Radio Immuno Assay

Radio Immuno Assay

1. Prepared By
Dr.J.SwaminathanM.Pharm.,MBA., Ph.D.
Associate Professor,
ADHIPARASAKTHI COLLEGE OF PHARMACY,
MELMARUVATHUR –603 319
RIA
( Radio Immuno Assay)
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 1

2. RADIO IMMUNO ASSAY -
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3. RADIO IMMUNO ASSAY -DR.J.SAMINATHAN,
APCP, MLMR
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4. RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 4

5. Definition
Introduction
Principle and Theory
Methods in RIA
Other Immuno Assays
Application
Reference
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6. Radioimmunoassay(RIA)isascientificmethodusedtotestantigens(for
example,hormonelevelsintheblood)withouttheneedtouseabioassays.
Radioimmunoassay(RIA)isaRadio-analyticaltechniquewithremarkable
sensitivityandahighdegreeofspecificitythatiswidelyusedfortheestimation
ofavarietyofmoleculespresentincomplexmatrices.AlsoknownasRadio
tracertechniqueandbestexampleofinvitrodiagnosistechniqueusingradio
isotopes.
Thistechniqueisusedoverawidespectraofsubstancessuchashormones,
steroids,vitamins,drugs,tumormarkersandviralantigens.
Radio Immuno Assay
Use of radio
active material
Antigen antibody
binding theory
Detection of
compoundRADIO IMMUNO ASSAY -
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8. Thisisotopicmeasuringmethodwasdevelopedin1959bytwoAmericans,
biophysicistRosalynYalowandphysicianSolomonA.Berson.
RIAcombinesthespecificityofanantigen-antibodyreactionwithsensitivityof
radioactivitymeasurements.
Thisisatechniqueusedfordetectionofmicroquantitiesofprotein,viralantigens,
antibodies,structuralproteins,vitaminsanddrugandtheirmetabolites.
Itcanalsobeusedfordetectionofpictogramquantities(10
−12
g)ofbiological
constituentspresentinbiologicalfluid.
RIAisusedinplaceofbioassayinvariousbranchesofsciencelikeBiochemistry,
Microbiology,andHematologyandClinicalpharmacology.
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10. RIAworksonbasicprincipleofbiochemistrythatcompetitivebindingbetween
antigensforsameantibodybindingsite.
Thecompetitionofananalytewithitsradioisotopicallylabeledcounterpartfora
limitedamountofantibody,thespecificreagent,istheunderlyingprincipleofthis
technique.Increasingtheanalyteconcentrationinhibitsthebindingofthelabeled
analytetotheantibody.
Ag+Ag*+AbAgAb+Ag*Ab+Ag
Unbound Ag* and Ag washed out
Radioactivity of bound residue measured
Ligand conc. is inversely related to radioactivity
Ag:ligandtobemeasured;
Ag*:radiolabelledligand
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11. A
B
C
Antibody
Unlabeled antigen
Labeled antigen
Nowhowthecompetitionoccurwithincreasetheconcentrationofunlabeled
antigeninthesystemofRIAinthreedifferentcasesA,BandC.
Herefirstantibodiesboundwithlabeledantigenareputintotheknown
concentrationofanalytesolutionanditisobservedhowthelabeledantigenfree
fromantibodyandunlabeledwillbindinplaceofthere.
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12. Theconcentrationoftheunknownanalyteisthusobtainedbycomparingits
inhibitoryEffectonthebindingofthelabeledanalytetothatofaknownstandard.
Afterdeterminingtheratioofboundtofreeantigenineachunknown,theantigen
concentrationscanbereaddirectlyfromthestandardcurve(asshownabove
Afterdeterminingtheratioofboundtofreeantigenineachunknown,theantigen
concentrationscanbereaddirectlyfromthestandardcurve(asshownabove).
1
2
1. –Ratio in unknown and
2. -Antigen in unknown
Red line –binding line
Green line –free labeled antigen
RADIO IMMUNO ASSAY -DR.J.SAMINATHAN, APCP, MLMR
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13. Fromgraphwecanalsocalculate%F(fractionoffreelabeledantigen)and%B
(fractionofboundlabeledantigen).
&
•F–amountoffreelabeledantigen
•B-amountofboundlabeledantigen
Hereastheconcentrationofunlabeledincreaseitreplacesthelabeledbound
antigenbycompetitivebindingitinhibitingthebindingoflabeledone.
Antigen-antibodycomplexdependonantibodyaffinity(Ka)–
The affinity with which antibody binds antigen results from a balance between
the attractive and repulsive forces
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14. Advantages
Highlyspecific:Immunereactionsarespecific,thegreaterthespecificityofthe
antiserum,thegreaterthespecificityoftheassay.
Highsensitivity:Immunereactionsaresensitive,Usingantibodiesofhigh
affinityitispossibletodetectafewpicograms(10
−12
g)ofantigeninthetube.
AccuracyandPrecision
Disadvantages
Radiationhazards:Usesradiolabelledreagents
Requiresspeciallytrainedpersons
Labsrequirespeciallicensetohandleradioactivematerial
Requiresspecialarrangementsfor
Requisition,storageofradioactivematerial
radioactivewastedisposal.
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15. Development of the
Assay System
Development of the Assay System
1.Acrucialstepisseparationofunboundantigens
2.Thisachievedbybindingtheantibodiestothemicrotitrewellsurface
[SolidphaseRIA]
3.Antigensboundtothefixedantibodiesremainstucktotheinnersurface
4.Decanting&washingthewellremovesunboundantigens
5.Othertechniquesofseparation:Centrifugation
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16. Assay Procedure
1.Addknownamountsofthetestsample+labelled
antigenintothemicrotitrewells
2.Incubateallowthereactiontoreachcompletion
3.Decant&washcontentsofthewellremovesall
unboundantigens
4.RadioactivityremainingintheMicrotitrewells
measuredbyaCounter[GMcounter,Scintillation
counteretc]
5.Intensityofradioactivityisinverselycorrelatedwith
theconcofantigensinthetestsample
6.Sensitivetoverylowconcofantigens
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20. 1.Radio labelling of the Antigen or radio labelled production
2.Preparation & characterisation of the Antigen [Ligand to be
analysed]
3.Preparation of the Specific Antibody
4.Development of Assay System or separation techniques
Methods in RIA :
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21. 1.Preparation and radio labeling of antigen
Antigenpreparation:
Synthesisofthemolecule
Isolationfromnaturalsources
Radiolabelling [Tagging procedure] :
TwomostcommonlyusedradiolabelsinRIA
3
Hand
125
IalthoughSe,P,Co
14
Cand
131
Ihavealsobeenused.butthesehavesomelimitationsthatare–

32
Pand
57
Co=limitedbystereochemicalaspectsbecausedrug
naturallycontainphosphorousandcobalt

14
C =lowspecificactivityfield

131
I =Shortdecayhalflifeandradiodegradationcharacter
ontheotherhand
3
Hand
125
Ihaveadvantagesasfollows:

3
H=Directincorporateintomolecularstructure,halflives(12years)

125
I=Highactivityandeaseofcounting(halflives60days)
75 3257
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22. Techniqueoflabelingofdrugorantigenwith
3
Hand
125
I:
Specifictritiumlabelsaregenerallyobtainedbyreducinganappropriate
precursorinpresenceof
3
H.
Labelingofdrugwith
125
Iincludechloramines-T,monochlorideexchange
andenzymaticiodinationmethods.Themostsuitableiodination
procedureisdependingonthestabilityofthedrugandthespecificactivity
thatissufficienttomeetthesensitivityrequirementsofassy.
Comparisonof
3
Hand
125
Iisasfollow:
Sr no. Tritium (
3
H) Iodine (
125
I)
1 Itismoreefficientwhenrelativelysmall
numbersofsamplesareassayed.
Itismoreefficientasthenumbersofsamplesare
increased.
2
Suchtypeofproblemdoesnotoccur.
It’shavingqualitycontrolproblemssuchas
damageduringthereactionandradiationdamage
followingsynthesis.
3 Havingadvantagesoflonghalflife,higher
affinity.
Ithasshorthalflifedictatesfrequentpreparation.
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23. itispossibletofindthespecificantibodyfordrug(antigen)asaresultof
sensitivityreaction
Severaldrugswhichhaveinducedantibodiesproductionduetheirinherent
antigenicitylike–penicillin,strychnine,tetracycline,sulphonamideand
procainamide.Howevertheirlowspecificityandlimitedavailabilitymakestheir
useratherimprobable.
Inmostcasesthedrugs(analysedantigen)arebindwithsuitablecarrierprotein
tomakeconjugatedantigenimmunogenic.
Thereareseveralreactivegroupsonproteincarrierwhichcanusedforthe
purposeofconjugationofstandarddrug(antigen).thesegroupsincludethe
terminalaminoandcarboxylgroups,ε-aminogroupoflysine,thecarboxylgroup
ofasparticandglutamicacid,thephenolicgroupoftyrosine.
2.Preparationpurificationofdrug-proteinconjugate(ligand-antigen)tobe
analyzed:
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24. Themostcommonlyusedmethodsforconjugationareasfollows:
Carbodimideandglutaraldehydereaction
Carbony-diimidazolereaction
Scotten-baumannreaction
Mannichreaction
Diazotizationreaction
Themethodchosenforconjugatedwilldependonthefunctionalgroups
availableforcouplingthedrugtoproteinandno.ofdrugsmolecules
whicharetobecoupledtoprotein.
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25. Preparation:
Oncethepureantigenpreparedthenitisemulsifiedintoequalvolumesof
salineandFreud’sadjuvant(containalumppts,naturaldetergents,mineraloils,
killedmycobacterium)togetfinalconcentrationof10to50mg/ml.
Onemloftheemulsionisinjectedintradermally,subcutaneously,and/or
intramuscularlyatweeklyormonthlyintervalsintomultiplesitesofasuitable
animalspeciessuchasrat,guineapigsorrabbits.Asuitableanimalspeciesis
usuallydictatedbythesizeoftheanimalfacilities.
Animalsaretestedafter3-4weeksandthenbloodiscollectedandseparated.
Theresultingbloodcontainingantibodycalledantiserum.Itisdirectlyusedin
assayandshouldbestoredat4°c.
Characterization:
Characterizationofantiserumdonebyfractionation,immunoadsorptionor
immunosaturationtechnique.
3.Preparation&characterizationoftheSpecificandhighaffinityAntibodies:
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27. 4.Developmentofsuitableseparationtechniquestoseparatefreefrom
boundstandarddrug:
Severalmethodsthatemployphysiochemicalandimmunologicalseparationhavebeen
devisedasfollows–
Physicalmethods:Filtration,Chromatography,Electrophoresis,charcoal-dextran
adsorption,andionexchangeresin.
Disadvantages:itistendtobetimedependentandharshsotheymayremovebounddrug
fromantibodyduringseparation.
Chemicalmethod:organicsolventssuchasethanol,dioxaneandpolyethyleneglycol
(PEG),andsaltssuchassodium,zinc,ammoniumsulfate.
Disadvantages:chemicalprecipitationmayprecipitatefreeaswellasbounddrugduring
separationdependingonphysicochemicalnatureofdrug.
Secondantibodymethod:itismostphysiologicproceduretoprecipitateboundantigen.
Thismethodemploys,anantibodyagainstgammaglobulinoftheanimalspeciesusedto
produceantidrugantibodytotheplasmaserumtoprecipitatedrug-antibodycomplex.
Disadvantages:Thistechniquehaverequiredprolongincubationtime.
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29. Other related Immuno Assay:
Relatedmethodsincludetheuseofantibodiestospecificallybindantigenic
substances.
Theydonotincludecompetitiveproteinbindingassays(CPBA)orreceptorsite
bindingassay(RSBA).
ThemostcommonlyrelatedmethodisImmunoradiometric(IRM)–the
techniquewhichemploysanexcessoflabeledantibodytoquantifythedruginthe
system.Theprincipleofthesystemdisplayedinfigureasbelow.
S + Ab٭ SAb٭+ Ab٭ S Ab٭+ SPAb٭
SP
Sisunknownorstandardantigen,Ab٭islabeledantibody,andSPistheantigen
coupledtoasolidsupport.
SAb٭fractionwillremaininthesupernatant,whereastheSPAb٭willbecoupled
tothesolidsupport.
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30. •Advantages:
Theantibodycanbemorereadilylabeledwithhighspecificactivityusing
125
I.
NoneedofanyspecificseparationtechniqueasinRIA.
Insteadofusingradiotracersseveralothermethodsrelyonalternatelabeling
procedures.Theyincludehemaglutination-inhibition,fluorescence,andfreeradical
orspinlabelandlastEnzymeImmuno-Assays.
OnlyEnzymeImmuno-Assays(EIA)–hasgainedwideacceptance.The
theoreticalconceptofEIAissameasRIA,principleofEIAisasfollows:
P S٭+ Ab S٭Ab
+
S SAb
SandS٭arepureandenzymelinked
drug,respectively;Abisantibody;andPis
themeasurablereactionproductthatcan
resultonlywhenfreeS٭reactwithan
appropriateantibody.
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31. •AdvantagesofEIAoverRIA:
•Noradiationhazard
•Economyofquantitation
•HomogenousEIAdoesnotrequireseparationoffreeandboundligands
(antigen)
•Disadvantages:
•Relativelyinsensitiveduetothelowcatalyticactivityofenzymes
•Alinkagespecificityproblemwhensamelinkageusedforantibodyconjugates
andenzymeconjugates.
SeveraldrugEIAsystemsarecurrentlyinuseintheclinicalchemistry
laboratory,primarilyforthedetectionofdrugsofabuseandquantificationof
Anticonvulsantanddigoxinplasmaconcentration.
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32. Application of RIA:
Analysisofhormones,vitamins,metabolites,diagnosticmarkers:
E.g.ACTH,FSH,triiodothyronine(T
3)andthyroxine(T
4),Glucagons,Insulin,
Testosterone,vitaminB12,prostaglandins,glucocorticoids,
Therapeuticdrugmonitoring:
Barbiturates,morphine,digoxin,
Diagnosticproceduresfordetectinginfection:
HIV,HepatitisA,Betc
Tumourmarkers:
•RIAoftumourmarkerssuchasalpha-fetoprotein(AFP),carcinoembrionic
antigen(CEA),b-HCGforchoroid-carcinoma,prostatespecificantigen(PSA)for
prostatecancer,areavailablefordetectionandmanagementofcancer
Nonclinicalapplication:suchasveterinaryscience,foodprocessingindustry,
drugindustry,forensicscienceandenvironmentalmonitoring.
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