Serological-Techniquues for testing of disease

Serological-Techniques

1. SEROLOGICAL TECHNIQUES –II
(Primary Binding Tests)
RAKESH SHARDA
Department of Veterinary Microbiology
NDVSU College of Veterinary Science & A.H.,
MHOW

2. Antigen antibody reactions
Ag-Ab reaction occurs in three stages:
Primary Stage
Formation of Ag-Ab complex combined by weaker
intermolecular forces
Secondary stage
leads precipitation
agglutination
lysis of cells etc.
Tertiary stage (reaction):
Leads to tissue damage
Destruction of Ag or itsNeutralization

3. CLASSIFICATION
Primarybindingtest-directlymeasurethe
bindingofantigentoantibodye.g.RIA,IF,ELISA.
Secondarybindingtest-measuretheresultsof
antigen–antibodyinteractioninvitro,e.g.
precipitation,complementfixation.
Invivotest-measurestheactualprotectiveeffectof
antibodiesinahost,e.g.passivecutaneous
anaphylaxis.

5. RADIO IMMUNO ASSAY
(RIA)

6. RADIO IMMUNO ASSAY
RIAwasdevelopedbyBarsonandYalowin1960
Thetestuseradioactiveisotopesfordetectingantigenor
antibody.ThecommonlyusedisotopesareH
3
,C
14
,orI
125
RIAisoneofthemostsensitiveimmunologicaltechnique
(Usingantibodiesofhighaffinity-K
0=10
8
–10
11
M
−1
,itispossibleto
detectafewpicograms(10
−12
g)ofantigeninthetube.
UsesofRadioimmunoassay
The test can be used to determine very small quantities of antigens
and antibodies in the serum (anti DNA Abs in SLE).
The test is used for quantitation of hormones, drugs, HBsAg, and other
viral antigens.
Analyze nanomolar and picomolar concentrations of hormones in
biological fluids.
LimitationsofRadioimmunoassay–expensive(gamma
counters),hazardsofradioactivity,limitedshelf-lifeoflabeled
reagents,availabilityofradioisotopesandenvironmental
concernswithsafedisposalofradioactivematerial

7. Principle of Radioimmunoassay
It involves a combination of three principles.
An immune reaction i.e.antigen, antibody binding.
A competitive binding or competitive displacement reaction. (It gives
specificity)
Measurement of radio emission. (It gives sensitivity)
TheclassicalRIAmethodsarebasedontheprincipleof
competitivebinding:
anunlabeledantigencompeteswitharadiolabeledantigenforbinding
toanantibodywiththeappropriatespecificity
theamountoffree(notboundtoantibody)radiolabeledantigenis
directlyproportionaltothequantityofunlabeledantigeninthe
mixture.

8. Technique of Radioimmunoassay
Amixtureispreparedof
radioactiveantigen(“hot")
antibodies("First"antibody)againstthatantigen.
Knownamountsofunlabeled("cold")antigenareaddedtosamplesof
themixture.Thesecompeteforthebindingsitesoftheantibodies.
Atincreasingconcentrationsofunlabeledantigen,anincreasing
amountofradioactiveantigenisdisplacedfromtheantibody
molecules.
Theantibody-boundantigenisseparatedfromthefreeantigeninthe
supernatantfluid,andtheradioactivityofeachismeasured.
Fromthesedata,astandardbindingcurvecanbedrawn
The samples to be assayed (theunknowns) are run in parallel.
After determining the ratio of bound to free antigen ("cpm Bound/cpm
Free") in each unknown, the antigen concentrations can be read
directly from the standard curve

9. Technique of Radioimmunoassay
Separating Bound from Free Antigen
Precipitatetheantigen-antibodycomplexesbyaddinga
"second"antibodydirectedagainstthefirst.Forexample,
rabbitIgGandantirabbit-IgG
Theantigen-specificantibodiescanbecoupledtotheinnerwalls
ofatesttubeandafterincubation:
thecontents("free")areremoved;
thetubeiswashed("bound"),and
theradioactiveofbothismeasured.
Theantigen-specificantibodiescanbecoupledtoparticles,
likeSephadex.Centrifugationofthereactionmixtureseparates
theboundcounts(inthepellet)from
thefreecountsinthesupernatantfluid.

10. Technique of RIA
Standard curve
Separating antigens by precipitation

11. Competitive RIA for Ag detection
Determine amount of Ab
needed to bind to a known
amount of labeled Ag
Use predetermined
amounts of labeled Ag and
Ab and add a sample
containing unlabeled Ag as
a competitor
Determine amount of
labeled Ag bound to Ab
Concentration of Ag in
sample is determined from
a standard curve using
known amounts of
unlabeled Ag
+
Prior to Test
Labeled Ag
+ 
Test
+
Patient’s
sample
Labeled
Ag
+

12. Non-Competitive RIA for Ab detection
Immobilize Ag
Incubate with patient’s
serum sample
Add labeled anti-Ig
Amount of labeled Ab
bound is proportional to
amount of Ab in the
sample
Solid
Phase
AgImmobilized
Ab in
Patient’s
sample
Labeled
Anti-Ig

13. Non-Competitive RIA for Ag detection
Immobilize Ab
Incubate with patient’s
sample containing antigen
Add labeled antibody
Amount of labeled Ab bound
is proportional to the
amount of Ag in the sample
Solid
Phase
Ag
Immobilized
Ag in
Patient’s
sample
Labeled
Ab

14. IMMUNO FLUORESCENCE ASSAY
(IFA)

15. INTRODUCTION
Immunofluorescenceassay(IFA)isastandardserological
techniquetoidentifythepresenceofantigensininfectedcellsby
theirabilitytoreactwithspecificantibodies,whichconjugatedwith
arevisualizedbyincubationwithFluorescentdyes.
Fluorescentmoleculesareorganicdyesthatabsorblightofone
(shorter)wavelength(excitation)andemitlightofanother(larger)
wavelength.
Theemittedlightcanbeviewedwithafluorescentmicroscope,
whichisequippedwithaUVlightsource.
Themostcommonlyusedfluorescentdyesarefluorescein
isothiocyanate(FITC)andlissaminerhodamine
FAtestsareperformedonfrozensectionsoftissues,bloodsmears,
tissueimprintsandscrapingsorincellcultures.

16. Slide 29t:/classes/BMS524/524lect003.ppt© 1993-2008 J. Paul Robinson -Purdue University Cytometry Laboratories
Fluorescent Microscope
Dichroic Filter
Objective
Arc Lamp
Emission Filter
Excitation Diaphragm
Ocular
Excitation Filter
EPI-Illumination

18. INTRODUCTION
Advantages:
Themethodisrapidandspecific.
IFassayscanalsobeusedtodetectco-localisationoftwoor
moredifferentantigenswithinsamespecimen
Themethodcanalsobeusedforvisualizationofintracellular
localizationofantigens,suchasviruses.
Limitations:
expensive fluorescence microscope and reagents,
trained personnel
have a factor of subjectivity that may result in erroneous results

21. TYPES OF IFA
TherearetwobasictypesofIFtechniques:
direct(DFT)-isusedtodetectunknownantigeninacellortissueby
employingaknownlabeledantibodythatinteractsdirectlywithunknown
antigen.
indirect(IFT)-isadoubleantibodytechniqueandhastheadvantageof
usingasinglelabeledantiglobulin(antibodytoIgG)asa“universalreagent”
todetectmanydifferentspecificantigen–antibodyreactions.
Indirectimmunofluorescencetestisusedwidelyto:
detectspecificantibodiesforserodiagnosisofsyphilis,amoebiasis
leptospirosis,,toxoplasmosis,andmanyotherinfectiousdiseases;
Identifytheclassofagivenantibodybyusingfluorescentantibodiesspecificfor
differentimmunoglobulinisotypes;
Identifyandenumeratelymphocytesubpopulationsbyemployingmonoclonal
antibodiesandcytofluorographs(FACS);and
Detectautoantibodies,suchasantinuclearantibodiesinautoimmunediseases.
Diagnosisofdiseasessuchasrabies
Demonstrationofnon-cytopathicvirusesincellcultures

22. DIRECT IMMUNOFLUORESCENCE
–Ab to tissue Ag is labeled with fluorochrome
–quicker to perform
Ag
Fluorochrome
Labeled Ab
Tissue Section

23. Thespecimenwithsuspectedantigenisplacedonaslideand
fixedbyacetone
Fluorescentlabeledspecificantibodyisthenaddedtoitand
incubated
Thepreparationisthenwashedwhichwillallowtheremovalof
othercomponentsexceptthecomplexofantigenandfluorescent
labeledantibody.
Onmicroscopy(FluorescenceMicroscopy),antigen-antibody
complexareobservedfluorescingduetothedyeattachedto
antibody.
Theneedforpreparationofseparatelabeledantibodyforeach
pathogenisthemajordisadvantageofthedirect
immunofluorescencetest.
Procedure of DFA

24. INDIRECT IMMUNOFLUORESCENCE
PrimaryAbtotissueAgisunlabeled
Fluorochrome-labeledanti-Ig(Secondaryordetecting
antibody)isusedtodetectbindingofthefirstAb.
Moresensitive(sinceseveralmoleculesoffluorochrome-
labeleddetectingreagentbindtoeachprimaryAbmolecule)
Specific(ifmonoclonalantibodiesareused)
Moretimeconsuming
Economicalasitrequiresonlyoneanti-speciesIgGlabeled
antibodyfordetectingallpathogens
IFTischoiceoftestfordiagnosisofRabiesasperWHOand
OIE

25. Ag
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled
Ab
Indirect Immunofluoroscence Test

26. Procedure of IFT
Firstofall,thevirusinfectedcellsaregrownoncoverglass.
Thecellsarethenfixedbyparaformaldehydesothatthe
proteinsinthecellsarepoisedtoantibodybinding.
Cellsarethenpermeabilizedbyaproperdetergentsuchas
tritonX-100.
Aspecificantibodyisappliedonthesurfacesothattheviral
antigenisrecognizedbytheantibody.
Then,thesecondaryantibody,whichisspecifictoFc
fragmentofIgGmoleculeoftheprimaryantibody,isapplied.
Sinceafluorescencedyeislinkedtothesecondaryantibody,the
antigencanbevisualizedunderafluorescencemicroscope.

28. New flurochrome reagents
Fluorochrome-labeledproteinA,whichbindstothe
FcregionofprimaryIgGantibodymolecules
Fluorochrome-conjugatedavidin,whichbindsto
biotinmoleculeofsecondarybiotinylatedanti-
speciesantibody
Fluorochromeconjugatedanti-c3antibody,which
bindstomultiplec3bmoleculesthatbindstoAg-Ab
complexduetoactivationofclassicalcomplement
pathwayfollowinginteractionbetweenantigenand
antibody.

31. IMMUNOPRECIPITATION

32. Immunoprecipitation
Immunoprecipitation(IP)isamethodtoisolateaspecificantigen
fromamixture,usingtheantigen-antibodyinteraction.Antigens
isolatedbyIPareanalyzedbySDS-PAGEorWesternblotting.
Itisamodificationoftraditionalprecipitationtechniqueandisuseful
todeterminethepresenceandquantityoftheviralantigen,
particularlywhenitsconcentrationisverylowininfectedtissuesor
cellcultures.
The principle
InIP,anantibodyisaddedfirsttoamixturecontaininganantigen,
andincubatedtoallowantigen-antibodycomplexestoform.
Subsequently,theantigen-antibodycomplexesareincubatedwithan
immobilizedantibodyagainsttheprimaryantibody(secondary
antibody)orwithproteinA/G-coatedbeadstoallowthemtoabsorb
thecomplexes.Thebeadsarethenthoroughlywashed,andthe
antigeniselutedfromthebeadsbyanacidicsolutionorSDS.
Theuseofanantibodywithhighbindingspecificityandaffinityfor
theantigeniscriticalforsuccessfulIP.

33. Principle of Immunoprecipitation

34. Procedure of Immunoprecipitation
Theimmunoprecipitationprocedurecanbedividedinto
followingsteps:
labelingtheantigen(optional)(witharadioactiveprecursor
oravidin);
lysesofthecellstoreleasetheantigen;
additionofprimaryantibodyandformationofAg-Ab
complexes;
purificationoftheimmunecomplexbytheadditionofco-
precipitatingagents,suchasbiotinylatedanti-
immunoglobulinantibodies,synthetic(agarose)beads
coupledtoproteinAorproteinGorsupermagneticbeads
theunboundantigenisremovedbywashingandthe
immunecomplexarethencollectedbycentrifugation.
SDS-PAGEorWesternBlotting

36. Types of IP
Individualproteinimmunoprecipitation(IP)-Involvesusingan
antibodythatisspecificforaknownproteintoisolatethatparticular
proteinoutofasolutioncontainingmanydifferentproteins.
Compleximmunoprecipitation(Co-IP)-Immunoprecipitationof
intactproteincomplexes,i.e.antigenalongwithanyproteinsor
ligandsthatareboundtoit.
Chromatinimmunoprecipitation(ChIP)-anantibodythatisspecific
toaputativeDNAbindingproteinisusedtoimmunoprecipitatethe
protein–DNAcomplexoutofcellularlysates.
RNPImmunoprecipitation(RIP)- ribonucleoproteinsare
immunoprecipitatedusinganantibodytargetingtheribonucleoprotein
ofinterest.
Tagged-proteinIP–proteinsofinterestaretaggedoneithertheC-or
N-terminalendwithanepitopetowhichahigh-affinityantibodyis
available.ThemostcommonlyusedtagsincludeFLAG,c-Myc,
Hemagglutinin(HA),V5andGreenfluorescentprotein(GFP).

37. Tag strategies for co-IP (A), ChIP (B) and RNP-IP (C)

38. Advantages and Limitations of IP
Advantages
IPisidealforsmall-scaleenrichmentofproteins;proteinata
verylowconcentrationcanbeconcentratedfromtherelatively
largevolumeof1–2mL.
Itisfastandrelativelyeasy
antigensareallowedtoreactwiththeantibodiesintheirnative
conformationpriortotheirsubsequentseparationand
quantification.
Co-IP is considered as the golden standard assay for protein-
protein interaction.
Limitations
mightnotbeabletocapturelowaffinityandtransientprotein
interactions.
limitedbytheavailabilityofantibodiesthatrecognizethebait
protein

39. ENZYME LINKED IMMUNOSORBENT ASSAY
(ELISA)

40. What is ELISA?
•Animmunologicaltechniqueusinganenzymeasalabelto
detect(assay)presenceofatargetprotein(antigenorantibody).
•AconjugateinELISAisanenzyme(label)boundtoanantibody
whichbindtotargetprotein
•Canbequalitativeorquantitative.
•Verysensitive.
•Commonlyusedinmedicineandscientificresearch.

41. Principle of ELISA
Enzyme Linked ImmunosorbentAssay
1.Antigenofinterestisadsorbedontoplasticsurface(‘sorbent’).
2.Antigenisrecognisedbyspecificantibody(‘immuno’).
3.Thisantibodyisrecognisedbysecondantibody(‘immuno’)
whichhasenzymeattached(‘enzyme-linked’).
4.Substratereactswithenzymetoproduceproduct,usually
coloured.
Irrespective of type and format, washing is mandatory after each
step of ELISA except after adding substrate
Coloured product
=
measure (assay) of antigen present

42. Substrate
Primary
antibody
Secondary
antibody
Different antigens in sample
Coloured
product
Principle of ELISA

43. 1. Add antigen
7. Add substrate
for enzyme
2. Wash with
PBST
4. Wash with
PBST
3. Add primary
antibody
6. Wash with
PBST
5. Add secondary
antibody
8. Observe
colour
development
Incubationfora
predetermined time
aftereachstepis
required
Washingaftereachstep
ismandatorytoremove
unboundreactants

45. Enzymes used in ELISA
Peroxidase from horseradish
Alkaline phosphatase from E. coli
b-galactosidase from E. coli
•React with a colourless substrate to produce a coloured product.
•Must work fast at room temperature so the colour develops
quickly.
•Have minimal interference from factors in sample.

46. Colorimetric ELISA substrates
Enzyme Product
Absorbance
and Color
Alkaline
Phosphatase(AP)
PNPP(p-Nitrophenyl
Phosphate)
405nm
Yellow
Horse Radish
Peroxidase(HRPO)
ABTS(2,2'-Azinobis [3-
ethylbenzothiazoline-6-sulfonic
acid])
410nm(650nm)
Green
HRPO OPD(o-phenylenediamine
dihydrochloride)
492nm
Yellow orange
HRPO TMB(3,3',5,5'-
tetramethylbenzidine)
450nm(652nm)
Yellow
Β-galactosidaseONPG 410nm
Yellow

47. Types of ELISA
DirectELISA
IndirectELISA
SandwichELISA
CompetitiveELISA

49. Direct ELISA
AdirectELISAisonewhichtheantigenisbounddirectlytothe
wellofamicroplateandthedetecting(orbinding)antibodyis
directlyconjugatedwithenzyme.
Thestepsare:
CoatingofELISAplatewithantigeninabuffer,incubatedand
washed
Blockingofnon-specificbindingsites,incubatedandwashed
Additionoftheenzymeconjugateddetectingantibody,which
bindsspecificallytothetestantigencoatingthewell,incubated
andwashed
Asubstrateforthisenzymeisthenaddedwhichchangescolor
uponreactionwiththeenzyme.
Aftershortincubationstopsolutionisaddedandthesignalis
measuredonaplatereader.

50. Direct ELISA

51. Indirect ELISA
IndirectELISAisatwo-stepELISAwhichinvolvestwoantibodies
-primaryunlabeledantibody(testsample)andalabeled,
secondaryantibody.Theprimaryantibodyisincubatedwiththe
antigenfollowedbytheincubationwiththesecondary
antibody,whichisenzymeconjugatedanti-speciesIg.
Thestepsare:
CoatingofELISAplatewithantigeninabuffer,incubatedandwashed
Blockingofnon-specificbindingsites,incubatedandwashed
Additionofserumsamplecontainingprimaryantibody,whichbinds
specificallytotheantigencoatingthewell,incubatedandwashed
Enzymelinkedsecondaryantibodyareadded,incubatedandwashed.
Asubstrateforthisenzymeisthenaddedwhichchangescolorupon
reactionwiththeenzyme.
Aftershortincubationstopsolutionisaddedandthesignalismeasured
onaplatereader.

52. Indirect ELISA

53. Indirect ELISA
TheindirectELISAisusedforthequantitativeestimationof
antibodiesintheserumandotherbodyfluids.
Advantages
High sensitivity:More than one labeled antibody is bound per
antigen molecule;
Flexible: Different primary antibodies can be used with a single
labeled secondary antibody;
Cost-saving: Fewer labeled antibodies are required.
Disadvantages
Cross-reactivitymightoccurwiththesecondaryantibody,
resultinginnon-specificsignal(noise).
Anextraincubationstepisrequiredintheprocedure.

54. Sandwich ELISA
Thesandwichassayusestwodifferentantibodies(captureand
detection)thatarereactivewithdifferentepitopesonthe
antigen;itisnamedsoasantigenissandwichedbetweentwo
antibodies.
Theantigentobemeasuredmustcontainatleasttwodifferent
antigenicepitopes.
Eithermonoclonalorpolyclonalantibodiescanbeusedasthe
captureanddetectionantibodiesinSandwichELISAsystems;
mAbsimprovessensitivityandspecificityoftest
ApolyclonalisoftenusedasthecaptureantibodyandmAbas
detectionantibody.
SandwichELISAisusedasaqualitativeandquantitativetestfor
antigendetection.
Astandardcurveisdrawnwithabsorbancereadingsagainst
knownconcentrationsofantigenandthetestantigenisquantified
byextrapolation.

55. Sandwich ELISA
SandwichELISAisatwo-steptestwhichinvolvestwoantibodies
–capturinganddetection.Thecapturingantibodyisincubated
withtheantigenfollowedbytheincubationwiththedetecting
antibody,whicheitheritselfmaybelabeledorananti-speices
Iglabeledsecondaryantibodycanbeused.
Thestepsare:
CoatingofELISAplatewithcaptureantibodyinabuffer,incubatedand
washed
Blockingofnon-specificbindingsitesbyblockingagents,incubatedand
washed
Additionoftestantigensample,whichbindsspecificallytothecapture
antibodiescoatingthewell,incubatedandwashed
Additionofdetectingantibody,incubatedandwashed
Asubstrateforenzymeisthenaddedwhichchangescoloruponreaction
withtheenzyme.
Aftershortincubationstopsolutionisaddedandthesignalismeasured
onaplatereader.

56. Sandwich ELISA

57. Sandwich ELISA
ThesandwichELISAismostpreferredELISAformatusedforthe
qualitativeandquantitativedetectionofviralpathogens,such
asFMDV,BTV,PPRV,etc.
Advantages
Highspecificitysincetwoantibodiesareusedandantigenis
specificallycapturedanddetected.
Highsensitivitysincetwoantibodiesareused
Purificationofantigenfromacomplexmixtureisnotrequired
Flexible,sincebothdirectandindirectdetectionmethodscan
beused.
Disadvantages
Anextraincubationstepisrequiredintheprocedure.

58. Comparison of Direct, Indirect and Sandwich ELISA

59. Competitive ELISA (c-ELISA)
AcompetitiveELISA,alsoknownasaninhibitionorblocking
ELISA,measurestheamountofanalyteinasampleby
quantificationofitsinterferencewithanexpectedsignal.
Thesignalinverselycorrelateswiththeamountofanalytesuch
that,iftheconcentrationofthetargetanalyteishigh,thenthe
referencesignalisdiminishedthroughitscompetitivebinding
toalimitedamountoflabeledantibody.
Lessismore-moretargetanalyteinyoursamplewillmean
moreantibodycompetedaway,whichwillleadtolesssignal
andvice-versa.
Thetestcanbeusedtomeasuretheconcentrationofan
antigenorantibodyinasample.
TheELISAcurrentlybeingusedforassayofvaccine-induced
seroconversioninFMDcontrolprogrammeisanexampleof
c-ELISA.

60. Competitive ELISA for Antigen -Principle
Asamplecontainingunknownantigen(testantigen)isfirst
incubatedwithantibodiesspecificforreferenceantigenina
solution(liquidphase);thetwocombinetoformimmune
complexes,ifspecificforeachother.
Theantigen-antibodymixtureisthenaddedtotheELISAplate
whichhaswellspre-coatedwithknownantigen(reference
antigen).
Afterwashing,enzymeconjugatedsecondaryantibodyspecific
forisotypeoftheprimaryantibodyisaddedfollowedbycolor
development
Ifbothantigensarehomologousthenlessfreeantibodywillbe
availabletobindtothereferenceantigencoatedonwell
(hencecompetition)
Thusthehighertheconcentrationofantigeninthesample,the
lowertheabsorbance.

61. Competitive ELISA for Antibody -Principle
Principleofthetestisthattwospecificantibodiesforsame
antigen,oneconjugatedwithenzymeandtheotherpresentin
testserum(unlabeled),areused.
Asamplecontainingunknownantibodies(testserum)isfirst
incubatedwithaknownantigeninasolution(liquidphase);the
two,ifspecific,combinetoformimmunecomplexes.
Theantigen-antibodymixtureisthenaddedtotheELISAplate
whichhaswellspre-coatedwithsameantigen.
Afterwashing,enzymeconjugatedantibodyspecificforantigenis
addedfollowedbycolordevelopment.
Competitionoccursbetweenthetwoantibodiesforthesame
antigen.
Appearanceofcolorindicatesanegativetest(absenceof
antibodies),whiletheabsenceofcolorindicatesapositivetest
(presenceofantibodies);thecolorbeingblocked

62. Competitive/Blocking ELISA

63. Competitive ELISA
Advantages
Highspecificity,sincetwoantibodiesareusedandthe
antigen/analyteisspecificallycapturedanddetected
Suitableforcomplexsamples,sincetheantigendoesnotrequire
purificationpriortomeasurement
Highsensitivitysincetwoantibodiesareused
Flexible,sincebothdirectandindirectdetectionmethodscanbe
used.
Disadvantages
Anextraincubationstepisrequiredintheprocedure.

64. Modifications of ELISA
DotELISA
Avidin-BiotinELISA(AB-ELISA)
MultiplexELISA
ELISPOT/FLUOROSPOTAssay
ChemiluminescenceImmunoassay

65. Dot ELISA
DotELISAisatypeofsolidphasemicroenzyme-linked
immunosorbentassayutilizingantigendottedonto
nitrocellulosefilterdiscs.
Itisarapidimmunochemicaltestwhichisextensivelyusedas
animmunologicaltoolinresearchaswellas
analytical/diagnosticlaboratories
Themostsignificantfeatureofdot-ELISAistheprecipitationof
thechromogenicsubstrateonlyintotheareawiththe
enzymaticactivity,hencethenameDot-ELISA
Theenzymeactivityisindicatedbyintensityofspot,whichis
directlyproportionaltotheantigenconcentration.
DotELISAprotocolsusuallyareofsandwichELISA

66. Dot ELISA
InsandwichDot‐ELISA,theantigenissandwicheddirectly
betweentwoantibodieswhichreactwithtwodifferent
epitopesonthesameantigen.
Herethecapturingantibodyisimmobilizedontoasolid
support,whichisanitrocellulosemembraneandthesecond
(detecting)antibodyislinkedtoanenzyme.
Antigeninthetestsamplefirstreactswiththeimmobilized
antibodyandthenwiththesecondenzyme‐linkedantibody.
Theamountofenzymelinkedantibodyboundisassayedby
incubatingthestripwithanappropriatechromogenicsubstrate,
whichisconvertedtoacoloured,insolubleproduct.

68. Avidin Biotin ELISA (AB-ELISA)
Avidinisaproteinderivedfrombothavians(whitesofeggs)
andamphibiansthatshowsahighaffinityforbiotin.
Biotinisasmallvitaminmolecule(VitaminH,VitaminB7,
CoenzymeR)thatiseasilymodifiedsothatitcanbechemically
attachedtoproteins,antibodiesandotherbiomolecular
probesofinterest.
Streptavidinisanotherproteinsthatoriginatefromdifferent
sourcebutbindverystronglyandspecificallytothebiotin
molecule
Theavidin-biotinaffinitysystemisfrequentlyemployedinthe
designoftagginganddetectingsystemsforELISA
InABsystemtheantibodiesarebiotinylatedand
avidin/streptavidinisconjugatedwithenzymes.
Avidin/streptavidinproteinsaretetramericandhavefour
biotin-bindingsitespermolecule.

69. Avidin, Streptavidin or NeutrAvidin proteins can bind up to four biotin molecules, which
are normally conjugated to an enzyme or antibody to form an Avidin-biotin complex.
† denotes that Avidin is also often conjugated to an antibody, target protein or immobilized support
Avidin-biotin interaction

70. Avidin Biotin ELISA (AB-ELISA)
Theavidin-biotinchemistryresultsinamplificationofsignalinELISA
inindirectdetection.Therearetwoapproaches:
Thefirstmethodinvolvesusingabiotinylateddetectionantibody,whichis
probedusingavidinorstreptavidinproteinconjugatedtoeitherhorseradish
peroxidase(HRP)oralkalinephosphatase(AP)enzymes.
Thesecondapproachalsoemploysabiotinylateddetectionantibody,butitis
probedwithapre-incubatedmixtureofavidinandbiotinylatedenzyme,a
processknownas“avidin-biotincomplex”(ABC)signalamplification.
Signalamplificationoccursthroughtwomechanisms:
First,biotinylationresultsinmultiplebiotintagsperantibodymolecule,thus
allowingmorethanonestreptavidinmoleculetobindtoeachantibody.
Second,theprocessofeitherlabelingthestreptavidinmoleculewithenzymes
orusingapre-incubatedmixtureofstreptavidinplusbiotinylatedenzyme
resultsinconjugateshavingmorethanoneenzyme.
Thecombinedeffectofthismultiplelabelingistoincreasethe
numberofenzymemoleculesinthefinalimmunecomplex.This
increasesthecatalysisofappropriatesubstrateandgivesastronger
signalcomparedtoaconventionalenzyme-labeledsecondary
antibody.

71. Signal Amplification by AB-ELISA

72. Avidin Biotin ELISA (AB-ELISA)
Advantages of using Avidin-biotin systems
TheAvidin-biotincomplexisthestrongestknownnon-covalent
interaction(K
d=10
-15
M)betweenaproteinandligand.
ThebondformationbetweenAvidinandbiotinisveryrapid,
andonceformed,isunaffectedbyextremesofpH,
temperature,organicsolventsandotherdenaturingagents.
ThesefeaturesofbiotinandAvidinareusefulfordetecting
proteinsconjugatedtoeithercomponentoftheinteraction
withoutaffectingtheirstructure
TheAvidin-biotinsystemsamplifytheoriginalproteinsignalto
improvedetectionofproteinsexpressedatlowlevelsby
forminglargeAvidin-biotincomplexesandthusarehighly
sensitive.

73. Multiplex ELISA
AmultiplexELISAenablesaresearchertoassessthelevelsof
morethanoneproteintargetatatime.
Itiscommonlyusedtodeterminetheexpressionofmultiple
cytokinessimultaneously.
ThemultiplexELISAmethodsutilizesoneormoreofthe
followingtechniques:flowcytometry,fluorescence,
chemiluminescence,orelectrochemiluminescence.
Flowcytometricanalysisisthemostcommonlyemployed
multiplexELISAmethodandisperformedusingbead-
conjugatedantibodies.
Typically,agreaternumberofcytokinescanbedetectedand/or
measuredatatimebyusingfluorophores,flowcytometryand
bead-conjugatedantibodies,comparedtoothermethods.
Influorescenceassays,thedetectionantibodyiseitherlabeled
directlyorthesecondaryantibody(oroccasionallyavidin)is
labeledforindirectdetection

74. A multiplex array ELISA using fluorescence

75. ELISPOT
ELISPOTassayisbasedontheenzyme-linkedimmunosorbent
techniquedesignedtoenumeratecytokine-secretingcells
Itisbothaquantitativeandqualitativeassay,isextremely
sensitive(candetect1/300000cytokine-secretingcells-).
Thecytokinereleasedinresponsetoantigencanbemappedto
asinglecellcellshenceTcellresponderfrequenciescanbe
calculated.
Cellscanbestimulatedeitherintheanti-cytokineantibody
coatedplate(directassay)orpre-stimulatedandthen
transferredtothepre-coatedplate(indirectassay);
Oncetheassayiscomplete,theplatescanbeanalyzedona
platereader.
ThemainapplicationoftheELISPOTassayisinmonitoringof
immuneresponsesinbothhumansandanimals

76. ELISPOT
The steps are:
cellsareculturedonasurfacecoatedwithaspecificcapture
antibodyinthepresenceorabsenceofstimuli.
Proteins,suchascytokines,thataresecretedbythecellsare
capturedbythespecificantibodiesonthesurface.
Afteranappropriateincubationtime,cellsareremovedandthe
secretedmoleculeisdetectedusingadetectionantibodyina
similarproceduretothatemployedbytheELISA.
Thedetectionantibodyiseitherbiotinylatedandfollowedbya
streptavidin-enzymeconjugateortheantibodyisdirectly
conjugatedtoanenzyme.
Byusingasubstratewithaprecipitatingratherthanasoluble
product,theendresultisvisiblespotsonthesurface.Eachspot
correspondstoanindividualcytokine-secretingcell.

78. ELISPOT
Morerecently,theassayhasbeenadaptedtoaFluoroSpot
assaywhichutilizesfluorochrome-conjugateddetection
antibodiestherebyallowingthesimultaneousdetectionof
multipledistinctcytokinesandsubsequentTcellsub-
populationanalysis.
ThecytokineELISPOThasbeensuccessfullyusedacrossseveral
disciplinesofImmunology,includingorgan-transplantation,
cancerresearch,infectiousdiseases,vaccinedevelopmentand
autoimmunediseases.
BothELISPOTandFluoroSpotassaysarewidelyusedin
immuno-monitoringofclinicaltrialswherebothquantitative
informationandTcellphenotypeidentificationatasinglecell
levelishighlyinformative,e.ginSARSCoV-2immunology
studies

79. FLUOROSPOT ASSAY

80. Chemiluminescence Immunoassay (CLIA)
CLisdefinedastheemissionofelectromagneticradiationcausedbya
chemicalreactiontoproducelight.
CLIAisanassaythatcombinechemiluminescencetechniquewith
immunochemicalreactions.Itutilizechemicalprobeswhichcould
generatelightemissionthroughchemicalreactiontolabeltheAb
CLIAhashighsensitivity,goodspecificity,widerangeofapplications,
simpleequipmentandwidelinearrange.
CLIAhavethreedifferentlabelsystemsaccordingtothedifferenceof
physicalchemistrymechanismofthelightemission:
Labelchemicaldirectlyinvolvedinthelightemissionreaction-chemicalthatcan
transfertoanexcitedstatethroughchemicalreaction,e.g.acridiniumester
Enzymecatalyzedlightemissionreaction-utilizesenzymestolabelantibody,but
thesubstrateisaluminescentchemicalinsteadofchromogen,e.g.Luminol
insteadofOPDforHRPOenzyme
RedoxReactionMediatedLightEmissionReaction-utilizesrutheniumtris-
bipyridine(bpy)aslabelwhichbyelectrochemicalreactiongeneratesanexcited
stateofRu(bpy)
3
2+

82. CLIA Labels

83. IMMUNOCHROMATOGRAPHY ASSAY (ICA)
OR
LATERAL FLOW ASSAYS (LFA)

84. IMMUNOCHROMATOGRAPHY ASSAY
Immunochromatographyassay(ICA),namelylateralflowtest,isa
simpledeviceintendedtodetectthepresenceorabsenceofthe
targetanalyte(antigenorantibody)
Theconceptofimmune-chromatographyisacombinationof
chromatography(separationofcomponentsofasamplebasedon
differencesintheirmovementthroughasorbent)and
immunochemicalreactions,whichcanberecognizedaccordingto
thechangeofcolors.
Assaysusingthisformatarerapid,takingapproximately15minto
runandarealsosimpletouse,requiringonlythedilutionofthetest
agentinasamplebufferandapplyingseveraldrops(~200µl)tothe
teststrip.
StripsusedforICAcontainfourmaincomponents-sample
applicationpad(celluloseand/orglassfiber),conjugatepad(where
labeledantibodiesaredispensed),substrate(Nitrocellulose)
membrane,andadsorbentpad(worksassinkattheendofthestrip)

85. Typical layout of a lateral flow test strip.

86. Principle of ICA
Typicalhandheldassaydevicescontainacolloidalgold(or
other)labeledantibodydriedontoafilterpadaffixedtoa
nitrocellulosestrip.
Acaptureantibodyisappliedinalineonthestripanddried.
Toperformthetest,aspecimenissuspendedinbufferand
addedtothepadcontainingthecolloidalgoldlabeledantibody.
Theantibodyspecificallybindstoantigenpresentinthe
specimen,andtheresultingcomplexwicksdownthe
membranewhereitbindstothecaptureantibody.
Apositivereactionisvisualizedasaredlinecreatedbythe
boundcolloidalgold
Lateralflowimmunoassaysarechoiceoftestforpoint-of-care
tests(POCT)orfielduseapplications.

87. Steps in ICA
Major steps in ICA are:
Preparationoflabeledantibodyandcaptureantibodyagainst
targetanalyte
Immobilizingthelabeledantibodyontoconjugatepad,andthe
captureantibodyontothestripmembranetoformthetest/
controlline.
Assemblingofallcomponentsontoabackingcardafter
dispensingofreagentsattheirproperpads.
Addsamplesandbufferontosamplepad.
Waitthesampleflowthroughthetestandcontrollinefor5-10
minutes.
Readtheresultwhenthecolorreveal.

89. Types of ICA
ThreeformatsareincommonuseinICA.Theseare:
1.SandwichAssay-Inthisassay,labelcoatedantibodyisimmobilized
atconjugatepad.Acaptureantibodyagainsttargetantigenis
immobilizedovertestline.Asecondaryantibodyagainstlabeled
antibodyisimmobilizedatcontrolzone.
2.CompetitiveAssay–Inthisassay,solutioncontainingtarget
antigenisappliedontothesampleapplicationpad,whichalsohas
prefixedlabeledantibody.Testlinecontainspre-immobilized
antigenwhichbindsspecificallytolabelconjugate.Controlline
containspre-immobilizedsecondaryantibodywhichhastheability
tobindwithlabeledprimaryantibody.Antigeninthesample
solutionandtheonewhichisimmobilizedattestlineofstrip
competetobindwithlabeledconjugate.
3.MultiplexDetectionAssay–Thisassayisusedfordetectionof
morethanonetargetspecies.Itisperformedoverthestrip
containingtestlinesequaltonumberoftargetantigenstobe
analyzed.

90. Types of ICA

91. Advantages and Limitations of ICA
ADVANTAGES
Relative ease of manufacture
Easily scalable to high-volume production.
Stable: shelf-lives of 12-24 months often without refrigeration.
Ease of use: minimal operator-dependent steps and interpretation
Can handle small volumes of multiple sample types
Can be integrated with onboard electronics, reader systems, and
information systems (POCT)
Relatively low cost and short timeline for development and approval
Market presence and acceptance -minimal education required for
users and regulators
LIMITATIONS
Sensitivity issues in some systems
Simultaneous analysis of multiple markers may difficult
Test-to-test reproducibility challenging

92. Applications of ICA