ELISA: Enzyme-linked Immunosorbent Assay

ELISA or Enzyme-linked Immunosorbent Assay is a qualitative and quantitative assay for detecting the presence of antigens (virus, hormones, enzymes, etc.) in a sample.

1. S Y E D M U H A M M A D K H A N
BSH O N S. Z O O L O G Y
ELISA: ENZYME-LINKED
IMMUNOSORBENT ASSAY

2. INTRODUCTION
•Immunoassaysarebiochemicalteststhatdetect
thepresenceofagivensubstance(ormeasureits
concentration)inasolution.
•Theyuseanantibody(usually)oranantigen
(sometimes).
•Twoimportantcomponents:
1.Antigen(Ag)
2.Antibody(Ab)

5. INTRODUCTION
TYPESOFIMMUNOASSAYS:Thereareseveraldifferenttypesof
immunoassays,someofthemare:
•PrecipitinTest→precipitin(antibodiesthatprecipitateoutofa
solutionuponbindingwithanantigen)
•AgglutinationTest→agglutinationreactions(clumpingofcells
duetoexposuretospecificantibodies).Itisusedtoperform
bloodgrouptestsandevenindetectingdiseasesliketyphoid
fever,brucellosis,salmonellosis,etc.
•ComplementFixationTest→complementfixingantibodiesinthe
serumofthesubject,whichareproducedinresponseto
bacterialantigens.
•Radio-immunoassay(RIA)→radiolabeledmolecules.Itisusedfor
thedetectionofhormones,serumproteins,infectiousagents,
vitamins,etc.
•Enzyme-linkedImmunosorbentAssay(ELISA)→componentsof
theimmunesystem(antigensandantibodies)coupledwith
enzymes.

6. ENZYME-LINKED IMMUNOSORBENT
ASSAY
•ELISA(Enzyme-linkedImmunosorbent Assay)orEnzyme
Immunoassay(EIA)isanimmunoassaythatusescomponents
oftheimmunesystem(antigensandantibodies)coupledwith
enzymesforthedetectionofimmuneresponsesinthebody.
•Itissocalledbecause:
1.Enzyme-linked:Enzymesareattachedwitheitherthe
antigenortheantibody.
2.Immune:Antigenisrecognizedbyspecificantibodyand
sometimestheantibodyisrecognizedbyasecondone.
3.Immunosorbent:Antigen/antibodyofinterestisabsorbed
ontotheplasticsurface.
4.Assay:Substratereactswithenzymetoproduceproduct,
usuallycolored.

7. HISTORY OF ELISA
•ThetermELISAwasfirstusedbyEngvall&Perlmanin
1971.
•FirstscreeningtestcommonlyemployedforHIV.
•Formerly,theonlyoptionforconducting an
immunoassay was radioimmunoassay and as
radioactivityposesapotentialhealththreat,asafer
alternativewassought,i.e.enzymes.
•Buttheenzymehastobelinkedtoanappropriate
antibody.Thislinkingprocesswasindependently
developedbyStratisAvrameasandG.B.Pierce.

8. HISTORY OF ELISA
•Theantibodyorantigenhastobefixedtothe
surfaceofthecontainer;i.e.,theimmunosorbent
mustbeprepared(WideandJerkerPorathin1966).
•In1971,PeterPerlmannandEvaEngvallat
StockholmUniversityinSweden,andAntonSchuurs
andBaukevanWeemen intheNetherlands
independentlypublishedpapersthatsynthesized
thisknowledgeintomethodstoperformEIA/ELISA.

9. PRINCIPLES OF ELISA
•Antibodiesandsomeantigenscanattachtoplastic
surfaces.
•Antigensandantibodiescanbebondedto
enzymes.
•Theenzymedetectsthebindingofantigen(Ag)
withanantibody(Ab).
•Theenzyme convertsacolorlesssubstrate
(chromogen)toacoloredproduct.
•AnELISAcanbeusedtodetecteitherthepresence
ofantigensorantibodiesinasampledepending
howthetestisdesigned.

10. APPLICATIONS OF ELISA
•Detection of hormones
•Vaccine quality control
•GMO (Genetically modified organism)
•Detection of infectious agents
•Detection of drug markers
•Detection of immunoglobins
•Detection of tumor markers
•In new born screening
•Detection of serum proteins
•In clinical research

11. SENSITIVITY OF ELISA
•ELISA is one of the most sensitive immunoassays
available.
•It has a typical detection range between 0.01 ngand
0.1 ng.
•Sensitivity relies on the specific characteristics of the
interaction between the antibody and the antigen.
•A number of substrates, like the ones yielding fluorescent
signal or enhanced chemi-luminescent, can be utilized
to enhance results.

12. BASIC STEPS OF ELISA
ThefollowingarethebasicstepsforanELISAtest:
•Thesample(antigen/antibody),whichmayormaynotbe
labeled,isaddedtopolystyrenemicro-titerwellswhich
alreadyhasareagent(antigen/antibody)boundtoits
surface.
•Thesampleisincubatedforasuitabletimeandatasuitable
temperaturetoallowbindingofthetworeagents.
•Excessorunboundreagentiswashedoffwithawashing
buffersolution.
•Ifthefirstreagent(antibody)wasnottaggedthenasecond
enzyme-linkedantibodyisalsoaddedtobindwiththefirst
one.Thissecondantibodyistermedas“antihumanantibody”,
“antiglobulin”or“anti-antibody”.Itisalsoallowedtoincubate.

13. BASIC STEPS OF ELISA
•Asubstrateisaddedfortheenzyme(whichmaybe
boundwitheitherthefirstorthesecondreagent
added).
•Theenzymeconvertsthesubstrate(chromogen)intoa
coloredsubstance,oranyothertypeofindicating
signal.
•ImportanceofIncubation:Duringthetestperformance,
incubationtimeandmentionedtemperatureisessential
for:(1)properbindingbetweenantigenandantibody,
(2)bindingofantibodywiththeconjugateand(3)color
developmentofsubstrate.
•ImportanceofWashing:Itisdonetoensuretheremoval
ofanyunboundantibody/antigen.Properwashingis
requiredotherwisetheresultsmaybecomeinaccurate.

14. Colored wells of the Micro-titer plate indicates a positive result

15. QUALITATIVE & QUANTITATIVE
ELISA
•QualitativeELISA:Itdeterminesthepresenceor
absenceofantigenorantibody(asimplepresent/
absent).
•QuantitativeELISA:Itdeterminesthequantityofthe
antigenorantibody,theopticaldensity(OD)ofthe
sampleiscomparedtoastandardcurve,whichis
typicallyaserialdilutionofaknown-concentration
solutionofthetargetmolecule.

16. Standard curve of human IgMat OD 450nm

17. MATERIALS NEEDED FOR ELISA
The following materials are needed to perform an
ELISA test:
1. Testing sample 2. Polystyrene micro-titer plate
3. Pipettes 4. Incubator
5. ELISA reader 6. Antibody (1st & 2nd)
7. Antigen 8. Enzyme
9. Substrate 10. Blocking buffer
11. Stopping solution 12. Washing buffer

18. ELISA test kit for Human IgM

19. MATERIALS NEEDED FOR ELISA
•SpecimenSampleForELISA:serum,cerebrospinal
fluid,sputum,urine,semen,supernatantofculture
stool,etc.
•Polystyrenemicro-titerplate:The96-wellplates,
madeofpolystyreneandarecoatedwitheither
inactivatedantigenorantibody.
•ELISAreader:Itisaninstrumentusedtodetect
biological,chemicalorphysicaleventsofsamples
inmicro-titerplates.

20. MATERIALS NEEDED FOR ELISA
•Enzyme:SeveralenzymesareusedinELISA,someof
themare:(1)Horseradishperoxidase(mostcommonly
used),(2)Alkalinephosphatase,(3)β-galactosidase,(4)
Lacto-peroxidaseand(5)Tetra-methylbenzidine.
•Incaseofperoxidase,thesubstratehydrogenperoxide
isconvertedintowaterandO
2inthepresenceof
electrondonors,i.e.diaminobenzidine or4-
chloronaphthol,etc.
•Theelectrondonorsarethemselvesoxidized.
•Oxidationofdiaminobenzidineproducesdarkbrown
colorwhilethatof4-chlorornaphtholyieldspurplecolor
whichisthebasisofELISA

21. MATERIALS NEEDED FOR ELISA
•Substrate:Initiallycolorlessbutstronglycoloredor
fluorescentafterenzymeaction.
•Blockingbuffer:Itisasolutionofirrelevantprotein,
mixtureofproteins,orothercompound that
passivelyadsorbstoallremainingbindingsurfaces
oftheplate.
•Stoppingsolution:Itstopstheenzymesubstrate
reactionandcolordevelopment.
•Washingbuffer:Itactsasabufferedsolution
containingdetergenttowashunboundmaterial
fromtheplate.

22. Polystyrene Micro-titer Plate (96 wells)

23. ELISA
Reader

24. TYPES OF ELISA
•There are five basic variants of ELISA test, these are:
1.Direct ELISA
2.Indirect ELISA
3.Sandwich ELISA
4.Competitive ELISA
5.Reverse ELISA

25. DIRECT ELISA
•DirectELISAisatypeofELISAinwhichonlyalabeled
primaryantibody(boundwithanenzyme)isusedto
detectthepresenceofanantigen.
•Ithasthefollowingsteps:
•Coatingsurfacewithantigens:Abufferedsolutionofthe
antigentobetestedisaddedtoeachwell(usually96-
wellplates)ofamicro-titerplate.
•Adhesion:Itisgiventimetoadheretotheplastic
throughchargeinteractions.
•Blockingunoccupiedsites:Asolutionofnon-reacting
protein,suchasbovineserumalbuminorcasein,is
addedtoeachwellinordertocoveranyplasticsurface
inthewellwhichremainsuncoatedbytheantigen.
•Washing:Excessorunboundantigensarewashedoff.

26. DIRECT ELISA
•Additionofprimaryantibody:Theprimaryantibodywith
anattached(conjugated)enzymeisadded,which
bindsspecificallytothetestantigencoatingthewell.
•Washing:Excessorunboundantibodiesarewashedoff.
•Additionofsubstrateandcolorchange:Asubstratefor
thisenzymeisthenadded,whichchangescolorupon
reactionwiththeenzyme.Thehighertheconcentration
oftheprimaryantibodyintheserum,thestrongerthe
colorchange.
•Quantificationviaspectrometry:Often,aspectrometeris
usedtogivequantitativevaluesforcolorstrength.

27. Basic steps and mechanism of direct ELISA

28. DIRECT ELISA
Advantages
•Itisashortprotocol.
•Itsavestimeandreagents.
•Thereisnocross-reactivity
fromsecondaryantibody.
Disadvantages
•Themethod ofantigen
immobilization isnot
specific;whenserumisused
asthesourceoftest
antigen,allproteinsinthe
samplemaysticktothe
micro-titerplatewell.
•Smallconcentrationsof
analyte (antibody or
antiserum)inserummust
competewithotherserum
proteinswhenbindingto
thewellsurface.

29. INDIRECT ELISA
•Aprimary,unlabeledantibodybindswithimmobilized
antigenandisinturnboundwithasecondaryenzyme
linkedantibody,whichwillgivecolorchangeuponthe
additionofasuitablesubstrate.Itisusedtotestfor
antibodies.
•Ithasthefollowingsteps:
•Coatingsurfacewithantigens:Thepolystyrenemicro-
titerwellsarecoatedwithantigens.
•Washing:Excessorunboundantigensarewashedoff.
•Additionofprimaryantibody:Testantiserum(containing
primaryunlabeledantibody)isaddedandallowedto
incubate.

30. INDIRECT ELISA
•Bindingofantigenandprimaryantibody:Theprimary
antibodywillbindwiththeantigen(giventhatitwas
presentintheantiserum).
•Washing:Excessorunboundprimaryantibodiesare
washedoff.
•Additionofsecondaryantibody:Asecondaryenzyme-
linkedantibodyisaddedwhichbindswiththeprimary
antibody.
•Washing:Excessorunboundantibodiesarewashedoff.
•Additionofsubstrateandcolorchange:Thesubstratefor
theenzymeisaddedandacolorchangeisobserved
duetotheactionoftheenzyme.

32. INDIRECT ELISA
Advantages
•Itexhibitssignal
amplification, i.e.
several secondary
antibodieswillbindto
theprimaryantibody.
•Ithasahighflexibility,
i.e. the same
secondary antibody
may beusedfor
several primary
antibodies.
Disadvantages
•Itisalongprotocolif
compared todirect
ELISA.
•Thereisapossibilityof
cross-reactivityfrom
secondaryantibody.

33. SANDWICH ELISA
•Theantigenissandwichedbetweenacapture
antibody(boundtothewell)andtheprimary
antibodywhichisaddedlateron.
•Thesecondaryantibody,whichislabeled(bound
withenzyme)thenbindswiththeprimaryantibody
andaftertheadditionofsubstrate.
•Itcanbebothdirectandindirect.

34. Basic steps and mechanism
of Direct Sandwich ELISA

35. Basic steps and mechanism
of Indirect Sandwich ELISA

36. SANDWICH ELISA
Advantages
•Highspecificity:twoantibodies
detectingdifferentepitopesonthe
sameantigen.
•Itissuitableforcomplexsamples.
•Ithasahighflexibilityand
sensitivity,i.e.bothdirectand
indirectmethodscanbeused.
•Useofthepurifiedspecific
antibodytoattachtheantigento
theplasticeliminatesaneedto
purify the antigen from
complicatedmixturesbeforethe
measurement.
Disadvantages
•Ithasademandingdesign.
•Findingtwoantibodiesagainstthe
same targetthatrecognize
differentepitopesandworkwell
togethercanbechallengingat
times.

37. COMPETITIVE ELISA
•Inthistest,acompetitionisgeneratedbetweenthe
antigens(betweenthoseboundtothemicro-titerwell
andthosewhicharealreadyboundwiththeantibody).
•Itsstepsareasfollows:
•Incubationofantibodyanditsantigen:Unlabeled
antibodyisincubatedinthepresenceofitsantigen
(sample).
•Additionoftheantigen-antibodycomplex:Thesebound
antibody-antigencomplexesarethenaddedtoan
antigen-coatedwell.
•Competition:Themoreantigensinthesample,themore
antigen-antibodycomplexesareformedandsothere
arelessunboundantibodiesavailabletobindtothe
antigeninthewell,hence"competition".

38. COMPETITIVE ELISA
•Washing:Theplateiswashed,sounbound
antibodiesareremoved.
•Additionofsecondaryantibody:Thesecondary
antibody(enzyme-linked),specifictotheprimary
antibody,isadded.
•Washing:Excessorunboundsecondaryantibodies
arewashedoff.
•Additionofsubstrate:Asubstrateisaddedwhich
producesacolor(chromogenic)orfluorescent
signal(duetotheactivityoftheenzyme).

39. Basic steps and mechanism of competitive ELISA

40. COMPETITIVE ELISA
AlternativeformofCompetitiveELISA:
•SomecompetitiveELISAkitsincludeenzyme-linked
antigenratherthanenzyme-linkedantibody.
•Thelabeledantigencompetesforprimaryantibody
bindingsiteswiththesampleantigen(unlabeled).
•Thelessantigeninthesample,themorelabeled
antigenisretainedinthewellandthestrongerthe
signal.
•Commonly,theantigenisnotfirstpositionedinthe
well.

41. REVERSE ELISA
•Thistestleavestheantigenssuspendedinthetest
fluidfromwhichalloftheexcessantibodiesare
pulledoutby“scavengerantigens”andthe
remaindersolutionispassedthroughadetectorfor
analysis.
•Ithasthefollowingsteps:
•Incubationofunlabeledantibodywiththesample
(antigen):Unlabeledantibodyisincubatedinthe
presenceofitsantigen(sample).Sufficient
incubationperiodisprovidedtoallowthe
antibodiestobindtotheantigens.

42. REVERSE ELISA
•Passingthroughscavengerchannel:Thesampleisthen
passedthrougha“scavengerchannel”whichisatest
tubeoraspecificallydesignedflowthroughchannel
thathas“scavengerantigens”boundtoitssurface.
Thesecanbeidenticalorsufficientlysimilartothe
primaryantigensthatthefreeantibodieswillbind.
•Bindingwithexcessantibodies:Thescavengerantigens
bindwillalltheexcessantibodiesintroducedintothe
sample,ifpropertimeandsurfaceareaisprovided.
•Detector:Thesample,thatnowcontainsthetagged
andboundantibodies,ispassedthroughadetector.This
devicecanbeaflowcytometerorotherdevicethat
illuminatesthetagsandregisterstheresponse.

43. REVERSE ELISA
Advantages
•Allowsmultipleantigensto
betaggedandcountedat
thesametime.
•Thisallowsspecificstrainsof
bacteriatobeidentifiedby
two(ormore)differentcolor
tags.Ifbothtagsare
presentonacell,thenthe
cellisthatspecificstrain.If
onlyoneispresent,itisnot.
•Theequipmentneededis
usuallylesscomplicated
andcanbeusedinthe
field.
Disadvantages
•Thistestisdone,generally,
onetestatatimeand
cannotbedonewiththe
micro-titerplate.

44. ADVANTAGES OF ELISA
•Reagentsarerelativelycheap.
•Reagentshavealongshelflife.
•ELISAishighlyspecificandsensitive.
•Noradiationhazardsoccurduringlabelingor
disposalofwaste.
•Easytoperform.
•Proceduresarequick(notverytimeconsuming).
•Homekitsareinexpensiveandwidelyavailable.
•ELISAcanbeusedtodetectavarietyofinfections.

45. DISADVANTAGES OF ELISA
•Measurementofenzymeactivitycanbemorecomplex
thanmeasurement ofactivityofsometypeof
radioisotopes.
•Enzyme activitymaybeaffectedbyplasma
constituents.
•Kitsarecommerciallyavailable,butnotcheap.
•Veryspecifictoaparticularantigen,i.e.itwon’t
recognizeanyotherantigen.
•Falsepositive/negativeresultsarepossible,especially
withmutated/alteredantigen.
•Resultsmaynotbeabsolute.
•Concentrationmaybeunclear.
•Appropriateantibodiesmustbeavailable.

46. FINAL VERDICT ON ELISA
ELISAtestshavetheirdrawbacksandflawsthatmay
overshadowtheirmeritsatsomepointsbutoverall
thetestsareverypracticalandareprettypopularas
wellrangingfromsimplehouse-holdpregnancytest
kitstocomplexbloodprofiletests(HIV,hepatitis,
Mycobacterium,etc.)