ELISA

ELISA

1. Dr. J. P. Saranraj
ELISA

2. ENZYME LINKED IMMUNOSORBANT
ASSAY (ELISA)
In1972,EngvallandPerlmanndiscovered
ELISA.
AlsocalledEnzymeImmunoassay(EIA)or
Solid–phaseImmunoassay.
ELISAissimilarinprincipletoRIAbutdifferson
anEnzymeratherthanaRadioactivelabel.
Anenzymeconjugatedwithanantibody
reactswithacolorlesssubstratetogeneratea
coloredreactionproduct.Suchasubstrateis
calledaChromogenicsubstrate.
Numberofenzymeshavebeenemployedfor
ELISA,includingAlkalinephosphatase,
HorseradishperoxidaseandGalactosidase.

3. 1. Indirect ELISA
Antibodycanbedetectedorquantitatively
determinedwithanIndirectELISA.
Serumorsomeothersamplecontainingprimary
antibody(Ab1)isaddedtoanantigen-coated
microtiterwellandallowedtoreactwiththe
antigenattachedtothewell.
AfteranyfreeAb1iswashedaway,thepresence
ofantibodyboundtotheantigenisdetectedby
addinganenzyme-conjugatedsecondaryanti-
isotypeantibody(Ab2),whichbindstothe
primaryantibody.
AnyfreeAb2theniswashedaway,anda
SubstratefortheEnzymeisadded.Theenzyme
convertsthesubstratetoadetectablecolored
compound.

4. Theamountofcoloredreactionproductthat
forms ismeasured by specialized
spectrophotometricplatereaders(ELISA
Reader),whichcanmeasuretheabsorbanceof
allofthewellsofa96-wellplateinseconds.
IndirectELISAisthemethodofchoicetodetect
thepresenceofserumantibodiesagainstHIV.
Inthisassay,recombinantenvelopeandcore
proteinsofHIVareadsorbedassolid-phase
antigenstomicrotiterwells.Individualsinfected
withHIVwillproduceserumantibodiesto
epitopesontheseviralproteins.
Generally,serumantibodiestoHIVcanbe
detectedbyindirectELISAwithin6weeksof
infection.

6. 2. Sandwich ELISA
Antigencanbedetectedormeasuredbya
SandwichELISA.
Inthistechnique,theantibody(ratherthanthe
antigen)isimmobilizedonamicrotiterwell.
Asamplecontainingantigenisaddedand
allowedtoreactwiththeimmobilizedantibody.
Afterthewelliswashed,asecondenzyme-
linkedantibodyspecificforadifferentepitopeon
theantigenisaddedandallowedtoreactwith
theboundantigen.
Afteranyfreesecondantibodyisremovedby
washing,substrateisadded,andthecolored
reactionproductismeasured.

8. 3. Competitive ELISA
Anothervariationformeasuringamountsof
antigeniscompetitiveELISA.
Inthistechnique,antibodyisfirstincubatedin
solutionwithasamplecontainingantigen.
Theantigen-antibodymixtureisthenaddedtoan
antigencoatedmicrotiterwell.
Themoreantigenpresentinthesample,theless
freeantibodywillbeavailabletobindtothe
antigen-coatedwell.
Additionofanenzyme-conjugatedsecondary
antibody(Ab2)specificfortheisotypeofthe
primaryantibodycanbeusedtodeterminethe
amountofprimaryantibodyboundtothewellas
inanindirectELISA.

9. Inthecompetitiveassay,however,thehigherthe
concentrationofantigenintheoriginalsample,
thelowertheabsorbance.

11. Substrates used in ELISA
PNPP(p-NitrophenylPhosphate-DisodiumSalt)
isawidelyusedsubstratefordetectingAlkaline
phosphatase.
ABTS(2,2'-Azinobis[3-ethylbenzothiazoline-6-
sulfonicacid]-Diammoniumsalt)isusedto
detectHorseRadishPeroxidase.
OPD(o-phenylenediaminedihydrochloride)is
usedtodetectHorseRadishPeroxidase.
TMB(3,3',5,5'-tetramethylbenzidine)usedto
detectHorseRadishPeroxidase.

12. Applications of ELISA
DetectionofHIVantibodiesinbloodsamples.
DetectionofHepatitis-Bmarkersinserum.
DetectionofRotavirusinfeces.
DetectionofMycobacteriumsp.antibodiesin
Tuberculosis.
DetectionofEnterotoxinofEscherichiacoliin
feces.
Detectvariouskindofdiseasessuch
as Malaria, Chagas disease
(Trypanosomiasis)andJohne'sdisease
(Paratuberculosis–Mycobacteriumavium).
foundapplicationsinthefoodindustryin
detecting food allergens such
asMilk,Peanuts,Walnuts,Almonds&Eggs.