BiochemicalMethodsGeneralidentificationmethods二生物檢測常用鑒別方法二

BiochemicalMethodsGeneralidentificationmethods二生物檢測常用鑒別方法二第1頁/共30頁Section4:Protein

DetectionTech蛋白質(zhì)檢測技術(shù)一、ELISA（Enzyme-linkedImmunosorbentAssays）酶聯(lián)免疫吸附法ELISAisabiochemicaltechniqueusedmainlyinimmunologytodetectthepresenceofanantibodyoranantigeninasample.

TheELISAcanbeusedtodetectthepresenceofantigensthatarerecognizedbyanantibodyoritcanbeusedtotestforantibodiesthatarerecognizedbyantigen.

ELISAcombinethespecificityofantibodieswiththesensitivityofsimpleenzymeassays,byusingantibodiesorantigenscoupledtoaneasily-assayedenzyme.

第2頁/共30頁Antigenisimmobilizedonasolidsupport(usuallyapolystyrene

(聚苯乙烯)microtiterplate).Aftertheantigenisimmobilized,theantibodysampleisadded,formingacomplexwiththeantigen,whichcanbedetectedbyasecondaryantibodywhichislinkedtoanenzymethroughbioconjugation.Betweeneachsteptheplateistypicallywashedwithamilddetergent(清潔劑)solutiontoremoveanyproteinsorantibodiesthatarenotspecificallybound.Afterthefinalwashsteptheplateisdevelopedbyaddinganenzymaticsubstratetoproduceavisiblesignal,whichindicatesthequantityofantibodyinthesample.A96-wellmicrotiterplatebeingusedforELISA第3頁/共30頁

ThePrincipleOfELISA

(1)Antigen(orantibody)isimmobilizedonasolidsupportandit’simmunityisremained.(2)Thedetectionantibody(orantigen)canbecovalently(共價)linkedtoanenzymethroughbioconjugation,whoseimmunityandenzymeactivityareremained.(3)Theplate(solidsupport)willcontainenzymeinproportiontotheamountofantigen/antibodycomplexes.Asubstratefortheenzymeisapplied,andcatalysisbytheenzymeleadstoachangeincolororfluorescence,whichindicatesthequantityofantigen(orantibody)inthesample.第4頁/共30頁

Immunosorbent:

固相的抗原或抗體，即“免疫吸附劑”

Conjugate:

酶標(biāo)記的抗原或抗體，即“酶聯(lián)物”

Substrate:

酶反應(yīng)的底物ThreenecessaryreagentsinElisa第5頁/共30頁TypesofELISASandwichELISA(雙抗體夾心法測抗原,雙抗原夾心法測抗體)IndirectELISA(間接法測抗體)CompetitiveELISA(競爭法測抗原/抗體)第6頁/共30頁SandwichELISA

工作原理：利用連接于固相載體上的抗體和酶標(biāo)抗體分別與樣品中被檢測抗原分子上兩個抗原決定簇結(jié)合，形成固相抗體-抗原-酶標(biāo)抗體免疫復(fù)合物。由于反應(yīng)系統(tǒng)中固相抗體和酶標(biāo)抗體的量相對于待測抗原是過量的,因此復(fù)合物的形成量與待測抗原的含量成正比(在方法可檢測范圍內(nèi))。測定復(fù)合物中的酶作用于加入的底物后生成的有色物質(zhì)量(OD值)，即可確定待測抗原含量。

是檢測抗原最常用的ELISA，適用于檢測分子中具有至少兩個抗原決定簇的多價抗原，而不能用于小分子半抗原的檢測。雙抗體夾心法測抗原乙肝標(biāo)志物中HBs-Ag(表面抗原)的檢測常采用本法第7頁/共30頁操作步驟：（1）將特異性抗體與固相載體聯(lián)結(jié)，形成固相抗體。洗滌除去未結(jié)合的抗體及雜質(zhì)。（2）加受檢標(biāo)本，保溫反應(yīng)。標(biāo)本中的抗原與固相抗體結(jié)合，形成固相抗原抗體復(fù)合物。洗滌除去其他未結(jié)合物質(zhì)。（3）加酶標(biāo)抗體，保溫反應(yīng)。固相免疫復(fù)合物上的抗原與酶標(biāo)抗體結(jié)合。徹底洗滌未結(jié)合的酶標(biāo)抗體。此時固相載體上帶有的酶量與標(biāo)本中受檢抗原的量相關(guān)。（4）加底物顯色。固相上的酶催化底物成為有色產(chǎn)物。通過比色，測知標(biāo)本中抗原的量。第8頁/共30頁AsandwichELISAProcedure:(1)Plateiscoatedwithacaptureantibody;(2)sampleisadded,andanyantigenpresentbindstocaptureantibody;(3)detectingantibody(enzyme-linkedantibody)isadded,andbindstoantigen;(4)substrateisadded,andisconvertedbyenzymetodetectableform.第9頁/共30頁"Indirect"ELISA(間接法測抗體)第10頁/共30頁"Indirect"ELISA

Applyasampleofknownantigenofknownconcentrationtoasurface,oftenthewellofamicrotiterplate.Theantigenisfixedtothesurfacetorenderitimmobile.Simpleadsorptionoftheproteintotheplasticsurfaceisusuallysufficient.Aconcentratedsolutionofnon-interactingprotein,suchasbovineserumalbumin(BSA)orcasein,isaddedtoallplatewells.Thisstepisknownasblocking,becausetheserumproteinsblocknon-specificadsorptionofotherproteinstotheplate.Theplatewellsorothersurfacearethencoatedwithserumsamplesofunknownantibodyofinterest.第11頁/共30頁Secondaryantibodies,whichwillbindtounknownantibodyofinterest,areaddedtothewells.Thesesecondaryantibodiesareconjugatedtothesubstrate-specificenzyme.Washtheplate,sothatexcessunboundenzyme-antibodyconjugatesareremoved.Applyasubstratewhichisconvertedbytheenzymetoelicit(引起)achromogenic(發(fā)色的)orfluorogenicorelectrochemicalsignal.View/quantifytheresultusingaspectrophotometer,spectrofluorometer,orotheroptical/electrochemicaldevice.第12頁/共30頁CompetitiveELISA

競爭法測抗原/抗體參考管待測管酶標(biāo)抗原酶標(biāo)抗原標(biāo)本(含待測抗原)參考品(不含抗原)第13頁/共30頁

以測定抗原為例，受檢抗原和酶標(biāo)抗原競爭與固相抗體結(jié)合，因此結(jié)合于固相的酶標(biāo)抗原量與受檢抗原的量呈反比。操作步驟如下：（1）將特異抗體與固相載體連接，形成固相抗體。洗滌。（2）待測管中加受檢標(biāo)本和一定量酶標(biāo)抗原的混合溶液，使之與固相抗體反應(yīng)。如受檢標(biāo)本中無抗原，則酶標(biāo)抗原能順利地與固相抗體結(jié)合。如受檢標(biāo)本中含有抗原，則與酶標(biāo)抗原以同樣的機會與固相抗體結(jié)合，競爭性地占去了酶標(biāo)抗原與固相載體結(jié)合的機會，使酶標(biāo)抗原與固相載體的結(jié)合量減少。參考管中只加酶標(biāo)抗原，保溫后，酶標(biāo)抗原與固相抗體的結(jié)合可達最充分的量。洗滌。（3）加底物顯色：參考管中由于結(jié)合的酶標(biāo)抗原最多，故顏色最深。參考管顏色深度與待測管顏色深度之差，代表受檢標(biāo)本抗原的量。待測管顏色越淡，表示標(biāo)本中抗原含量越多。參考管待測管酶標(biāo)抗原酶標(biāo)抗原參考品(不含抗原)標(biāo)本(含待測抗原)第14頁/共30頁CompetitiveELISAprocedure:Theantibodyisfixedtothesurfacetorenderitimmobile.Inreferencetube,Enzyme-linkedantigenareadded.Intesttube,mixturesofsampleantigenandenzyme-linkedantigenareadded,incubated.Themoreantigeninthesample,thelessantibodywillbeabletobindtotheEnzyme-linkedantigeninthewell,hence"competition."Substrateisadded,whichisconvertedbytheenzymetoelicit(引起)achromogenic(發(fā)色的)orfluorogenicorelectrochemicalsignal.ForcompetitiveELISA,thehighertheoriginalantigenconcentrationinsample,theweakertheeventualsignal.第15頁/共30頁ControldesignPositivecontrol(陽性對照)和Negativecontrol(陰性對照)陽性對照品應(yīng)與檢測標(biāo)本的組成相一致陰性對照品須先行檢測確定不含待測物第16頁/共30頁

BecausetheELISAcanbeperformedtoevaluateeitherthepresenceofantigenorthepresenceofantibodyinasample,itisausefultoolbothfordeterminingserumantibodyconcentrations(suchaswiththeHIVtest)andalsofordetectingthepresenceofantigen.itisalsousedtodetectcellgrowthfactor.Ithasalsofoundapplicationsinthefoodindustryindetectingpotentialfoodallergens（過敏原）.

TheApplicationOfELISA

第17頁/共30頁乙型肝炎血清標(biāo)志物

HepatitisBsurfaceantigen(HBs-Ag)：表面抗原

HepatitisBsurfaceantibody(HBs-Ab)：表面抗體

HBeAg:E-抗原

HBe-Ab:E-抗體

HBc-Ab-IgG:核心抗體IgG1.SandwichELISAdetectHepatitisBserumSigns(antibodyandantigen)

乙肝病毒標(biāo)志物，就是俗稱的乙肝“兩對半”第18頁/共30頁

雙抗體夾心ELISA法檢測HIV-1p24抗原近年來艾滋病病毒(HIV)感染在全世界迅速蔓延，我國自1985年發(fā)現(xiàn)艾滋病(AIDS)以來，HIV／AIDS的流行趨勢亦日趨嚴(yán)重，因此建立、發(fā)展與完善HIV檢測方法對于HIv／AIDS的防治尤為重要。HIV-1p24抗原的檢測日益受到世界各國的高度重視。而雙抗體夾心ELISA法不失為一種比較合適的方法。2.HIV-1p24antigen

detection(SandwichElisa)第19頁/共30頁TheELISAtestwasthefirstscreeningtestcommonlyemployedforHIV.Ithasahighsensitivity.InanELISAtest,aperson'sserumisdiluted400-foldandappliedtoaplatetowhichHIVantigenshavebeenattached.IfantibodiestoHIVarepresentintheserum,theymaybindtotheseHIVantigens.Theplateisthenwashedtoremoveallothercomponentsoftheserum.Aspeciallyprepared"secondaryantibody"—anantibodythatbindstootherantibodies—isthenappliedtotheplate,followedbyanotherwash.Thissecondaryantibodyischemicallylinkedinadvancetoanenzyme.Thustheplatewillcontainenzymeinproportiontotheamountofsecondaryantibodyboundtotheplate.Asubstratefortheenzymeisapplied,andcatalysisbytheenzymeleadstoachangeincolororfluorescence.3.HIVantibodydetection(indirectElisa)第20頁/共30頁二、Westernblot（免疫印跡技術(shù)）Thewesternblot(alternatively,immunoblot)isamethodofdetectingspecificproteinsinagivensampleoftissuehomogenateorextract.

Itusesgelelectrophoresistoseparatenativeordenaturedproteinsbythelengthofthepolypeptide,Theproteinsarethentransferredtoamembrane(typicallynitrocelluloseorPVDF),wheretheyareprobed(detected)usingantibodiesspecifictothetargetprotein.Thismethodisusedinthefieldsofmolecularbiology,biochemistry,immunogeneticsandothermolecularbiologydisciplines.ThemethodoriginatedfromthelaboratoryofGeorgeStarkatStanford.ThenamewesternblotwasgiventothetechniquebyW.NealBurnetteandisaplayonthenameSouthernblot.

Principles:第21頁/共30頁第22頁/共30頁WesternblotprocedureSDS(蛋白電泳)

Theproteinsofthesampleareseparatedusinggelelectrophoresis.Separationofproteinsmaybebyisoelectricpoint(pI),molecularweight,electriccharge,oracombinationofthesefactors.Thenatureoftheseparationdependsonthetreatmentofthesampleandthenatureofthegel.Byfarthemostcommontypeofgelelectrophoresisemployspolyacrylamidegelsandbuffersloadedwithsodiumdodecylsulfate(SDS).SDS(SDSpolyacrylamidegelelectrophoresis)maintainspolypeptidesinadenaturedstateoncetheyhavebeentreatedwithstrongreducingagentstoremovesecondaryandtertiarystructureandthusallowsseparationofproteinsbytheirmolecularweight.

第23頁/共30頁2.Transfer（轉(zhuǎn)膜）Inordertomaketheproteinsaccessibletoantibodydetection,theyaremovedfromwithinthegelontoamembranemadeofnitrocelluloseorpolyvinylidenefluoride(PVDF).

Transfermethods:Capillaryblotting:

Themembraneisplacedontopofthegel,andastackoftissuepapersplacedontopofthat.Theentirestackisplacedinabuffersolutionwhichmovesupthepaperbycapillaryaction,bringingtheproteinswithit.

Electroblotting:

AnothermethodfortransferringtheproteinsiscalledelectroblottingandusesanelectriccurrenttopullproteinsfromthegelintothePVDFornitrocellulosemembrane.

將凝膠中的蛋白質(zhì)轉(zhuǎn)印至膜上的方法很多。目前常用方法是毛細管虹吸法和電印跡法。其主要優(yōu)點是轉(zhuǎn)印迅速、完全。第24頁/共30頁3.Blocking（封閉）Sincethemembranehasbeenchosenforitsabilitytobindprotein,andbothantibodiesandthetargetareproteins,stepsmustbetakentopreventinteractionsbetweenthemembraneandtheantibodyusedfordetectionofthetargetprotein.Blockingofnon-specificbindingisachievedbyplacingthemembraneinadilutesolutionofprotein-typicallyBovineserumalbumin(BSA)ornon-fatdrymilk(bothareinexpensive)Theproteininthedilutesolutionattachestothemembraneinallplaceswherethetargetproteinshavenotattached.Thus,whentheantibodyisadded,thereisnoroomonthemembraneforittoattachotherthanonthebindingsitesofthespecifictargetprotein.第25頁/共30頁Duringthedetectionprocessthemembraneis"probed"fortheproteinofinterestwithamodifiedantibodywhichislinkedtoareporterenzyme,whichwhenexposedtoanappropriatesubstratedrivesacolourimetricreactionandproducesacolour.4.Detection(檢測)第26頁/共30頁

Twostepdetection(間接檢測):Primaryantibody,