Antigen/ Antibody reactions Diagnostic Immunology

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 agglutination

��OccursOccurs whenwhen thetheantigenicantigenicdeterminantdeterminant isisinherentinherent toto thetheparticleparticle itselfitself..particleparticle itselfitself..(naturally)(naturally)

��ExampleExample ##11 –– UsingUsinggroupgroup AA rbc’srbc’s 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 agglutination

��ResultsResults whenwhen inertinertparticlesparticles areare coatedcoatedwithwith solublesoluble AgsAgs whichwhichmaymay reactreact withwith AbAb..ParticlesParticles includeinclude latex,latex,rbc’s,rbc’s, charcoal,charcoal, etcetc..

ExampleExample –– AgAg attachedattached��ExampleExample –– 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 agglutination

��InIn 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..



��InIn 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..



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


HemagglutinationHemagglutination

��AgglutinationAgglutination ofof rbc’srbc’sasas aa resultresult ofof AbAbinteractioninteraction withwithantigenicantigenic determinantsdeterminantsonon rbc’srbc’s surfacessurfaces..onon rbc’srbc’s surfacessurfaces..

��ExampleExample –– usingusinggroupgroup AA rbc’srbc’s toto detectdetectantianti--AA inin serumserum..


Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests

• Incomplete Ab• Direct Coombs Test

– Detects antibodies on erythrocytes

+ �

Patient’s RBCs Coombs Reagent(Antiglobulin)



�� Indirect Coombs TestIndirect Coombs Test

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

+ �Step 1

Patient’s Serum

TargetRBCs

+ �

+ �

Coombs Reagent(Antiglobulin)

Step 2



�� ApplicationsApplications

•• Detection of antiDetection of anti--Rh AbRh Ab

•• Autoimmune hemolytic anemiaAutoimmune hemolytic anemia


Flocculation testsFlocculation tests

�� FlocculationFlocculation teststests forfor AbAb detectiondetectionareare 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 && RPR’sRPR’s..



PrecipitationPrecipitation

�� PrecipitationPrecipitation :: MeansMeans aa depositdeposit ononthethe 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 precipitation

1.1. 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

Dia

met

er2


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

• Qualitative–Rapid

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 for

agglutination/precipitation

• Can detect presence based on ability to deplete

complement fromserum (complement fixation)

• Antigen added to serumwith complement

• If antibodies against antigen present, activates and

depleted 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 rbc’srbc’s 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 rbc’srbc’s.. (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 rbc’srbc’s.. (Hemolysis(Hemolysisindicatesindicates aa negativenegative testtest;; negativenegative forfor thethe unknownunknownAbAb inin thethe patientspatientsserumserum.. TheThe onlyonly thingsthings reactingreactingAbAb inin thethe patientspatientsserumserum.. TheThe onlyonly thingsthings reactingreactingareare thethe knownsknowns..))

•• Ag/Ab/CAg/Ab/C ++ AbAb--coatedcoated rbc’srbc’s == nono hemolysishemolysis(positive)(positive)

•• Ag/CAg/C ++ AbAb--coatedcoated rbc’srbc’s == 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

Patient’sserum

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 wonwonthethe 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

�� “The“The 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 Formats

DirectDirect 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 washedwashedtoto removeremoveanyany excessexcess(Ab(Ab--E)E).. SubstrateSubstrate isisareare washedwashedtoto removeremoveanyany excessexcess(Ab(Ab--E)E).. SubstrateSubstrate isisaddedadded andand colorcolor developmentdevelopment isis observedobserved.. TheTheenzymeenzyme conjugateconjugate bindsbinds ‘directly’‘directly’ 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


ComponentsComponents

EnzymeEnzyme::

••AlkalineAlkaline phosphatasephosphatase

••HorseHorse radishradish peroxidaseperoxidase••HorseHorse 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 inPatient’ssample

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 inPatient’ssample

LabeledAb

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

• Quantitative

SolidPhase

Ag

Immobilized


Competitive RIA/ELISA for AgCompetitive RIA/ELISA for Ag

�� MethodMethod•• DetermineDetermine amountamount

ofof AbAb neededneeded totobindbind toto aa knownknownamountamount ofof labeledlabeledAgAg

+ �

Prior to Test

LabeledAgAg

+ �

Test

+Patient’ssample

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

+Patient’ssample

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 fluorochrome–Fluorescen 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



• 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



• Flow Cytometry cont.– Data displayed

One Parameter Histogram

Gre

en F

luor

esce

nce

Inte

nsity

Two Parameter Histogram

Green Fluorescence Intensity

Num

ber

of C

ells

Unstained cells

FITC-labeled cells

Red Fluorescence Intensity

Gre

en F

luor

esce

nce

Inte

nsity


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


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Antigen/ Antibody reactions Diagnostic Immunology