Immuno asaay

7/28/2019 Immuno asaay

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IMMUNOASSAYIMMUNOASSAYAA briefbrief guideguide throughthrough itsits historyhistory,, principlesprinciples,,

practicepractice andand futurefuture trendstrends

HelenaHelena FingerovFingerovPalack UniversityPalack University MedicalMedical SchoolSchool, Olomouc,, Olomouc, CzechCzech RepublicRepublic


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IMMUNOASSAYSIMMUNOASSAYSHighly specific in vitrotests that use antigen-antibodyreaction to detect extremely low concentrations

of a broad range of biologically important substancesin blood and other body fluids

Antigen-antibody reaction - known since the end of the

19th ct, precipitation in gel, agglutination or turbidimetryassays gradually developed until

their potential has fully been appreciated since 1960when higher sensitivity was achieved by

labeling one of the components


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WHY?WHY?

BecauseBecause thethe principleprinciple mademade possiblepossible toto developdevelop

simplesimple

preciseprecise

sensitivesensitive ((nanonano-- andand picomolarpicomolar rangerange)) highhigh throughputthroughput measurementmeasurement

ofof moremore substancessubstances thanthan anyany otherother analyticalanalytical techniquetechnique


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AllAll immunoassayimmunoassay requirerequire thethe samesamekeykey reagentsreagents

OneOne oror moremore antibodiesantibodies raisedraised againstagainstepitopesepitopes believedbelieved toto bebe specificspecific toto thethe analyteanalyte inin

questionquestion

AA labellabel ((tracertracer)) producingproducing aa measurablemeasurable signalsignal

CalibratorsCalibrators in a fluid (in a fluid (matrixmatrix)) similarsimilar toto thethepatientpatientss samplesample


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AntibodyAntibody ((antiserumantiserum))TheThe antibodyantibody == immunoglobulinimmunoglobulin producedproduced byby thethe body inbody in

response toresponse to anan invadinginvading ( (foreignforeign) substance as a part) substance as a partofof immuneimmune responseresponse

GoodGood antibodiesantibodies possesspossess highhigh specificityspecificity andand affinityaffinity forfor

aa specificspecific antigenantigen

TheThe antibodyantibody usedused inin immunoassayimmunoassay isis usuallyusually ofof thethe IgGIgG

classclass

Antibody

polyclonal monoclonal engineered


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IgGIgG structurestructure


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NaturalNatural AntigenAntigen

SubstanceSubstance thatthat naturallynaturally elicitelicit immuneimmune responseresponse

UsuallyUsually aa largerlarger moleculemolecule ((overover 1010 kDkD)) withwith severalseveral

epitopesepitopes ((antigenicantigenic determinantsdeterminants))

rhFSH


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PolyclonalPolyclonal antibodiesantibodies

raisedraised inin animalsanimals ((rabbitsrabbits,, sheepsheep,, goatgoat) by) by repeatedrepeatedimmunizationimmunization

aa mixturemixture ofof antibodiesantibodies whichwhich maymay bindbind toto differentdifferent

epitopesepitopes ofof thethe immunogenimmunogen withwith differentdifferent aviditiesavidities


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DoubleDouble antibodyantibodyRaisedRaised inin anotheranother speciesspecies toto thethe primaryprimary antibodyantibody, e.g., e.g.

sheepsheep ((goatgoat,, donkeydonkey)) antianti rabbitrabbit ((mousemouse, ), ) IgGIgG addedadded in muchin much higherhigher concentrationsconcentrations thanthan primaryprimary antibodyantibody ++

normalnormal rabbitrabbit ((mousemouse,..),..) gammagamma globulinglobulin


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ProductionProduction ofof monoclonalmonoclonal antibodiesantibodies

InjectingInjecting anan antigenantigen intointo a hosta host

animalanimal ((typicallytypically aa mousemouse))IsolatingIsolating antibodyantibody--producingproducing

cellscells (B(B lymphocyteslymphocytes))

FusingFusing immuneimmune cellscells toto mousemousemyelomamyeloma cellscells

HybridomasHybridomas areare growngrown inin cultureculture

andand produceproduce antibodiesantibodies

SelectingSelecting hybridomashybridomas thatthat

produceproduce desireddesired antibodiesantibodies


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MonoclonalMonoclonal antibodiesantibodiesderivedderived fromfrom aa single cell line,single cell line, monoclonalsmonoclonals

areare specificspecific for afor a singlesingle epitopeepitope on aon amultivalentmultivalent antigenantigen

hybridomahybridoma cellcell lineslines cancan produceproduce thethe samesameantibodyantibody consistentlyconsistently andand indefinitelyindefinitely,,

monoclonalmonoclonal antibodiesantibodies facilitatedfacilitated::

manufacturingmanufacturing ofof immunodiagnosticsimmunodiagnostics

furtherfurther developmentdevelopment andand automationautomation ofof

immunoassaysimmunoassays


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HowHow doesdoes itit workwork??


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HowHow doesdoes itit workwork??

Photo: Z. Putz


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In 1960In 1960 thethe firstfirst RIA forRIA for insulininsulin ((BersonBerson && YalowYalow))

usedused 131131

II--insulin as ainsulin as a

tracertracer

,,

andand gelgel filtrationfiltration toto separateseparate thethe boundbound andand freefree fractionfraction PaperPaper waswas originallyoriginally rejectedrejected by Scienceby Science andand JJ ClinClin InvestInvest,, butbut laterlater acceptedaccepted..

InIn 19771977 R.R. YalowYalow, R., R. GuilleminGuillemin andand A.V.A.V. SchallySchally sharedshared NobelNobel prizeprize forfor thethedevelopmentdevelopment ofof RIAsRIAs for peptidefor peptide hormoneshormones

PrinciplePrinciple:: competitioncompetition ofof unlabeledunlabeled analyteanalyte inin samplesample withwithfixedfixed amountamount ofof radioradio--labeledlabeled analyteanalyte forfor limitedlimited bindingbindingsitessites on aon a specificspecific AbAb

ProbablyProbably thethe mostmost importantimportant advanceadvance inin biologicalbiologicalmeasurementmeasurement inin thethe secondsecond halfhalf ofof thethe 2O2Othth centurycentury

DISCOVERY OF RIADISCOVERY OF RIA


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CompetitiveCompetitive immunoassayimmunoassayTheThe lessless AgAg inin thethe samplesample,,

thethe moremore labeledlabeled AgAg cancan bebe boundbound byby AbAb

a) Isotope label (RIA)b) Enzyme label (EIA)

Calibration curve

Precision profile


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CompetitiveCompetitive assayassay forfor antibodyantibody testingtesting


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NoncompetitiveNoncompetitive immunoassayimmunoassayaa newnew assayassay formatformat in 1968in 1968

AnalyteAnalyte inin thethe samplesample isis boundbound toto excessexcess ofof capturecapture antibodyantibody

immobilizedimmobilized on solidon solid phasephase ((testtubestesttubes,, microplatesmicroplates,, etcetc.).)

SoSo thatthat therethere alwaysalways remainremain unoccupiedunoccupied bindingbinding sitessites

OnlyOnly thethe occupiedoccupied bindingbinding sitessites cancan bebe detecteddetected byby labeledlabeled

antibodyantibody

TheThe amountamount ofof AgAg inin thethe samplesample isis directlydirectly relatedrelated toto thethe signalsignal,,

e.g.e.g. thethe amountamount ofof boundbound labeledlabeled AbAb


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NoncompetitiveNoncompetitive immunoassayimmunoassay((alsoalso knownknown asas sandwichsandwich,, immunometricimmunometric,, excessexcess reagentreagent assaysassays))

Competitive format


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WhyWhy noncompetitivenoncompetitive immunoassaysimmunoassays areare

betterbetter thanthan competitivecompetitive assaysassays??

higherhigher sensitivitysensitivity andand

specificityspecificity

universaluniversal labelinglabeling

procedureprocedure ((IgGsIgGs))

generallygenerally longerlonger shelfshelf--lifelife

ofof labeledlabeled antibodiesantibodies

extendedextended workingworking rangerange


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LimitationsLimitations ofof noncompetitivenoncompetitive immunoassaysimmunoassays

notnot applicableapplicable toto smallsmall moleculesmolecules

moremore expensiveexpensive ((higherhigher consumptionconsumption ofof antibodiesantibodies,, isolationisolationofof purepure immunoglobulinimmunoglobulin necessarynecessary))

hookhook effecteffect ((highhigh dosedose hookhook effecteffect) in) in somesome assaysassays

HAMA interferenceHAMA interference


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CategoriesCategories ((formatsformats)) ofof immunoassayimmunoassay

CompetitiveCompetitive immunoassaysimmunoassays(limited(limited reagentreagent assaysassays))

NoncompetitiveNoncompetitive oror immunometricimmunometric

assaysassays((excessexcess reagentreagent assayassay,, sandwichsandwich assayassay))


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HeterogeneousHeterogeneous AssaysAssaysAlwaysAlways requirerequire separationseparation ofof thethe labellabel boundbound inin

thethe immuneimmune complexcomplex andand thethe freefree labellabel DoubleDouble antibodyantibody + PEG+ PEG

SolidSolid phasephase systemssystems

CoatedCoated tubestubes,, microplatesmicroplates,, beadsbeads,, etcetcMagneticMagnetic particlesparticles


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SolidSolid phasephase

RIARIA kitkit (DHEA(DHEA--S)S)

procedureprocedure

standardstandard curvecurveexpectedexpected valuesvalues

inin ageage andand sexsex groupsgroups


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LabelsLabels,, tracerstracers

RadioactiveRadioactive isotopesisotopes

33HH usedused inin competitivecompetitive bindingbinding assaysassays beforebefore

thethe eraera ofof immunoassaysimmunoassays

131131II usedused inin thethe firstfirst RIAsRIAs (t(t1/21/2 88 daysdays))

125125II withwith aa longerlonger halfhalf--lifelife (60(60 daysdays)) soonsoon

replacedreplaced 131131I isotope for use inI isotope for use in RIAsRIAs


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RADIOACTIVE LABELS (RADIOACTIVE LABELS (125125I,I, 33H)H)

HighHigh sensitivitysensitivity ofof

detectiondetection

NoNo interferencesinterferences

SmallSmall molecularmolecular sizesize

SimpleSimple labelinglabeling

LowLow costcost

EnviromentalEnviromental risk (risk (wastewaste

disposaldisposal))

DedicatedDedicated instrumentationinstrumentation

SeparationSeparation stepstep necessarynecessary

ShortShort shelfshelf--lifelife

DifficultDifficult to automateto automate


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ALTERNATIVE LABELSALTERNATIVE LABELS

EnzymesEnzymes ((alkalinealkaline phosphatasephosphatase,, horsehorse raddishraddishperoxidaseperoxidase andand othersothers))

FluorescentFluorescent substancessubstances (fluorescein,(fluorescein,lanthanidelanthanide chelateschelates))

LuminiscentLuminiscent substancessubstances ((substitutedsubstitutedisoluminolisoluminol,, acridiniumacridinium estersesters))

ParticlesParticles (latex(latex particlesparticles,, colloidalcolloidal goldgold,, EuEu chelatechelatenanoparticlesnanoparticles))


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DetectionDetection ofof enzymeenzyme labelslabels

LowLow sensitivity:sensitivity: AbsorbanceAbsorbance measurementmeasurement

chromogenicchromogenic substratessubstrates

HighHigh sensitivitysensitivity:: LightLight emissionemission measurementmeasurement

chemiluminiscentchemiluminiscent substratessubstrates ((peroxidaseperoxidase ++ luminolluminol ++ enhancerenhancer))oror alkalinealkaline phosphatasephosphatase ++ adamantyladamantyl--1,21,2--dioxetanedioxetane phenylphenylphosphatephosphate))

nonfluorescentnonfluorescent substratessubstrates thatthat areare convertedconverted toto fluorescentfluorescentproductsproducts (4(4--methylumbelliferylmethylumbelliferyl phosphatephosphate))

Sensitivity gain


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ELISAELISAEEnzymenzyme LLabeledabeled IImmunommunoSSorbentorbent AAssayssayprobablyprobably thethe mostmost popularpopular formatformat

microplateindividual strips or wellsin a frame


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ELISAELISA readersreaders


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FluorogenicFluorogenic ELISAELISA

Test specific module


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FLUORESCENT LABELSFLUORESCENT LABELS

FluoresceinFluorescein andand fluoresceinfluorescein isothiocyanateisothiocyanate

(FITC)(FITC)usedused forfor labelinglabeling antibodiesantibodies inin histochemistryhistochemistry

BackgroundBackground fluorescencefluorescence isis aa problemproblem inin biologicalbiological

specimensspecimens

SolutionSolution:: timetime--resolvedresolved fluoroimmunoassaysfluoroimmunoassays

usingusing longlong--livedlived fluorescencefluorescence ofof lanthanidelanthanide chelateschelates ((lifetimelifetimeinin micromicrosecondsseconds))labellabel signalsignal isis measuredmeasured afterafter backgroundbackground

fluorescence hasfluorescence has decayeddecayed ((lifetimelifetime inin nanonanosecondsseconds))


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DELFIADELFIA

DDissociationissociation EEnhancednhanced LLanthanideanthanide FFluoroluoroIImmunommunoAAssayssay


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LUMINISCENCE HISTORYLUMINISCENCE HISTORY16671667 bioluminiscencebioluminiscence waswas recognizedrecognized

18871887 firstfirst luminiscentluminiscent substancessubstances knownknown19471947 firstfirst applicationapplication ofof fireflyfirefly luciferaseluciferase

19671967 firstfirst immunoassayimmunoassay utilizingutilizing luminolluminol

19821982 acridiniumacridinium esterester usedused forfor thethe firstfirst timetime as aas alabellabel in ain a manualmanual assayassay

MAGICLITE by Ciba Corning


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InIn 1990,1990, CibaCiba CorningCorning launchedlaunched thethe worldworldss firstfirstfullyfully automatedautomated immunoassayimmunoassay systemsystem

withwith RandomRandom Access,Access, ACS:180ACS:180


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ADVIA CENTAURADVIA CENTAURoutputoutput 240240 teststests//hourhour


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MODERN PARTICLE LABELSMODERN PARTICLE LABELSImmunochromatographicImmunochromatographic teststests,, membranemembrane teststests,,

laterallateral flowflow oror oneone stepstep teststests(in dipstick or device format)


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How does it work?

Ab coated particle

(coloured latex, colloidalgold) binds Ag and iscaptured by immobilised

Ab

Excess of labeled Ab

binds to the second Ab(anti- mouse Ab)


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SomeSome alternativealternative labelslabelsmademade itit possiblepossible toto developdevelop

HomogeneousHomogeneous immunoassaysimmunoassays

HeterogeneousHeterogeneous assaysassays ((requirerequire

separationseparation ofof boundbound AbAb--AgAg complexcomplex))

HomogeneousHomogeneous assaysassays (do not(do not requirerequirethisthis separationseparation, as, as signalsignal isis changedchanged whenwhen thethe

labellabel isis boundbound inin thethe AbAb--AgAg complexcomplex


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MorelessMoreless outdatedoutdated

HOMOGENEOUS ENZYME IMMUNOASSAYSHOMOGENEOUS ENZYME IMMUNOASSAYS

EMITEMIT ((EnzymeEnzyme ModifiedModified ImmunoassayImmunoassay Technology),Technology), thethe

activeactive sitesite ofof thethe enzymeenzyme labellabel isis blockedblocked whenwhen boundbound

FPIAFPIA (Fluorescence(Fluorescence PolarizationPolarization ImmunoAssayImmunoAssay)) rotationrotationofof fluorescentfluorescent labellabel isis slowerslower whenwhen boundbound

CompetitiveCompetitive assaysassays forfor smallsmall moleculesmolecules inin relativelyrelatively highhighconcentrationconcentrationTDMTDM

TheThe onlyonly isotopicisotopic homogenoushomogenous immunoassayimmunoassay::

SPASPA ((ScintillationScintillation ProximityProximity AssayAssay),), 33HH labelledlabelled smallsmallhaptenhapten getsgets intointo thethe proximityproximity ofof aa scintilatorscintilator encapsuledencapsuled

inin

thethe

solidsolid

phasephase

withwith

immobilizedimmobilized

antibodyantibody


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ModernModern

HOMOGENEOUS ENZYME IMMUNOASSAYHOMOGENEOUS ENZYME IMMUNOASSAY

automateCryptor


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MESSAGE: IMMUNOASSAYSMESSAGE: IMMUNOASSAYS

areare uniqueunique inin usingusing antibodiesantibodies asas analyticalanalytical reagentsreagents

areare indirectindirect analyticalanalytical teststests

thethe intensityintensity ofof aa signalsignal in ain a samplesample isis comparedcompared withwith thethe signalsignal

generatedgenerated by aby a simultaneouslysimultaneously measuredmeasured calibratorcalibrator

calibratorscalibrators shouldshould bebe in a properin a proper matrixmatrix toto mimicmimic thethe

samplesample traceabilitytraceability ofof calibratorcalibrator to referenceto reference preparationpreparation shouldshould bebe

documenteddocumented

InternationalInternational ReferenceReference PreparationsPreparations

referencereference methodsmethods do notdo not existexist in manyin many casescases


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11stst

generationgeneration immunoassaysimmunoassays competitivecompetitive immunoassayimmunoassay

RIA, EIA, FIA, LIA,RIA, EIA, FIA, LIA, oftenoften inin--house,house, manualmanual doubledouble antibodyantibody andand

solidsolid phasephase assaysassays,, commercialcommercial kitskits .).)

22ndnd generationgeneration immunoassaysimmunoassays noncompetitivenoncompetitive immunometricimmunometric assaysassays

(IRMA, ELISA,(IRMA, ELISA, laterallateral flowflow assaysassays,, automatedautomated assaysassays,,randomrandom accessaccess automatesautomates,, consolidationconsolidation withwith clinicalclinicalchemistrychemistry .).)

33rdrd generationgeneration immunoassaysimmunoassays microspotmicrospot analysisanalysis,, biochipbiochip arraysarrays,,

multiplemultiple parameterparameter testingtesting


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3rd3rd generationgeneration immunoassaysimmunoassays

multiplemultiple parameterparameter

testingtesting

nanotechnologiesnanotechnologies


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FactorsFactors impactingimpacting analyticalanalytical performanceperformance

AntibodiesAntibodies

specificityspecificity,, avidityavidity, type, typeLabelsLabels

sensitivitysensitivity ofof detectiondetection,, sizesize, stability,, stability, interferencesinterferences,,

backgroundbackground noisenoiseFormatFormat ((competitivecompetitive oror noncompetitivenoncompetitive))

SeparationSeparation systemsystem (in(in heterogeneousheterogeneous formatsformats))

AutomationAutomation

eliminatingeliminating humanhuman errorerror


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MAIN INTERFERENCES IN IMMUNOASSAYSMAIN INTERFERENCES IN IMMUNOASSAYS

CompetitiveCompetitive assaysassays

falsefalse positivepositive resultsresults duedue toto crosscross--reactingreacting moleculesmolecules((typicaltypical exampleexample: E: E22 oror testosteronetestosterone directdirect assaysassays))

remedyremedy:: improvingimproving sensitivitysensitivity ofof primaryprimary antibodyantibody

NoncompetitiveNoncompetitive assaysassays

HAMA, autoHAMA, auto-- andand heterophilicheterophilic antibodiesantibodies,, rheumatoidrheumatoid

factorsfactors -- bothboth falsefalse positivepositive andand falsefalse negativenegative resultsresults((typicaltypical examplesexamples:: hCGhCG,, myoglobinmyoglobin))

remedyremedy:: heterophilheterophil blockingblocking reagentreagent


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ComplexedComplexed (PSA),(PSA), dimersdimers ((prolactinprolactin),), fragmentsfragments ((hCGhCG),),

differentdifferent degreedegree ofof glycosylationglycosylation,, etcetc

ManyMany analytesanalytes areare heterogenicheterogenic

It may result in discrepant findingsbetween assays from differentmanufacturers

Sometimes ability to measuredifferent molecular forms canincrease diagnostic value of the test(PSA/freePSA, hCG subunits or

hyperglycosylated hCG)


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ParametersParameters ofof AssayAssay QualityQualityAnalyticalAnalytical:: AccuracyAccuracy:: abilityability toto measuremeasure thethe correctcorrect concentrationconcentration

((comparisoncomparison withwith referencereference methodmethod?)?) PrecisionPrecision ((reproducibilityreproducibility):): acceptableacceptable coeficientcoeficient ofof variancevariance

DetectionDetection limitlimit (sensitivity):(sensitivity): lowestlowest concentrationconcentration differentdifferent fromfromzerozero

DiagnosticDiagnostic:: Sensitivity:Sensitivity: %% ofof truetrue positivepositive resultsresults

IfIf highhigh,, thenthen a negativea negative resultresult practicallypractically excludeexclude thethe diagnosisdiagnosis

((SnNSnN

outout

))

specificityspecificity:: %% ofof truetrue negativenegative resultsresults

IfIf highhigh,, thenthen a positivea positive resultresult practicallypractically includeinclude thethe diagnosisdiagnosis((SpPSpPinin))


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ImmunoassaysImmunoassays inin thethe pastpast


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AndAnd todaytoday

CommunicationCommunication betweenbetween cliniciansclinicians andand

laboratorianslaboratorians isis crucialcrucial forfor thethe benefitbenefit ofof patientspatients!!

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Immuno asaay