient 热膨胀系数thermal insulation 隔热thermal insulation 隔热thermal insulation 隔热thermal insulation and Aluminium-sacrificial anode system 隔热和牺牲铝阳极系统thermal insulation material 隔热材料thermal insulation system 隔热系统thermal insulation system 隔热系统thermal shrink-fit method 热收缩-配合法Thermo-hydraulic fit method 热-液压配合法Thermo-Mechanical Controlled Processing 热机械控制工艺Thermoplastic friction sheath 热塑耐磨护层thermoplastic hose 热塑软管。
Bio-Plex悬液芯片系统简明使用教程Bio-Plex 悬液芯片系统一、仪器名称：Bio-Plex 悬液芯片系统 二、规格型号：Bio-Plex 200 System 三、生产厂家：Bio-Rad Laboratories, Inc 四、产品简介 人类基因组计划（HGP）的完成和蛋白质组计划（HPP）的启动，获得了数量巨大的基因和蛋 白质信息，而要对如此庞大的信息进行全面的处理和研究，必须设计和利用更为高效的硬件和软件 技术，建立新型、高效、快速的检测分析方法。
生物芯片正是在这种背景下应运而生，其不仅在高 通量基因测序、基因表达研究、蛋白质相互作用等方面发挥重要作用，也将在临床诊断中占据重要 地位[1]。
液相芯片（liquichip）是新一代生物芯片技术，既能为后基因组时代科学研究提供强大的技 术支持，又能提供高通量的新一代分子诊断技术平台。
Bio-Plex 悬液芯片系统将先进的软件包、系统检验工具、微球耦联试剂和即用型细胞因子与磷 酸化蛋白测试试剂整合在一起，使硬件、软件和检测试剂形成一个功能强大的芯片技术平台，大大 提高了结果的精确性和可重复性，使用更加简便高效。
该系统为蛋白与核酸研究人员提供了灵活的 复合测试方案，可在单个样品中同时分析多达 100 个生物分子。
利用 100 种不同颜色微球（xMAP 技术）标记生物分子配体，每个微球可耦联一个对应不同靶分子的特异反应物。
反应物可以是酶底 物、受体、抗原或者抗体。
检测范围 0.2-3200pg/ml 或 1.95-32000 pg/ml， 自动的校正和校验工具可 以保证样品间差异、板间差、系统间差异控制在 10%以下，30 分钟可以完成 96 个样品的检测并获 得多达 10,000 个分析数据。
多重检测获得的数据可以完全揭示生命分子的相互关系及信号传导途径。
[1] Hulse R E, Kunkler PE, Fedynyshyn J P, et al. Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue. J Neurosci Method, 2004, 136: 87-98. 五、技术原理 1、芯片原理：Bio-Plex 悬液芯片的核心技术是把微小的颗粒亦称微球（bead 或 microsphere）分 别染成不同的荧光色，然后再把针对不同检测物的蛋白质或寡核苷酸探针以共价的方式吸附到不同 颜色的微球上。
我科学家实现聚噻吩主链垂直于基底生长引起世界关注
佚名
【期刊名称】《中国西部科技》
【年(卷),期】2008(7)2
【摘要】在国家自然科学基金委和中国科学院的支持下,中国科学院长春应用化学研究所高分子物理和化学国家重点实验室杨小牛课题组通过使用可控溶剂气氛处理的方法成功制备出由聚噻吩平躺片晶构成的大面积均匀薄膜,其中聚合物主链垂直基底取向,相关工作发表在国际著名杂志德国《先进材料》
（Adv.Mater.2007,19,3594-3598）上。
【总页数】1页(P83-83)
【关键词】中国科学院长春应用化学研究所;聚噻吩;基底;垂直;主链;科学家;国家自然科学基金;国家重点实验室
【正文语种】中文
【中图分类】TQ320.721;TQ324.8
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1.不同微观形貌所引起的聚二氧乙撑噻吩的光电性能改变 [J], 李瑀;封伟;吴隽
2.电活性和生物活性多巴-胰岛素样生长因子-1@聚(乙交酯-丙交酯)/聚(3-己基噻吩)静电纺丝纤维的制备及神经组织工程应用 [J], 张守燕;胡江磊;史新翠;章培标;伊藤嘉浩
3.主链含3-吡啶基-2,5-二噻基噻吩的电化学聚合及其电致变色性能研究 [J], 帕提古丽·艾散; 阿布都克尤木·阿布都热西提; 吐尼莎古丽·阿吾提
4.主链含苯并二噻吩的D-A结构甲壳型共轭聚合物的合成与表征 [J], 万里鹰;李佩杰;易凡
5.我科学家实现聚噻吩主链垂直于基底生长引起世界关注 [J],
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重点实验室获国家自然科学基金资助项目
佚名
【期刊名称】《今日科技》
【年(卷),期】2012(000)009
【摘要】近日前，接国家自然科学基金委员会通知，重点实验室杨章女博士今年负责申报的题为“莱姆螺旋体保守基因簇bba68-bba73的生物学功能研究”项目获国家青年科学基金项目资助，批准号为81201319。
该项目为该重点实验室继朱函坪主任技师、姚苹苹副主任技师相继在2007年、2011年获国家基金项目资助后的第3个主持的资助项目。
【总页数】1页(P57-57)
【正文语种】中文
【中图分类】S88-55
【相关文献】
1.我院一科技项目获国家自然科学基金项目资助 [J], ;
2.复旦医科461个项目获2019年度国家自然科学基金项目立项资助 [J], 无
3.我校6项国家自然科学基金项目、3项国家社科基金项目获资助立项 [J],
4.2006年度国家自然科学基金资助项目及项目负责人专栏介绍华中科技大学服务计算技术与系统教育部重点实验室——陶文兵 [J],
5.我校8个项目获2013年度国家自然科学基金项目立项资助 [J],
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专利名称：一种小分子抑制剂及其制备方法与其在多发性骨髓瘤治疗中的应用
专利类型：发明专利
发明人：饶燏,刘万里,杨茂君,孙永汇
申请号：CN201711000708.7
申请日：20171024
公开号：CN107973754A
公开日：
20180501
专利内容由知识产权出版社提供
摘要：本发明公开了一种小分子抑制剂及其制备方法与其在多发性骨髓瘤治疗中的应用。
本发明小分子抑制剂的结构式如式Ⅰ、式Ⅱ、式Ⅲ或式Ⅳ所示，本发明也提供了所述小分子抑制剂的制备方法。
本发明小分子抑制剂能够抑制布鲁顿酪氨酸蛋白激酶的活性，因此能够应用于多发性骨髓瘤的治疗中，如IgE型多发性骨髓瘤。
申请人：清华大学
地址：100084 北京市海淀区北京市100084信箱82分箱清华大学专利办公室
国籍：CN
代理机构：北京纪凯知识产权代理有限公司
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TechNote 201AMicrosphere Selection9025 Technology Dr. • Fishers, IN 46038-2886800.387.0672 • 317.570.7020 • Fax 317.570.7034info@ • ContentsI. Introduction II. Diameter III. Composition IV. CoatingA. AdsorptionB. Covalent CouplingC. Affinity Binding V. Special PropertiesI. IntroduCtIonMicrospheres offer a highly convenient and flexible system for developing reagents for assays and bioseparations, and for use as instrument standards. As there are many varieties of microspheres available, it is important to think about the demands the application will place on them when making a base bead selection. Physical and optical properties should be considered in the context of handling and detection, and thought should also be given to requirements for diameter and size distribution, composition, surface chemistry, and any other needed properties.II. dIameterMicrosphere size may be critical to the proper function of an assay, or it may be secondary to other characteristics. Considering traditional diagnosticmethods, the test or assay format commonly dictates particle size, such as the use of very small spheres (~0.1-0.4µm) to ensure satisfactory wicking in lateral flow tests, or the use of larger, cell-sized spheres (~4-10µm) for bead-based flow cytometric assays. Also see application-specific TechNotes 301-304.In magnetic separations, particularly those involving capture and elution of the target, the exact size of the magnetic particle may be unimportant provided that the particles are in some general size range, and offer desired separation characteristics. See TechNote 102 for additional details regarding our three magnetic particle lines.Diameter also determines surface area. Small-diameter spheres present more surface area per unit weight, while larger spheres present more surface area per bead. Size also affects ease of handling, processing considerations (such as the method used for separations [centrifugation, dialysis, filtration]), and the amount of reagent needed for coating.III. ComposItIonCommon microsphere compositions include polystyrene (PS), poly(methyl methacrylate) (PMMA), and silica. These materials possess different physical and optical properties, which may present advantages or limitations for different applications.Polymer beads are generally hydrophobic, and as such, have high protein binding abilities. However, they often require the use of some surfactant (e.g. 0.01-0.1% Tween ® 20 or SDS) in the storage buffer to ensure ease of handling. During synthesis, functional monomers may be co-polymerized with styrene or methyl methacrylate to develop beads with surface reactive groups. Functional groups may be used in covalent binding reactions, and also aid in stabilizing the suspension.Silica microspheres are inherently hydrophilic and negatively charged. Consequently, aqueous silica suspensions rarely require use of surfactants or other stabilizers. Carboxyl- and amine-functionalized silica spheres are available for use in common covalent coating protocols, and plain silica microspheres may be modified using a variety of silanes to generate functional groups or alter surface properties.IV. CoatIngMicrospheres may be coated with capture molecules, such as antibodies, oligonucleotides, peptides, etc. for use in diagnostic or separation applications. Microsphere coatings are typically optimized to achieve desired specific activity, while minimizing nonspecific interactions. Consideration should also be given to the required stability, development time frame and budget, and the specific biomolecule to be coated. These factors will aid in determining the most fitting coating strategy for both short- and long-term objectives.Standard microsphere products support three basic coating strategies: adsorption, covalent coupling, and affinity binding.A. AdsorptionAdsorption relies primarily on hydrophobic interactions between the biomolecule and the polymer particle. Such coatings are fairly simpleto conduct, involving incubation of the microspheres with the purified biomolecule. They typically require little optimization, and reagents may be developed relatively quickly. However, as adsorption relies on the formation of multiple attachment points between the molecule and particle, this strategy is typically reserved for use with proteins and non-functionalized polymer spheres. Adsorption is generally not suitable for hormones, peptides, or nucleic acids in hybridization-based applications, and protein adsorption to silica is expected to be less efficient than to polymer. See TechNotes 201 and 204.B. Covalent CouplingCovalent coupling results in the permanent attachment of the molecule to the functionalized (e.g. carboxyl or amine) microsphere. It can provide needed stability when developing a commercial reagent, and for multiplexed assays, where analyte-specific bead populations are mixed. Additionally, specialized chemical linkers may be employed to address steric effects or to optimally orient the molecule. Although covalent binding protocols often involve a higher level of optimization than other approaches, coupling kits are available to simplify the process. See TechNotes 201 and 205.C. Affinity BindingAffinity binding is a straightforward method for immobilizing primary antibodies or biotinylated molecules. Proteins A and G and Fc-specific antibody coatings permit the directed immobilization of primary antibodies, and streptavidin is used extensively for the binding of biotinylated molecules, such as antibodies, peptides, and oligonucleotides. See TechNotes 101 and 302.It is important to note that each binding strategy has benefits and limitations, which should be weighed in the context of study objectives and the demands that will be placed on the finished reagent.V. speCIal propertIesMany applications in the life sciences demand added properties, suchas fluorescence or a visible color, or iron oxide inclusions for magnetic separations. Polymer spheres (and polymer-based magnetic spheres)are often internally dyed via organic solvent swelling, and many standard products are available. Dye concentrations can be adjusted to produce beads with different intensities to meet special needs, such as QuantumPlex™for multiplexed flow cytometric assays, or our Dragon Green or Flash Red Intensity Standards, which support imaging applications and associated instrument QC. Many surface- or internally-labeled fluorescent beads are also available as specialized flow cytometry standards.Various types of superparamagnetic microparticles are available as well– with different matrices, magnetite content, surface groups, etc. For new assays or applications, magnetic beads should be evaluated with application demands in mind.The following tables provide product suggestions for common microsphere applications. These are offered as general guidelines only. Further literature research and screening experiments may be appropriate.BioMag® and ProMag™ are trademarks or registered trademarks of Polysciences, Inc.COMPEL™ and QuantumPlex™ are trademarks of Bangs Laboratories, Inc. Tween® is a registered trademark of ICI Americas, Inc.Copyright 2008, Bangs Laboratories, Inc.All Rights Reserved。
Luminex液态芯片在临床及科研中的应用张保强;张晓【摘要】Luminex 液态芯片是一个多功能、多指标并行分析系统,集编码微球、激光技术、流式细胞、数字信号处理等技术于一体,具有高通量、既能检测蛋白,又能检测核算等特点,可广泛应用于免疫分析、核酸研究、酶学分析、受体和配体识别分析等研究.本文就液态芯片技术的原理、特点及在临床和科研中的应用进行阐述.【期刊名称】《当代医学》【年(卷),期】2012(018)004【总页数】3页(P18-20)【关键词】液态芯片;悬浮阵列;肿瘤标志物;HPV基因分型;移植配型【作者】张保强;张晓【作者单位】261061,潍坊市疾病预防控制中心检验科;261061,潍坊市疾病预防控制中心检验科【正文语种】中文液态芯片是美国纳斯达克上市公司Luminex于本世纪初研制出的后基因组时代的技术平台，又称悬浮阵列、流式荧光技术，是基于多功能流式点阵仪（Luminex 100）开发的多功能生物芯片平台，是一个多功能、多指标并行分析系统（见图1）。
它有机地整和了编码微球（color-codedbeads）、激光技术、应用流体学、最新的高速数字信号处理器和计算机运算法则，造就了高度的检测特异性和灵敏度，可广泛应用于免疫分析、核酸研究、酶学分析、受体和配体识别分析等研究，也是目前唯一得到权威机构和医学界共同认可用于临床诊断的生物芯片平台。
2003年美国食品与药品管理局(FDA)批准该项技术用于临床检验诊断。
图1 Luminex 100 多功能流式点阵仪1 Luminex液态芯片技术的概况1.1 工作原理该技术的核心是采用聚苯乙烯(polystyrene)制作微球，把微小的聚苯乙烯小球（5.6 μm）包覆以不同比例的红光及红外光染色剂，制成100种不同颜色的微球[1]（见图2）。
将每种颜色的微球（或称为荧光编码微球）共价交联上针对特定检测物的探针、抗原或抗体。
不同的微球结合了针对不同待检测物的蛋白（抗体或抗原，用于免疫检测）或核酸（DNA或RNA用于基因检测），检测抗体中以生物素标记，并用高灵敏的荧光染料染色。
专利名称：一种具有癌细胞及线粒体双靶向性的核酸纳米器件专利类型：发明专利
发明人：吴再生,孙树娟,阳娅,江豪
申请号：CN202111335608.6
申请日：20211111
公开号：CN114053425A
公开日：
20220218
专利内容由知识产权出版社提供
摘要：本发明提供了一种具有癌细胞及线粒体双靶向性的核酸纳米器件。
所述核酸纳米器件是以纳米金为载体，将核酸适配体及线粒体靶向分子与DNA四面体相结合而制得的。
该纳米器件可将阿霉素定向递送至线粒体用于癌症治疗，且具有靶向性好、成本较低、效果显著的优势，在以线粒体为靶点的癌症精准治疗领域具有较高应用价值。
申请人：福州大学
地址：350108 福建省福州市闽侯县福州大学城乌龙江北大道2号福州大学
国籍：CN
代理机构：福州元创专利商标代理有限公司
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中科大开发纳米给药系统可同时杀伤乳腺癌细胞和癌症干细胞佚名
【期刊名称】《肿瘤防治研究》
【年(卷),期】2016(43)8
【摘要】近日，来自中国科学技术大学的王均教授实验室在国际学术期刊Biomaterials上发表了一项最新研究，他们发现借助纳米颗粒运输系统将自噬抑制药物和化疗药物联合使用可以有效杀伤乳腺癌细胞和乳腺癌干细胞，该研究为解决因乳腺癌干细胞导致的治疗抵抗及复发等问题提供了一个新的治疗方法。
【总页数】1页(P732-732)
【关键词】乳腺癌细胞;纳米给药系统;癌症干细胞;杀伤;中国科学技术大学;乳腺癌干细胞;学术期刊;联合使用
【正文语种】中文
【中图分类】R737.9
【相关文献】
1.顺磁性铁纳米粒促进人淋巴细胞对乳腺癌细胞的体外杀伤 [J], 张亮;孙昭;段金虹;熊非;张宇;顾宁;许海燕;杨先达
2.纳米胶束共同递送 DOX和 Bcl-2 siRNA对 MCF-7乳腺癌细胞的杀伤作用 [J], 锁爱莉;王何静;钱军民;刘茸茸;姚煜
3.纳米羟基磷灰石携载lefty-1基因对人乳腺癌细胞的杀伤效应 [J], 钟云平;姚晨雪;宋国龙;赖文;陈岑;解纯刚;孔祥东
4.负载阿霉素的透明质酸修饰还原敏感纳米粒子对乳腺癌细胞株MDA-MB231的靶向杀伤及生物学意义 [J], 周春艳;钟伊南;刘文婷;王雪峰;钟志远;谢芳
5.功能化介孔二氧化硅负载顺铂纳米药物的制备及其对乳腺癌细胞的杀伤作用 [J], 刘伟坤; 张萌; 李炳龙; 管西栋; 孙明爽; 何玉静; 王春晓; 赵宝昌; 张吉梅
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ProtocolMultiplexed microsphere-based flow cytometricimmunoassays for human cytokinesKathryn L.Kellar *,Janine P.DouglassBiotechnology Core Facility Branch,Scientific Resources Program,National Center for Infectious Diseases,Centers for Disease Control and Prevention,MS D-34,1600Clifton Road,NE,Atlanta,GA 30333,USAReceived 5April 2003;accepted 2June 2003AbstractCytokines play a pivotal role in the regulation of immunologic,hematologic and wound-healing processes.They function to stimulate as well as inhibit the proliferation,differentiation and maturation of a variety of cell types.Thus,their functions are pleiotropic as well as interdependent to the extent that any cytokine may have effects that are synergistic or antagonistic with other cytokines.Cytokines also display redundancy when one mimics the functions of others.These characteristics imply that measuring the levels of one cytokine in a biologic system provides only a fraction of the information that is relevant to the existing physiologic state.A more realistic indication of the complexity of cellular interactions would include measurements of multiple cytokines at any time point.One method of multiplexed analysis can be performed by capture of the cytokines on an array of fluorescent microspheres for quantitation by flow cytometry.This technology has been applied to a variety of biomolecules,but simultaneous quantitation of multiple cytokines in a small sample volume has become rapid,inexpensive,reliable and informative.D 2003Elsevier B.V .All rights reserved.Keywords:Fluorescent microspheres;Multiplexed immunoassays;Microsphere-based flow cytometric assays;Luminex1.DescriptionCytokines,chemokines and growth factors (here-after referred to collectively as cytokines)form a complex network of interacting regulatory proteins that are of major importance for immune,hematopoi-etic,angiogenic and pathologic processes.Each cyto-kine has a variety of functions that may be additive,similar or inhibitory to the activities of other cyto-kines.Cytokine measurements and the ratios of dif-ferent cytokines in a biologic system have become critical indicators of normal and disease states.Immu-noassays such as ELISAs provide sensitive,specific and precise quantitation of cytokines,but when ap-plied to the expanding number of cytokines that are involved in defining a particular system,they become expensive and time-consuming to perform.A compa-rable methodology provides a more rapid and less costly means of measuring multiple cytokines and other analytes in small sample volumes simultaneous-ly (Oliver et al.,1998;Carson and Vignali,1999;Chen et al.,1999;Kellar et al.,2001;Camilla et al.,0022-1759/$-see front matter D 2003Elsevier B.V .All rights reserved.doi:10.1016/S0022-1759(03)00248-5*Corresponding author.E-mail address:klk1@ (K.L.Kellar)./locate/jimJournal of Immunological Methods 279(2003)277–2852001).Cytokines are captured on an array of fluores-cent microspheres and detected by the binding of fluorochrome-labeled detection antibodies.The multi-plexed assays are analyzed on a compact flow cytom-eter that is equipped with two lasers to excite the microspheres and reporter dye and digital signal processors to condense the thousands of signals intoa manageable data output.2.Type of research2.1.Quantitation of human IL-2,IL-4,IL-6,IL-8,IL-10,IL-12,TNF-a and IFN-g by multiplexed microsphere-based flow cytometric immunoas-say(MFCA).2.2.Measurement of cytokines in human serum andcell culture supernatants.2.3.Validation of a multiplexed MFCA for eighthuman cytokines.3.Time required3.1.Covalent coupling of cytokine antibodies tofluorescent microspheres:1day.3.2.Testing of the efficiency of coupling the micro-spheres:60min.3.3.MFCA procedure:2h,45min.a.Preparation of filter-bottom plate for assay:5min.b.Dilutions of cytokine standards for standardcurves:10min.c.Preparation,dilution and dispensing ofsamples and other reagents into filter plate:30min.d.First incubation of assay:45min.e.Washes of microspheres:5min.f.Addition of biotinylated detection antibodiesto filter plate:10min.g.Second incubation:30min.h.Addition of streptavidin R-phycoerythrin tofilter plate:5min.i.Third incubation:15min.j.Washes of microspheres:5min.k.Addition of fixation buffer to plate:5min.3.4.Measurement on Luminex100instrument:30–60min.4.Materials4.1.Covalent coupling of cytokine antibodies to fluorescent microspheresSpecial equipment and supplies:Luminex100(Luminex,Austin,TX)equipped with Luminex100FirmWare v.1.7.1Luminex100carboxylated microspheres(#132, 134,138,154,158,173,177,181).Microcentrifuge equipped with a horizontal rotor (Eppendorf5417C).Water bath-type sonicator(Cole Parmer#8851, Vernon Hills,IL).USA Scientific microtiter tubes,1.5ml(#1415–2500,Ocala,FL).Digital pipettors(Rainin,Woburn,MA).Digital multichannel pipettors(Rainin).Dialysis thimbles(Slide-A-Lyzer mini-dialysis units,#69570,Pierce,Rockford,IL).Black microtiter plates(#3792,Corning,Corning, NY).Costar reagent reservoirs(#4870,Corning).Microtiter plate shaker(Titer Plate Shaker,Lab-line Instruments,Melrose Park,IL).V ortex.Microbalance.Hematology rocker.Hemacytometer with improved Neubauer ruling. Microscope.Biological safety cabinet.Analytical software,SOFTMaxPRO(Molecular Devices,Sunnyvale,CA).Chemicals and reagents:N-hydroxy-sulfosuccinimide(sulfo-NHS,#24510;Pierce;dessicated and stored at4j C.).1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride(EDC;Sigma,St.Louis,MO;dessicated and stored atÀ20j C.).1Use of trade names and commercial sources is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention,Department of Health and Human Services.K.L.Kellar,J.P.Douglass/Journal of Immunological Methods279(2003)277–285 278Activation buffer:0.1M sodium phosphate buffer, pH 6.3(0.1M dibasic and0.1M monobasic sodium phosphate mixed to a pH of6.3).0.05M PBS,pH7.4(5.74gm.Na2PO4,1.31gm.NaPO4.H2O,8.76gm.NaCl in1L.dH2O).PBN:PBS,pH7.4,containing0.5%bovine serum albumin(BSA,#A4503;Sigma)and0.02% sodium azide.0.05M MES(Sigma),pH5.Biotinylated goat anti-mouse and anti-rat immuno-globulin(#M30015;Caltag Laboratories,Burlin-game,CA;and#112-065-143;Jackson Labora-tories,West Grove,PA).Streptavidin R-phycoerythrin(SA-PE;Molecular Probes,Eugene,OR).Monoclonal antibodies to human IL-4(MAB604), IL-6(MAB206),IL-8(MAB208)and IL-12 (MAB611)(R and D Systems,Minneapolis,MN) and to human IL-2(555051),IL-10(554705), TNF-a(551220)and IFN-g(550521)(Pharmin-gen/BD Biosciences,San Diego,CA).4.2.Multiplexed microsphere-based flow cytometric assay(MFCA)Additional equipment and supplies:MultiScreen MABVN1.2-A m filter plates(Milli-pore,Burlington,MA).MultiScreen Vacuum Manifold(Millipore).Vacuum grease.Titertubes(#223-9390,Bio-Rad Laboratories,Her-cules,CA).Plate sealers(Falcon#353073,Becton Dickinson Labware,Franklin Lakes,NJ).Additional reagents:Recombinant human IL-4(204-IL),IL-6(206-IL), IL-8(208-IL)and IL-12(219-IL)(R&D Systems) and human IL-2(554603),IL-10(554611),TNF-a (554618)and IFN-g(554616)(Pharmingen)recon-stituted or diluted to20A g/ml in PBN,respectively, and stored in25A l aliquots atÀ80j C.Goat polyclonal antibodies to human IL-4 (BAF204),IL-6(BAF206),IL-8(BAF208)and IL-12(BAF219)(R&D Systems)and mouse monoclonal antibodies to human IL-2(555040),IL-10(554499),TNF-a(554511)and IFN-g (554550)(Pharmingen).10%formaldehyde(Polysciences,Warrington,PA). PBS,pH7.4,containing2.5%BSA and0.02% sodium azide.Wash buffer:PBS,pH7.4,containing0.05% Tween20(Sigma).Normal human serum pool.Prepare normal serum from five to eight persons with Serum Separation Tubes(Becton Dickinson Labware),combine the sera,aliquot and store atÀ20j C.Normal mouse serum(#S2-1409,Lampire Bio-logical Laboratories,Pipersville,PA).Normal rat serum(#S2-1509,Lampire).5.Detailed procedures5.1.Covalent coupling of cytokine capture antibodies to Luminex fluorescent microspheresa.All steps are performed at room temperature.b.Prepare capture antibodies by dissolving in0.05MMES,pH5,at a concentration of0.5–1mg/ml or dialyzing for1h in300ml MES buffer in dialysis thimbles and repeating for1h after a buffer e only200A l of antibody per thimble.Concentrations of sodium azide greater than0.9% in the antibody preparation can inhibit the coupling reaction.c.Warm both EDC and sulfo-NHS to room temper-ature in their bottles.Weigh approximately10mg of each into labeled,capped microtubes.d.Perform following steps in low light and preferablyin a biological safety cabinet to maintain sterility of all reagents and exercise universal precautions for handling human biologic material.e.Sonicate each vial of microspheres for1min on ahigh setting and vortex on a high setting for30s.Immediately aliquot200A l microspheres(2.5Â106microspheres)into USA Scientific microtubes, add200A l activation buffer,and centrifuge at 10,000rpm for8min.f.Wash the microspheres twice with activation buf-fer,being careful not to disturb the pellets.Check that the microspheres are not adhering to the sides of the tubes.If this occurs,increase the time of centrifugation.K.L.Kellar,J.P.Douglass/Journal of Immunological Methods279(2003)277–285279g.Remove supernatants and resuspend the micro-spheres in80A l activation buffer.V ortex for5s and sonicate for30s.h.Add a volume of activation buffer to both EDCand sulfo-NHS to prepare a final concentration of 50mg/ml.Resuspend the chemicals by vortexing.i.To each tube of microspheres,add10A l sulfo-NHS solution,vortex5s,and then immediately add10A l EDC solution and vortex.j.Incubate microspheres for20min on a rocker. k.Pellet the microspheres by centrifugation at10,000 rpm for8min and wash twice with150A l0.05M MES,pH5,vortexing after each addition of buffer.l.Resuspend the microspheres in100A l MES buffer and sonicate30s.m.Add125A g of each capture antibody prepared in MES buffer to the respective microspheres.Any of the eight microsphere sets can be used with any of the capture antibodies.V ortex immediately for 10s on a medium high setting.n.Incubate microspheres for2h on a rocker.o.Centrifuge microspheres for8min,remove super-natant and resuspend in PBN to block unreacted sites.p.Incubate for30min on a rocker.q.Wash microspheres twice in200A l PBN,each time vortexing for10s to resuspend.Resuspend in500A l PBN.r.Dilute an aliquot of the microspheres1:5in PBN to count on the hemacytometer.Count two of the nine large squares on the grid.Calculate the concen-tration:Bead count/2(no.squares)Â5(dilution factor)Â10,000=microspheres/ml.s.Adjust the concentration of each microsphere set to2Â106/ml with PBN and store the stocks at4 j C in a light-safe container.5.2.Test for the efficiency of coupling to the microspheresa.Aliquot5A l of each microsphere set(10,000 microspheres)followed by either50A l PBN (blanks)or50A l of a biotinylated antibody,specific to the source of the capture antibody,at10A g/ml into the wells of a black microtiter plate,in duplicate.Test each microsphere set individually.b.Incubate for30min on a shaker.c.Add50A l SA-PE at a final concentration of50A g/ ml to each well.d.Incubate for15min on a shaker.e.Add50A l of2%formaldehyde in PBS to each well. Fixation will allow for storage overnight,if necessary,and ensure that the fluorescence of each well is stabilized at the same stop time.f.Shake for5min.g.Read the plate immediately on a Luminex100or store at4j C to read the next day.Count100total microspheres per well to obtain the median fluorescent intensity(MFI).h.Coupling to the microspheres is successful when readings are approximately20,000MFI.Back-grounds should be approximately100MFI.5.3.MFCA procedure for cell culture supernatant samplesa.Initial setup and incubation of assay plate:(1)All incubations are performed at room temper-ature with microspheres and SA-PE reagentprotected from light.(2)Prepare the filter plate by adding100A l PBS-2.5%BSA-0.02%sodium azide to each wellthat will be used for the assay.Cover unusedwells with plate sealer.Shake for5min(minimum).(3)Apply a small amount of vacuum grease to thevacuum manifold gasket.(4)Wash pretreated wells by adding100A l washbuffer and securing the plate on the vacuummanifold.Apply vacuum and release whenbuffer has been removed.(5)Add20A l PBN to each well and replace theplastic lid on the plate.(6)Add1A l of each cytokine to92A l PBN in aTitertube to prepare a concentration of200ng/ml each.V ortex on medium speed for5s.(7)Add10A l of mixed cytokines to390A l PBN inanother tube and vortex.Concentration of eachcytokine is5000pg/ml.(8)Perform three-fold serial dilutions of cytokinesby adding100A l of5000pg/ml solution to200A l of PBN,vortex,and repeat the dilutions toprepare a total of eight dilutions of the standards.(9)Prepare dilutions of the samples in PBN asneeded.K.L.Kellar,J.P.Douglass/Journal of Immunological Methods279(2003)277–285 280(10)Add20A l of each cytokine dilution to a seriesof eight wells on the filter plate,in duplicate.(11)For blanks,add20A l PBN to four to eightwells.(12)Add20A l of each sample in duplicate ortriplicate to the plate.(13)Prepare the microspheres by calculating thevolume needed for the assay:20A l per wellÂ(number of wells+5extra wells).Twothousand of each set of microspheres will beadded to each well in20A l.Determine thevolume of each microsphere stock to beadded to the calculated final volume in PBNand prepare the mixture.(14)V ortex the microsphere mixture for30s.Pipet20A l into each well of the filter plate.(15)Place the lid on the filter plate and incubatefor45min on a shaker.b.Addition of the biotinylated detection antibodies:(1)Prepare a mixture of the eight biotinylatedantibodies after calculating the total volumerequired:10A l per wellÂ(number of wells+5extra wells).Determine the volume of eachantibody stock to add to PBN to obtain therequired concentration shown below.To re-optimize the assay,these amounts may bedetermined by titration.(i)anti-IL-2from500A g/ml stock,3A g/mlrequired(ii)anti-IL-4from200A g/ml,3A g/ml required(iii)anti-IL-6from200A g/ml,3A g/ml required(iv)anti-IL-8from200A g/ml,1A g/ml required(v)anti-IL-10from500A g/ml,0.5A g/ml required(vi)anti-IL-12from200A g/ml,10A g ml required(vii)anti-TNF-a from500A g/ml,3A g/ml required(viii)anti-IFN-g from500A g/ml,18A g/ml required(2)Wash the wells with100A l wash buffer.Repeatthe wash twice with200A l wash buffer.(3)Immediately add50A l PBN to the wells.(4)V ortex the biotinylated antibody mixture for10s and add10A l to all wells.(5)Place the lid on the plate and incubate for30min on a shaker.c.Addition of fluorescent reporter:(1)Prepare a dilution of SA-PE based on a5:1(ng/ng)ratio of the total biotinylated antibody.Forexample,if415ng total biotinylated antibodyis added per well,then2075ng SA-PE shouldbe added per well in a volume of10A l.Makethe same volume of SA-PE as biotinylatedantibody mixture.(2)V ortex and add10A l diluted SA-PE per well.(3)Place the lid on the plate and incubate for15min on a shaker.d.Termination and reading of the assay:(1)Wash the plate three times as described abovein b(2).(2)Add50A l PBN to each well.Then,add50A l2%formaldehyde.Replace the lid and shakethe plate briefly.(3)Read the plate on the Luminex100or storeovernight at4j C.Count75A l and100of eachset of microspheres per well.(4)Perform data analysis on SOFTMaxPROsoftware(Molecular Devices)by importingdata saved in MS Excel by the LuminexSoftware.5.4.MCF A for human serum samplesa.Perform steps5.3.a(1)–(8).(1)Prepare dilutions of the serum samples in PBN.A minimum dilution of1:5is recommended,butundiluted serum has been tested extensively.(2)To block false positives and negatives occur-ring with human serum samples,make a1:1mixture of normal mouse and rat serum.Add20A l to each well of the filter plate.(3)To simulate a serum matrix for the standardcurve,add20A l of a normal serum pool towells that will contain standards and blanks.(4)Add20A l of each cytokine dilution to a seriesof eight wells on the filter plate,in duplicate.(5)For blanks,add20A l PBN to four to eight wells.(6)Add20A l of each sample in duplicate ortriplicate to the plate.(7)Incubate to block for30min on a shaker.b.Continue with the MFCA procedure as described in5.3.a(13)–d(3).K.L.Kellar,J.P.Douglass/Journal of Immunological Methods279(2003)277–285281Fig.1.Standard curves from a multiplexed microsphere-based flow cytometric immunoassay (MFCA)for eight human cytokines in a serum matrix.Median fluorescent intensities were measured on a Luminex 100and the values were transformed by linear regression (A)or a four-parameter formula (B)for representation on a log –log or log –linear plot,respectively.Median fluorescent intensity was measured on a Luminex 100with Luminex 100Firmware 1.7and data transformed with SigmaPlot 2000software (SPSS,Chicago,IL).K.L.Kellar,J.P .Douglass /Journal of Immunological Methods 279(2003)277–2852826.Results6.1.Standardization of the MFCAStandard curves for all eight cytokines are repre-sented in Fig.1.Linear regression analysis of a log–log transformation of the data is useful for observation of the lower range of sensitivity of the standard curves (Fig.1A).However,data points often do not align with the curves at the higher range.Four-parameter (Fig.1B)and five-parameter formulas provide better transformation of standard values and interpolation of unknowns for these assays.Optimization of the standard curves is achieved primarily by titration of the biotinylated antibody required for each cytokine.The ranges of the stan-dard curves are defined by the affinities of the specific capture and detection antibodies and may not be the same for each analyte.The ratio of SA-PE to total biotinylated antibody should also be titrated. An optimal signal may be achieved with a low ratio when detection antibody levels are low.During initial assay development,a pool of serum from five to eight healthy individuals should be prepared to provide a complimentary matrix for the standards in assays of human serum samples.Serum often shifts or reduces the slopes of the standard curves to adversely affect the assay compared with buffer alone(Kellar et al.,2001).Conversely,pro-spective antibody pairs should be tested for accept-able performance with human serum.Once the MFCA is developed,individual normal serum can be tested as serum blanks and compared with buffer blanks to screen out those sera with higher MFIs that indicate detectable levels of cytokines.Optimally,the values of the serum assay blanks(MFIs)should be 100or lower for each cytokine.6.2.Validation of the MFCAThe primary purpose for the development of the MFCA was to reduce the time and costs for the measurement of multiple cytokines by ELISAs and to determine if the data obtained with both assay formats were comparable.Table1includes49exam-ples of data from cell culture supernatant or serum samples assayed by ELISA and MFCA.The same antibodies and standards were used in both assay Table1Validation of eight-bead multiplexed microsphere-based assays by comparison with ELISAsCytokines Beadsupernatantassay(pg/ml)ELISA(pg/ml)Beadserumassay(pg/ml)ELISA(pg/ml)IL-2252,305238,9001729158,757143,4002830117,807169,700241650,74652,000202929,21921,600IL-478<3153<31IL-619011669146153313361138206392004089<1058<1033<10 IL-821,20826,3411931528881156836104783016267837884984773523232974992512581291026381 IL-10760465IL-1243474122IFN-g23,91524,60536832,18627,228311650,54277,3933091629 TNF-a3142846794073356287561329470365234982097556162923612,3458718Cell culture supernatants and human serum samples were assayed with the multiplexed microsphere-based flow cytometric assays (MFCAs)and commercial ELISA kits to compare the microsphere methodology to the‘‘gold standard’’for sandwich immunologic assays,the mercial ELISAs were obtained from Pharmingen/BD Biosciences and R&D Systems.MFCAs were read on a Luminex100.ELISAs were read on a SpectraMAX Plus spectrophotometer(Molecular Devices).Data was transformed with SOFTMaxPRO software(Molecular Devices).A correlation coefficient of0.981(n=44)was calculated for all values within range of both assays by the Pearson Product Moment Correlation Parametric Test performed with Sigma Stat2.0statistical software (SPSS Science,Chicago,IL).K.L.Kellar,J.P.Douglass/Journal of Immunological Methods279(2003)277–285283formats.The MFCA detected a higher level of cytokine than the ELISAs in the majority of the comparisons and also detected IL-4and IL-6levels that were not detectable by ELISA.The correlation coefficient (R )for all values within range of both assays was 0.981(n =44).Similar and more exten-sive validations of the MFCA have been performed,primarily on two multiplexed assays for four cyto-kines (4-plex assays)that were combined to form the 8-plex assay detailed above (Kellar et al.,2001).Data in Table 2demonstrates the specificity of the 8-plex immunoassay.No cross-reactivity was ob-served at the highest standard concentration (5000pg/ml)when an individual cytokine was incubated with the multiplexed antibodies for detection of all eight cytokines.7.DiscussionMultiplexed microsphere-based flow cytometric immunoassays (MFCAs)have a number of advan-tages for quantitation of multiple human cytokines compared with ELISAs.An MFCA provides a more rapid,less-expensive method that has a three to four logarithmic range of sensitivity compared with one to two logs for ELISAs,as well as the specificityand reproducibility expected from the latter (Kellar et al.,2001;Kellar and Iannone,2002).These param-eters are achieved with high affinity antibodies that define the lower end of sensitivity of the assay and the rapid reaction kinetics.Multiplexed assay perfor-mance is totally dependent on the quality of the antibodies.Therefore,potential antibodies should be screened initially by comparing the slopes of single and multiplexed standard curves to detect possible cross-reactivity problems with the multiplexed rea-gents.Cross-reactivity can be identified more specif-ically by testing single cytokines with all the antibodies for a multiplexed assay (see Table 2)and by performing spike and recovery experiments (Kel-lar et al.,2001).The latter can also be used to iden-tify interference from the components of the sample itself.Human serum samples present more of a problem with multiplexed assays than with traditional one-analyte immunoassays (Boscato and Stuart,1988;De Groote,1998;Hennig et al.,2000;Kaplan and Levinson,1999;Kellar et al.,2001).The false positives and negatives that are often seen with sandwich immunoassays are multiplied when several pairs of antibodies are combined.In the MCFA outlined above,false positives were attributable to human anti-animal (HAA)antibodies (Kellar et al.,2001).The most effective blocking agent was the undiluted serum from the mammalian source of the capture and detection antibodies,which was a com-bination of normal rat and mouse sera.Goat serum did not improve the blocking activity.Potential blocking agents should be evaluated by performing spike and recovery studies with potential samples.Although seven different personnel have per-formed this protocol for almost three years with little or no variation,many variables in the MFCA can be modified.Incubation times and antibody pairs can be changed,washes can be eliminated,steps can be combined and reagents modified to further improve the time and costs of the assay.For example,the eight microsphere sets,cytokine standards and de-tection antibodies can be prepared as stock solutions for use in repeat experiments.Most importantly,the format of this assay can be readily applied to the development of multiplexed assays for other analy-tes,with the caveat that the reproducibility and reliability of the assay will be dependent on theTable 2Cross-reactivity of cytokines and detection antibodies in a multi-plexed microsphere-based immunoassay Microsphere specificity IL-2IL-4IL-6IL-8IL-10IL-12TNF-a IFN-c Blanks42663315133562747Cytokine addedIL-25759À20À9À9À7À12À7À13IL-4À85821À6À31À3À22À9IL-6À9À13935À31À6À8À8À10IL-8À9À14À910,313À7À15À8À11IL-10À3À6À3À228353À6À2À5IL-12À9À9À7À24À23135À5À6TNF-a À9À15À10À24À4À132301À8IFN-g À2À7À3À2473À1941High concentrations of only one cytokine (5000pg/ml)were added to reagents for a multiplexed standard curve for all eight cytokines in a serum matrix.The average of triplicate determinations of the median fluorescent intensities (MFIs)for each reaction minus the blank values are shown.MFIs were measured on a Luminex 100.K.L.Kellar,J.P .Douglass /Journal of Immunological Methods 279(2003)277–285284specificities and affinities of the antibodies for each analyte.8.Quick protocol8.1.Covalently couple cytokine capture antibodiesto microspheres.8.2.Test the efficiency of coupling the captureantibodies to microspheres.8.3.Preincubate the filter plate to block nonspecificbinding.8.4.Prepare the cytokine standards and dilute thesamples.8.5.Add20A l each of the standards and samples tothe filter plate.8.6.Prepare the microspheres,add20A l to each wellof the filter plate and incubate for45min in thedark.8.7.Wash the plate three times with100–200A lwash buffer before addition of10A l of thebiotinylated antibodies to each well.8.8.Incubate30min and then add10A l of SA-PE toeach well.8.9.Incubate15min,wash the plate three times,andadd a total of50A l of both PBN and fixationbuffer to each well.8.10.Read the plate on the Luminex100.Count75A l from each well and100of each set ofmicrospheres.8.11.Perform data analysis on SOFTMaxPRO.ReferencesBoscato,L.M.,Stuart,M.C.,1988.Heterophilic antibodies:a prob-lem for all immunoassays.Clin.Chem.34,27.Camilla,C.,Me´ly,L.,Magnan,A.,Casano,B.,Prato,S.,Debono, S.,Montero,F.,Defoort,J.-P.,Martin,M.,Fert,V.,2001.Flow cytometric microsphere-based immunoassay:analysis of se-creted cytokines in whole-blood samples from asthmatics.Clin.b.Immunol.8,776.Carson,R.T.,Vignali,D.A.A.,1999.Simultaneous quantitation of 15cytokines using a multiplexed flow cytometric assay.J.Im-munol.Methods227,41.Chen,R.,Lowe,L.,Wilson,J.D.,Crowther,E.,Tzeggai,K., Bishop,J.E.,Varro,R.,1999.Simultaneous quantification of six human cytokines in a single sample using microparticle-based flow cytometric technology.Clin.Chem.45,1693.De Groote,D.,1998.Cytokine measurement in serum or plasma with ELISAs:getting around the b.17,12. Hennig,C.,Rink,L.,Fagin,U.,Jabs,W.J.,Kirchner,H.,2000.The influence of naturally occurring heterophilic anti-immunoglobu-lin antibodies on direct measurement of serum proteins using sandwich ELISAs.J.Immunol.Methods235,71.Kaplan,I.V.,Levinson,S.S.,1999.When is a heterophile antibody not a heterophile antibody?When it is an antibody against a specific immunogen.Clin.Chem.45,616.Kellar,K.L.,Iannone,M.A.,2002.Multiplexed microsphere-based flow cytometric methods.Exp.Hematol.30,1227.Kellar,K.L.,Kalwar,R.R.,Dubois,K.A.,Crouse,D.,Chafin,W.D., Kane,B.-E.,2001.Multiplexed fluorescent bead-based immu-noassays for quantitation of human cytokines in serum and culture supernatants.Cytometry45,27.Oliver,K.G.,Kettman,J.R.,Fulton,R.J.,1998.Multiplexed analy-sis of human cytokines by use of the FlowMetrix system.Clin.Chem.44,2057.K.L.Kellar,J.P.Douglass/Journal of Immunological Methods279(2003)277–285285。
