蛋白质组学技术：液相芯片系统 Liquichip

液相芯片系统Liquichip蛋白质组学和细胞因子炎症的中心环节—血管细胞反应炎症的关键环节—炎症细胞因子IntensiveCareMed2012;30:1702-14细胞因子与炎症反应SIRSInfectiontraumaburnshemorrhageMODSDeathCytokinesMonocyte/macrophageNeutrophilFibroblastTlymphocyteMastcellBlymphocyte细胞因子的复杂性—来源和靶细胞细胞因子的复杂性—急性和慢性炎症反应细胞因子的复杂性—促炎因子和抗炎因子细胞因子的检测方法

ELISAMultiplexArrays—Luminex液相芯片11BALB/c小鼠内毒素休克后3h肝脏组织中细胞因子的表达谱改变0IP-10KCMCP-1MIGMIP-1

GM-CSFIFN-

IL-10IL-12IL-1

IL-2IL-4IL-5IL-6TNF-

40008000120001600020000ControlLPSMFI0IP-10KCMCP-1MIGMIP-1

GM-CSFIFN-

IL-10IL-12IL-1

IL-2IL-4IL-5IL-6TNF-

ControlLPSBALB/c小鼠内毒素休克后3h血浆中细胞因子的表达谱改变500010000150002000025000MFI12Liquichip液相芯片系统orLuminex®

MAP

液相芯片系统有机地整和了有色微球（color-codedmicrosphereorbead）、激光技术、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LuminexMAP无与伦比的检测特异性和灵敏度。13BenefitsApplicationsFutureTechnology14PrincipleDetectionandQuantitationofthesebiologicalinteractionsismadebyfluorescentdetectionreagentsBeads球形基质Capturemolecule探针分子Analyte待检测物Reportermolecule报告分子15TwointernalDyesforBeadClassificationDefinedMixture Dyingin ShrinkinginofbothDyes OrganicSolventAqueousSolvent16Two-ColorBead-CodingBasedonxMAPtechnologyByusing10differentlevelsofeachoftwofluorescentdyes,anarrayof100beads,eachwithauniquecolorsignature,iscreated17反应主要包括3个步骤探针分子的固定将这种标记好探针的球形基质与样品反应。反应结果的检测18“LiquidArray”TechnologyDifferentbeadcodes(colors)areusedtoallowtheidentificationofmultiplereactionsinasinglewell19“LiquidArray”TechnologyDifferentbeadcodes(colors)areusedtoallowtheidentificationofmultiplereactionsinasinglewell20“LiquidArray”TechnologyDifferentbeadcodes(colors)areusedtoallowtheidentificationofmultiplereactionsinasinglewell21LiquiChipWorkstationFlowcytometer-basedtechnologyMeasuresfluorescenceComponents:ReaderMicroplateHandlerFluidModule22FlowCytometerbasedAnalysisBeadsarealignedinasinglefilestreambyhydrodynamicfocusingTwolasersareusedforexcitationoffluorescence23PrincipleofDetectionTheredlaserisusedtoidentifythebeadcodeThegreenlaserisusedtoidentifythereportersignalCytokineCapture/Sandwich

IL-2

IL-4

TNFa

GmCSF25

利用这100种球形基质，可以标记上100种不同的探针分子，同时对一个样本中的100种不同的目的分子进行检测。

26Benefits27系统的优点灵活性好，可适用于各种蛋白质分析，可以接受实验室已有的实验方案，使用者可以自行设计分析方案，也可使用成套试剂盒。

通量大，可对同一样本中的多种不同目的分子同时进行分析；在35-60分钟内可对96个不同样本进行检测液相环境更有利于保持蛋白质的天然构象，也更有利于探针和被检测物的反应灵敏度高，信噪比好，只需要微量的样品即可进行检测操作简便，耗时短，成本低

28Liquichip和ELISA灵敏度的比较

分别用ELISA和liquichip对硫氧还蛋白进行定量测定得到的标准曲线

I:使用liquichip测定得到的标准曲线

II、III：使用ELISA测定得到的标准曲线

29ComparedtoELISA:lesssampleneededlesspipettinglesswashingfasterproceduremoreeconomic30Ready-to-useBroad-RangeKinasekitsformeasuringkinaseactivity

Ser/ThrKinase,TyrKinaseKit

Kinase/PhosphorylatedProtein31Applications32WhentousetheLiquiChipSystem?TheLiquiChipSystemisusedforalargevarietyofproteinassays:ImmunoassaysEnzymeAssaysProtein-proteininteractionAssaysReceptorligandAssaysProteinnucleicacidassays33AssayTypesImmunoassaysEnzymeassays(kinase,protease)DNA-hybridizationassays

Interactionassays34CytokinesBDNFHuman

CREBpS133General

CREBTotalGeneral

DR-5Human

EGFHuman

Mouse

ENA-78Human

EotaxinMouse

Human

FattyAcidBindingProteinHuman

FGF-basicHuman

Mouse

G-CSFHuman

Mouse

GCP-2Mouse

GM-CSFHuman

Mouse

Rat

GRO-KCRat

HGFHuman

Mouse

ICAM-1Human

IFN-alphaHuman

IFN-gammaHuman

Mouse

Rat

IL-10Human

Mouse

Rat

IL-11Mouse

IL-12Human

Mouse

IL-12p40Human

Mouse

IL-12p40/p70Mouse

Rat

IL-12p70Human

Mouse

Rat

IL-13Human

Mouse

IL-15Human

Mouse

IL-16Human

IL-17Human

Mouse

IL-18Rat

IL-1alphaMouse

Human

Rat

IL-1betaHuman

Mouse

Rat

IL-1raHuman

IL-1ra/IL-1F3Human

IL-2Human

Mouse

Rat

IL-3Human

Mouse

IL-4Human

Mouse

Rat

IL-5Human

Mouse

IL-6Human

Mouse

Rat

IL-7Human

Mouse

IL-8Human

IL-9Mouse

IP-10Human

Mouse

JE/MCP-1Mouse

KCMouse

KC/GROaMouse

LIFMouse

LymphotacinHuman

Mouse

M-CSFMouse

MCP-1Human

Mouse

Rat

MCP-1(MCAF)Human

MCP-3Mouse

MCP-5Mouse

MDCHuman

Mouse

MIGHuman

Mouse

MIP-1alphaHuman

Mouse

MIP-1betaHuman

Mouse

MIP-1gammaMouse

MIP-2Mouse

MIP-3betaMouse

OSMMouse

PDGF-BBMouse

RANTESHuman

Mouse

Rb(pT821)Human

Rb(total)Human

RbpSpT249/252Human

Tau(pS214)Human

Tau(pS396)Human

Tau(total)Human

TissueFactorHuman

Mouse

TNF-alphaHuman

Mouse

Rat

TNF-betaHuman

TNF-RIHuman

TNF-RIIHuman

VCAM-1Mouse

VEGFHuman

Mouse

35InfectiousdiseaseAdenovirusHuman

Mouse

BordetellapertussisHuman

CampylobacterjejuniHuman

ChlamydiapneumoniaeHuman

ChlamydiatrachomatisHuman

CholeraToxinHuman

CholeraToxinbHuman

Clostridiumpiliforme(Tyzzer's)Mouse

CytomegalovirusHuman

Mouse

DiphtheriaToxinHuman

EctromeliavirusMouse

EDIM(Epidemicdiarrheaofinfantmice)Mouse

EncephalitozooncuniculiMouse

Epstein-BarrEAHuman

Epstein-BarrNAHuman

Epstein-BarrVCAHuman

HBVCoreHuman

HBVEnvelopeHuman

HBVSurface(Ad)Human

HBVSurface(Ay)Human

HCVCoreHuman

HCVNS3Human

HCVNS4Human

HCVNS5Human

HelicobacterpyloriHuman

HepatitisAHuman

HepatitisDHuman

HIV-1gp41Human

HIV-1p24Human

HPVHuman

HSV-1gDHuman

HSV-1/2Human

HSV-2gGHuman

HTLV-1/2Human

InfluenzaAHuman

InfluenzaAH3N2Human

InfluenzaBHuman

LeishmaniadonovaniHuman

LymediseaseHuman

LymphocyticchoriomeningitisvirusMouse

M.pneumoniaeHuman

M.tuberculosisHuman

MinutevirusMouse

MumpsHuman

MycoplasmapulmonisMouse

Parainfluenza1Human

Parainfluenza2Human

Parainfluenza3Human

ParvovirusMouse

PneumoniavirusofmiceMouse

PolioVirusHuman

PolyomavirusMouse

Reovirus-3Mouse

RSVHuman

RubellaHuman

RubeolaHuman

SendaivirusMouse

T.cruziHuman

T.pallidum15kdHuman

T.pallidump47Human

TetanusToxinHuman

Theiler'smouseencephalomyelitisvirusMouse

ToxoplasmaHuman

VaricellazosterHuman

HEVorf23KDHuman

HEVorf26KDHuman

HEVorf33KDHuman

HIV-1gp120Human

36

Autoimmune

ASCAHuman

beta-2MicroglobulinHuman

Mouse

CentromereBHuman

ChromatinHuman

ENAProfile4(SSA,SSB,Sm,RNP)Human

ENAProfile5(SSA,SSB,Sm,RNP,Scl-70)Human

ENAProfile6(SSA,SSB,Sm,RNP,Scl-70,Jo-1)Human

HistoneHuman

HistoneH1Human

HistoneH2AHuman

HistoneH2BHuman

HistoneH3Human

HistoneH4Human

HSP-27pS82General

HSP-27TotalGeneral

HSP-32Human

HSP-65Human

HSP-71Human

HSP-90aHuman

HSP-90bHuman

Jo-1Human

PCNAHuman

Mouse

PR3Human

PR3(cANCA)Mouse

RibosomalPHuman

Mouse

RNPHuman

Mouse

RNP-AHuman

RNP-CHuman

SCFHuman

Mouse

Scl-70Human

Mouse

SerumAmyloidPHuman

SLEProfile8(SSA,SSB,Sm,RNP,Scl-70,Jo-1,Ribosome-P,chromatin)Human

SmGeneral

Human

SmithMouse

Human

SSAHuman

Mouse

SSBHuman

Mouse

StreptolysinOHuman

TPOHuman

Mouse

37Kinase/PhosphorylatedProteinAktGeneral

Akt/PKB(total)General

Akt/PKBpS473General

ATF2(Thr71)General

Erk-2General

Erk1(Thr202/Tyr204)General

Erk1/Erk2(Thr202/Tyr204)General

Erk2(Thr202/Tyr204)General

GSK-3betaGeneral

IkappaB-alphapS32General

IkappaB-alphaTotalGeneral

JNK(pTpY183/185)General

JNKTotalGeneral

JNKp(Thr183/Y182)General

MAPKAPK2General

p38(total)General

p38MAPKpT180/pY182General

p53(total)General

p53pS15General

PKB-alphaGeneral

PKCGeneral

SAPK1General

SAPK1a/JNK2General

SAPK4General

STAT1pY701General

STAT1TotalGeneral

STAT3(Tyr705)General

ZAP-70General

38CancerMarkersalphaFetoproteinHuman

CancerAntigen125Human

CarcinoembryonicAntigenHuman

PSA,FreeHuman

CardiacMarkersCreatineKinase-MBHuman

Endothelin-1Mouse

PAPHuman

SGOTHuman

Mouse

TIMP-1Human

Mouse

IsotypingIgAHuman

Mouse

IgEHuman

Mouse

IgG1Mouse

IgG2alphaMouse

IgG2betaMouse

IgG3Mouse

IgMHuman

Mouse

lightchain(kappaorgamma)Mouse

GenotypingFlexMAP™General

MitochondrialDNAScreeningGeneral

Tag-It™MutationDetectionKitGeneral

Y-SNPIdentificationGeneral

39EndocrineAdiponectinHuman

Rat

AmylinMouse

Rat

Human

C-PeptideHuman

CalcitoninHuman

CRFHuman

FGF-9Mouse

GLP-1Human

Mouse

Rat

GlucagonMouse

Rat

Human

GrowthHormoneHuman

Mouse

InsulinHuman

Mouse

Rat

LeptinHuman

Mouse

Rat

Lipoprotein(a)Human

ResistinHuman

Mouse

Rat

T3Human

T4Human

TBGHuman

ThyroglobulinHuman

TSHHuman

AllergyTestingAlternaria(Mold)Human

BermudaGrassHuman

CatDanderHuman

EggWhiteHuman

MilkHuman

MitePternoyssinusHuman

MountainCedarHuman

ShortRagweedHuman

TimothyGrassHuman

Wheat(food)Human

40040008000120001600020000ControlLPSIP-10KCMCP-1MIGMIP-1

GM-CSFIFN-

IL-10IL-12IL-1

IL-2IL-4IL-5IL-6TNF-

图1.BALB/c小鼠内毒素休克后3h肝脏组织中细胞因子的表达谱改变MFI410500010000150002000025000IP-10KCMCP-1MIGMIP-1

GM-CSFIFN-

IL-10IL-12IL-1

IL-2IL-4IL-5IL-6TNF-

ControlLPS图2.BALB/c小鼠内毒素休克后3h血浆中细胞因子的表达谱改变MFI42Future434445Spectral-notSpatialSeparation谢谢各位！Mrp8andMrp14belongtotheS100familyof

calcium-binding.MRP8/MRP14isanendogenousDAMPinvolvedinvariousinflammatorydiseases.Trauma/hemorrhagicisassociatedwithmorbidityandmortalityduetodysregulatedinflammation,whichisdriveninpartbymonocytes/macrophagesstimulatedbyinjury-inducedreleaseofDAMPmolecules.MRP8/14asNovelDAMPsMRP8/MRP14inpatientplasmawasmeasuredusingspecificELISAkit.THP-1cellswerechallengedwith3mg/mlrecombinantMRP8/MRP14.CytokineprofilesintheculturesupernatantswerescreenedusingmultiplexcytokineassaykitbyLuminexsystem.MRP8/MRP14inducesIP-10expressionattranscriptionallevel.TLR4-TRIFpathway,butnotRAGE,MyD88,andCD14pathway,mediatesinductionofIP-10byMRP8/MRP14.FunctionalactivationofNF-kBinresponsetoMRP8/MRP14.ActivationofIRF3inducedbyMRP8/MRP14isinvolvedintra