Ag Ab Interactionn II 2009

8/3/2019 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

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Ag Ab Interactionn II 2009