Ag-Ab Reactions in Diagnosis

Antigen/ Antibody reactions Antigen/ Antibody reactions Diagnostic Immunology Diagnostic Immunology

Professor Md. Professor Md. AkramAkram HossainHossain

MMCMMC

12/21/1312/21/13 11Prof. Md. Akram, MMCProf. Md. Akram, MMC

Types of antigenTypes of antigen-- Antibody reactions in Antibody reactions in vivovivo

1.1. AgglutinationAgglutination

2.2. PrecipitationPrecipitation

3.3. Complement fixationComplement fixation

4.4. NeutralizationNeutralization4.4. NeutralizationNeutralization

5.5. Antibody dependant cell mediated Antibody dependant cell mediated cytotoxicity (ADCC)cytotoxicity (ADCC)

6.6. ImmobilizationImmobilization


Types of antigen antibody Types of antigen antibody reactions used in vitroreactions used in vitro

1.1. AgglutinationAgglutination

2.2. Precipitation Precipitation

3.3. NeutralizationNeutralization

4.4. Complement fixationComplement fixation4.4. Complement fixationComplement fixation

5.5. FluorescentFluorescent--antibody techniqueantibody technique

6.6. ELISAELISA-- Enzyme linked immunosorbent Enzyme linked immunosorbent assayassay

7.7. Radio immunoassayRadio immunoassay

8.8. ImmunochromatographY (ICT)ImmunochromatographY (ICT)12/21/1312/21/13 33Prof. Md. Akram, MMCProf. Md. Akram, MMC

Applications / use in vitroApplications / use in vitro Diagnosis of many diseasesDiagnosis of many diseases

Severity or stage of diseasesSeverity or stage of diseases

Respond to treatment Respond to treatment

EpidemiologyEpidemiology


How antigen How antigen antibody reactions in vitro antibody reactions in vitro helps in Dx?helps in Dx?

InfectiousInfectious diseasedisease ByBy determiningdetermining whetherwhether anan individualindividual hashas developeddeveloped

antibodiesantibodies inin responseresponse toto infectioninfection.. ByBy detectingdetecting antigenantigen ofof aa particularparticular infectiousinfectious agentagent

fromfrom bloodblood oror otherother bodybody fluidsfluids

AutoimmuneAutoimmune diseasediseaseAutoimmuneAutoimmune diseasedisease ByBy detectingdetecting antibodiesantibodies againstagainst particularparticular selfself antigenantigen inin

casecase ofof autoimmuneautoimmune diseasesdiseases

TumorsTumors ByBy detectingdetecting tumortumor markersmarkers..

MetabolicMetabolic diseasesdiseases

PhysiologicalPhysiological conditionsconditions


Which diseases can be diagnosed by Which diseases can be diagnosed by antigenantigen-- antibody reactions?antibody reactions?

Infectious diseasesInfectious diseases BacterialBacterial

ViralViral

ProtozoaProtozoa ProtozoaProtozoa

FungalFungal

ParasiticParasitic

Autoimmune diseasesAutoimmune diseases

TumorsTumors


Other examples of how immunology can be Other examples of how immunology can be used in the diagnostic laboratoryused in the diagnostic laboratory

Occasionally,Occasionally, bacteriologybacteriology andand virusesviruses needneed toto bebeidentifiedidentified fromfrom culturescultures..

PositivePositive culturescultures appliedapplied toto slidesslides cancan bebe examinedexaminedbyby immunofluorescenceimmunofluorescence..

ThisThis isis howhow wewe identifyidentify herpesherpes simplexsimplex virusvirus inintissuetissue cultureculture andand howhow wewe recognizerecognize thethe presencepresenceofof respiratoryrespiratory virusesviruses inin tissuetissue cultureculture..

GonorrhoeaGonorrhoea andand LegionellaLegionella cancan bebe identifiedidentified fromfromisolatedisolated coloniescolonies byby thethe samesame methodmethod

Sometimes,Sometimes, specificspecific antibodiesantibodies cancan helphelp totodeterminedetermine thethe exactexact speciesspecies..


What is the basis of AgWhat is the basis of Ag-- Ab Ab reactions?reactions?

Specificity between antigen and Specificity between antigen and antibody is the basis of diagnosis.antibody is the basis of diagnosis.


What are the limitations?What are the limitations?

CrossCross reactionreaction betweenbetween similarsimilarantigens/antigens/ antibodiesantibodies

TimeTime forfor developmentdevelopment ofof antibodiesantibodies TimeTime forfor developmentdevelopment ofof antibodiesantibodiesagainstagainst anyany infectiousinfectious agentagent

PresencePresence ofof antibodiesantibodies eveneven afteraftercurecure ofof diseasedisease


How antigen How antigen antibody reactions in vitro helps antibody reactions in vitro helps in Dx of infectious disease?in Dx of infectious disease?

ByBy determiningdetermining whetherwhether anan individualindividual hashasdevelopeddeveloped antibodiesantibodies inin responseresponse totoinfectioninfection

IgMIgM antibodiesantibodies areare usuallyusually aa reflectionreflection IgMIgM antibodiesantibodies areare usuallyusually aa reflectionreflectionofof aa recentrecent infectioninfection..

RisingRising levelslevels ofof IgGIgG antibodiesantibodies oftenoftenindicateindicate recentrecent infectioninfection

SometimesSometimes aa veryvery highhigh titretitre ofof antibodyantibodywillwill signalsignal recentrecent infectioninfection


AgglutinationAgglutination TheThe termterm agglutinationagglutination camecame fromfrom gluglu--

whichwhich meansmeans adhesionadhesion..

TheThe actact ofof adhesionadhesion ofof differentdifferent partsparts isisagglutinationagglutination..

WhenWhen anan antibodyantibody reactsreacts withwith aa WhenWhen anan antibodyantibody reactsreacts withwith aamultivalentmultivalent particulateparticulate (insoluble)(insoluble)antigenantigen,, latticelattice formationformation occursoccurs duedue totocrosscross linkinglinking ofof variousvarious antigenantigen particlesparticlesbyby thethe antibodyantibody..


Types of AgglutinationTypes of Agglutination Direct agglutinationDirect agglutination

1.1. Slide Slide Blood grouping, Serotyping of bacteriaBlood grouping, Serotyping of bacteria

2.2. Tube Tube Widal test (Classical)Widal test (Classical)

Indirect or Passive agglutinationIndirect or Passive agglutination1.1. HemagglutinationHemagglutination

2.2. Latex agglutinationLatex agglutination2.2. Latex agglutinationLatex agglutination

3.3. Particle agglutinationParticle agglutination

4.4. CoCo--agglutinationagglutination

Flocculation testsFlocculation tests

Coombs testCoombs test Direct Direct to detect antibody bound to fetal to detect antibody bound to fetal

RBC surfaceRBC surface

Indirect Indirect To detect circulating antibody in To detect circulating antibody in serum in motherserum in mother


Advantages and disadvantages of Advantages and disadvantages of agglutinationagglutination

AdvantagesAdvantages

Most widely usedMost widely used

Very simpleVery simple

No instrument is requiredNo instrument is required

CheapCheap CheapCheap

Fairly sensitiveFairly sensitive

DisadvantagesDisadvantages

Not highly specificNot highly specific

Not highly sensitiveNot highly sensitive


Direct agglutinationDirect agglutinationOccursOccurs whenwhen thetheantigenicantigenicdeterminantdeterminant isisinherentinherent toto thetheparticleparticle itselfitself..particleparticle itselfitself..(naturally)(naturally)

ExampleExample ##11 UsingUsinggroupgroup AA rbcsrbcs totodetectdetect antianti--AA ininserumserum..


Direct agglutination..2Direct agglutination..2

Example # 2 Example # 2 Using Using bacteria (Ag) looking bacteria (Ag) looking for Ab in serum.for Ab in serum.


Indirect or Passive agglutinationIndirect or Passive agglutinationResultsResults whenwhen inertinertparticlesparticles areare coatedcoatedwithwith solublesoluble AgsAgs whichwhichmaymay reactreact withwith AbAb..ParticlesParticles includeinclude latex,latex,rbcs,rbcs, charcoal,charcoal, etcetc..

ExampleExample AgAg attachedattachedExampleExample AgAg attachedattachedtoto latexlatex particleparticle(known)(known) ++ serumserumlookinglooking forfor (unknown)(unknown)AbAb.. IfIf AbAb present,present, youyougetget visiblevisibleagglutinationagglutination..


Passive Passive Agglutination/HemagglutinationAgglutination/Hemagglutination

Definition Definition -- agglutination test done agglutination test done with a soluble antigen coated onto with a soluble antigen coated onto a particlea particle

+

Applications Measurement of antibodies to soluble antigens12/21/1312/21/13 1717Prof. Md. Akram, MMCProf. Md. Akram, MMC

Latex agglutinationLatex agglutinationInIn latexlatex agglutinationagglutinationprocedures,procedures, AgAgmoleculesmolecules cancan bebeboundbound toto thethe surfacesurface ofoflatexlatex beadsbeads..latexlatex beadsbeads..

IfIf AbAb isis presentpresent inin thethetesttest specimen,specimen, thethe AgAgwillwill combinecombine withwith thetheAbAb andand formform visiblevisibleaggregatesaggregates..


Latex agglutinationLatex agglutination

LatexLatex particlesparticles cancan bebecoatedcoated withwith Ab,Ab, andand ininthethe presencepresence ofof AgAgcancan formform visiblevisibleaggregatesaggregates..aggregatesaggregates..


HemagglutinationHemagglutination

AgglutinationAgglutination ofof rbcsrbcsasas aa resultresult ofof AbAbinteractioninteraction withwithantigenicantigenic determinantsdeterminantsonon rbcsrbcs surfacessurfaces..onon rbcsrbcs surfacessurfaces..

ExampleExample usingusinggroupgroup AA rbcsrbcs toto detectdetectantianti--AA inin serumserum..


Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test

Detects antibodies on erythrocytes

+

Patients RBCs Coombs Reagent(Antiglobulin)


Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests Indirect Coombs TestIndirect Coombs Test

Detects antiDetects anti--erythrocyte antibodies in erythrocyte antibodies in serumserum

+ Step 1

Patients Serum

TargetRBCs

+

+

Coombs Reagent(Antiglobulin)

Step 2


Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests ApplicationsApplications

Detection of antiDetection of anti--Rh AbRh Ab

Autoimmune hemolytic anemiaAutoimmune hemolytic anemia


Flocculation testsFlocculation tests FlocculationFlocculation teststests forfor AbAb detectiondetection

areare basedbased onon thethe interactioninteraction ofofsolublesoluble AgAg withwith Ab,Ab, whichwhich resultsresultsinin thethe formationformation ofof aa precipitateprecipitate ofofinin thethe formationformation ofof aa precipitateprecipitate ofoffinefine particlesparticles.. (Ag(Ag consistsconsists ofof lipidlipidtypetype particles)particles)

ExamplesExamples VDRLVDRL && RPRsRPRs..


PrecipitationPrecipitation PrecipitationPrecipitation :: MeansMeans aa depositdeposit onon

thethe earthearth ofof hail,hail, mist,mist, rain,rain, sleet,sleet, ororsnowsnow;; also,also, thethe quantityquantity ofof waterwaterdepositeddeposited..depositeddeposited..

WhenWhen solublesoluble antigensantigens andandantibodiesantibodies areare mixedmixed togethertogether atatoptimumoptimum concentration,concentration, latticelatticeformationformation occursoccurs..


Types of precipitationTypes of precipitation1.1. Precipitation in gelPrecipitation in gel

Single radial immunodiffusionSingle radial immunodiffusion

Double diffusionDouble diffusion

2.2. Precipitation in ElectrophoresisPrecipitation in Electrophoresis2.2. Precipitation in ElectrophoresisPrecipitation in Electrophoresis

Immune electrophoresisImmune electrophoresis

Counter current Immune Counter current Immune electrophoresis (CIE)electrophoresis (CIE)


Advantages and disadvantages of Advantages and disadvantages of precipitationprecipitation


Fairly sensitiveFairly sensitive

High specificityHigh specificity


Time consumingTime consuming

Some costly instruments are requiredSome costly instruments are required

High technical skill requiredHigh technical skill required


Radial Immunodiffusion (Mancini)Radial Immunodiffusion (Mancini)

InterpretationInterpretation

Method Ab in gel Ag in a well

AgAgAgAg

Ab in gel

InterpretationInterpretation Diameter of ring Diameter of ring

is proportional is proportional to the to the concentrationconcentration

QuantitativeQuantitative Ig levelsIg levels

Ag Concentration

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IImmunoelectrophoresismmunoelectrophoresis MethodMethod

Ags are separated by electrophoresisAgs are separated by electrophoresis

Ag-+

Ag

Ab is placed in trough cut in the agar

Interpretation Precipitin arc represent individual antigens

Ag

Ab

Ab


IImmunoelectrophoresismmunoelectrophoresis MethodMethod

InterpretationInterpretation

QualitativeQualitative

Relative concentrationRelative concentration Relative concentrationRelative concentration


Countercurrent electrophoresisCountercurrent electrophoresis MethodMethod

Ag and Ab migrate toward each other by Ag and Ab migrate toward each other by electrophoresiselectrophoresis

Used only when Ag and Ab have opposite Used only when Ag and Ab have opposite chargescharges

QualitativeRapid

Ag Ab- +


Complement fixation test Complement fixation test (CFT)(CFT)

Lattice formation not requiredLattice formation not required


CFTCFT

PrinciplePrinciple:: AntigenAntigen-- antibodyantibody (IgG,(IgG, IgM)IgM)complexcomplex activatesactivates thethe complementcomplement whichwhichcancan lyselyse targettarget (RBC)(RBC)..

ComponentsComponents ofof testtest::

1.1. SensitisedSensitised sheepsheep RBCRBC (Sheep(Sheep RBC+RBC+ AntiAntisheepsheep RBC)RBC)

2.2. ComplementComplement-- (( GunieaGuniea pigpig serum)serum)

3.3. KnownKnown AgAg // knownknown AbAb

MovieMovie


Complement Fixation Reaction

Antibody titer may be too low foragglutination/precipitation

Can detect presence based on ability to depletecomplement from serum (complement fixation)

Antigen added to serum with complement

If antibodies against antigen present, activates anddepleted complement12/21/1312/21/13 3636Prof. Md. Akram, MMCProf. Md. Akram, MMC

StepsSteps ofof CFTCFT::

1.1. HeatHeat inactivateinactivate thethe testtest serumserum (to(todetectdetect presencepresence oror absenceabsence ofof Ab)Ab) totogetget ridrid ofof thethe nativenative complementcomplement.. ((565600

CC forfor 3030 minutes)minutes)

2.2. ThenThen addadd measuredmeasured amountsamounts ofof AgAg2.2. ThenThen addadd measuredmeasured amountsamounts ofof AgAg(known)(known) andand complementcomplement (known),(known), totothethe serumserum (unknown(unknown Ab)Ab)..

3.3. IfIf AbAb specificspecific forfor thethe knownknown AgAg isispresentpresent inin thethe serum,serum, AgAg--AbAb complexescomplexeswillwill formform andand bindbind allall complementcomplement..(reaction(reaction isis invisible)invisible)


StepsSteps IfIf AbAb (unknown)(unknown) specificspecific forfor thethe knownknown AgAg isis

notnot presentpresent inin thethe serum,serum, thenthen thethe knownknown AgAgandand complementcomplement remainremain unboundunbound..

IndicatorIndicator systemsystem:: addadd sheepsheep rbcsrbcs coatedcoated withwithknownknown AbAb specificspecific forfor knownknown AgAg..

ResultsResults:: ResultsResults::

IfIf allall ofof thethe complementcomplement hashas beenbeen fixed,fixed, nonenonewillwill bebe freefree toto lyselyse thethe sheepsheep rbcsrbcs.. (No(Nohemolysis,hemolysis, indicatesindicates aa positivepositive complementcomplementfixationfixation testtest;; positivepositive forfor thethe unknownunknown AbAb ininthethe serum)serum)


InterpretationInterpretation ofof CFTCFT IfIf nono AbAb isis presentpresent inin thethe patientspatients serum,serum, thethe

complementcomplement isis notnot fixedfixed andand isis freefree toto interactinteract inin thetheindicatorindicator systemsystem andand lyselyse thethe rbcsrbcs.. (Hemolysis(Hemolysisindicatesindicates aa negativenegative testtest;; negativenegative forfor thethe unknownunknownAbAb inin thethe patientspatients serumserum.. TheThe onlyonly thingsthings reactingreactingAbAb inin thethe patientspatients serumserum.. TheThe onlyonly thingsthings reactingreactingareare thethe knownsknowns..))

Ag/Ab/CAg/Ab/C ++ AbAb--coatedcoated rbcsrbcs == nono hemolysishemolysis(positive)(positive)

Ag/CAg/C ++ AbAb--coatedcoated rbcsrbcs == hemolysishemolysis(negative)(negative)


Complement fixation testComplement fixation test

PosPos NegNeg


Advantages and disadvantages of CFTAdvantages and disadvantages of CFT

UsesUses

CFT for kalazar, Filaria, Gonoccal CFTCFT for kalazar, Filaria, Gonoccal CFT

CFT for many viral infectionsCFT for many viral infections


Fairly sensitiveFairly sensitive Fairly sensitiveFairly sensitive

Wide applicationWide application-- can be used for variety of can be used for variety of diseasesdiseases


Time consumingTime consuming

Very difficult to standardizeVery difficult to standardize

High technical skill requiredHigh technical skill required


Complement FixationComplement Fixation

Ag mixed with test serum to be assayed Ag mixed with test serum to be assayed for Abfor Ab

Erythrocytes coated with Abs is added Amount of erythrocyte lysis is determined

Methodology

Ag

Patientsserum

Ag No Ag

Ag


Radioimmuoassays (RIA)Radioimmuoassays (RIA)EnzymeEnzyme--Linked Immunosorbent Linked Immunosorbent

AssaysAssays (ELISA)(ELISA)AssaysAssays (ELISA)(ELISA)


Detection principlesDetection principles

Radiolabelled isotopesRadiolabelled isotopes

125125I, I, 1414C, C, 3232P, P, 3535SS

EnzymesEnzymes EnzymesEnzymes

PeroxydasePeroxydase

ChromophoresChromophores

Fluorogenic probes, fluorescent proteinsFluorogenic probes, fluorescent proteins


Nobel Prize WinnersNobel Prize Winners

Rosalyn YalowRosalyn Yalow--discovered radio discovered radio immunoimmuno--assay (RAI) by studying the assay (RAI) by studying the reaction of insulin with reaction of insulin with antibodiesantibodies

Presented to the world in Presented to the world in Presented to the world in Presented to the world in 1959 (Dash 55)1959 (Dash 55)

RIA used in endocinology, RIA used in endocinology, virology (Dash 56)virology (Dash 56)


Rosalyn S. YalowRosalyn S. Yalow AmericanAmerican physicistphysicist whowho wonwon

thethe NobelNobel prizeprize forfordevelopmentdevelopment ofofradioimmunoassaysradioimmunoassays ofof peptidepeptidehormoneshormonesTheThe processprocess mademade itit possiblepossible TheThe processprocess mademade itit possiblepossibletoto detectdetect andand measuremeasure minuteminuteamountsamounts ofof hormones,hormones, drugs,drugs,enzymes,enzymes, andand antibodiesantibodies

TheThe introductionintroduction ofof radioradio--immunoassayimmunoassay isis probablyprobably thethesinglesingle mostmost importantimportantadvanceadvance inin biologicalbiologicalmeasurementmeasurement ofof thethe pastpast twotwodecadesdecades.. ItIt hashas revolutionizedrevolutionizedoneone majormajor disciplinediscipline andandinfluencedinfluenced severalseveral othersothers..


Improved DiagnosticsImproved Diagnostics

RadioimmunoassayRadioimmunoassay:: AA veryvery sensitive,sensitive,specificspecific laboratorylaboratory testtest (assay)(assay) usingusingradiolabeledradiolabeled (and(and unlabeled)unlabeled) substancessubstances ininanan immunologicalimmunological (antibody(antibody--antigen)antigen)anan immunologicalimmunological (antibody(antibody--antigen)antigen)reactionreaction..


RIA: radio immuno assay RIA: radio immuno assay


ELISA FormatsELISA FormatsDirectDirect sandwichsandwich ELISAELISA antibodiesantibodies (Ab)(Ab) arearecoatedcoated toto micromicro wellswells.. AntigenAntigen (Ag)(Ag) isis addedadded andandbindsbinds withwith antibodyantibody.. ExcessExcess antigenantigen isis washedwashed awayaway..EnzymeEnzyme conjugateconjugate (Ab(Ab--E)E) isis addedadded andand bindsbinds withwithantigenantigen toto formform thethe doubledouble antibodyantibody sandwichsandwich.. WellsWellsareare washedwashed toto removeremove anyany excessexcess (Ab(Ab--E)E).. SubstrateSubstrate isisareare washedwashed toto removeremove anyany excessexcess (Ab(Ab--E)E).. SubstrateSubstrate isisaddedadded andand colorcolor developmentdevelopment isis observedobserved.. TheTheenzymeenzyme conjugateconjugate bindsbinds directlydirectly toto thethe antigenantigen..

AbAb AgAg AbAb--EE++ ++12/21/1312/21/13 5050Prof. Md. Akram, MMCProf. Md. Akram, MMC

Types of ELISA

Three different methods used to perform ELISAs

Direct method (different from book)

Indirect method

Capture method (called direct method in book)


Direct ELISA (According to Dr. Nika) Antigen attached to well

Unbound antigen removed by washing

Enzyme conjugated antibody added to well

Unbound antibody washed away

Substrate to enzyme conjugated to antibody added

If antibody bound, substrate is cleaved

Color develops, allows identification of organism

Requires production of conjugated antibody for each bacterial species12/21/1312/21/13 5252Prof. Md. Akram, MMCProf. Md. Akram, MMC

Indirect ELISA Antigen attached to well

Primary antibody added, unbound antibody removed

Enzyme conjugated secondary antibody added, recognizes primary antibody

Unbound secondary antibody removed Unbound secondary antibody removed

Substrate for enzyme conjugated to secondary antibody added

Color develops only if primary antibody bound

More sensitive than direct ELISA, does not require production of numerous conjugated antibodies12/21/1312/21/13 5353Prof. Md. Akram, MMCProf. Md. Akram, MMC

Capture ELISA Antibody attached to well

Sample added to well, antigen captured by antibody

Enzyme conjugated second antibody against antigen added to well - may be against second epitope or same epitope as antibody used to capture antigen

Unbound antibody removed

Substrate added, color develops if antigen present in sample applied to well

Useful for detecting antigens present as very minor species in sample


Elisa: Elisa: EnzymeEnzyme--linked immunosorbent assaylinked immunosorbent assay


Sandwich ElisaSandwich Elisa


ComponentsComponentsEnzymeEnzyme::

AlkalineAlkaline phosphatasephosphatase

HorseHorse radishradish peroxidaseperoxidaseHorseHorse radishradish peroxidaseperoxidase

SubstrateSubstrate ::

HydrogenHydrogen peroxideperoxide


Solid Phase NonSolid Phase Non--Competitive Competitive RIA/ELISARIA/ELISA

AbAb detectiondetection

ImmobilizeImmobilize AgAg

IncubateIncubate withwithsamplesample

AddAdd labeledlabeled antianti-- AgImmobilized

Ab inPatientssample

LabeledAnti-Ig

AddAdd labeledlabeled antianti--IgIg

AmountAmount ofof labeledlabeledAbAb boundbound isisproportionalproportional totoamountamount ofof AbAb ininthethe samplesample Quantitative

SolidPhase

AgImmobilized


Solid Phase NonSolid Phase Non--Competitive Competitive RIA/ELISARIA/ELISA

AgAg detectiondetection

ImmobilizeImmobilize AbAb

IncubateIncubate withwith samplesample

AddAdd labeledlabeled antibodyantibody Ag

Ag inPatientssample

LabeledAb

AmountAmount ofof labeledlabeled AbAbboundbound isis proportionalproportionaltoto thethe amountamount ofof AgAginin thethe samplesample

Quantitative

SolidPhase

AgImmobilized


Competitive RIA/ELISA for AgCompetitive RIA/ELISA for Ag MethodMethod

DetermineDetermine amountamountofof AbAb neededneeded totobindbind toto aa knownknownamountamount ofof labeledlabeledAgAg

+

Prior to Test

LabeledAgAg

+

Test

+Patientssample

LabeledAg

+ Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor


Competitive RIA/ELISA for AgCompetitive RIA/ELISA for Ag Method cont.Method cont.

Determine Determine amount of amount of labeled Ag labeled Ag bound to Abbound to Ab

+

Test

+Patientssample

LabeledAg

+SolidPhase

SolidPhase

Quantitative Most sensitive test

Concentration determined from a standard curve using known amounts of unlabeled Ag


ImmunofluorescenceImmunofluorescence Direct

Ab to tissue Ag is labeled with fluorochromeFluorescen isothiocyanate (FITC), Tetramethy Rhodamine isothiocyanate (TRITC)

Ag

FluorochromeLabeled Ab

Tissue Section12/21/1312/21/13 6262Prof. Md. Akram, MMCProf. Md. Akram, MMC

Direct Immunofluorescent Test Requires production of species specific antibody

Fluorescent group (FITC) directly conjugated to species specific antibody

Bacteria attached to slide, antibody added to bacteria, unbound antibody removed

Bacteria observed at wavelength of light that causes conjugate to fluoresce


Indirect Immunofluorescent Test Primary antibody added to specimen, unbound washed away

Secondary conjugated antibody added, recognizes primary antibody

Unbound secondary antibody removed, specimen observed at wavelength of light that produces fluorescence

More sensitive, secondary antibody amplifies signal

Also more time consuming


ImmunofluorescenceImmunofluorescence

IndirectIndirect Ab to tissue Ag is Ab to tissue Ag is

unlabeledunlabeled

FluorochromeFluorochrome--labeled labeled antianti--Ig is used to Ig is used to

FluorochromeLabeled Anti-Ig

UnlabeledAb

antianti--Ig is used to Ig is used to detect binding of the detect binding of the first Ab.first Ab. Ag

Tissue Section

Ab

Qualitative to Semi-Quantitative


ImmunofluorescenceImmunofluorescence Flow Cytometry

Cells in suspension are labeld with fluorescent tag Direct or Indirect Fluorescence

Cells analyzed on a flow cytometer

FlowTip

Laser

FLDetector

LightScatter

Detector


ImmunofluorescenceImmunofluorescence Flow Cytometry cont.

Data displayed

One Parameter Histogram

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Two Parameter Histogram

Green Fluorescence Intensity

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Unstained cells

FITC-labeled cells

Red Fluorescence Intensity

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use the cellular immune response to use the cellular immune response to diagnose infections?diagnose infections?

SkinSkin testingtesting isis usedused mostmost oftenoften (the(the TBTBskinskin testtest isis thethe mostmost common)common) (Mantoux(Mantouxtest)test)

TBTB antigensantigens areare injectedinjected underunder thethe skinskin ((55 TBTB antigensantigens areare injectedinjected underunder thethe skinskin ((55TU)TU)

OverOver 4848 hours,hours, cellscells migratemigrate towardstowards thetheinjectedinjected antigenantigen

ThisThis producesproduces locallocal swellingswelling (induration)(induration).. TheThediameterdiameter ofof thethe indurationinduration isis measuredmeasured..

IndividualsIndividuals withoutwithout pastpast TBTB havehave nonoindurationinduration12/21/1312/21/13 6868Prof. Md. Akram, MMCProf. Md. Akram, MMC

Documents

Ag-Ab Reactions in Diagnosis