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AntigenandAntibodyReactions

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1.1.AntigenandAntibodyReactionsbasicsDr.T.V.RaoMDDr.T.V.RaoMD1
2.2.Classificationofantigenantibodyinteractions1.Primaryserologicaltests:(Markertechniques)e.g.
Enzymelinkedimmuonosorbentassay(ELISA)Immunoflorescentantibodytechnique(IFAT)Radio
immunoassay(RIA)2.Secondaryserologicaltests:e.g.AgglutinationtestsComplementfixationtests
(CFT)PrecipitationtestsSerumneutralizationtests(SNT)Toxinantitoxintest3.Tertiaryserological
test:e.g.Determinationoftheprotectivevalueofanantiseruminananimal.Dr.T.V.RaoMD2
3.3.TypesofAntigenandAntibodyreactions1.Agglutinationtests2.Doublediffusionprecipitationtests3.
Immunoelectrophoresis4.Westernblottests5.Complementfixationtests6.Immunofluorescencetesting7.
ImmunoassaysDr.T.V.RaoMD3
4.4.SerologicalTestsAntigenandantibodyreactionsinvitroareknownasserologicaltestsWhatHappens
canbestudiedin3stages1stAntigenandantibodyreactwithvisibleeffects,obeysthelawsofphysical
dynamics.ButreversibleThereactioniseffectedbyweakerintermolecularforcesVanderWaalsforces
Ionicbonds,HydrogenbondingnotbycovalentbondingCanbedetectedbyRadioactiveisotopes,
fluorescentdyesDr.T.V.RaoMD4
5.5.WhathappensinAntigenandAntibodyreactionsReactwitheachotherinaobservablemanner.Uses
1Helpsantibodymediatedimmunityininfection,andtissueinjury2HelpsdiagnosisofInfections.3In
epidemiologicalsurveys4DetectionsandquantizationofantigensandantibodiesDr.T.V.RaoMD5
6.6.Detectablereactions2ndStageReactioncanoccuras1Precipitation2Agglutination3Lysisand
killingofliveantigens4Neutralizatiobnoftoxins5Fixationofcomplement6Immobilizationofmotile
microbes7EnhancementofPhagocytosis.Dr.T.V.RaoMD6
7.7.GeneralfeaturesofAntigenandantibodyreactions.Specificreactioncombineswithspecific
antigenEntiremoleculereactsnotfragmentsNodenaturationofantigenorantibodyCombination
occursassurfaceantigenstosurfaceofantibodiesComminationisfirmbutreversibledependson
affinityandavidityBothantigensandantibodiesparticipateCombineinvaryingproportionsBivalent
andmultivalentDr.T.V.RaoMD7
8.8.TermsusedinevaluatingtestMethodologySensitivityAnalyticalSensitivityabilityofatestto
detectverysmallamountsofasubstanceClinicalSensitivityabilityoftesttogivepositiveresultif
patienthasthedisease(nofalsenegativeresults)Dr.T.V.RaoMD8
9.9.SpecificityAnalyticalSpecificityabilityoftesttodetectsubstancewithoutinterferencefromcross
reactingsubstancesClinicalSpecificityabilityoftesttogivenegativeresultifpatientdoesnothave
disease(nofalsepositiveresults)Dr.T.V.RaoMD9
10.10.AffinityAntibodyaffinityisthestrengthofthereactionbetweenasingleantigenicdeterminantanda
singlecombiningsiteontheantibody.Itisthesumoftheattractiveandrepulsiveforcesoperating
betweentheantigenicdeterminantandthecombiningsite.Dr.T.V.RaoMD10
11.11.AvidityAvidityisameasureoftheoverallstrengthofbindingofanantigenwithmanyantigenic
determinantsandmultivalentantibodiesAvidityisinfluencedbyboththevalenceoftheantibodyand
thevalenceoftheantigen.Avidityismorethanthesumoftheindividualaffinities.Dr.T.V.RaoMD11
12.12.Dr.T.V.RaoMD12
13.13.DilutionEstimatingtheantibodybydeterminingthegreatestdegreetowhichtheserummaybe
dilutedwithoutlosingthepowertogivenanobservableeffectinamixturewithspecificantigen
Dr.T.V.RaoMD13
14.14.MeasurementofAntigenandAntibodyreactionsMeasuredasMassNitrogen(microgram)As
UnitsAstiterDr.T.V.RaoMD14
15.15.TiterDifferentdilutionsofserumaretestedinmixturewithaconstantamountofantigenand
greatestreactingdilutionistakenasthemeasureorTiterDr.T.V.RaoMD15
16.16.CommonmethodsincreatingdilutionsDr.T.V.RaoMD16
17.17.ExpressionofTitersExpressedintermofthewasinwhichtheyaremadeDilution1in8isa
dilutionmadebymixingonevolumeofserumwithsevenvolumesofdiluents(NormalSaline)Incorrect
toexpressdilutionas1/8Dr.T.V.RaoMD17
18.18.MajorityDiagnostictestsareSerologicaltestsThereareseveralserologytechniquesthatcanbe
useddependingontheantibodiesbeingstudied.Theseinclude:ELISA,agglutination,precipitation,
complementfixation,andfluorescentDr.T.V.RaoMDantibodies.
19.19.AntigensxAntibodyDr.T.V.RaoMD19
20.20.HowAntigenAntibodyreactionoccursPrimarystageInteractionbetweenantigenandantibody
occurswithoutanyvisibleeffectsThereactionisrapidevenatlowtemperaturebutthereactionis
reversibleWeaker_intermolecularforcesVanderwallsforceandionicbondsCanbemeasuredwith
radioactiveisotopesandFlorescentdyes.Dr.T.V.RaoMD20
21.21.AgAbinteractionsBonds:HydrogenIonicHydrophobicinteractionsVanderWaalsforces
EachbondisweakmanyarestrongToholdtheymustbeclose requiringhighamtsof
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complementarity!Dr.T.V.RaoMD21
22.22.NatureofAg/AbReactionsFourtypesofnoncovalentforcesoperatesoveraveryshortdistance
Dr.T.V.RaoMD22(generally1angstrom)
23.23.Whathappenfuther?1Precipitation2Lysisofcells.3Killingofliveantigen4Neutralizationof
toxins5Complementfixation6Immobilizationofmotilemicrobes7EnhancementofPhagocytosis8
Entiremoleculereactnofragmented9Nodenaturation10Combinationoccursonsurface11Firmbut
reversibleDr.T.V.RaoMD23
24.24.KarlLandsteinar(18681943)AnAustrianphysicianbytraining,Landsteinerplayedanintegralpart
intheidentificationofbloodgroups.Hedemonstratedthecatastrophiceffectoftransfusingwiththe
wrongtypeofblood,Dr.T.V.RaoMD24
25.25.SerologyThebranchoflaboratorymedicinethatstudiesbloodserumforevidenceofinfectionand
otherparametersbyevaluatingantigenantibodyreactionsinvitroDr.T.V.RaoMD25
26.26.SerologySerologyisthescientificstudyofbloodserum.Inpractice,thetermusuallyreferstothe
diagnosticidentificationofantibodiesintheserumWecandetectantigenstooDr.T.V.RaoMD26
27.27.Typesofserologicaltests1.Agglutinationtests2.Doublediffusionprecipitationtests3.
Immunoelectrophoresis4.Westernblottests5.Complementfixationtests6.Immunofluorescencetesting7.
ImmunoassaysDr.T.V.RaoMD27
28.28.Immunology/Serology?PrecipitationReactionsCapillarytubeprecipitation(RingTest)
OuchterlonyDoubleDiffusion(Immunodiffusion)Radialimmunodiffusion(RID)
Immunoelectrophoresis(IEP)RocketElectroimmunodiffusion(EID)Counterimmunoelectrophoresis
(CIEP)Dr.T.V.RaoMD
29.29.AntigenAntibodyreactionspresentingwithprecipitationDr.T.V.RaoMD29
30.30.PrecipitationReactionInprecipitationantigencombineswithitsantibodyinthepresenceof
electrolytes(Nacl)atasuitabletemperatureandPhtheantigenandantibodycomplexesformainsoluble
precipitatesuspendedasfloccules.Reactioncantakeplaceinliquidmedium,gels,agar,agarose,
polyacrylamideDr.T.V.RaoMD30
31.31.PrecipitationPrincipleSolubleantigen+antibody(inproperproportions)>visibleprecipitate
Latticeformation(antigenbindswithFabsitesof2antibodies)ExamplesDoublediffusion
(Ouchterlony)Singlediffusion(radialImmunodiffusion)ImunoelectrphoresisImmunofixation
Dr.T.V.RaoMD31
32.32.HowprecipitationoccursPrecipitationoccurswithmostantigensbecausetheantigenismultivalent
(i.e.hasseveralantigenicdeterminantspermoleculetowhichantibodiescanbind).Antibodieshaveat
leasttwoantigenbindingsites(andinthecaseofIgMthereisamultimericcomplexwithupto10
antigenbindingsites),thuslargeaggregatesorgellikelatticesofantigenandantibodyareformed
SpecificreactionAntigencombineswithhomologusantibodyviceversaDr.T.V.RaoMD32
33.33.MechanismofPrecepitationLatticehypothesisMultivalentantigenscombinewithbivalent
antibodiesinvaryingproportionsdependingontheantigenandantibodyratiointhereactingmixture
Dr.T.V.RaoMD33
34.34.PrecipitationReactions(noprecipitateisformed(LatticesorifanAgcontainsonlyalargeaggregates
)singlecopyofeachepitope)FIGURE64PrecipitationreactionsinDr.T.V.RaoMDfluidsyielda
precipitincurve.34
35.35.ApplicationsofprecipitationreactionsQualitativeandquantitativeMoreusefulforantigen
detectiontoasleastas1microgramBlood,seminalstains,FoodadulterationDr.T.V.RaoMD35
36.36.SlidetestFlocculationtestVDRLtestVDRLTestadropofVDRLantigentoadropofpatients
serum,ShakeThereactionobservedundermicroscopeObserveforflocculationreactionAKhan
testisdoneinatesttubeDr.T.V.RaoMD36
37.37.NonreactiveandReactiveVDRLTestsDr.T.V.RaoMD37
38.38.Agglutination+:veRPRAgglutination:Dr.T.V.RaoMD38
39.39.TubetestPrecipitationKahntestforSyphilisatubeflocculationtest.Quantitativetube
flocculationtestusedinstandardarisationoftoxin/toxoidSerumdilutionoftoxinortoxoidisaddedto
tubescontainingafixedquantityofantitoxinTheamountoftoxinthatflocculatesoptimallywithone
unitofantitoxinisdefinedasLfdoseDr.T.V.RaoMD39
40.40.SinglediffusioninoneDirectionOOudinProcedureDr.T.V.RaoMD40
41.41.RingTestThereactionisdemonstratedbylayeringantigensolutionoverthecolumnofantigenina
narrowtube,Theprecipitateformsatthejunctionoftwoliquids.EgAscolisthermoprecipitatetest
GroupingofstreptococciCreactiveproteinDr.T.V.RaoMD41
42.42.OudinProcedureAntibodiesinagargelAboveantigenislayeredSinglediffusioninonedirection
CalledQudinprocedureAntibodyisincorporatedinagarAntigendiffusesdownPrecipitation
concentrationofantigenatthesiteincreasesduetodiffusionDr.T.V.RaoMD42
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43.43.Dr.T.V.RaoMD43
44.44.DoublediffusioninonedimensionOakleyFulthropeprocedureAntibodiesingelAgarlayer
AntigenlayeredAntigensandantibodiesmovestowardseachotherFormsprecipitateDr.T.V.RaoMD
44
45.45.SingleDiffusionIntwoDimensionsRadialImmunodiffusionRadialImmunodiffusionisan
Immunodiffusiontechniqueusedinimmunologytodetectquantityofantigenbymeasuringtheradius
surroundingsamplesoftheantigen,markingtheboundarybetweenitandantibodyDr.T.V.RaoMD45
46.46.SingleDiffusionIntwoDimensionsRadialImmunodiffusionAntigenisaddedtothewellscuton
thesurfaceofthegelItdiffusesradiallyfromwellandformsaringshapedbandofprecipitationThe
haloofprecipitationdiametergivestheestimateofconcentrationofantigenUsedinestimationof46
Dr.T.V.RaoMDImmunoglobulins
47.47.PrecipitationinAgarThesetestsaredoneinagarTestedforpassiveimmunodiffusioninAgarose
ThereareseveralmethodsintestingthisprocedureDr.T.V.RaoMD47
48.48.GelelectrophoresisallowstoseparatepiecesofDNA.Dr.T.V.RaoMD48
49.49.EleksImmunodiffusionTestElekImmunodiffusiontest.Sterilefilterpaperimpregnatedwith
diphtheriaantitoxinisimbeddedinagarculturemedium.IsolatesofCdiphtheriaarethenstreakedacross
theplateatanangleof90totheantitoxinstrip.ToxigenicCdiphtheriaisdetectedbecausesecreted
toxindiffusesfromtheareaofgrowthandreactswithantitoxintoformlinesofprecipitinDr.T.V.RaoMD
49
50.50.ImmunoDiffusionTestCandemonstratetheimmunologicalidentity(ornot)oftwoantigensamples
radialImmunodiffusionDr.T.V.RaoMD50
51.51.RadialImmunodiffusionRadialImmunodiffusion,avariationoftheagarprecipitationtechnique,is
usedinclinicalimmunologyforthedetectionandquantitationofallclassesofImmunoglobulins,
complement,ceroplastic,transferring,andotherserumcomponentsDr.T.V.RaoMD51
52.52.DoubleDiffusioninoneDimensionOakleyFulthropeprocedureInDoublediffusioninone
directionantibodyinagargelmovesthroughthelayerofplainagartotheantigenaboveTheyreactto
eachotherFormabandofprecipitationVirusantigenisplacedinthecentralwellanddiffuses
outwards.WellsAandCcontainpositivesera,wellBcontainsanegativesample.Theblackareasshow
whereantibodyinthepositiveserahaveboundtovirusantigenandformedaprecipitateDr.T.V.RaoMD
52
53.53.DoublediffusionintwodimensionsOuchterlonyprocedureBothantigenandantibodydiffuses
independentlythroughagargelintwodimensionshorizontallyandverticallyDoneinaslideor
petridishIn1248hourslineofprecipitatesareformedUsefulinserologicalidentificationDr.T.V.Rao
MD53
54.54.Immunoelectrophoresis:InImmunoelectrophoresis,acomplexmixtureofantigensisplacedinawell
punchedoutofanagargelandtheantigensareelectrophoresedsothattheantigenareseparated
accordingtotheircharge.Afterelectrophoresis,atroughiscutinthegelandantibodiesareadded.Asthe
antibodiesdiffuseintotheagar,precipitinlinesareproducedintheequivalencezonewhenan
antigen/antibodyreactionoccurs.Dr.T.V.RaoMD54
55.55.ImmunoelectrophoresisMixturescontainingmultipleantigenspecieswhichcrossreactwiththe
sameantiserummaybeanalysedbyrunningthemfirstonananalyticalgel,thencuttingastripfromthat
gelandlayingitinaslitcutintotheimmunoelectrophoresisgeltoformalargewell.Theresultisa
patternwhichshowsthepositionsofstronglyreactingantigenspecies.Dr.T.V.RaoMD55
56.56.ImmunoelectrophoresisImmunoelectrophoresismigrationofmoleculesduetoelectriccharge
PositiveparticlestraveltocathodeNegativeparticlestravelanodePrecipitinspecificityprovidesacritical
indicatorofidentityDr.T.V.RaoMD56
57.57.ImmunoElectrophoresisInthisproceduretheelectrophoreticseparationofcompatibleantigeninto
constituentproteinfollowedbyImmunodiffusionagainstitsantiserumresultinginseparateprecipitation
line,indicatingrelationbetweeneachindividualproteinintheantibodyDr.T.V.RaoMD57
58.58.ElectricalgelImmunoelectrophoresisThereactioniselectricallydriven,differentantigensare
separatedaccordingtotheirchargesunderelectricalfieldsNumberofantigenscanbeidentified
Dr.T.V.RaoMD58
59.59.CountercurrentelectrophoresisMethodAgandAbmigratetowardeachotherbyelectrophoresis
UsedonlywhenAgandAbhaveoppositecharges+AgAbQualitativeRapidDr.T.V.RaoMD59
60.60.CountercurrentImmunoelectrophoresisInthisproceduremovementofantigenstowardstheanode
andantibodytowardsthecathodethroughagarunderelectricfieldUsefulstudiesonCSFHepatitisB
surfaceantigendetectionDetectionoffetoproteinsDr.T.V.RaoMD60
61.61.Imunoelectrphoresis(IEP)QualitativeAserumsampleiselectrophoresedthroughanagarmedium.
AtroughiscutintheagarandfilledwithAb.Aprecipitinarcisthenformed.BecauseAgdiffuses
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radiallyandAbfromatroughdiffuses,thereactantsmeetinoptimalproportionsforprecipitation.
Dr.T.V.RaoMD61
62.62.RocketelectrophoresisLaurelThereactionsappearasrocketDrivenbyelectriccurrentRocket
ImmunoElectrophoresisisusedasarapidwaytoquantitateantigenincomplexsamples.Dr.T.V.Rao
MD62
63.63.ImmunoelectrophoresisgelMixturescontainingmultipleantigenspecieswhichcrossreactwiththe
sameantiserummaybeanalyzedbyrunningthemfirstonananalyticalgel,thencuttingastripfromthat
gelandlayingitinaslitcutintotheimmunoelectrophoresisgeltoformalargewell.Theresultisa
patternwhichshowsthepositionsofstronglyreactingantigenspecies.Dr.T.V.RaoMD63
64.64.CounterimmunoelectrophoresisDr.T.V.RaoMD64
65.65.ElectroImmunodiffusionDr.T.V.RaoMD65
66.66.Dr.T.V.RaoMD66
67.67.RocketelectrophoresisCrossedImmunoelectrophoresisofantigensandantiserum.Inthefirst
dimension,proteinsareseparatedbystandardelectrophoresis.Theseparatedproteinsarethenrunintothe
seconddimensiongelatanangleof90fromthefirstdimension.Dr.T.V.RaoMD67
68.68.SerologycanbedoneonvariousspecimensSomeserologicaltestsarenotlimitedtobloodserum,
butcanalsobeperformedonotherbodilyfluidssuchassemenandsaliva,whichhave(roughly)similar
propertiestoserum.Serologicaltestsmayalsobeusedforensically,generallytolinkaperpetratortoa
pieceofevidence(e.g.,linkingarapisttoasemensample).Dr.T.V.RaoMD68
69.69.MeasurementofPrecipitationbyLightAntigenantibodycomplexes,whenformedatahighrate,
willprecipitateoutofasolutionresultinginaturbidorcloudyappearance.Turbidimetrymeasuresthe
turbidityorcloudinessofasolutionbymeasuringamountoflightdirectlypassingthroughasolution.
Nephelometryindirectmeasurement,measuresamountoflightscatteredbytheantigenantibody
complexes.Dr.T.V.RaoMD69
70.70.CrossReactivityTheabilityofanindividualAbcombiningsitetoreactwithmorethanoneantigenic
determinant.TheabilityofapopulationofAbmoleculestoreactwithmorethanoneAgCrossreactions
AntiAAntiAAntiAAbAbAbAgAAgBAgCSharedepitopeDr.T.V.RaoMDSimilarepitope70
71.71.ForArticlesofInterestonmicrobiologyfollowmeon..Dr.T.V.RaoMD71
72.72.ProgrammecreatedbyDr.T.V.RaoMDforMedicalandParamedicalstudentsintheDevelopingWorld
Emaildoctortvrao@gmail.comDr.T.V.RaoMD72
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SerologySerologyisthescientificstudyofbloodserum.Inpractice,theter...

Typesofserologicaltests1.Agglutinationtests2.Doublediffusionprecipitationtests3.

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Immunoelectrophoresis4....

Immunology/Serology?PrecipitationReactionsCapillarytubeprecipitation(RingTest)OuchterlonyDouble
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Diffusion...

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AntigenAntibodyreactionspresentingwithprecipitationDr.T.V.RaoMD29

PrecipitationReactionInprecipitationantigencombineswithitsantibodyinthepresenceofelectrolytes(

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Nacl)a...

PrecipitationPrincipleSolubleantigen+antibody(inproperproportions)>visibleprecipitateLattice
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format...

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HowprecipitationoccursPrecipitationoccurswithmostantigensbecausetheantigenismultivalent(i.e.has

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MechanismofPrecepitationLatticehypothesisMultivalentantigenscombinewithbivalentantibodiesin

varyingprop...

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PrecipitationReactions(noprecipitateisformed(LatticesorifanAgcontainsonlyalargeaggre...

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ApplicationsofprecipitationreactionsQualitativeandquantitativeMoreusefulforantigendetectionto...

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SlidetestFlocculationtestVDRLtestVDRLTestadropofVDRLan...

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NonreactiveandReactiveVDRLTestsDr.T.V.RaoMD37

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Agglutination+:veRPRAgglutination:Dr.T.V.RaoMD38

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usedin...

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SinglediffusioninoneDirectionOOudinProcedureDr.T.V.RaoMD40

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tube,The...

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Qudi...

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Dr.T.V.RaoMD43

DoublediffusioninonedimensionOakleyFulthropeprocedureAntibodiesingelAgarlayerAntigen

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layeredAntigen...

SingleDiffusionIntwoDimensionsRadialImmunodiffusionRadialImmunodiffusionisanImmunodiffusion
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techniqu...

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SingleDiffusionIntwoDimensionsRadialImmunodiffusionAntigenisaddedtothe...

PrecipitationinAgarThesetestsaredoneinagarTestedforpassiveimmunodiffusioninAgaroseThereare

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several...

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GelelectrophoresisallowstoseparatepiecesofDNA.Dr.T.V.RaoMD48

EleksImmunodiffusionTestElekImmunodiffusiontest.Sterilefilterpaperimpregnatedwithdiphtheria

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antitoxinis...

ImmunoDiffusionTestCandemonstratetheimmunologicalidentity(ornot)oftwoantigensamplesradial
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Immunodiff...

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RadialImmunodiffusionRadialImmunodiffusion,avariationoftheagarprecipitationtechnique,isusedin

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DoubleDiffusioninoneDimensionOakleyFulthropeprocedureInDoublediffusioninone...

DoublediffusionintwodimensionsOuchterlonyprocedureBothantigenandantibodydiffusesindependently

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through...

Immunoelectrophoresis:InImmunoelectrophoresis,acomplexmixtureofantigensisplacedinawellpunched
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outofanagarg...

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ImmunoelectrophoresisMixturescontainingmultipleantigenspecieswhichcrossreactwiththesame

antiserummaybean...

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ImmunoelectrophoresisImmunoelectrophoresismigrationofmoleculesduetoelectricchargePositive

particlestrave...

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ImmunoElectrophoresisInthisproceduretheelectrophoreticseparationofcompatibleantigenintoconstituent

protein...

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ElectricalgelImmunoelectrophoresisThereactioniselectricallydriven,differentantigensareseparated

acco...

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CountercurrentelectrophoresisMethodAgandAbmigratetowardeachotherbyelectrophoresisUsed

onlywhenAga...

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CountercurrentImmunoelectrophoresisInthisproceduremovementofantigens...

Imunoelectrphoresis(IEP)QualitativeAserumsampleiselectrophoresedthroughanagarmedium.Atrough

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is...

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RocketelectrophoresisLaurelThereactionsappearasrocketDrivenbyelectriccurrentRocketImmuno...

ImmunoelectrophoresisgelMixturescontainingmultipleantigenspecieswhichcrossreactwiththesame

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antiserummayb...

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CounterimmunoelectrophoresisDr.T.V.RaoMD64

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ElectroImmunodiffusionDr.T.V.RaoMD65

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Dr.T.V.RaoMD66

RocketelectrophoresisCrossedImmunoelectrophoresisofantigensandantiserum.Inthefirstdimension,

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proteinsare...

SerologycanbedoneonvariousspecimensSomeserologicaltestsarenotlimitedtobloodserum,butcanalso
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b...

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MeasurementofPrecipitationbyLightAntigenantibodycomplexes,whenformedatahighrate,will

precipitate...

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CrossReactivityTheabilityofanindividualAbcombiningsitetoreactwithmorethanoneantigenic

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ForArticlesofInterestonmicrobiologyfollowmeon..Dr.T.V.RaoMD71

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UpcomingSlideShare

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