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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
12/21/1312/21/13 22Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 44Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 55Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 66Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 77Prof. Md. Akram, MMCProf. Md. Akram, MMC
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.
12/21/1312/21/13 88Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 99Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 1010Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 1111Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 1212Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 1313Prof. Md. Akram, MMCProf. Md. Akram, MMC
Direct agglutinationDirect agglutinationOccursOccurs whenwhen thetheantigenicantigenicdeterminantdeterminant isisinherentinherent toto thetheparticleparticle itselfitself..particleparticle itselfitself..(naturally)(naturally)
ExampleExample ##11 UsingUsinggroupgroup AA rbcsrbcs totodetectdetect antianti--AA ininserumserum..
12/21/1312/21/13 1414Prof. Md. Akram, MMCProf. Md. Akram, MMC
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.
12/21/1312/21/13 1515Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 1616Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 1818Prof. 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..
12/21/1312/21/13 1919Prof. Md. Akram, MMCProf. Md. Akram, MMC
Latex agglutinationLatex agglutination
LatexLatex particlesparticles cancan bebecoatedcoated withwith Ab,Ab, andand ininthethe presencepresence ofof AgAgcancan formform visiblevisibleaggregatesaggregates..aggregatesaggregates..
12/21/1312/21/13 2020Prof. Md. Akram, MMCProf. Md. Akram, MMC
HemagglutinationHemagglutination
AgglutinationAgglutination ofof rbcsrbcsasas aa resultresult ofof AbAbinteractioninteraction withwithantigenicantigenic determinantsdeterminantsonon rbcsrbcs surfacessurfaces..onon rbcsrbcs surfacessurfaces..
ExampleExample usingusinggroupgroup AA rbcsrbcs toto detectdetectantianti--AA inin serumserum..
12/21/1312/21/13 2121Prof. Md. Akram, MMCProf. Md. Akram, MMC
Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test
Detects antibodies on erythrocytes
+
Patients RBCs Coombs Reagent(Antiglobulin)
12/21/1312/21/13 2222Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 2323Prof. Md. Akram, MMCProf. Md. Akram, MMC
Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests ApplicationsApplications
Detection of antiDetection of anti--Rh AbRh Ab
Autoimmune hemolytic anemiaAutoimmune hemolytic anemia
12/21/1312/21/13 2424Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 2525Prof. Md. Akram, MMCProf. Md. Akram, MMC
12/21/1312/21/13 2626Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 2727Prof. Md. Akram, MMCProf. Md. Akram, MMC
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)
12/21/1312/21/13 2828Prof. Md. Akram, MMCProf. Md. Akram, MMC
Advantages and disadvantages of Advantages and disadvantages of precipitationprecipitation
AdvantagesAdvantages
Fairly sensitiveFairly sensitive
High specificityHigh specificity
DisadvantagesDisadvantages
Time consumingTime consuming
Some costly instruments are requiredSome costly instruments are required
High technical skill requiredHigh technical skill required
12/21/1312/21/13 2929Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
D
i
a
m
e
t
e
r
2
12/21/1312/21/13 3030Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 3131Prof. Md. Akram, MMCProf. Md. Akram, MMC
IImmunoelectrophoresismmunoelectrophoresis MethodMethod
InterpretationInterpretation
QualitativeQualitative
Relative concentrationRelative concentration Relative concentrationRelative concentration
12/21/1312/21/13 3232Prof. Md. Akram, MMCProf. Md. Akram, MMC
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- +
12/21/1312/21/13 3333Prof. Md. Akram, MMCProf. Md. Akram, MMC
Complement fixation test Complement fixation test (CFT)(CFT)
Lattice formation not requiredLattice formation not required
12/21/1312/21/13 3434Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 3535Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 3737Prof. 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)
12/21/1312/21/13 3838Prof. Md. Akram, MMCProf. Md. Akram, MMC
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)
12/21/1312/21/13 3939Prof. Md. Akram, MMCProf. Md. Akram, MMC
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)
12/21/1312/21/13 4040Prof. Md. Akram, MMCProf. Md. Akram, MMC
Complement fixation testComplement fixation test
PosPos NegNeg
12/21/1312/21/13 4141Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
AdvantagesAdvantages
Fairly sensitiveFairly sensitive Fairly sensitiveFairly sensitive
Wide applicationWide application-- can be used for variety of can be used for variety of diseasesdiseases
DisadvantagesDisadvantages
Time consumingTime consuming
Very difficult to standardizeVery difficult to standardize
High technical skill requiredHigh technical skill required
12/21/1312/21/13 4242Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 4343Prof. Md. Akram, MMCProf. Md. Akram, MMC
Radioimmuoassays (RIA)Radioimmuoassays (RIA)EnzymeEnzyme--Linked Immunosorbent Linked Immunosorbent
AssaysAssays (ELISA)(ELISA)AssaysAssays (ELISA)(ELISA)
12/21/1312/21/13 4444Prof. Md. Akram, MMCProf. Md. Akram, MMC
Detection principlesDetection principles
Radiolabelled isotopesRadiolabelled isotopes
125125I, I, 1414C, C, 3232P, P, 3535SS
EnzymesEnzymes EnzymesEnzymes
PeroxydasePeroxydase
ChromophoresChromophores
Fluorogenic probes, fluorescent proteinsFluorogenic probes, fluorescent proteins
12/21/1312/21/13 4545Prof. Md. Akram, MMCProf. Md. Akram, MMC
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)
12/21/1312/21/13 4646Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 4747Prof. Md. Akram, MMCProf. Md. Akram, MMC
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..
12/21/1312/21/13 4848Prof. Md. Akram, MMCProf. Md. Akram, MMC
RIA: radio immuno assay RIA: radio immuno assay
12/21/1312/21/13 4949Prof. Md. Akram, MMCProf. Md. Akram, MMC
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)
12/21/1312/21/13 5151Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 5454Prof. Md. Akram, MMCProf. Md. Akram, MMC
Elisa: Elisa: EnzymeEnzyme--linked immunosorbent assaylinked immunosorbent assay
12/21/1312/21/13 5555Prof. Md. Akram, MMCProf. Md. Akram, MMC
Sandwich ElisaSandwich Elisa
12/21/1312/21/13 5656Prof. Md. Akram, MMCProf. Md. Akram, MMC
ComponentsComponentsEnzymeEnzyme::
AlkalineAlkaline phosphatasephosphatase
HorseHorse radishradish peroxidaseperoxidaseHorseHorse radishradish peroxidaseperoxidase
SubstrateSubstrate ::
HydrogenHydrogen peroxideperoxide
12/21/1312/21/13 5757Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 5858Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 5959Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 6060Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 6161Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 6363Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 6464Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 6565Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 6666Prof. Md. Akram, MMCProf. Md. Akram, MMC
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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12/21/1312/21/13 6767Prof. Md. Akram, MMCProf. Md. Akram, MMC
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
12/21/1312/21/13 6969Prof. Md. Akram, MMCProf. Md. Akram, MMC