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(93"#3"       ` #32  A  .3  0   Tertiaryphenomenon#3M݌L)'+ % % Ќ   )F  0  0%%0` %%0 ` %` %)F 1  )0 % %  Antigen/antibodyreactionisnotvisiblebutisdetectedbytheaffectthe $+)- reactionhasontissuesorcells.)F| ی,*.%% Ќ   -h,0 > >% >  _)F  0  0%%0` %%0 ` %` %)F  2  )0 % %  Thesetypesofreactionsinclude:inflammation,phagocytosis,deposition  ofimmunecomplexes,immuneadherence,depositionofimmune l complexes,andchemotaxis.)F  یX%% Ќ  0  0%%0` %%0 ` %` %0 % %0h%%0h%h% %%  )  0  0%% )! 3  .0` %%  Thesecondaryphenomenaisamethodofchoiceformanyserologicalteststodetectin 0 vitropresenceofantigenorantibody. )!! ی` %` % Ќ  +  0  0%%0` %%+#a  .0 ` %` %  Precipitation involvescombinationof soluble antibodywith soluble antigento t produceinsolublecomplexes.+#m#ی` % % Ќ  +  0  0%%0` %%+$b  .0 ` %` %   Agglutination istheprocessbywhichparticulateantigensuchascellsare  8  aggregatedtoformlarge,visibleaggregatesifthespecificantibodyispresent.+$%ی $  % % Ќ  +  0  0%%0` %%+n&c  .0 ` %` %   Complementfixation 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Antigensandantibodieswiththeperfectlockandkeyfitwillhavethestrongest l! affinity.+1R1ی X" % % Ќ  0  0%%0` %% ` %` %  )  0  0%% )2 3  .0` %%  Avidity isthesumofallattractiveforcesbetweenanantigenandanantibody. )22 ی"0!$` %` % Ќ  +  0  0%%0` %%+4a  .0 ` %` %  Itistheforcethatstabilizestheantigenantibodyreaction,keepingthemolecules $#& together.+4^4یt%#' % % Ќ  +  0  0%%0` %%+y5b  .0 ` %` %  Thestrongerthechemicalbondswhichformbetweentheantigenandantibody, L'%) thelesslikelythatthereactionwillreverse.+y55ی8(&* % % Ќ   )  0  0%% )7 4  .0` %%   LawofMassAction governsthereversibilityoftheantigenantibodyreaction. )7<7 ی*(,` %` % Ќ  +  0  0%%0` %%+Z8a  .0 ` %` %  Freereactantsareinequilibriumwithboundreactants.+Z88ی*|)- % % Ќ  0  0%%0` %% ` %` % +  0  0%%0` %%+9b  .0 ` %` %  Theequilibriumconstantrepresentstheratesofantigen/antibodybindingand ,T+/ dissociating.+9":ی-@,0 % % Ќ   .,-1 +  0  0%%0` %%+N;c  .0 ` %` %  Measuresthe goodnessoffitofthereaction,astheavidityincreasestherateof  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methodemploysmeasurementstakenbeforethezoneofequivalenceis %$' reached.+Cیp&$( % % Ќ  0  0%% %%  )  0  0%% ) 6  .0` %%  Technicalsourcesoferror: )҃ یH(&*` %` % Ќ  +  0  0%%0` %%+a  .0 ` %` %  Overfillingorunderfillingthewell.+ی *(, % % Ќ  +  0  0%%0` %%+υb  .0 ` %` %  Spillingsampleoutsideofthewell.+υی +)- % % Ќ  +  0  0%%0` %%+c  .0 ` %` %  Nickingthewell.+9ی+x*. % % Ќ  +  0  0%%0` %%+d  .0 ` %` %  Improperincubationtimeortemperature.+Aی,d+/ % % Ќ  0  0%%0` %%.<-1` %` % *  0  *eD  .0%%  OuchterlonyGelDiffusion(doublediffusion/doubledimension)*eی%% Ќ  0   %% 0  0%%1.0` %%OuchterlonyImmunodiffusionisamethodusedforcomparisonofantigens.X` %` % 0  0%% %%   )  0  0%% ) 2  .0` %%  Holesarecutintheagar,onecentralholesurroundedbyotherwells. ) ی0` %` % Ќ   0  0%%3.0` %%Antibodyisaddedtothecentralwell,antigensareaddedtotheouterwells,theposition  ofthebandsformedbetweentheantigensallowsforcomparisonoftheantigenstoeach t other.`` %` % 0  0%%4.0` %%Threepossibilities: 8 ` %` % +   0  0%%0` %%+a  .0 ` %` %  Identitythebandsformanarc.+ی   % % Ќ  +  0  0%%0` %%+ڏb  .0 ` %` %  Partialidentityfusionof2lineswithaspur.+ڏ)ی|  % % Ќ  0  0%%0` %%c.0 ` %` %Nonidentitypatternoflineswhichcrosseachother.h  % % 0  0%%0` %%0 ` %` %  % % *n  *nIV  .0    ElectrophoreticTechniques*n5ی@%% Ќ  *  0  * A  .0%%  Introduction* 4ی%% Ќ   )  0  0%% ) 1  .0` %%  Immunodiffusioncanbecombinedwithelectricalcurrenttospeedthingsup. )$ یp` %` % Ќ   )  0  0%% ) 2  .0` %%  Electrophoresisisatechniquewhichseparatesmoleculesaccordingtodifferencesintheir H electricalchargewhentheyareexposedtoanelectriccurrent. )Y ی4` %` % Ќ   )  0  0%% ) 3  .0` %%  Adirectcurrentisappliedtothegel,theantigenandantibodymigratethroughthegel,as   diffusiontakesplaceprecipitinbandsareformed. ) یx` %` % Ќ   )  0  0%% )7 4  .0` %%  Canbeappliedbothasasingleordoublediffusionmethod. )7r یP` %` % Ќ  0  0%% %% *  0  *B  .0%%  RocketImmunolectrophoresis*ی(%% Ќ       (#39"#3"  0  0%%#32#  1  .3  0` %%  Description#3]݌ ` %` % Ќ  0  0%% %% "0;"  0  0%%0;C0` %%2#  a  .3  0 ` %` %  Onedimensionalelectroimmunodiffusion,itisanadaptationofradial  X" immunodiffusion(RID).0;C݌!D # % % Ќ  0  0%% %% "0;"  0  0%%0;0` %%2#  b  .3  0 ` %` %  Aquatitativemethodformeasuringserumproteins.0;<݌#"% % % Ќ  0  0%% %% "0;"  0  0%%0;]0` %%2#  c  .3  0 ` %` %  Involveselectrophoresisofantigenintoagelcontainsantibody.0;]݌t%#' % % Ќ  0  0%% %% "0;"  0  0%%0;ڠ0` %%2#  d  .3  0 ` %` %  Thetechniqueisrestrictedtodetectionofantigensthatmovetothepositivepole L'%) duringelectrophoresis.0;ڠ'݌8(&* % % Ќ  0  0%% %% "#3"  0  0%%#32#  2  .3  0` %%  Procedure#3݌*(,` %` % Ќ  0  0%% %% "0;"  0  0%%0;ƣ0` %%2#  a  .3  0 ` %` %  Agarmixedwithantibodyandpouredintoaplate,wells(holes)cutintoagar.0;ƣ݌+h*. % % Ќ  0  0%% %% "0;"  0  0%%0;P0` %%2#  b  .3  0 ` %` %  Antigenisplacedinthewells,anelectricalcurrentisappliedandprecipitation -@,0 begins.0;P݌.,-1 % % Ќ  "0;"  0  0%%0;æ0` %%2#  c  .3  0 ` %` %  Astheconcentrationofantigenchanges,thereisdissolutionandreformationof  theprecipitateateverincreasingintervals.0;æ݌l % % Ќ  0  0%% %% "0;"  0  0%%0;0` %%2#  d  .3  0 ` %` %  Theendresultisaprecipitinlinewithaconicalshaperesemblingarocket.0;ި݌D % % Ќ  0  0%% %% "0;"  0  0%%0;0` %%2#  e  .3  0 ` %` %  Theheightismeasuredandisdirectlyproportionaltotheconcentrationof  antigen.0;g݌ % % Ќ  0  0%% %% "0;"  0  0%%0;0` %%2#  f  .3  0 ` %` %  Ifstandardsarerunastandardcurveisconstructedandconcentrationdetermined.0; ݌` % % Ќ  "#3"  0  0%%#32#  3  .3  0` %%  AdvantageoverRIDistheresultsareobtainedinafewhours.#3c݌ 8 ` %` % Ќ  0  0%% %% "#3"  0  0%%#3|2#  4  .3  0` %%  Primarilyusedtoquantitateimmunoglobulinsandtoassayproteinswhoseconcentrations    aretoolowfornephelometrybuttoohighforRID#3|ɮ݌| ` %` % Ќ  *  0  * C  .0%%  Immunoelectrophoresis(IEP)* 2یT %% Ќ  ! ! (#33"#3"   0  0%%#32#  1  .3  0` %%  Description.#3݌,` %` % Ќ  "0;"  0  0%%0;0` %%2#  a  .3  0 ` %` %  Atwostepdoublediffusiontechniquewhichfirstinvolvestheelectrophoretic  separationofproteins.0;݌p % % Ќ  "0;"  0  0%%0;20` %%2#  b  .3  0 ` %` %  Followedbythelineardiffusionofantibodiesintotheelectrophoreticgelfroma H troughwhichextendsthroughthelengthofthegeladjacenttotheelectrophoretic 4 path.0;2݌  % % Ќ  0  0%% %% "#3"  0  0%%#3A2#  2  .3  0` %%  Procedure#3A݌x` %` % Ќ  "0;"  0  0%%0;<0` %%2#  a  .3  0 ` %` %  Proteinsarefirstelectrophoresedtoseparatethem.0;<݌P % % Ќ  "0;"  0  0%%0;v0` %%2#  b  .3  0 ` %` %  Atroughiscutinthegelparalleltothelineofseparation,antiserumisaddedto ( thetroughandthegelisincubatedovernight.0;vø݌ % % Ќ  "0;"  0  0%%0;0` %%2#  c  .3  0 ` %` %  Doublediffusionoccursastheantibodyandseparatedproteinsdiffusetowards l! oneanotherinthegelandformprecipitinlinesinthegel.0;a݌ X" % % Ќ  "0;"  0  0%%0;0` %%2#  d  .3  0 ` %` %  Thelinescanbecomparedinshape,intensityandlocationtothatofnormalserum "0!$ controltodetectabnormalitiesandissemiquantitative.0;݌#"% % % Ќ  "#3"  0  0%%#3^2#  3  .3  0` %%  Excellentscreeningtesttodifferentiateserumproteinsanddetectabnormalitiessuchasin t%#' myelomas,Waldenstromsmacroglobulinemia,malignantlymphomasandother `&$( lymphoproliferativedisorders.#3^݌L'%)` %` % Ќ  "#3"  0  0%%#3?2#  4  .3  0` %%  Canalsoidentifyimmunodeficiencies.#3?݌$)'+` %` % Ќ  _ .,-1 )* )  )  *  0  *D  .0%%  ImmunofixationElectrophoresis(IFE)*ی%% Ќ  ! ! (#33"#3"  0  0%%#3h2#  1  .3  0` %%  Description#3h݌X` %` % Ќ  "0;"  0  0%%0;e0` %%2#  a  .3  0 ` %` %  SimilartoIEPexceptthatafterelectrophoresisisperformedtheantiserumis 0 applieddirectlytothesurfaceofthegel.0;e݌ % % Ќ  "0;"  0  0%%0;0` %%2#  b  .3  0 ` %` %  Themostsensitivemethodusedtodetect,confirmandcharacterizemonoclonal t gammopathiessuchasthosefoundinplasmacell_dyscrasias_Ԁsuchasmultiple ` myeloma_Waldenstrom_s_macroglobulinemia_,monoclonal_gammopathy_Ԁof  L  unknownsignificance,lightorheavychaindisease.0;F݌ 8  % % Ќ  "0;"  0  0%%0;0` %%2#  c  .3  0 ` %` %  Particularlyusefulinidentifyingminormonoclonalproteins,thelightchain    componentofmonoclonalproteins,andunknownbandsseeninserumprotein |  electrophoresis.0;݌h  % % Ќ  "#3"  0  0%%#32#  2  .3  0` %%  Procedure#3݌@` %` % Ќ  "0;"  0  0%%0;0` %%2#  a  .3  0 ` %` %  Gelwithantibodyiselectrophoresed0;݌ % % Ќ  "0;"  0  0%%0;0` %%2#  b  .3  0 ` %` %  Theantibodyisimpregnatedintoacelluloseacetatestripwhichisplacedoverthe p gel.0;݌\ % % Ќ  "0;"  0  0%%0;;0` %%2#  c  .3  0 ` %` %  Theantibodyfromthestripdiffusesdownintothegelandaprecipitatewillform 4 ifthespecificantigenispresent.0;;݌  % % Ќ  "0;"  0  0%%0;0` %%2#  d  .3  0 ` %` %  Afterwashingthestriptoremoveextraneousproteinsastainisapplied.0;݌x % % Ќ  "#3"  0  0%%#32#  3  .3  0` %%  ThebestadaptationofthistestistheWesterBlottesttodetectantibodiestotheHuman P Immunodeficiencyvirus1(HIV1).#3g݌<` %` % Ќ  +  0  0%%0` %%+a  .0 ` %` %  AmixtureofHIVantigensisplacedonagelandelectrophoresedtoseparatethe  componentsoftheHIVantigen.+ی  % % Ќ  +  0  0%%0` %%+3b  .0 ` %` %  Thecomponentsaretransferredtonitrocellulosepaper.+3ی X" % % Ќ  +  0  0%%0` %%+bc  .0 ` %` %  Patientserumisaddedtothepaperandallowedtoreact.+bی"0!$ % % Ќ  +  0  0%%0` %%+d  .0 ` %` %  Thestripiswashedandstainedtodetectprecipitinbandsindicatingthepresence $#& ofantibodiestotheHIVantigen.+یt%#' % % Ќ  +  0  0%%0` %%+e  .0 ` %` %  Musthaveseveralbandspresenttobepositive.+bیL'%) % % Ќ  *  0  *:E  .0%%  SourcesofErrorinElectrophoresis*:aی$)'+%% Ќ   )  0  0%% )- 1  .0` %%  Applyingcurrentinwrongdirection. )-h ی*|)-` %` % Ќ   )  0  0%% )4 2  .0` %%  IncorrectbufferpH. )4o ی+h*.` %` % Ќ   )  0  0%% )+ 3  .0` %%  Incorrecttiming. h      p      x  )+f ی,T+/` %` % Ќ   )  0  0%% )s 4  .0` %%  Amountofcurrentapplied. )s ی-@,0` %` % Ќ   .,-1 Ї*n  *nV  .0    Agglutination*nی%% Ќ  ! ! (#330  A.0%%_Overview_X%% "#3"      #3[2#  1  .3  0 `   Definitiontheclumpingtogetherinsuspensionofantigenbearingcells, 0 microorganisms,orparticlesintthepresenceofspecificantibodies(agglutinins).#3[݌` %` % Ќ  "#3"      #32#  2  .3  0 `   Twostepprocessinvolving sensitization and latticeformation .#34݌t` %` % Ќ  "#3"      #3(2#  3  .3  0 `   Particlesusedinthetestcanberedbloodcells(hemagglutination),bacterialcells,orinert  L  particlessuchaslatexorcharcoal.  #3(m݌ 8 ` %` % Ќ  0  B.0%%StepsinAgglutination  %% ! ! (#33"#3"      #32#  1  .3  0 `   Sensitization#3݌h ` %` % Ќ  "0;"      0;0 ` 2#  a  .3  0 ` %` %  Firststepinvolvesattachmentofantibodytoitsspecificantigen.0;݌@ % % Ќ  "0;"      0;0 ` 2#  b  .3  0 ` %` %  Rapidandreversiblereaction.0;݌ % % Ќ  "0;"      0;0 ` 2#  c  .3  0 ` %` %  Thisstepaffectedbyantibodyaffinityandaviditywhichwilldeterminehow p muchantibodyremainsattached.0;-݌\ % % Ќ  "0;"      0;^0 ` 2#  d  .3  0 ` %` %  _IgM_ԀmuchmoreefficientthanIgGinagglutinationreactions.0;^݌4 % % Ќ  "0;"      0;0 ` 2#  e  .3  0 ` %` %  Antigenbearingsurfacesmusthavesufficientquantitiesofthe_epitope_Ԁpresent,if   sparseorobscuredlesslikelytointeractwithantibody.0;݌x % % Ќ  "#3"      #3g2#  2  .3  0 `   LatticesFormation#3g݌P` %` % Ќ  "0;"      0;[0 ` 2#  a  .3  0 ` %` %  Secondstagerepresentssumofinteractionbetweenantigenandantibody.0;[݌( % % Ќ  "0;"      0;0 ` 2#  b  .3  0 ` %` %  Dependentuponenvironmentalconditionsandconcentrationsofantigenand   antibody.0;݌l! % % Ќ  "0;"      0;0 ` 2#  c  .3  0 ` %` %  Physicochemicalfactorssuchasionicstrength,pHandtemperatureaffect !D # reaction.0;;݌"0!$ % % Ќ  "0;"      0;S0 ` 2#  d  .3  0 ` %` %  Antibodymustbeabletobridgegapbetweentwoparticlestoformlattice.0;S݌$#& % % Ќ  "0;"      0;0 ` 2#  e  .3  0 ` %` %  Electricalchargeofparticlesmayhinderlatticeformation,especiallyimportantif `&$( reactioninvolvesIgG,_IgM_Ԁusuallyabletoovercome.0;݌L'%) % % Ќ  "#3"      #3F2#  3  .3  0 `   EnhancementofAgglutination#3F݌$)'+` %` % Ќ  "0;"      0;D0 ` 2#  a  .3  0 ` %` %  Surfacechargemustbecontrolled,canbeaccomplishedby_additve_Ԁwhich *|)- neutralizessurface_hcarge_.0;D݌+h*. % % Ќ  "0;"      0;0 ` 2#  b  .3  0 ` %` %  Increasingviscositycanalsoneutralizecharge.0;6݌-@,0 % % Ќ   .,-1 "0;"      0;+0 ` 2#  c  .3  0 ` %` %  Treatmentofantigenwithcertainenzymescanremovesubstancesoncellsurface  toreduceinterferenceofelectricalcharge.0;+p݌l % % Ќ  "0;"      0;0 ` 2#  d  .3  0 ` %` %  Agitationandcentrifugationprovidesphysicalmeanstoincreasecelltocell D contactandenhancestrengthofreaction.0;݌0 % % Ќ  "0;"      0;20 ` 2#  e  .3  0 ` %` %  Temperatureiscriticalanddependsonantibodyclassinvolved:_IgM_Ԁprefersroom  temperatureorlower,IgGreactsbestatbodytemperature37C.0;2w݌t % % Ќ  "0;"      0;0 ` 2#  f  .3  0 ` %` %  OptimalpHof6.7to7.2.0;0݌ L  % % Ќ  0  B.0%%TypesofAgglutinationReactions $ %% ! ! (#33"#3"      #32#  1  .3  0 `   Introduction#3C݌| ` %` % Ќ  "0;"      0;0 ` 2#  a  .3  0 ` %` %  Agglutinationreactionshavemanyadvantages:easytocarryout,nocomplicated T  equipmentandcanbeperformedasneeded.0;1݌@ % % Ќ  "0;"      0;o0 ` 2#  b  .3  0 ` %` %  Mostagglutinationreactionsareavailableasprepackagedkitswithallreagents  andcontrolsneededpresent.0;o݌ % % Ќ  "0;"      0;0 ` 2#  c  .3  0 ` %` %  Candetecteitherantigenorantibody,criticaltoknowprincipleofprocedure.0;+݌\ % % Ќ  "0;"      0;+0 ` 2#  d  .3  0 ` %` %  Mostreactionsarequalitative,eitherpositiveornegative,whichindicates 4 presenceorabsence,althoughtiterscanbeperformedtogivesemiquantitative   results.0;+p݌  % % Ќ  "#3"      #32#  2  .3  0 `   DirectAgglutination#33݌d` %` % Ќ  "0;"      0;0 ` 2#  a  .3  0 ` %` %  Agglutinationofanantigenfoundnaturallyonaparticle.0;)݌< % % Ќ  "0;"      0;0 ` 2#  b  .3  0 ` %` %  Canbeusedtoidentifybacteriaaswhen_serotyping_ԀSalmonellaspecies.0;_݌ % % Ќ  "0;"      0;w 0 ` 2#  c  .3  0 ` %` %  Testpatientserumagainstknownbacteriainsuspensionswhencultivationof l! bacteriaisdifficult:Tularemia,rickettsialdiseases,andtyphoidfever.0;w  ݌ X" % % Ќ  "0;"      0; 0 ` 2#  d  .3  0 ` %` %  HemagglutinationutilizedtodetermineABObloodgrouping.0; ] ݌"0!$ % % Ќ  "0;"      0;H 0 ` 2#  e  .3  0 ` %` %  HemagglutinationkitsavailablefordetectionofantibodiestohepatitisB,Cand $#& HIVIandII0;H  ݌t%#' % % Ќ  "#3"      #3 2#  3  .3  0 `   PassiveAgglutination#3  ݌L'%)` %` % Ќ  "0;"      0;0 ` 2#  a  .3  0 ` %` %  Theagglutinationofinertparticlesbyantibodydirectedagainstantigenboundto $)'+ theirsurface.0;݌*(, % % Ќ  "0;"      0;0 ` 2#  b  .3  0 ` %` %  EmploysparticlesthatarecoatedwithantigenssuchasRBCs,polystyrenelatex, +h*. bentoniteorcharcoal.0;]݌,T+/ % % Ќ  "0;"      0;0 ` 2#  c  .3  0 ` %` %  Artificialparticleshaveadvantageofconsistency,uniformityandstability.0;݌.,-1 % % Ќ  __"0;"      0;0 ` 2#  d  .3  0 ` %` %  Reactionsaregenerallyveryeasytoreadmacroscopically.0;0݌ % % Ќ  "0;"      0;0 ` 2#  e  .3  0 ` %` %  ManyantigensadsorbtoRBCsspontaneously,tannedsheepRBCsfrequently X used..0;`݌D % % Ќ  "0;"      0;t0 ` 2#  f  .3  0 ` %` %  IgGnaturallyadsorbstosurfaceoflatexparticles.0;t݌ % % Ќ  "0;"      0;0 ` 2#  g  .3  0 ` %` %  Passiveagglutinationroutinelyusedforthefollowingtests:antinuclear t antibodies,groupAstrep,Rheumatoidfactor,andantibodiestovirusessuchas ` cytomegalovirus(CMV),rubellaandvaricellazoster.0;݌ L  % % Ќ  "#3"      #32#  4  .3  0 `   ReversePassiveAgglutination#3݌ $ ` %` % Ќ  "0;"      0;0 ` 2#  a  .3  0 ` %` %   Antibody attachedtocarrierparticleinsteadofantigen.0;݌|  % % Ќ  "0;"      0;0 ` 2#  b  .3  0 ` %` %  Antibodymustbeactiveandbeorientedsuchthattheactivesiteisfacing T  outward.0; ݌@ % % Ќ  "0;"      0;"0 ` 2#  c  .3  0 ` %` %  Oftenusedfordetectionofmicrobialantigenssuchas:GroupAandB  Streptococcus,Staphylococcusaureus,NeisseriaMeningitidis,Haemophilus  influenzae,_Cryptococcus_Ԁ_neoformans_,MycoplasmapneumoniaeandCandida p albicans.0;"g݌\ % % Ќ  "0;"      0;0 ` 2#  d  .3  0 ` %` %  Someoftheseorganismsverydifficulttogrowand/orquickdiagnosisneededso 4 correcttreatmentcanbestarted.0;݌  % % Ќ  "0;"      0; 0 ` 2#  e  .3  0 ` %` %  Widestapplicationisindetectingsolubleantigensinurine,spinalfluidand x serum.0; ^ ݌d % % Ќ  "0;"      0;x!0 ` 2#  f  .3  0 ` %` %  Antigenspresentinthesefluidswillattachtoantibodiesonparticles.0;x!!݌< % % Ќ  "0;"      0;"0 ` 2#  g  .3  0 ` %` %  Extractionstepmaybenecessary.0;""݌ % % Ќ  "#3"      #3#2#  5  .3  0 `   AgglutinationInhibition#3#$݌l!` %` % Ќ  "0;"      0;$0 ` 2#  a  .3  0 ` %` %  Basedoncompetitionbetweenparticulateandsolubleantigensforlimited !D # antibodycombiningsites.0;$ %݌"0!$ % % Ќ  "0;"      0;4&0 ` 2#  b  .3  0 ` %` %  Patientsampleaddedtoreagentantibodyspecificforantigenbeingtested,if $#& antigenispresentitbindstoreagentantibody.0;4&y&݌t%#' % % Ќ  "0;"      0;'0 ` 2#  c  .3  0 ` %` %  Reagentparticles(latexorRBCs)coatedwiththesameantigenareadded,if L'%) antigenwaspresentinthesampleallreagentantibodybindstoitsonoantibodyis 8(&* presenttoreactwithantigenscoatingtheparticles.0;'(݌$)'+ % % Ќ  "0;"      0;)0 ` 2#  d  .3  0 ` %` %  Usedfordetectingantibodiestocertainvirusessuchas:rubella,mumps,measles *|)- andinfluenzaforwhichRBCshave_neaturally_Ԁoccurringviralreceptors.0;))݌+h*. % % Ќ  "0;"      0;u+0 ` 2#  e  .3  0 ` %` %  Test:incubatepatientserumwithviralpreparation,addRBCswhichthevirusis -@,0 knowntoagglutinate,ifpatientantibodypresentitwillbindtofree_viurs_Ԁand .,-1 cannotattachtoRBCs,noagglutinationisapositiveresult.0;u++݌ % % Ќ  "0;"      0;-0 ` 2#  f  .3  0 ` %` %  Usedasaturbidimetricprocedurefortherapeuticdrugmonitoring0;--݌X % % Ќ  "#3"      #3.2#  6  .3  0 `   _Coagglutination_#3./݌0` %` % Ќ  "0;"      0;/0 ` 2#  a  .3  0 ` %` %  Namegiventosystemsusingbacteriaastheinertparticlestowhichantibodyis  attached.0;/0݌t % % Ќ  "0;"      0;>10 ` 2#  b  .3  0 ` %` %  Directlydetectssolublebacterialantigens.0;>11݌ L  % % Ќ  "0;"      0;`20 ` 2#  c  .3  0 ` %` %  Staphylococcusaureusmostfrequentlyused.0;`22݌ $  % % Ќ  "0;"      0;30 ` 2#  d  .3  0 ` %` %  Bacteriaarenotcoloredandreactionsaredifficulttoread.0;33݌|  % % Ќ  "0;"      0;40 ` 2#  e  .3  0 ` %` %  Notassensitivefordetectingsmallquantitiesofantigen.0;44݌T  % % Ќ  "0;"      0;50 ` 2#  f  .3  0 ` %` %  UsedtoidentifyStreptococci,Neisseriameningitidis,Neisseriagonorrheaand , Haemophilusinfluenzae.0;506݌ % % Ќ  "0;"      0;s70 ` 2#  g  .3  0 ` %` %  Thesemayprovidetruepositiveresultswhencultureandgramstainresultsare p negativeandforpatientswhohavealreadyreceivedantimicrobialtherapy.0;s77݌\ % % Ќ  "0;"      0;90 ` 2#  h  .3  0 ` %` %  Antigendetectionmethodsshouldneverbesubstitutedforcultureandgramstain. @H%  @H%  @H%  @H% 0;9\9݌4 % % Ќ   $(f03*n  *n;VI  .0    LabeledImmunoassays*n;);ی %% Ќ       `    *  0  *7<A  .0%%  Introduction*7<^<یx%% Ќ  "1"  1=0  0%%2f  1  .3  0` %%  Needrapid,specific,sensitiveassays.1=@=݌P` %` % Ќ  "1"  1,>0  0%%2f  2  .3  0` %%  Labeledimmunoassays1,>Y>݌(` %` % Ќ  " ir"   ir2?0  0%%0` %%2f  a  .3  0 ` %` %  Someantigen/antibodyreactionsnotdetectedbyprecipitationoragglutination. ir2?_?݌  % % Ќ  " ir"   ir@0  0%%0` %%2f  b  .3  0 ` %` %  Measuredindirectlyusingalabeledreactant. ir@@݌ X" % % Ќ  " ir"   irA0  0%%0` %%2f  c  .3  0 ` %` %  Referredtoasreceptorligandassays. irAA݌"0!$ % % Ќ  " ir"   irB0  0%%0` %%2f  d  .3  0 ` %` %   Ligand isthesubstancetobemeasuredandisdefinedasamoleculethatbindsto $#& anothermoleculeofacomplementaryconfiguration,usuallyitbindstothe t%#' substancethetestistryingtodetect. irBC݌`&$( % % Ќ  " ir"   irD0  0%%0` %%2f  e  .3  0 ` %` %  The receptor iswhatbindsthespecifictargetmolecule. irDE݌8(&* % % Ќ  "1"  15F0  0%%2f  3  .3  0` %%  Sometimesreferredtoasasandwichtechnique.15FbF݌*(,` %` % Ќ  " ir"   irUG0  0%%0` %%2f  a  .3  0 ` %` %  Haveareceptorofsomekindthatwillbindtotheantigenorantibodythesystem +h*. istryingtodetect. irUGG݌,T+/ % % Ќ   .,-1 2H  2,, 2  " ir"   irKI0  0%%0` %%2f  b  .3  0 ` %` %  Ifthesubstanceispresentitwillbindandawashingprocesseliminates X extraneoussubstancespresent. irKIxI݌D % % Ќ  " ir"   irJ0  0%%0` %%2f  c  .3  0 ` %` %  Alabeledligandisaddedwhichalsohasspecificityforthesubstancebeing  detectedandresultsinalabeledproduct. irJJ݌ % % Ќ  " ir"   ir^L0  0%%0` %%2f  d  .3  0 ` %` %  Thisresultsinasandwich,receptor,substancetobedetectedandligand. ir^LL݌ % % Ќ  " ir"   irM0  0%%0` %%2f  e  .3  0 ` %` %  Dependingupontheligandlabelavisibleordetectablereactionwilloccur. irMM݌ ` % % Ќ  *  0  *OB  .0%%  ConstituentsofLabeledAssays*O'Oی 8 %% Ќ    )  0  0%% ) P 1  .0` %%  Labelswhichmaybeusedinclude: fluorescent,radioactive,chemiluminescentand h   enzymes.  ) PEP یT ` %` % Ќ   )  0  0%% )gQ 2  .0` %%  Theassayincludestheuseof: )gQQ ی( ` %` % Ќ  +  0  0%%0` %%+iRa  .0 ` %` %  Labeledandnonlabeled_ligands_+iRRی % % Ќ  +  0  0%%0` %%+Sb  .0 ` %` %  Specificantibody+SSی % % Ќ  +  0  0%%0` %%+Tc  .0 ` %` %  Standardsandcalibrators+TTی % % Ќ  +  0  0%%0` %%+Ud  .0 ` %` %  Meansofseparatingboundfromfreecomponents+UVیl % % Ќ  +  0  0%%0` %%+Ve  .0 ` %` %  Meansoflabeldetection.+V+WیX % % Ќ   )  0  0%% )W 3  .0` %%  Competitivebinding )W(X ی0` %` % Ќ  +  0  0%%0` %%+Ya  .0 ` %` %  Alabeledligandismixedwiththepatientserum.+YOYی` % % Ќ  +  0  0%%0` %%+)Zb  .0 ` %` %  Itisaddedtothereceptor.+)ZxZی8 % % Ќ  +  0  0%%0` %%+=[c  .0 ` %` %  Competitivebindingoccursinwhichpatientsampleandlabeledligand compete  forantibodysites.+=[[ی % % Ќ  +  0  0%%0` %%+\d  .0 ` %` %  Iflittleornoantigenispresentinthepatientseraastrongpositiveoccurs.+\]ی|  % % Ќ  +  0  0%%0` %%+]e  .0 ` %` %  Ifthereisalotofantigeninthepatientsera,itwillsuccessfullybindinlarge !T" quantities,causingadecreaseinthebindingofthelabeledligand,causinga "@ # decreaseincolor,radioactivity,etc.+]I^ی#,!$ % % Ќ  0  0%% %%  )  0  0%% )` 4  .0` %%  Antibodiesusedinlabeled_immunoassay_Ԁreactionsmusthavehighaffinity. )`U` ی\%#&` %` % Ќ   )  0  0%% )ga 5  .0` %%   Standards or calibrators aresubstancesof knownconcentration . )gaa ی4'$(` %` % Ќ  +  0  0%%0` %%+ba  .0 ` %` %  Standardsarerunatthesametimeaspatientsample.+bcی)&* % % Ќ  +  0  0%%0` %%+cb  .0 ` %` %  Usuallythreestandardsarerun,theresultsaregraphed,andastandardcurveis *(, drawn.+c@dی+|)- % % Ќ  +  0  0%%0` %%+Uec  .0 ` %` %  Theconcentrationoftheunknownpatientsamplecanbedeterminedfromthe -T+/ standardcurve.+Ueeی.@,0 % % Ќ  _ )  0  0%% )f 6  .0` %%  Onceareactionhasoccurredtheremustbeawaytoremoveunboundanalytesfromthe X reaction,thisisdonethrougha separationmethod . )fg یD` %` % Ќ  +  0  0%%0` %%+sha  .0 ` %` %  Canmeasureeitherboundligandorfreeligandremainingafterseparation  method.+shhی % % Ќ  +  0  0%%0` %%+ib  .0 ` %` %  Unreactedligandcanberemovedby adsorption ontoinertparticlesaddedtothe  system.+ijیt % % Ќ  +  0  0%%0` %%+?kc  .0 ` %` %   Precipitation oftheantigenantibodycomplexes.+?kkی L  % % Ќ  +  0  0%%0` %%+sld  .0 ` %` %  Usea secondantibody toprecipitateouttheantigenantibodycomplexes.+sllی| $  % % Ќ  +  0  0%%0` %%+me  .0 ` %` %   Solidphase isthemostpopularmethod,antigenorantibodyisphysicallyattached T  toatube,plate,etc,thesubstancebeingdetectedwillbind,theexcessiswashed @  away.+mnی,  % % Ќ  +  0  0%%0` %%+of  .0 ` %` %  Inallprocedures,theseparationmethodisalimitingfactor.+ooی % % Ќ  +  0  0%%0` %%+pg  .0 ` %` %  Separationproceduremustbepreciseandreproducible.+pqی % % Ќ  0  0%%0` %% ` %` %  )  0  0%% )Er 7  .0` %%  Detectionofthelabeledanalyte. )Err ی\` %` % Ќ  +  0  0%%0` %%+fsa  .0 ` %` %  Theremustbeanaccuratesystemfordetectingandmeasuringthelabeledproduct 4 produced.+fssیx  % % Ќ  +  0  0%%0` %%+tb  .0 ` %` %  Forradioimmunoassay,radioactivityismeasured.+tuیP % % Ќ  +  0  0%%0` %%+uc  .0 ` %` %  Forlabelssuchasenzymes,fluorescenceorchemiluminescence,changesin ( absorbancyonaspectrophotometerareused.+uCvی % % Ќ  0  0%%0` %% ` %` %  )  0  0%% )w 8  .0` %%  Qualitycontrolproceduresmustalwaysbeperformedtoensuretheaccuracyoftheresults  obtained. )ww ی ` %` % Ќ  +  0  0%%0` %%+6ya  .0 ` %` %   Blanks aretubesfilledwithaclearsolutionor,ifthesolutionsaddedhave !X" color,withthesolutiononly,todeterminethe background,anysubstances "D # presentintheoriginalsolutionmustbecalculatedout.+6yyی#0!$ % % Ќ  +  0  0%%0` %%+C{b  .0 ` %` %   Controls aresubstanceswithaknownrangeofvaluesandgenerallythreelevels `%#& arerun:normal,highandlow.+C{{یL&#' % % Ќ  +  0  0%%0` %%+|c  .0 ` %` %  Ifcontrolsdonotgivetheexpectedvaluestheresultscannotbereportedout.+|}ی$(%) % % Ќ  *  0  *~C  .0%%  Radioimmunoassay (RIA)Techniques*~7~ی)'+%% Ќ     )  0  0%% )# 1  .0` %%  CompetitiveBindingAssays )#^ ی+)-` %` % Ќ  +  0  0%%0` %%+=a  .0 ` %` %  Usesradioactivesubstanceasalabel,usuallyI125.+=ی-X+/ % % Ќ   .D,0 +  0  0%%0` %%+b  .0 ` %` %  Antibodyisboundtoatubeorothersolidmatrix.+ӁیX % % Ќ  +  0  0%%0` %%+c  .0 ` %` %  Ameasuredamountofpatientsampleisaddedtoameasuredamountof 0 radiolabeledanalyteorligand.+ی % % Ќ  +  0  0%%0` %%+d  .0 ` %` %  Theantigeninthepatientsampleandtheradiolabeledantigencompeteforthe  bindingtotheantibody.+mی % % Ќ  +  0  0%%0` %%+e  .0 ` %` %  Ifthereisnoantigeninthesamplethentherewillbeahighlevelofradiation  ` sincetheradiolabeledantigencanbindtoallantibodysites.+ی L  % % Ќ  +  0  0%%0` %%+-f  .0 ` %` %  Ifantigenispresentinthepatientsamplethenradioactivitywillbedecreased | $  proportionallytotheamountofantigenpresent.+-|یh   % % Ќ  +  0  0%%0` %%+g  .0 ` %` %  Standardsareruntocreateastandardcurve.+ ی@  % % Ќ  +  0  0%%0` %%+߉h  .0 ` %` %  Controlsareruntoensurepropertechniqueandreagentreactivity.+߉.ی % % Ќ   )  0  0%% ) 2  .0` %%  ImmunoradiometricAssay (IRMA) )U ی` %` % Ќ   +  0  0%%0` %%+>a  .0 ` %` %  Excesslabeledantibodyisaddedtoatubewithpatientantigen.+>یt % % Ќ  +  0  0%%0` %%+vb  .0 ` %` %  Allpatientantigenisboundbytheantibody.+vōیL % % Ќ  +  0  0%%0` %%+c  .0 ` %` %  Solidphaseantigenisaddedwhichwillthenbindupallexcesslabeledantibody.+ی|$ % % Ќ  +  0  0%%0` %%+d  .0 ` %` %  Thetubeisspunandthesolidphaseantigenwillgotothebottom,allantibody T boundtopatientantigenremainsinsolution.+3ی@ % % Ќ  +  0  0%%0` %%+ne  .0 ` %` %  Theradioactivityofthesupernatantsolutionisdetermined.+nی % % Ќ  +  0  0%%0` %%+f  .0 ` %` %  Thecountobtainedisdirectlyproportionaltotheamountofpatientantigen  presentinthespecimen.+ی  % % Ќ  +  0  0%%0` %%+g  .0 ` %` %  Advantages:faster,reaction,increasedsensitivityandspecificity.+bی!\" % % Ќ  +  0  0%%0` %%+Oh  .0 ` %` %  Disadvantage:pureantigenandantibodyareneeded.+Oی#4!$ % % Ќ   )  0  0%% )z 3  .0` %%  ProcedureswhichutilizeRIAare:humanchorionicgonadotropin(HCG),follicle d% #& stimulatinghormone(FSH),gastrin,insulin,carcinoembryonicantigen(CEA),thyroxine, P&#' estrogens,androgens,IgEanderythropoietin. )z ی<'$(` %` % Ќ   )  0  0%% )[ 4  .0` %%  DisadvantagesofusingRIA: )[ ی)&*` %` % Ќ   +  0  0%%0` %%+va  .0 ` %` %  Healthhazard+vřی*(, % % Ќ  +  0  0%%0` %%+zb  .0 ` %` %  Disposalofradioactivewaste+zɚی+)- % % Ќ  +  0  0%%0` %%+c  .0 ` %` %  Shortshelflife+ݛی,l*. % % Ќ  +  0  0%%0` %%+d  .0 ` %` %  Expensiveequipment+ی-X+/ % % Ќ   .D,0 Ї*  0  *D  .0%%  EnzymeImmunoassay(PerformELISAVirutalLabSerologyOnlineActivities)*ܝیX%% Ќ    )  0  0%% ) 1  .0` %%  Introduction )' ی0` %` % Ќ  0  0%%0` %%a.0 ` %` % Advantages ofenzymeimmunoassay: % % 0  0%%0` %%0 ` %` %1)0 % %labelscheapandplentiful.%% 0  0%%0` %%0 ` %` %2)0 % %labelshavealongshelflifet%% 0  0%%0` %%0 ` %` %3)0 % %easilyadaptedtoautomation `%% 0  0%%0` %%0 ` %` %4)0 % %reactionmeasuredusinginexpensiveequipment L %% 0  0%%0` %%0 ` %` %5)0 % %verysensitive 8 %% 0  0%%0` %%0 ` %` %6)0 % %nohealthhazardsassociatedwithreagents| $ %%      `  7)0  canbeusedforqualitativeORquantitativeprocedures.h  %% +  0  0%%0` %%+ˤb  .0 ` %` %  Enzymeschosenforusaslabelsaccordingtothefollowing:+ˤی@  % % Ќ   )F  0  0%%0` %%0 ` %` %)F 1  )0 % %  numberofsubstratemoleculesconvertedpermoleculeofenzyme)F} ی%% Ќ  )F  0  0%%0` %%0 ` %` %)Fc 2  )0 % %  purity)FcƧ ی%% Ќ  )F  0  0%%0` %%0 ` %` %)Ft 3  )0 % %  sensitivity)Ftר ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 4  )0 % %  easeandspeedofdetection)F ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 5  )0 % %  stability)F یp%% Ќ  )F  0  0%%0` %%0 ` %` %)Fī 6  )0 % %  absenceofinterferingsubstances)Fī' ی\%% Ќ  )F  0  0%%0` %%0 ` %` %)F 7  )0 % %  availability)FS یH%% Ќ  )F  0  0%%0` %%0 ` %` %)F 8  )0 % %  cost)Fj ی4%% Ќ  0  0%%0` %%0 ` %` %  % % +  0  0%%0` %%+uc  .0 ` %` %  Typicalenzymesusedinclude:+uįیd  % % Ќ  0  0%%0` %%0 ` %` %  % %   )F  0  0%%0` %%0 ` %` %)F 1  )0 % %  horseradishperoxidasecheap,verypopular,reactswithanumberof < chromogens)Fg ی(%% Ќ  )F  0  0%%0` %%0 ` %` %)Fs 2  )0 % %  glucoseoxidase)Fsֲ ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 3  )0 % %  glucose6phosphatedehydrogenaseusesfluorimetricmeans)F ی%% Ќ  )F  0  0%%0` %%0 ` %` %)FӴ 4  )0 % %  alkalinephosphataseexpensive)FӴ6 ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 5  )0 % %  %XY XXXMb#XMX X%XY#ԄDgalactosidase)Fa ی %% Ќ  0  0%%0` %%0 ` %` %  % % +  0  0%%0` %%+d  .0 ` %` %  Theenzymelabelislinkedtoantibodyorligand.+ ی!d" % % Ќ  +  0  0%%0` %%+e  .0 ` %` %  Twoclassificationofenzymeassays:+3ی# 2  )0 % %  Antigenisboundtosolidphase,unlabeledpatientantibodyisadded.)F> یh  %% Ќ  )F  0  0%%0` %%0 ` %` %)F 3  )0 % %  Afterincubationawashstepisperformedandanenzymelabeledsubstrate @  isadded.)F ی, %% Ќ  )F  0  0%%0` %%0 ` %` %)F 4  )0 % %  Theamountofenzymelabeldetectedisdirectlyproportionaltothe  amountofantibodyinthespecimen.)Fd ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 5  )0 % %  Indirect_ELISAs_Ԁareverysensitive.)F ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 6  )0 % %  Disadvantageisthereismoremanipulationofthetest.)F: ی\%% Ќ  +  0  0%%0` %%+c  .0 ` %` %   _Immunoenzymometric_ԀAssay +iی4 % % Ќ   )F  0  0%%0` %%0 ` %` %)Fr 1  )0 % %  Noncompetitive_ELISA_)Fr یd %% Ќ  )F  0  0%%0` %%0 ` %` %)F 2  )0 % %  Detectsunknownantigenbymeansofexcesslabeledantibody.)F ی<%% Ќ  )F  0  0%%0` %%0 ` %` %)F 3  )0 % %  Antibodyandpatientsampleallowedtoreact.)F^ ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F4 4  )0 % %  Antigenattachedtosolidphase,suchasglassbeads,addedtotestsystem.)F4 ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 5  )0 % %  Solidphaseantigencombineswith_unreacted_Ԁlabeledantibody.)F ی l!%% Ќ  )F  0  0%%0` %%0 ` %` %)F 6  )0 % %  Centrifugetubeandmeasuresupernatantwhichcontainsantigenantibody "D # complexes.)FW ی#0!$%% Ќ  )F  0  0%%0` %%0 ` %` %)Ff 7  )0 % %  Thismethodrequireslesstime,eliminatesnonspecificreactivityandhas `%#& greatersensitivitythanothercompetitive_ELISAs_.)Ff یL&#'%% Ќ  0  0%%0` %% ` %` % +  0  0%%0` %%+ld  .0 ` %` %  SandwichorCaptureAssays +lی$(%) % % Ќ   )F  0  0%%0` %%0 ` %` %)F 1  )0 % %  Usedwithantigenshavingmultiple_epitopes_.)F  ی)'+%% Ќ  )F  0  0%%0` %%0 ` %` %)F 2  )0 % %  Excessantibodyattachedtosolidphase,allowedtocombinewithtest +|)- sample.)Fa ی,h*.%% Ќ  )F  0  0%%0` %%0 ` %` %)Fk 3  )0 % %  Afterincubation,enzymelabeledantibodyisadded.)Fk ی.@,0%% Ќ  _)F  0  0%%0` %%0 ` %` %)F 4  )0 % %  Secondantibodymayrecognizesameordifferentepitopethansolidphase D antibody.)F ی0%% Ќ  )F  0  0%%0` %%0 ` %` %)F, 5  )0 % %  Enzymeactivitydirectlyrelatedtoconcentrationofantigen.)F, ی%% Ќ  )F  0  0%%0` %%0 ` %` %)Fu 6  )0 % %  Detectsantigenspresentinlowconcentrations.)Fu ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 7  )0 % %  Suitedtoantigenswithmultipleepitopes.)F ی `%% Ќ  )F  0  0%%0` %%0 ` %` %)F 8  )0 % %  Epitopemustbeuniquetotheorganismandpresentinallstrains.)FI ی 8 %% Ќ  )F  0  0%%0` %%0 ` %` %)F4 9  )0 % %  Majoruseofthistechniqueisformeasurementofimmunoglobulins,the h   immunoglobulinistheantigenandanantiantibodyisaddedtothetest T  system.)F4  ی@ %% Ќ  )F  0  0%%0` %%0 ` %` %)F 10  )0 % %  Disadvantage:linearrelationshipdoesnotexist,mustpreparestandard  curve,testconditionsmustbecarefullycontrolled.)Fa  ی%% Ќ     )  0  0%% ) 4  .0` %%  HomogeneousEnzymeImmunoassay  ) ی` %` % Ќ  +  0  0%%0` %%+a  .0 ` %` %  Reagentantigenislabeledwithenzymetag,patientsampleisadded.+0ی\ % % Ќ  +  0  0%%0` %%+b  .0 ` %` %  Whenantibodybindstoantigen,stearichindrancetotheenzymeoccurswhich 4 inactivatestheenzyme.+lیx  % % Ќ  +  0  0%%0` %%+c  .0 ` %` %  Antigencompeteswithenzymelabeledanalyteforlimitednumberofantibody P bindingsites,thisisacompetitiveassay.+ی< % % Ќ  +  0  0%%0` %%+d  .0 ` %` %  Enzymeactivityisdirectlyproportionaltoconcentrationofpatientantigen.+_ی % % Ќ  +  0  0%%0` %%+Ue  .0 ` %` %  Noseparationstepnecessary.+Uی % % Ќ  +  0  0%%0` %%+jf  .0 ` %` %  Sensitivityoftheprocedureisdeterminedby:+jی l! % % Ќ   )F  0  0%%0` %%0 ` %` %)F 1  )0 % %  detectabilityofenzymaticactivity)F ی"D #%% Ќ  )F  0  0%%0` %%0 ` %` %)F 2  )0 % %  changeinactivitywhenantibodybindstoantigen)F= ی#0!$%% Ќ  )F  0  0%%0` %%0 ` %` %)F 3  )0 % %  strengthofantibodybond)Fy یt$"%%% Ќ  )F  0  0%%0` %%0 ` %` %)F: 4  )0 % %  susceptibilityoftheassaytointerferencessuchascrossreactivity.)F: ی`%#&%% Ќ  +  0  0%%0` %%+g  .0 ` %` %  Sensitivityfarlessthanheterogenousenzymeassays.+ی8'$( % % Ќ  0  0%%0` %% ` %` %  )  0  0%% ) 5  .0` %%  AdvantagesofEnzymeImmunoassay )> ی)&*` %` % Ќ  +  0  0%%0` %%+#a  .0 ` %` %  SensitivitysimilartoRIAwithouthealthhazardsandreagentdisposalproblems.+#rی*(, % % Ќ  +  0  0%%0` %%+jb  .0 ` %` %  & %XXMExpensiveequipmentnotnecessary,spectrophotometersornotationofcolorchange#XMX %&#.+jی+|)- % % Ќ  +  0  0%%0` %%+c  .0 ` %` %  Mostarefairlysimple+Dی,h*. % % Ќ   .@,0  )  0  0%% ) 6  .0` %%  Disadvantages )S یX` %` % Ќ  +  0  0%%0` %%+%a  .0 ` %` %  Somesamplesmayhavenaturalinhibitors.+%tی0 % % Ќ  +  0  0%%0` %%+Eb  .0 ` %` %  Sizeofenzymelabellimitingfactorindesigningsomeassays.+Eی % % Ќ  +  0  0%%0` %%+zc  .0 ` %` %  Nonspecificproteinbindingmayoccur.+zی % % Ќ  +  0  0%%0` %%+d  .0 ` %` %  Enzymereactionsverysensitivetotemperature.+ی % % Ќ  0   %% *  0  *E  .0%%  FluorescentImmunoassay *یt%% Ќ    )  0  0%% ) 1  .0` %%  Introduction )$ ی P ` %` % Ќ  +  0  0%%0` %%+a  .0 ` %` %  Usefluorescentcompoundscalledfluorophoresorfluorochromesasmarkers.+Dی (  % % Ќ  +  0  0%%0` %%+7 b  .0 ` %` %  Markershavetheabilitytoabsorbenergyandconvertitintolight.+7  یX  % % Ќ  +  0  0%%0` %%+s c  .0 ` %` %  Fluorescentprobemustexhibithighintensitytodistinguishfrombackground.+s  ی0  % % Ќ  +  0  0%%0` %%+ d  .0 ` %` %  Twocompoundsmostfrequentlyusedarefluoresceinandrhodamine.+  ی % % Ќ   )F  0  0%%0` %%0 ` %` %)F  1  )0 % %  Fluoresceinemitsgreencolor,ithashighintensityandgood  photostablility.)F p  یt%% Ќ  )F  0  0%%0` %%0 ` %` %)F{ 2  )0 % %  Tetramethylrhodamineemitsredlight.)F{ یL%% Ќ  )F  0  0%%0` %%0 ` %` %)F 3  )0 % %  Newercompoundsincludealgae,porphyrinsandchlorophylls.)F ی|$%% Ќ  +  0  0%%0` %%+e  .0 ` %` %  Fluorescenttagsfirstusedforlocalizationofantigenintissues.+BیT % % Ќ   )F  0  0%%0` %%0 ` %` %)FJ 1  )0 % %  Immunofluorescentassay(IFA)isqualitativeobservationoffluorescent , reactionusingfluorescentscopeforvisualization.)FJ ی%% Ќ  )F  0  0%%0` %%0 ` %` %)F 2  )0 % %  Candetectantigensinfixedcellsorlivecellsuspensions.)FH ی%% Ќ   )  0  0%% )- 3  .0` %%  FluorescentMicroscopy )-h ی p!` %` % Ќ  +  0  0%%0` %%+Ca  .0 ` %` %  Useslightsourcethatemitslightoftheappropriatewavelengthnecessaryto "H # excitefluorochromeused.+Cی#4!$ % % Ќ  +  0  0%%0` %%+b  .0 ` %` %  Thepresenceofaspecificantigenisdeterminedbytheappearanceoflocalized d% #& coloragainstablackbackground.+یP&#' % % Ќ  +  0  0%%0` %%+3c  .0 ` %` %  Fluorescentstaining+3ی((%) % % Ќ   )F  0  0%%0` %%0 ` %` %)F[ 1  )0 % %   Directimmunofluorescent assaytheantibodyhasafluorescenttags, *'+ addeddirectlytounknownantigenfixedtoslide,afterincubationand *(, washingobserveforfluorescence.)F[ ی+)-%% Ќ   .D,0 )F  0  0%%0` %%0 ` %` %)F] 2  )0 % %   Indirectimmunofluorescent assayreactpatientserumwithknown X antigenattachedtosolidphase,wash,addantihumanantibodyattachedto D flourescenttag,wash,observeforfluorescence.)F] ی0%% Ќ  0  0%%0` %%0 ` %` %  % %  )  0  0%% ) 3  .0` %%  HeterogeneousFluorescentImmunoassays ) ی` %` % Ќ  +  0  0%%0` %%+a  .0 ` %` %  Fluorescentimmunoassays(FIA)areclassifiedasheterogeneousorhomogeneous.+, ی % % Ќ  +  0  0%%0` %%+#!b  .0 ` %` %  Heterogeneousassaysrequireaseparationstepandinclude:+#!r!ی ` % % Ќ   )F  0  0%%0` %%0 ` %` %)Fr" 1  )0 % %  indirect)Fr"" ی 8 %% Ќ  )F  0  0%%0` %%0 ` %` %)F# 2  )0 % %  competitive)F## ی| $ %% Ќ  )F  0  0%%0` %%0 ` %` %)F$ 3  )0 % %  sandwich)F$$ یh  %% Ќ  )F  0  0%%0` %%0 ` %` %)F% 4  )0 % %  capture)F%& یT %% Ќ  +  0  0%%0` %%+&c  .0 ` %` %  SameprincipleasEIAexceptfluorescentlabelisused.+&'ی,  % % Ќ  +  0  0%%0` %%+'d  .0 ` %` %  Candetecteitherantigenorantibody.+'?(ی % % Ќ  +  0  0%%0` %%+)e  .0 ` %` %  Solidphaseusesantigenorantibodyattachedtobeadswhichmaybemagnetized.+)])ی % % Ќ  +  0  0%%0` %%+U*f  .0 ` %` %  Whenbeadsareutilizedthereactionoccurs,thetubecentrifuged,supernatant \ discarded,andthebeadsobservedforfluorescence.+U**یH % % Ќ  +  0  0%%0` %%++g  .0 ` %` %  Magnetizedbeadsareplacedonamagneticsurfacetoattractbeadstothebottom x  ofthetube.++2,یd  % % Ќ  +  0  0%%0` %%+L-h  .0 ` %` %  Solidphasecanalsoemployadipstickcoatedwithantigenorantibody,reactwith < patientsamplethenaddalabeledantibody.+L--ی( % % Ќ   )  0  0%% ). 5  .0` %%  HomogenousFluorescentImmunoassays )./ ی` %` % Ќ   +  0  0%%0` %%+/a  .0 ` %` %  JustlikeEIA,noseparationnecessary,oneincubationsstepandnowashstep.+/H0ی  % % Ќ  +  0  0%%0` %%+?1b  .0 ` %` %  Usuallyinvolvescompetitivebinding.+?11ی!X" % % Ќ  +  0  0%%0` %%+\2c  .0 ` %` %  Directrelationshipbetweenamountoffluorescenceandquantityofantigenin #0!$ patientsample.+\22یt$"% % % Ќ  +  0  0%%0` %%+3d  .0 ` %` %  Thistechniquesuffersfromlackofsensitivity.+34یL&#' % % Ќ   )  0  0%% )4 6  .0` %%  FluorescencePolarizationImmunoassay(FPIA) )4(5 ی$(%)` %` % Ќ  +  0  0%%0` %%+6a  .0 ` %` %  Basedonchangeinpolarizationoffluorescentlightemittedfromlabeled )'+ moleculeboundbyantibody.+6h6ی*(, % % Ќ  +  0  0%%0` %%+7b  .0 ` %` %  Whenfluorophoresareexcited,emitpartiallypolarizedfluorescence.+77ی,h*. % % Ќ  +  0  0%%0` %%+8c  .0 ` %` %  Antibodyboundlabeledantigenswillemitmorepolarizedlight.+89ی.@,0 % % Ќ  _+  0  0%%0` %%+ :d  .0 ` %` %  Competitivebinding.+ :\:یX % % Ќ  +  0  0%%0` %%+;e  .0 ` %` %  Degreeoffluorescenceinverselyrelatedtoconcentrationof_analyte_.+;g;یD % % Ќ  +  0  0%%0` %%+t<f  .0 ` %` %  Techniquelimitedtosmallmolecules.+t<<ی0 % % Ќ  +  0  0%%0` %%+=g  .0 ` %` %  Usedmostfrequentlyfortherapeuticdrugsandhormones.+==ی % % Ќ  +  0  0%%0` %%+>h  .0 ` %` %  Requiressophisticatedinstrumentation.+>?ی % % Ќ   )  0  0%% )? 7  .0` %%  Advantagesoffluorescenttechniques: )?@ ی` %` % Ќ   +  0  0%%0` %%+Aa  .0 ` %` %  Methodsfairlysimple.+ARAی ` % % Ќ  +  0  0%%0` %%+Bb  .0 ` %` %  Nohazardousreagentstouseordiscard.+B_Bی L  % % Ќ  +  0  0%%0` %%+/Cc  .0 ` %` %  Increasedsensitivityover_radiolabeled_Ԁandenzymereactions.+/C~Cی 8  % % Ќ   )  0  0%% )D 8  .0` %%  Disadvantagesinclude: )DD یh  ` %` % Ќ  +  0  0%%0` %%+Ea  .0 ` %` %  Fluorescentcompoundsaresensitivetoenvironmentalchanges.+EEی@  % % Ќ  +  0  0%%0` %%+Fb  .0 ` %` %  Stabilityoflabels+FGی,  % % Ќ  +  0  0%%0` %%+Gc  .0 ` %` %  Nonspecificbindingcandiminishsignal.+G'Hی % % Ќ  +  0  0%%0` %%+Hd  .0 ` %` %  Bilirubinorhemoglobincanabsorbtheexcitationoremissionenergy.+HFIی % % Ќ  +  0  0%%0` %%+3Je  .0 ` %` %  Requiresexpensivededicatedinstrumentation.+3JJی % % Ќ   )  0  0%% )XK 9  .0` %%  ChemiluminescentImmunoassays. )XKK  یp` %` % Ќ  +  0  0%%0` %%+vLa  .0 ` %` %  Chemiluminescenceistheproductionoflightenergyduetoachemicalreaction.+vLLیH % % Ќ  +  0  0%%0` %%+Mb  .0 ` %` %  Mostcommonsubstancesusedare:_luminol_,_acridium_Ԁestersand_dioxetane_ x  phosphate.+M Nیd  % % Ќ  +  0  0%%0` %%+{Oc  .0 ` %` %  Thesubstanceisoxidized,usuallywithhydrogenperoxideandanenzymefora < catalyst.+{OOی( % % Ќ  +  0  0%%0` %%+Pd  .0 ` %` %  Energygivenoffintheformoflight.+P-Qی % % Ќ  +  0  0%%0` %%+Qe  .0 ` %` %  Heterogeneousandhomogenousassaysavailable.+QKRی  % % Ќ  +  0  0%%0` %%+"Sf  .0 ` %` %  Disadvantages:+"SqSی!X" % % Ќ    )F  0  0%%0` %%0 ` %` %)FDT 1  )0 % %  Falseresultsmaybeobtainediflackofprecisioninadditionofhydrogen #0!$ peroxide.)FDTT یt$"%%% Ќ  )F  0  0%%0` %%0 ` %` %)FU 2  )0 % %  Somebiologicmaterialscausequenching.)FUV ی`%#&%% Ќ  )F  0  0%%0` %%0 ` %` %)FV 3  )0 % %  Needdedicatedinstrumentation.)FVMW یL&#'%% Ќ  +    0  0%%0` %%+Xg  .0 ` %` %  Advantages:+XXی$(%) % % Ќ    )F  0  0%%0` %%0 ` %` %)FPY 1  )0 % %  reactivestabilityoflabel)FPYY ی)'+%% Ќ  )F  0  0%%0` %%0 ` %` %)FvZ 2  )0 % %  speedofdetection)FvZZ ی*(,%% Ќ  )F  0  0%%0` %%0 ` %` %)F[ 3  )0 % %  sensitivitycomparableto_RIA_ԀandEIA)F[[ ی+|)-%% Ќ  )F  0  0%%0` %%0 ` %` %)F\ 4  )0 % %  lowcost     p      x   (# )F\F] ی,h*.%% Ќ   .@,0 0  G.0%%SummaryX%% ! ! (#30$"#3"      #3V_2#  1  .3  0 `   LabeledassaysareusedtomeasureantigenORantibody.#3V__݌D` %` % Ќ  "#3"      #3n`2#  2  .3  0 `   Differentlabelscanbeused.#3n``݌0` %` % Ќ  "#3"      #3la2#  3  .3  0 `   Twoclassificationofenzymeassays:#3laa݌` %` % Ќ  "0;"      0;qb0 ` 2#  a  .3  0 ` %` %   Heterogenou srequiresasteptophysicallyseparateboundligandfromfree.0;qbb݌ % % Ќ  "0;"      0;c0 ` 2#  b  .3  0 ` %` %   Homogeneous assaysrequirenoseparationstep.0;cd݌ % % Ќ  "#3"      #3d2#  4  .3  0 `   _Immunoassay_Ԁlabelsmustbeveryspecificandhavehighaffinity.#3d1e݌` %` % Ќ  "#3"      #3-f2#  5  .3  0 `   Radioactivelabels#3-frf݌t` %` % Ќ  "0;"      0; g0 ` 2#  a  .3  0 ` %` %  _RIA_ԀisCOMPETITIVEknownantibodyaddedinspecificamounttounknown  ` sample,competeswithpatientantibodytoattach.Indirectmeasurement.0; geg݌ L  % % Ќ  "0;"      0;h0 ` 2#  b  .3  0 ` %` %  IRMAallunknownantigenreactsduetoexcessantibodybeingadded.0;hi݌ 8  % % Ќ  "#3"      #3j2#  6  .3  0 `   Enzymescanbeusedaslabels,resultsinacolorreaction.#3jXj݌| $ ` %` % Ќ    "0;"      0;5'_exonuclease_Ԁproofreading  capacitytoreplaceanaccidentalmismatchinthenewlysynthesizedDNAstrand.#XMXX8XMy# % %      ` j.0 WithPCR,itisroutinelypossibletoamplifyenoughDNAfromasinglehair t follicleorsinglespermcellforDNAtyping. ` % %      ` k.0 ThePCRtechniquehasevenmadeitpossibletoanalyzeDNAfrommicroscope  L  slidesoftissuepreservedyearsbefore. 8  % %      ` l.0 ThegreatsensitivityofPCRmakescontaminationbyextraneousDNAaconstant | $  problemh   % %     3.0 ` TranscriptionMediatedAmplificationT ` %` %      ` a.0 _TMA_Ԁisthenextgenerationofnucleicacidamplificationtechnology.@  % %      ` b.0 _TMA_ԀisanRNAtranscriptionamplificationsystemusingtwoenzymestodrive ,  thereaction:RNApolymeraseandreversetranscriptase. % %      ` c.0 _TMA_Ԁisisothermal;theentirereactionisperformedatthesametemperatureina  waterbathorheatblock.Thisisincontrasttootheramplificationreactionssuch  asPCRorLCRthatrequireathermal_cycler_Ԁinstrumenttorapidlychangethe  temperaturetodrivethereaction.p % %      ` d.0 _TMA_ԀcanamplifyeitherDNAorRNA,andproducesRNA_amplicon_,incontrast \ tomostothernucleicacidamplificationmethodsthatonlyproduceDNA.H % %      ` e.0 _TMA_Ԁhasveryrapidkineticsresultinginabillionfoldamplificationwithin15-30 4 minutes.x  % % "0;"  0;0  2  E  .3  0%%  ProbeAmplification0;F݌P%% Ќ      1.0 ` Amplifydetectionofmoleculeorprobeinsteadoftargetmolecule.<` %` %     2.0 ` QB_Replicase_(` %` %      ` a.0 usesanRNAdirectedRNApolymerasethatreplicatesthe_genomic_ԀRNAofa  bacteriophagenamedQB. % %      ` b.0 TheRNAgenomeofQBisessentiallytheonlysubstraterecognizedbythe  polymerase.  % %      ` c.0 BecauseashortprobecanbeinsertedintotheQBRNAthisbecomesthesystem  l! foramplification.!X" % %      ` d.0 Aftertheprobehasannealedtothetarget,unboundprobeistreatedwith_RNase_ "D # andwashedaway.#0!$ % %      ` e.0 Thehybridizedprobeis_RNase_Ԁresistant.t$"% % %      ` f.0 WhenQB_replicase_Ԁisaddedtheprobeisenzymaticallyreplicatedtodetectable `%#& levels.L&#' % %     3.0 ` _Ligase_ԀChainReactionLCR8'$(` %` %      ` a.0 TheLCRtestemploysfoursynthetic_oligonucleotide_Ԁprobes(twoperDNA $(%) strand)annealatspecifictargetsitesonthecryptic_plasmid_.)&* % %      ` b.0 EachpairofprobeshybridizeclosetogetheronthetargetDNAtemplate,with1- )'+ to2-nucleotidegapbetween.*(, % %      ` c.0 Oncetheprobesareannealed,thegapisfilledbyDNA_polimerase_Ԁandcloseby +|)- the_ligase_Ԁenzyme.,h*. % %      ` d.0 Thistwo-stepprocessofclosingthegapbetweenannealedprobesmakesthe -T+/ LCR,intheory,morespecificthanPCRtechnology..@,0 % %      ` e.0 Theligatedprobepairsannealtoeachotherand,upondenaturation,fromthe X templateforsuccessivereactioncycles,thusproducingalogarithmic D amplificationofthetargetsequence.0 % %      ` f.0 LikePCR,LCRismadeina_thermocycler_. % %      ` g.0 TheLCRproductisdetectedinanautomatedinstrumentthatusesan  _immunocolorimetric_Ԁbeadcapturesystem. % %      ` h.0 AttheendoftheLCRassay,amplifiedproductsareinactivatedbytheautomatic  additionofachelatedmetalcomplexandaoxidizingagent.t % % "0;"  0;0  2  F  .3  0%%  SignalAmplification0;݌ `%% Ќ      1.0 ` Replicatessignalratherthaneitherthetargetortheprobe. L ` %` %     2.0 ` Basedonthereportergroup(thelabeledtag)beingattachedingreaternumberstothe  8  probemoleculeorincreasingtheintensitygeneratedbyeachlabeledtag.| $ ` %` %     3.0 ` Patientnucleicacidnotreplicatedoramplifiedtechniqueislesspronetocontamination.h  ` %` %     4.0 ` Sensitivityislower.T ` %` %     5.0 ` Branchedchainsignalamplificationemploysseveralsimultaneoushybridizationsteps.@ ` %` %     6.0 ` AuthorstatessimilartodecoratingaChristmastreeandinvolvesseveralsandwich ,  hybridizations.` %` %      ` a.0 first,targetspecific_oligonucleotide_Ԁprobescapturestargetsequencetosolid  support. % %      ` b.0 Secondsetoftargetspecificprobescalledextendershybridizetoadjoining  sequencesandactasbindingsiteforlargepiececalledbranchedamplification p _multimer_.\ % %      ` c.0 Eachbranchhasmultiplesidebranchescapableofbindingnumerous H _oligonucleotides_.4 % %      ` d.0 Branchedchainsarewellsuitedtodetectionofnucleicacidtargetswithsequence x  heterogeneitysuchashepatitisCandHIV.d  % % "0;"  0;f0  2  G  .3  0%%  DrawbacksofAmplificationSystems0;f݌P%% Ќ      1.0 ` Potentialforfalsepositiveresultsduetocontaminatingnucleicacids.<` %` %     2.0 ` PCRandLCR,DNAproductsmainsourceofcontamination.(` %` %     3.0 ` QB_replicase_Ԁand_TMA_,RNAproductsarepossiblecontaminants.` %` %     4.0 ` MusthaveproductinactivationaspartofQCprogram.` %` %     5.0 ` Separatepreparationareasfromamplificationareasanduseofinactivationsystemssuch  asUVlighthelpalleviatecontamination. ` %` %     6.0 ` Veryexpensive. l!` %` %     7.0 ` Closedsystem,automationwillalsodecreasenumberofproblems.!X"` %` % "0;"  0;^0  2  H  .3  0%%  FutureofMolecularDiagnosticTechniques0;^݌"D #%% Ќ      1.0 ` Despiteexpensemaybetimesthatrapiddiagnosiswillresultindecreasedcost.#0!$` %` %     2.0 ` Example:Mycobacteriaquickdiagnosisnoneedforexpensiverespiratoryisolation.t$"%` %` %     3.0 ` DetectionofmultidrugresistantM.Tuberculosiswillleadtomoretimelypublichealth `%#& measures.L&#'` %` %     4.0 ` Incrediblyusefulinserologyandmicrobiology.8'$(` %` %     5.0 ` Increasedspecificityandsensitivityofmoleculartestingwillbecomethestandardof $(%) practiceinimmunologyandmicrobiology.)&*` %` %     6.0 ` Testingwillcontinuetobecomemorerapidasassaysareautomatedwhichwillalsobring )'+ downthecosts.*(,` %` %     7.0 ` Authorstateswillnotreplacecultureforroutineorganisms,butitalreadyis,andasDNA +|)- chiptechnologyimproves,theabilitytotestformultipleorganismswillbecome_easier._