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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!!