免疫学精要免疫学精要1

Chapter13Immunotechnology

(免疫学技术)

SectionAAntibodiesasresearchanddiagnostictools

抗体作为研究和诊断的工具

Keynotes

Avarietyofassayshavebeendevelopedwhichprovidsspecificqualitativeand

quantitativemeasurementofAgorAb,bothofwhicharcoftencfconsiderableresearchand

clinicalrelevance.Abtoanorganismintheserumofapatientdemonstratesinfe-ctionby

theorganism.Ahwithdefinedspecificityisusedtodeterminethepresenceofdisease

associatedantigensinapatient.Astoolsinmolecularandcellularresearch,Abspermit

localizationandcharacterizationofAgs.

Antibodyandassays

Methodsformeasuringantigen-antibodyreactionshavebeenwellestablishedandinclude

those(hathavedirectbiologicrelevance(llihle1).ThecombinationofAbwithbiologically

activeAg(virus,toxin,enzymeandhormone)canbedetectedbyneutralizationofthevirus

infection,toxicity,enzymaticandhormoneactivity,respectively.Precipitationand

agglutinationhavealsobeenadaptedfodevelopmentofseveralusefulassays.

Table1.Effectsofcombinationofantigenandantibody

AgglutinationAntigenicparticle+specificAbresultsinaggregationofparticles

PrecipitationSolubleAg+specificAbresultsinlatticeformationandprecipitationC

ActivationAginsolutionoronparticle+specificAbresultsinactivationofC

CytolysisCell+anti-cellAb+Cmayresultinlysisofthecell

OpsonizationAntigenicparticle+Ab+CenhancesphagocytosisbyMo,MO,PMNs

NeutralizationToxins,viruses,enzymes,etc.+specificAbsmayresultintheirinactivation

*C,Complement；Mo,monocytes；MO,macrophages:Ab,antibody；PMNs,

polymorpho-nuclearcells

Avarietyofotherassayshavebeendevelopedwhichprovidespecificqualitativeand

quantitativemeasurementofAgorAbforbothresearchanddiagnosticpurposes.

Diagnosticpurpose

SincetheimmunesystemrecognizesandremembersvirtuallyallAgthatareintroducedinto

anindividual,assayswhichdemonstratethepresenceofAbtoanorganismintheserumofa

patienthavebecomeastandardwayofdeterminingthatthepatienthashadcontactwith,was

infectedby,theorganism

DiagnoseforAIDs

e.gthepresenceofAbtoHIVintheserumofapatientusuallymeansthatthepatienthasbeen

infectedwithHIV.

Alternatively,Abswithdefinedspecificity(e.g.(toAgsassociatedwithcancercells)canbe

usedtodeterminethepresenceofdiseaseassociatedAgsinapatient.

Researchpurpose

Absarealsoextremelyimportanttoolsinmolecularandcellularresearchastheypermitthe

localizationandcharacterizationofAgs.

SectionBprecipitationandagglutination

沉淀和凝集

Keynotes

Precipitationassays

CombinationofAbwithAgresultsinlatticeformationandprecipitationifthereis

sufficientAgandAb(equivalence).Thesereactionsarethebasisforqualitativeand

quantitativeassaysforAgorAb,includingradialimmunodiffusion(辐射免疫扩散)and

immunoclcctropliorcsis(免疫电泳).

Agglutinationassays

TheinteractionofsurfaceAgsoninsolubleparticles(e.g.cells)withspecificAbtothese

Agsresultsinagglutinationoftheparticles.Agglutinationcanbeusedtodetermineblood

types;thepresenceofAbtobacteriainserumisanindicationofpreviousorcurrentinfection;

andintheCoombstestautoantibodiestoerythrocytescanbeassayed.

1.Precipitationassays

Aspreviouslydescribed,whenthereisbothsufficientAgandsufficientAb.thecombination

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ofAgandAbproceedsuntillargeaggregatesareformedwhichareinsolubleinwaterand

precipitate(equivalence).Theextenttowhichalatticeformsdependsontherelativeamounts

ofAgandAbpresent.Latticefonnation,andprecipitationarethebasisforseveralqualitative

andquantitativeassaysforAgorAb.Theseassaysaredoneinsemisolidgelsintowhichholes

arecutforAgand/orforAbanddiffusionoccursuntilAgandAbareatequivalenceand

precipitate.

1.1Radialimmunodiffusion

InradialimmunodifTusion,Ab(e.g.horseanti-humanIgG)isincorporatedintothegeland

Ag(e.g.humanserum)isplacedinaholecutinthegel.Agdiffusesradiallyoutofthewell

intothegelandinteractswiththeAbformingaringofprecipitation,thediameterofwhichis

relatedto(heconcentrationoftheAg(Fig.1).

Fig.I.MeasurementofAgbyprecipitationIngels.Ab-containinggelisplacedonaglassor

plasticsurface.HolesarecutinthegelandfilledwithAgwhichdiffusesradiallyoutofthe

wellandinteractswiththeAbinthegel.Solublecomplexesareinitiallyformedbutasmore

Agdiffusesequivalenceisreachedresultinginalatticeandprecipitation.Thediameterofthe

precipitationringIsrelatedtotheconcentraiionoftheAgand,usingknownstandards,canbe

quantitatedandcomparedwiththelevelsofAginothersamples.

Similarassayshavebeendevelopedinwhichavoltagegradient(electrophoresis)isusedto

speedupmovementofAgintotheAhcontaininggel(rocketImmunoelectrophoresis火箭免

疫电泳).

1.2immunoelectrophoresis

Inimmunoelectrophoresis,Ags(e.g.scrum)arcplacedinawellcutinagel(withoutAb)

andelectrophorcscd,afterwhichatroughiscutinthegelintowhichAbs(e.g.horse

anti-human)arcplaced.TheAbsdiffuselaterally(横向扩散)tomeetdiffusingAg,and

presenceofcirculatingAbtoacellsurfaceAgisdemonstratedbyaddingthepatient'sserum

totestcells(e.g.erythrocytes)followedbyadditionofAbtohumanAb.

Agglutination:Antigeninsolublebeforeaddingantibody

Precipitation:Antigenisinitiallysoluble;antibodybindingtoitcreatesalatticeand

makesitinsoluble

Agglutination

Antibody

SectionCImmunoassay

免疫测定

Keynotes

ELISA,RIA

ThepresenceandconcentrationofaspecificAgorofanAbtoaspecificAginsolutioncan

bedeterminedbyradioimmunoassays(RIA,放射免疫测定法)orenzyme-linked

immunoabsorbentassays(ELISA,酶联免疫吸附测定法).Agattachedtoasolidsurface

capturestheAbwithwhichitreactsandisquantitatedusingalabeledsecondAbreactiveto

thefirs!.TheseassayspermitmeasurementofawidevarietyofAgsaswellasthe

concentrationofAbsspecificfbragivenAg,suchas(hosereactivewithaninfectious

organism.

Immunofluorescenceandflowcytometry

UsingafluorescencemicroscopeandAbslabeledwithafluorescentmolecule,tissue

sectionscanbeexaminedforcellsexpressingparticularAgs(e.g.thosewhicharetumor

associated).Directorindirectimmunofluorescencetechniquespermitqualitativeand

quantitativeevaluationofseveraldifferentcellassociatedmoleculesatthesametime.Flow

cytometersrapidlyanalyzelargenumbersofcellsinsuspension,providingamolecular

fingerprintoftheceils.Fluorescenceactivatedcellsortersseparatecellsubpopulationsfbr

moredetailedstudy.

immunoblotting

Immunoblottingisusedtoassayforthepresenceofmoleculesinamixture.Westernblot

analysisinvolvesseparatingmoleculesbysodiumdcdccylsulfatepolyacrylamidegel

electrophoresis(SDS),transferringthemtoanothermatrixanddelectingthemolecule

ofinterestusingELISAorRIA.ThisassayisoftenusedtoconfirmthepresenceofAbsto

infectiousagents(e.g.HIV)inpatientserum.Immunoblottingcanalsobeusedtoanalyze

productsofsinglecells(e.g.cytokines)andthenatureoftheproducingcell.

1.ELISA,RIA

ThepresenceofAbtoaparticularAgintheserumofapatientcanbedeterminedusing

verysensitiveradioimmunoassays(RIA)orenzyme-linkedirnmuno-absorbentassays

(ELISA).Suchassays(Fig.)areofparticularvalueindemonstratingAbtoAgsofinfectious

agents,e.g.virus,bacteria,etc.Theradicallergosorbenttest(RAST,放射变应原吸附测定法)

usesaradiolabeledAbtohumanIgEasdetectingligandandpermitsthemeasurementof

specificIgEAbtoanallergen.ELISAandRIAalsoprovideveryspecificandsensitive

measurementoftoxins,drugs,hormones,pesticides,etc.,notonlyinserum,butalsoinwater,

foodsandotherconsumerproducts.Basedontheseprocedures,assaysfornearlyanyAgor

Abcanbereadilydeveloped.

1.1Radioimmunoassay(RIA)

Addantigen

andwashAntigen

AddtestAb

'Ab

andwash

Addradiolabeled

ligand,washandYigand

couni/

/

/

plastic

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Radioimmunoassay(RIA).Antigenisincubatedonplasticandsmallquantitiesareadsorbed.

Freeantigeniswashedaway.Testantibodyisadded,whichmaybindtotheAg,andunbound

Abwashedaway.AbremainingboundtotheAgisdetectedbyaradiolabeledligand(e.g.an

AbspecificfortheisotypeofthetestAb,orstaphylococ-calproteinAwhichbindstotheFc

regionoflgG

1.2Radioallergosorbenttest(RAST)

ThisissimilartoRIAexceptthattheligand(e.g.theAbthatbindsthetestAg)iscovalently

coupledtoanenzymesuchasperoxidase.ThisligandbindsthetestAbandafterfreeligandis

washedawaytheboundligandisdetectedbytheadditionofsubstratewhichisactedonby

theenzymetoyieldacoloredanddetectableendproduct.

Addantigen

(ategen)andwash'An舸

Addlestserum

IgEAb

andwash

Addradiolabeled0K*

ligand,washandLgand印ecdic

CCUfltlorIgEAb

ThismeasuresA^-specificIgEinanRIAwheretheligandisalabeledanti-lgEAbandisvery

similartothestandardRIA.

1.3Enzyme-linkediminunoabsorbantassay(ELISA)

Addantigen

andwash

AddtestAb

andwash

Addligand

andwash

Add$ubs:rate

andread

1.4SandwichELISA

ThisisbasicallythesameasdescribedinRIAandELISAexceptthatAbtotheAgisfirst

usedtocoattheplasticinordertospecificallycapturetheAgfromamixture.Asecond

enzymeorradioisotopicallylabeledAb,whichreactswithanepitopeontheAgwhichis

differentfromthatofthefirstAb,isthenaddedforquantitationoftheantigen.

Addantibody

andwash'Antibody・

AddtestAgr皿^Antigen

andwash

AddenzymelinkedY黑dtnzyme

Abtodifferentd^lerm-

inantonAgandwash

AddsubstrateY(。Subslralp

andread

2.Immunofluorescence免疫荧光

ComparisonwithELISAandRIA

AlthoughitispossibletouseELISAandRIAtoevaluatethepresenceofanAgonacell,

thisisusuallymoreconvenientlydoneusingAbstowhichafluorescentmarkerhasbeen

covalentlyattached.Moreover,inmostcasesainAbisusedandthusishighlyspecificfora

particularmoleculeandaparticularepitopeonthatmolccplc.

Directandindirectimmunofluorescence

ThistypeofassaycanbedoneusinganAbto由eAgwhichisdirectlyfluorescentlabeled

(directimmunofluorescence)orbyfirstincubatingtheunlabclcdAbwiththecells(e.g.a

mousemAbtohumanTcells)andthen,afterwashingawayunboundAb,addingasecond

fluorescentlabeledAbthatreactswiththefirstAb(e.g.agoatAbtomouseimmunoglobulin).

Animaltissues[rat)arefrequentlyusedtoidentifyhumanautoantibodiessincethe

autoantigensrecognizedaregenerallyconservedacrossthespecies.Patientserumisaddedto

thetissuesectionsandtheautoantibodiesbindtoparticularautoantigen(s).Afterwashing,

fluorescentantibodiestohumanIgGareaddedandviewedunderafluorescencemicroscope.

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Agreencolorshowswherethehumanantibodieshaveboundtothetissueautoantigens.

+Auorescen-labeled

mouseantihumanIgG

HPatternserum

(IgGantibodies)

Tissueantigens

△八1A

Advantagesofindirectimmunofluorescene

Thisindirectimmunofluorescentassayhastwoadvantages,ithashighersensitivity

andrequireslabelingofonlyoneAb,(hesecondAb,because,intheexamplegiven,itcan

delect(reactwith)anymouseAb.

Applications

(1)FluorescentAbs(ocellsurfacemolecules(e.g.thosewhicharetumorassociated)arevery

usefulinexaminingtissuesectionsfbrcellsexpressingtheAg.

ThisassayisdonebyincubatingthetissuesectionwiththelabeledAb(fordirect

immunoAuorescence(IF))orunlabeledAb,followedbylabeledsecondAband(hen

examiningthe(issuesectionusingafluorescentmicroscope.Thesemicroscopesirradiate(he

tissuewithawavelengthoflightthatexcites(hefluorescentlabelontheAbloemitlightata

differentwavelength.Thisemittedlightcanbedirectlyvisualized,photographedandeven

quantitated.

(2)Moreover,i(ispossibletoanalyzeatissuesampleusingseveraldifferentAbsal(hesame

time,aseachAbcouldbelabeledwithadifferentfluorescentmoleculeeachofwhichemit

lightatawavelengthdistinctfromtheothers.

(3)Itisalsopossibletolookforintracellularmolecules(e.g.Abs)byfirstpenneabilizingthe

cellsandthendoingthestainingandfluorescencemicroscopy(先透化处理细胞,然后进行染

色和荧光镜检以寻找胞内分子).Thus,onecanusethisapproachtodevelopamolecular

fingerpriniofthecellsassociatedwithatissue.

3.Flowcytometry(流式细胞术)

Althoughfluorescencemicroscopycanbe,andis,appliedtotheanalysisofsinglecell

suspensions,anotherrathertechnologicallysophisticatedapproach,flowcytometry,ismost

oftenused.

Thisassayusesthesamebasicstainingproceduresasdescribedforfluorescencemicroscopy,

followedbyautomatedquantitationoftheamountoffluorescenceassociatedwithindividual

cells.(这种方法采用如用荧光显微镜所用的同一基本染色步骤，随之进行与单个细胞

相关的荧光量的自动定量。)

Flowcytometry

erlabelingwithfluorescentantibody,cellsarepassedoneatatimethroughalaserbeam.

Photodetectorsmeasuretheamountoffluorescencewhichisplottedasahistogramshowing

theproportionojnon-Jluoresceni(unstained)andJlnorescent(stained)cells.Otherdetectors

simultaneouslymeasurescatteredlaserlight,whichisusedtogenerateci'dotblot'inwhich

lymphocytes,monocytesandgranulocytescanbediscriminated.

Inparticular,thesuspensionofstainedcellsisfedtotheflowcytometerwhichdisperses

thecellssotheythenpasssinglefilethroughafocusedlaserbeam(细胞排成单一纵列通过

聚焦的激光束)whichexcitesanyfluorescentlabelassociatedwiththecells.Thosestainedby

thefluorescentAbemitlightthatisdetectedandquantitatedbyopticalsensorsand(he

intensityoffluorescenceisplottedinhistographIbrm(直方图形式)byacomputer.

Thismachinecananalyze1000cellspersecondandprovidequantitativedataonthe

numberofmoleculesofaparticularkindoneachcell.Itcanalsoanalyzemixturesofcells

andprovidedataontheirsizeandgranularityinadditiontotheirexpressionofspecific

molecules.

Someversionsofthismachine(fluorescenceactivatedcellsorter)arealsoableto

separateoutcellsintoinicrodropletsandsortthoseexpressingaselectedamountofa

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particularAgintoaseparatelubeforfurtheranalysisorculture.

4.Inimunoblotting

WesternblotanalysisinvolvesseparatingAgsbypolyacrylamidegelelectrophoresis(PAGE)

in(hepresenceofsodiumdodecylsulfate(SDS)whichresultsinseparationofmoleculeson

thebasisofsize.

Procedures

(1)WesternblotanalysisinvokesseparatingAgsbypolyacrylamidegelelectrophoresis

(PAGE)inthepresenceofsodiumdodecylsulfate(SDS)whichresultsinseparationof

moleculesonthebasisofsize.

(2)Thesemoleculesare(hentransferredtoanothermatrix(e.g.nitrocellulose)toforma

patternonthematrixidenticaltothatonthegel.

(3)EnzymelinkedAb(othemoleculeofinterestisthenadded,iheunboundAbwashedoff

andsubstrateadded(seeELISA)forvisualization.(stainingandvisualization)

applications

(1)Thisassaypermitsspecificidentificationofproteinsinamixture.

(2)Itisalsooftenusedtoconfir