ELISA and RIA

Enzyme linked immunosorbent assay and Radio immuno assay

1. Guided by: Presented By :
Dr. Mahesh Kumar Kataria NitishChugh
Professor and Head, Department of Pharmaceutics M.Pharmacy(Pharmaceutics)
SETH G. L. BIHANI S. D. COLLEGE OF TECHNICAL EDUCATION,
SRI GANGANAGAR (RAJ.)
Dr. Mahesh Kumar Kataria Email: [email protected] 1

2. ELISA(enzyme-linkedimmunosorbent assay)isaplate-basedassay
techniquedesignedfordetectingandquantifyingpeptides,proteins,antibodies
andhormones.InanELISA,anantigenmustbeimmobilizedtoasolidsurface
andthencomplexedwithanantibodythatislinkedtoanenzyme.Detectionis
accomplishedbyassessingtheconjugatedenzymeactivityviaincubationwitha
substratetoproduceameasureableproduct.Themostcrucialelementofthe
detectionstrategyisahighlyspecificantibody-antigeninteraction.
ELISAsaretypicallyperformedin96-well(or384-well)polystyreneplates,which
willpassivelybindantibodiesandproteins.Itisthisbindingandimmobilization
ofreagentsthatmakesELISAssoeasytodesignandperform.Havingthe
reactantsoftheELISAimmobilizedtothemicroplatesurfacemakesiteasyto
separateboundfromnon-boundmaterialduringtheassay.Thisabilitytowash
awaynonspecificallyboundmaterialsmakestheELISAapowerfultoolfor
measuringspecificanalyteswithinacrudepreparation.
ELISAisanantigenantibodyreaction.In1971,ELISAwasintroducedbyPeter
PerlmannandEvaEngvallatStockholmUniversityinSweden.Itisacommon
laboratorytechniquewhichisusuallyusedtomeasuretheconcentrationof
antibodiesorantigensinblood.Anenzymeconjugatedwithanantibodyreacts
withcolorlesssubstratetogenerateacoloredproduct.Suchsubstrateiscalled
chromogenicsubstrate.AnumberofenzymeshavebeenusedforELISAsuchas
alkalinephosphatase,horseradishperoxidaseandbetagalactosidase.Specific
substratesuchasortho-phenyldiaminedihydrochloride(forperoxidase),
paranitrophenylphosphate(foralkalinephosphatase)areusedwhichare
hydrolysedbyaboveenzymestogivecoloredendproduct.
Dr. Mahesh Kumar Kataria Email: [email protected]

3. Dr. Mahesh Kumar Kataria Email: [email protected]

4. ELISAsaretypicallyperformedin96-wellpolystyreneplates.The
serumisincubatedinawell,andeachwellcontainsadifferent
serum.Apositivecontrolserumandanegativecontrolserum
wouldbeincludedamongthe96samplesbeingtested.
Antibodiesorantigenspresentinserumarecapturedby
correspondingantigenorantibodycoatedontothesolid
surface.Aftersometime,theplateiswashedtoremoveserum
andunboundantibodiesorantigenswithaseriesofwash
buffer.Todetecttheboundantibodiesorantigens,asecondary
antibodiesthatareattachedtoanenzymesuchasperoxidase
oralkalinephosphataseareaddedtoeachwell.
Afteranincubationperiod,theunboundsecondaryantibodiesare
washedoff.Whenasuitablesubstrateisadded,theenzyme
reactswithittoproduceacolor.Thiscolorproducedis
measurableasafunctionorquantityofantigensorantibodies
presentinthegivensample.Theintensityofcolor/optical
densityismeasuredat450nm.Theintensityofthecolorgives
anindicationoftheamountofantigenorantibody.
Dr. Mahesh Kumar Kataria Email: [email protected]

5. Indirect ELISA
Sandwich ELISA
Competitive ELISA
Direct ELISA
Dr. Mahesh Kumar Kataria Email: [email protected]

6. Antibodycanbedetectedorquantitativelydeterminedbyindirect
ELISA.Inthistechnique,antigeniscoatedonthemicrotiterwell.
Serumorsomeothersamplecontainingprimaryantibodyisadded
tothemicrotiterwellandallowedtoreactwiththecoatedantigen.
Anyfreeprimaryantibodyiswashedawayandtheboundantibody
totheantigenisdetectedbyaddinganenzymeconjugated
secondaryantibodythatbindstotheprimaryantibody.
Dr. Mahesh Kumar Kataria Email: [email protected]

7. AntigencanbedetectedbysandwichELISA.Inthistechnique,
antibodyiscoatedonthemicrotiterwell.Asamplecontaining
antigenisaddedtothewellandallowedtoreactwiththe
antibodyattachedtothewell,formingantigen-antibody
complex.Afterthewelliswashed,asecondenzyme-linked
antibodyspecificforadifferentepitopeontheantigenis
addedandallowedtoreactwiththeboundantigen.Then
afterunboundsecondaryantibodyisremovedbywashing.
Finallysubstrateisaddedtotheplatewhichishydrolyzedby
enzymetoformcoloredproducts.
Dr. Mahesh Kumar Kataria Email: [email protected]

8. Inthistest,antibodyisfirstincubatedinsolutionwitha
samplecontainingantigen.Theantigen-antibodymixtureis
thenaddedtothemicrotitrewellwhichiscoatedwith
antigen.Themoretheantigenpresentinthesample,theless
freeantibodywillbeavailabletobindtotheantigen-coated
well.Afterthewelliswashed,enzymeconjugatedsecondary
antibodyspecificforisotypeoftheprimaryantibodyisadded
todeterminetheamountofprimaryantibodyboundtothe
well.Thehighertheconcentrationofantigeninthesample,
thelowertheabsorbance.
Dr. Mahesh Kumar Kataria Email: [email protected]

9. Fordirectdetection,anantigencoatedtoamulti-
wellplateisdetectedbyanantibodythathasbeen
directlyconjugatedtoanenzyme.Thisdetection
methodisagoodoptionifthereisno
commerciallyavailableELISAkitsforyourtarget
protein
Dr. Mahesh Kumar Kataria Email: [email protected]

10. Presenceofantigenorthepresenceof
antibodyinasamplecanbeevaluated.
Determination of serum antibody
concentrationsinavirustest.
Usedinfoodindustrywhendetecting
potentialfoodallergens.
Appliedindiseaseoutbreaks-trackingthe
spreadofdiseasee.g.HIV,birdflu,common,
colds,cholera,STDetc.
Dr. Mahesh Kumar Kataria Email: [email protected]

11. RIAorRadioimmunoassayisaninvitroassaythatmeasures
thepresenceofanantigenwithveryhighsensitivity.Basically
anybiologicalsubstanceforwhichaspecificantibodyexists
canbemeasured,eveninminuteconcentrations.RIAhas
beenthefirstimmunoassaytechniquedevelopedtoanalyze
nanomolarandpicomolarconcentrationsofhormonesin
biologicalfluids.
Dr. Mahesh Kumar Kataria Email: [email protected]

12. Radioimmunoassay isbaseduponthecompetitionbetween
labeledandunlabeledantigenforspecificantibodysites,
formingantigen-antibodycomplexes.Thisreactionis
describedbytheexpressionseejournalforformula.At
equilibirum,theradioactivecomplex(bound)isseparatedfrom
theradioactiveantigen(free).TheB/Fratioisdependentupon
theamountofnonradioactiveantigen.Antigenconcentrationin
unknownsamplesisdeterminedbycomparingtheB/Fratioto
theB/Fratiosobtainedbyincubatingvaryingamountsof
knownnonradioactiveantigenwiththesameamountof
antibodyasintheunknownsampleundersimilarassay
conditions.Sensitivityoftheorderof10-12moles/litermaybe
achievedthroughthepreparationanduseofalabeledantigen
ofhighspecificactivityandtheproductionandselectionof
antiserawithappropriatelyhighaffinityconstants.Specificityis
dependentupontheabilityoftheantiserumtorecognize
subtlestructuralfeaturesoftheantigenmolecule.Theabilityto
convenientlyassaylargenumbersofsampleswithgood
precisionhasledtotheapplicationofthistechniqueto
quantitatesubstances(suchassteroids)alreadymeasurable
butbymorecumbersomemethods.
Dr. Mahesh Kumar Kataria Email: [email protected]

13. Thetargetantigenislabeledradioactivelyandboundtoitsspecific
antibodies;alimitedandknownamountofthespecificantibodyhastobe
added.Asample,forexampleablood-serum,isthenaddedinorderto
initiateacompetitivereactionofthelabeledantigensfromthepreparation,
andtheunlabeledantigensfromtheserum-sample,withthespecific
antibodies.Thecompetitionfortheantibodieswillreleaseacertainamount
oflabeledantigen.Thisamountisproportionaltotheratiooflabeledto
unlabeledantigen.Abindingcurvecanthenbegeneratedwhichallowsthe
amountofantigeninthepatient’sserumtobederived.
Dr. Mahesh Kumar Kataria Email: [email protected]

14. Asanexampleofhowthistechniqueworks,
let’sapplyittoinsulin.Tomeasureinsulin,
thefirststepistomixknownamountsof
radioisotope-taggedinsulinandantibodies.
Thesecombinechemically.Next,asmall
amountofthepatient’sbloodisadded.The
insulincontainedintheblooddisplacessome
ofthetaggedinsulin.Thefree-taggedinsulin
isthenmeasuredwithisotopedetectorsand
thepatient’sinsulinleveliscalculated
Dr. Mahesh Kumar Kataria Email: [email protected]

15. Aplotofthedistributionofradioactivityasa
functionoftheamountofunlabeledantigenpresent
isknownasthestandardordoseresponsecurve.
Standardcurvescanbeplottedinavarietyofways.
Themostcommonlyusedresponsecurvesarethe
bound-free(B/F),free-bound(F/B),fractionbound
(B)ofoccasionallythepercentboundB/Boratios.
Thedosecanbeplottedoneitherthearithmaticor
logarithmicscale.Toobtainresponsecurvesthat
areessentiallylinearoveralargepartoftheantigen
concentration,thelogitfunctiondefinedasfollows
hasbeenused:Logit(y)=wherey=eitherB/Boor
(B/F)/(Bo/Fo)Whenlogit(y)isplottedagainstthe
logconcentrationoftheantigen,alineardose-
responsecurveresultsformostoftheassaysystem.
Dr. Mahesh Kumar Kataria Email: [email protected]

16. Dr. Mahesh Kumar Kataria Email: [email protected]

17. Radioimmunoassay(RIA)hasmanyuses,includingnarcotics(drug)
detection,bloodbankscreeningforthehepatitisahighlycontagious
conditionvirus,earlycancerdetection,measurementofgrowth
hormonelevels,trackingoftheleukemiavirus,diagnosisand
treatmentofpepticulcers,andresearchwithbrainchemicalscalled
neurotransmitters.
1)DetectionofNarcoticDrugs-Heroin&Morphinecanbedetectedin
hairwiththeuseofRadioimmunoassay (RIA).Inaresearchhair
samplesobtainedfrommorphinetreatedmiceandheroinuser
containedNanogramlevelsofdrugpermilligramofhair.Theresult
ofthehairanalysisforallsubjectadmittingtheuseofheroinwere
positivewhereastheresultofonly30%ofthinlayerchromatographic
urineanalysisofthesesamesubjectswerepositive.
2) RadioimmunoassayofHydromorphone &HydrocodoneinHuman
Plasma-Hydromorphone&Hydrocodonebelongstomorphinegroup
ofdrugsandareusedincombinationwithantitussive&analgesic
antipyreticmixture.TheRIAmethodiscapableofestimatingtheabove
drugwithinarangeof2.5to20ng/mLusingstandard100µlplasma
sample.RIAiscarriedoutusingmorphine-6-antiserum&
dihydromorphine.Freedrugisseparatedfromthebounddrugusing
dextrancoatedcharcoal&analiquotofthesupinatecontainingthe
antiserumbounddrugissubsequentlycountedforradioactivity.
Dr. Mahesh Kumar Kataria Email: [email protected]

18. 3)RadioimmunoassayofFlunisolideinhumanplasma-Flunisolideisa
fast-actingcorticoiddesignedforthetreatmentofallergicrhinitis,
asthma,andotheralliedrespiratorydisordersinhumans.Asthe
quantumofdrugdeliveredbyinhalation(i.e.,theusualrouteof
administrationofthedrug),isinvariablysmall,theplasma-levels
attainedcanalsobefairlysmall.Hence,thereisaneedforasensitive
methodofplasmaconcentrationevaluationwhichissatisfiedby
radioimmunoassay.
4)MeasurementofFerritin-Serumferritinlevelsareindicativeofiron
storespresentinapatient.Levelsareusefulindifferentiatingtrueiron
deficiencyfromthebody'sfailuretoutilizethesestores.
5) DetectionofDigoxin-Thisallowsdirectmeasurementofserum
digoxinlevelsquicklyandaccurately.ItisimportanttoruleoutDigi
toxicityquicklyandaccurately.WearealsoabletofilteroutDigibind
toletthephysicianknowhowmuchthelevelhasdroppedafterDigi
bindhasbeenadministered.
6)ThyroidTesting-Thisisusedtodeterminethepatient'sthyroidstatus
andtofollowpatientsafteriodine-131therapytoseeifthedosewas
indeedeffective.
Dr. Mahesh Kumar Kataria Email: [email protected]

19. • Prolonged reaction time (in days) as a
consequence highly diluted reagent is used.
• Radioisotopes are costly.
• Possible health hazards due to handling of
radioisotopes.
• Limited assay range.
• Lack of direct linear relationship between
analyte concentration and signal response.
• Difficult of automation.
• Lengthy counting time.
Dr. Mahesh Kumar Kataria Email: [email protected]

20. 1.Abraham,G.E.sndGrover,P.K.Covalentlinkageofhormonalhaptens
toproteincarriersforuseinradioimmunoassay.InOdell,W.D.and
Daughadsy,W.H.editors:Principlesofcompetitiveproteinbinding
assays.Philadelphia,J.B.LippincottCo.,1971.
2.Rodbsrd,D.,Rayford,P.L,Cooper,i.A.andRoss,G.T.Statistical
qualitycontrolandroutinedataprocessingforradioimmunoassays
(RIA)andimmunoradiometric assays(IRMA).din.Chem.,
1974;20:1255-70.
3. Rodbsrd,D.,Bridson,W.andRayford,P.LRapidcalculationof
radioimmunosssayresults.3.Lab.dIm.Med.,1969;74:770-81.
4.Garcia,E.J.andFiori,A.Radioimmunoasssyandcompetitivebinding
analysis.Intextbookofnuclearmedicine:BasicSciences(Eds.A.
Fernandosnd.1.C.Harbert):LeaandFebigerPublishers,1978;pp.
344360.
5.Hunter,W.M.andGreenwood,P.C.Preparationof1-131labeled
growthhormone ofhighspecificactivity.Nature(London),
1962;194:495-96.
Dr. Mahesh Kumar Kataria Email: [email protected]