Transcription of Pharmaceutical Microbiology ( 3T5)
1 Antigen and Antibody ReactionPharmaceutical Microbiology ( 3T5) ByDr. AbhishekPandeyAssistant ProfessorSchool of Studies in Pharmaceutical Sciences, Jiwaji University, : may be abbreviated as Ag -Ab reaction. Theseformthebasisforhumoralimmunityorant ibodymediatedimmunity. Thesereactionsformthebasisfordetectionof infectiousdiseasecausingagentsandalsosom enonspecificAg slikeenzymes. WhenAg-Abreactionsoccurinvitro, ThereactionsbetweenAgandAboccurinthreest ages. Infirststagethereactioninvolvesformation ofAg-Abcomplex. Thesecondstageleadstovisibleeventslikepr ecipitation,agglutinationetc. ThethirdstageincludesdestructionofAgorit sneutralizationSalient Features of Antigen -Antibody Reaction: SpecificityofAntigen-AntibodyReaction. Immunecomplex. BindingSiteofAntigen-AntibodyReaction.
2 Of Antigen -Antibody Reaction: antibody binds to a specific antigen; an interaction similar to a lock and key. Forexample, , : Animmunecomplexisformedfromtheintegralbi ndingofanantibodytoasolubleantigen. Theboundantigenactingasaspecificepitope, boundtoanantibodyisreferredtoasasingular immunecomplex. ,opsonizationoftargetcells,assemblyofmem braneattackcomplexesandreleaseofcompleme ntactivatorsforchemotaxis. Fcreceptormediatedcellactivationtriggers cellularresponses,suchasphagocytosis,ant ibody-dependentcellularcytotoxicity(ADCC ) +Ab- Ag-AbcomplexAntigen -Antibody ReactionBinding Site of Antigen -Antibody Reaction: Inantigen-antibodyreaction,theantibodyat tacheswiththeantigen. Thepartofantigenwhichcombineswithantibod yiscalledEpitope. Anepitope,alsoknownasantigenicdeterminan t,isthepartofanantigenthatisrecognizedby theimmunesystem,specificallybyantibodies ,Bcells,orTcells.
3 Of Antigen -Antibody Reaction:The types of antigen -antibody reactions are: Precipitation Reaction. Complement -Enzyme linked immunosorbent Reaction:.WhenasolubleAgcombineswithitsA binthepresenceofanelectrolyte(NaCl)atapa rticulartemperatureandpH, complex Function of precipitationoccurs in two media:reaction: Precipitation>Liquid. > in AddedPrecipitation curve shows three :RadialImmunodiffusion(Mancini):In these methods agar gel or similar gels are used on plates or petriplates. Both Ag and Ab diffuse freely in the gel system in all directions. At a certain point depending on the rate of diffusion and concentration of the reactants, a zone of equivalence will be formed, which is seen as a visible Ag or Ab preparations are complex, multiple bands form.
4 These are again of 2 types-Single diffusion methods and double diffusion IMMUNODIFFUSIONA ntigendiffusionAntibodyincorporatedAntig enin agarPrecipitateforms ringDOUBLE IMMUNODIFFUSIONA ntigenAgar matrixPrecipitatePrecipitation reactions in gelsAgglutination Reaction: WhenaparticularAgismixedwithitsAb sinthepresenceofelectrolytesatasuitablet emperatureandpH,theparticlesareclumpedor agglutinated. TheAboftheserumcausesthecellularAg stoformclumpsandthesearecalledAgglutinin s. Theparticulateantigensthatareaggregateda retermedAgglutinogens.> tae ot po** ccrtroi rwge* i to cede 1130* C>DC otl**l n *>each c* tx atcktit thornCHpOMtM iHt 4d itioolTEST CULTUQE2 One'Jrccdccr*o/ e*prt addedtocrfc*24 TwoocfflpMicobtmweqxtJyardcorrcMe^rimiaA edMbf#>agentncvdef)Slide AgglutinationToauniformsuspensionofparti culateAg, (Haemagglutination)andcrossmatching.
5 > , , sserum,ismeasuredbyaddingaconstantamount ofantigen(Solmonellatyphi) Agglutination> , (tannedRBC) , AgglutinationAgglutination :FirsthomepregnancytestHapten carrier conjugate Anti HCG antibodyObserve for visible clumpingUrine + Anti HCGPOSSIBLE REACTIONS(^)reaction: not pregnantVisible clumpingNovisibleclumpingurineComplement Fixation: LysisofRBCorbacteriarequiressomenon-spec ificunstablecomponentsoffreshserumwhicha recalledcomplement. Inthefirststage,thetestAgandtheantiserum (heatedto56oCtoinactivatecomplement) ,Ag-; Fixation TestSefiim viilhSerum v^tlioutantiuod lesAiitiqeii biruiR1J n UOIIIHIv *th anti bodiesAntigenCo mplemenl11 nu QttrwlbnikwithcomplementAg.'Ab complexH RmolysmH emoty-smSetts it I z ed ret*S eits rt i zedblood collsRHt-s serveAUindicatorasimliLdturRBCs lysed tryKliLs settle intounijoiinda pelletcomplementno lysislysisReactiveMon re activeELISA EnzymeLinkedImmunoSorbentAssay:In1971,en zymelabeledAg sandAb sweredevelopedasserologicalreagentsforth eassayofAb sandAg s.
6 Theseareverysimple,sensitive,economicand lesshazardwhencomparedtoRIA. Theligandusedhereisamoleculewhichcandete cttheAbandiscovalentlycoupledtoanenzymes uchasperoxidase,betagalactosidase, ,andunboundproteinsarewashedoff.> of > , more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well as in an indirect fluorescence: Fluorescenceisthepropertyofabsorbingligh traysofoneparticularwavelengthandemittin grayswithadifferentwavelength. Fluorescent dyes show up brightly under UV light as they convert into visible (1942)showedthatlabeleddyescanbeconjugat edtoAb sandtheselabeledantibodiescanbeusedtodet ectAg s.
7 Dyesthatarecommonlyusedinclude:Fluoresce in,anorganicdyethatisthemostwidelyusedla belforimmunofluorescenceprocedures,absor bsbluelight(490nm)andemitsanintenseyello w-greenfluorescence(517nm).Phycoerythrin isanefficientabsorberoflight(-30foldgrea terthanfluorescein)andabrilliantemittero fredfluorescence, and indirect ImmunofluorescenceApplicationofAntigen-A ntibodyReaction:Thechiefuseofantigen-ant ibodyreactionsare: Determinationofbloodgroupsfortransfusion . Serologicalascertainmentofexposuretoinfe ctiousagents. Developmentofimmunoassaysforthequantific ationofvarioussubstances. Todetectthepresenceorabsenceofproteinins erum. ,thetopichasbeenincluded, youYou
