Ag Ab Interactionn II 2009
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Transcript of Ag Ab Interactionn II 2009
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Introduction to serologicIntroduction to serologic
methods (II)methods (II)
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OutlineOutline
Indicator labeled immunoassayIndicator labeled immunoassay
EnzymeEnzyme
FluorescenceFluorescence ChemiluminescenceChemiluminescence
Molecular biological techniquesMolecular biological techniques
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IndicatorIndicator--Labeled ImmunoassaysLabeled Immunoassays
(conjugate)(conjugate) AgAg--AbAb
FluorochromesFluorochromes
EnzymeEnzyme
Radioactive IsotopesRadioactive Isotopes
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HeHe2+2+ ee--
Radioactive Isotopes: Molecules withRadioactive Isotopes: Molecules with
unstable nucleiunstable nuclei
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RadioisotopeRadioisotope
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EnzymeEnzyme--linked Approachlinked Approach
Enzymes are catalysts for chemical reactions.Enzymes are catalysts for chemical reactions. attachmentattachment
Enzyme activity manifest as colored product orEnzyme activity manifest as colored product or
chemiluminescene.chemiluminescene. Enzymes functionEnzymes function indirectlyindirectly as indicator labels.as indicator labels.
Better than Radioactive isotopesBetter than Radioactive isotopeslong halflong half--
life, no need of special precautions forlife, no need of special precautions forhandling.handling.
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Alkaline PhosphataseAlkaline Phosphatase
Instrumentation depend on the substrate selectInstrumentation depend on the substrate select
for the assay.for the assay.
Has wide pH stability , stable for at least 1Has wide pH stability , stable for at least 1
year in 4year in 4
Horseradish peroxidase
Narrow pH optimum , stable for months at4.
Many popular substrates are potentially
Carcinogenic.
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Enzyme immunoassay:EIAEnzyme immunoassay:EIA
AlkalineAlkaline--phosphatase ;ALPphosphatase ;ALP
pp--nitrophenyl phosphate(pNPP)nitrophenyl phosphate(pNPP)405nm405nm
horseradish peroxidase ;HRPhorseradish peroxidase ;HRP
oo--phenylenediamine(OPD)phenylenediamine(OPD)492nm492nm
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ImmunofluorescenceImmunofluorescence
Combines immunologic methods andCombines immunologic methods and
histochemical methodshistochemical methods
Use for detection of antigen & antibodyUse for detection of antigen & antibody Two types of fluorescent antibody techniques:Two types of fluorescent antibody techniques:
------direct methoddirect method
------indirect methodindirect method Very high sensitivity & specificityVery high sensitivity & specificity
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Direct immunofluorescent assayDirect immunofluorescent assay
Labeled Ab + Ag *detect antigenLabeled Ab + Ag *detect antigen
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Indirect immunofluorescent assayIndirect immunofluorescent assay
Ag + unlabeled Ab + labeled antihuman immunoglobulinAg + unlabeled Ab + labeled antihuman immunoglobulin
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DIF & IIFDIF & IIF
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FluorescenceFluorescence
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Absorption & emision spectraAbsorption & emision spectra
of a fluorescent com
poundof a fluorescent com
pound
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Fluorescent labelFluorescent label
fluorochrome, fluorophore, fluorfluorochrome, fluorophore, fluor
In immunofluorescence, the termIn immunofluorescence, the term
fluorochrome is used.fluorochrome is used. When an Ab is labeled with a fluorochrome,itWhen an Ab is labeled with a fluorochrome,it
is called a conjugateis called a conjugate
The most commonly used fluorochromes areThe most commonly used fluorochromes arefluorescein, rhodamine, acridine, andfluorescein, rhodamine, acridine, and
phycoerythrinphycoerythrin
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FluorochromeFluorochrome
FITC(green light):FITC(green light):
Adsorption :490nMAdsorption :490nM
Emition :517nMEmition :517nM
TMRITC(red light):TMRITC(red light):
Adsorption :550nMAdsorption :550nM
Emition :580nMEmition :580nM
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Fluorescence microscopeFluorescence microscope
with transmitted lightwith transmitted light
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Heterogeneous vs ho
mogeneous
Heterogeneous vs ho
mogeneous
heterogeneous:heterogeneous:
e.g. ELISAe.g. ELISA
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Heterogeneous FIAHeterogeneous FIA
::
----------Indirect methodIndirect method
----------Competitive methodCompetitive method----------Sandwich methodSandwich method
----------Avidin biotin methodAvidin biotin method
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EnzymeEnzyme--linked immunosorbentlinked immunosorbent
assay(ELISA)assay(ELISA)
EnzymeEnzyme--linkedlinked
ImmnosorbentImmnosorbent
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Competitive(limitedCompetitive(limited--reagent) vs nonreagent) vs non--
competitivecompetitive
::
//
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LabelLabel
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Sandwich assaySandwich assay
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BiotinBiotin--Avidin systemAvidin system
Avidin(68kD) or streptavidin(60kD)Avidin(68kD) or streptavidin(60kD)
biotinbiotin
StreptavidinStreptavidinStreptomyces avidiniiStreptomyces avidinii,,subunitsubunit,,subunitsubunitbiotinbiotin
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BiotinBiotin--labeled MHCFITC Avidinlabeled MHCFITC Avidin
system to detect antigen specific Tsystem to detect antigen specific T
cellcell
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Homogeneous immunoassayHomogeneous immunoassay
AgAg--Ab reactionAb reaction ,,complexcomplex
AgAg--Ab reactionAb reaction
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EnzymeEnzyme--multiplied immunoassaymultiplied immunoassay
tehcnique (EMIT)tehcnique (EMIT)
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Fluorescence polarizationFluorescence polarization
imm
unoassyimm
unoassy
AgAg--FF
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Fluorescence PolarizationFluorescence Polarization
Immunoassay (FPIA)Immunoassay (FPIA)
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ChemiluminescenceChemiluminescence
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Chemiluminescence (CL)Chemiluminescence (CL)
11~~1010%%
Luminol, Acridinium esters, PeroxyoxalatesLuminol, Acridinium esters, PeroxyoxalatesDioxetanes, Tris(Dioxetanes, Tris(22,,22bipyridyl) ruthenium(bipyridyl) ruthenium())
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LuminolLuminol HH22OO22
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Acridinium estersAcridinium esters
catalystcatalyst
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ChemiluminescenceChemiluminescence
imm
unoassaysimm
unoassays
(())
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Luminescence immunoassay (LIA)Luminescence immunoassay (LIA)
Luminescence enzyme immunoassayLuminescence enzyme immunoassay
Luminescence cofactor immunoassayLuminescence cofactor immunoassay
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SCIENCEIMAGING SYSTEMS
-- Introduction to LAS-3000
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Home workHome work
Indicator labeled antiIndicator labeled anti--human Ig antiobdy ishuman Ig antiobdy is
generally used in clinical diagnosis to detectgenerally used in clinical diagnosis to detect
patients antibody (secondary antibody). Pleasepatients antibody (secondary antibody). Please
describe different approaches to producedescribe different approaches to produce
Rabbit polyclonal antiRabbit polyclonal anti--human IgG specifichuman IgG specific
antibodyantibody
Mouse monoclonal antiMouse monoclonal anti--human IgM specifichuman IgM specificantibodyantibody
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Molecular Blotting TechniquesMolecular Blotting TechniquesSouthern blotSouthern blot
Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)
Restriction fragment lengthRestriction fragment lengthpolymorphism (RFLP)polymorphism (RFLP)
Northern blotNorthern blot
Western blot: HIVWestern blot: HIV
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Molecular Biology ToolsMolecular Biology Tools
Restriction endonucleasesRestriction endonucleases
Restriction nucleases or restrictionRestriction nucleases or restriction
enzymesenzymes
DNA methylated at restriction enzymeDNA methylated at restriction enzyme
sites is protectedsites is protected
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HybridizationHybridization of nucleic acids
is based on the following
complementary.
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Primers and ProbesPrimers and Probes
PrimerPrimer are short strands of nucleotidesare short strands of nucleotides
complementary to a particularcomplementary to a particularDNADNA sequencesequence
and usedand used to initiate DNA replicationto initiate DNA replication
ProbesProbes are used to target a particular sequenceare used to target a particular sequence
of complementaryof complementary RNA or DNARNA or DNA
Indicator labels to detect: Radioactive orIndicator labels to detect: Radioactive orfluorescent labelsfluorescent labels
S th Bl t D t t DNAD t t DNA
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Southern Blot: Detect DNADetect DNA
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Restriction fragment length polymorphismRestriction fragment length polymorphism
(RFLP)(RFLP)
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Detection Genetic Mutations In ClinicalDetection Genetic Mutations In Clinical
LaboratoriesLaboratories
Sickle cell disease: RFLP and Southern BlottingSickle cell disease: RFLP and Southern Blotting
Single point mutationSingle point mutation
in the globulin gene.in the globulin gene.
normal has 3 Mst normal has 3 Mst cleavage sites, mutatedcleavage sites, mutated
has only 2 RFLP has only 2 RFLP
Southern blottingSouthern blotting
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Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)
Denaturetion
90-95
Primer annealing
30-65
Primer extension
60-75
Repeat cycle
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Northern BlotNorthern Blot Detect mRNADetect mRNA
Detect by radiolabeled , singleDetect by radiolabeled , single--strandedstranded
DNA fragmentDNA fragment
Not routinely used in clinical molecularNot routinely used in clinical molecular
diagnosticsdiagnostics
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Developmental Northern blotting. (A-E) Procedure for
Northern blotting. (A) RNA is isolated from varioustissues and is separated by size using gelelectrophoresis. (B) The gel is then placed on a paperwick, which absorbs an ionic solution from a trough.(C) A filter that traps RNA is placed above the gel, andblotting paper is placed above that. Capillary actiondraws the solution through the gel, trapping the RNAon the filter. (D) The filter is incubated withradioactive single-stranded DNA complementary tothe mRNA of interest. (E) After washing off unboundDNA, autoradiography localizes the mRNA in thesamples that contain it. (F) Drawing of adevelopmental Northern blot showing the presence ofPax6 mRNA in the eye, brain, and pancreas of amammalian embryo. (F after Ton et al. 1991.)
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Western BlotWestern Blot
Detect protein (antibody)Detect protein (antibody)
Separated by molecular weightSeparated by molecular weight
Often used to confirm the specificity ofOften used to confirm the specificity ofantibodies detected by enzymeantibodies detected by enzyme--linkedlinkedimmunosorbent assay (ELISA) screenimmunosorbent assay (ELISA) screenproceduresprocedures
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Western blotWestern blot
Band pattern
Interpretation
Lane 1, HIV+ serum
(positive control)Lane 2, HIV- serum
(negative control)
Lane A, Patient A
Lane B, Patient B
Lane C, Patient C
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ELISAELISA Western blotWestern blot
substratesubstrate Water solubleWater soluble Water insolubleWater insoluble
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In Situ HybridizationIn Situ Hybridization
Labeled probes to bind target DNALabeled probes to bind target DNA
in thin tissue section or cytologicin thin tissue section or cytologicsmearssmears
Useful for detecting abundant RNAUseful for detecting abundant RNA
species or viral DNAspecies or viral DNA
In Situ : [];
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Tissue in situ hybridization. The example showsthe pattern of hybridization produced using a 35S-labeled F-myosin heavy chain antisense riboprobeagainst a transverse section of tissue from a 13 dayembryonic mouse. The dark areas represent strong
labeling, notably in the ventricles of the heart.
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MicroarraysMicroarrays
Affymetrix GenechipAffymetrix Genechip
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Figure 2. The Use
of Oligonucleotide
Arrays. mRNA is
extracted fromcells and amplified
through a process
that labels the RNA
for analysis. The
sample is then
applied to an array
and any bound
RNA stained.
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Industry advances in multiplex and parallel analysisIndustry advances in multiplex and parallel analysis
Test tubes Microwell plate MicroarrayMicrosphere-based
liquid array
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Microspheres are dyed to createMicrospheres are dyed to create100 distinct colors100 distinct colors
Each microsphere has spectralEach microsphere has spectraladdress based on red/infraredaddress based on red/infrared
contentcontent Microspheres are suspendableMicrospheres are suspendable
Microspheres are coated withMicrospheres are coated withcapture reagent (oligo orcapture reagent (oligo or
antibody)antibody) Sample is added to microspheresSample is added to microspheres
Analyte is captured toAnalyte is captured tomicrospheresmicrospheres
Fluorescent re orter ta addedFluorescent re orter ta added
MicrospheresMicrospheres
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Luminex systemLuminex system
Assays are read using aAssays are read using acompact microspherecompact microsphereanalyzeranalyzer
Analyzer samples wellAnalyzer samples well
Lasers excite fluorescent dyesLasers excite fluorescent dyes-- red laser for beadred laser for beadclassification and green laserclassification and green laserfor assay resultfor assay result
Multiple readings for eachMultiple readings for eachmicrosphere setmicrosphere set
Software reports results inSoftware reports results inrealreal--timetime
Up to 9600 results read in 1Up to 9600 results read in 1