Immunochemistry for medical revision.pdf

Biochemistry

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MBM 125: MEDICAL
BIOCHEMISTRY II
-Immunochemistry
Lecturer:Dr. Felix K. Biwott
Contacts: Email;[email protected]
Mobile No: 0697-529-032
KIUT
2
nd
Semester, 2023/2024

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1.Antibodystructureandbiologicalcharacterizations
2.Natureofantigens
3.Antigen-antibodyinteraction
•structureofantigen-bindingsite
•various techniquesusedtostudyantigen
binding
4.Primaryandsecondarybinding.
Specific Objectives

3. Antibodies
•Antibodies,alsoknownasimmunoglobulins(Ig).
•Theyareessentialcomponentsoftheimmune
system.Theyrecognizeandneutralizeforeign
substancessuchaspathogensandtoxins.
•Theyarepresentinbodilyfluids,secretionsandon
thesurfaceofB-cells.
•Antibodiesrecogniseandbindtouniqueepitopes,
whicharemolecularstructuresonthesurfaceoftheir
cognateantigens.
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4. Antibody Structure
•TheyareY-shapedglycoproteinsproducedby
differentiatedB-cellscalledplasmacells.
•Itiscomposedoffourpolypeptidechains:two
identicalheavy(H)chainsandtwoidenticallight
(L)chains.
•Thesechainsareheldtogetherbydisulfidebonds.
•Inaddition,theheavyandlightchainsconsistof
severalamino-acidsequences;eachcorresponding
toaproteindomain.
•Proteinsdomainsarethefunctionalunitsofthe
antibodyandcorrespondtoadiscrete,foldedregion
ofproteinstructure.
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5. Antibody Structure
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6. Antibody Structure
•Therearetwolightchaintypes:κ(kappa)andλ
(lambda)basedonthestructureandaminoacid
sequence.
•Eachantibodycanhaveeithertwoκortwoλchains
butnotoneofeach.Theratioofκandλis2:1.
However,therearenofunctionaldifferencesbetween
thetypes.
•Therearefiveheavychaintypes:
–μ(Mu)chaininimmunoglobulinIgM
–γ(Gamma)chaininimmunoglobulinIgG
–α(Alpha)chaininimmunoglobulinIgA
–ε(Epsilon)chaininimmunoglobulinIgEand
–δ(Delta)chaininimmunoglobulinIgD
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•Eachantibodycontainstwovariableregionsand
oneconstantregion.
•TheFabregions(fragmentantigenbinding)contain
thevariabledomainsofthelightandheavychains.
•Thevariabledomainsmakeupthevariable
regionsoftheantibodywhichgivetheantibodyits
antigenspecificity.
•Therefore,theseregionsdifferbetweenantibodies.
•EachFabregionalsocontainstwoconstant
domains;onefromtheheavychaincomponentand
onefromthelightchaincomponent.
Fab and Fc regions

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•TheFcregion(fragmentcrystallisable)consistsof
theremainingconstantdomainsfromthetwoheavy
chains.
•TheFcregioninteractswithdifferentimmunecells
andmediatesvariousfunctions.Forexample,
opsonisation.
•Theconstantregioninvolvestheconstantdomains
fromboththeFabandFcparts.Theheavy
chainconstantdomainsdetermineantibodyclass
andarethesameforallantibodiesofthesameclass.
•IgAandIgGantibodiesalsohavehingeregions,
whichareflexibleamino-acidchainsinthecentral
partoftheheavychains.
Fab and Fc regions

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Fab and Fc regions

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Majorimmunoglobulins
IgG
•IgGisthemostabundantantibodyclass.Itis
presentonthesurfaceofmatureB-cellsandin
serum.Therearefoursubclasses:IgG1,IgG2,IgG3
andIgG4;inorderofserumconcentration.IgGis
theonlyantibodytocrosstheplacentaand
consequently,ittransferspassiveimmunityfrom
mothertofoetus.
•Newborns,therefore,havehighIgGconcentrationsin
thefirst3-6monthsoflife.
Classification

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Majorimmunoglobulins
IgA
•IgAisthemostprevalentantibodyinsecretions,
suchassalivaandmucous.
•Therearetwosubclasses,IgA1andIgA2.
•IgAformsadimer,whereajoiningchainconnects2
Y-shapedmolecules,givingitfourantigen-binding
sitesintotal.
•IgAantibodiesareresistanttoenzymaticdigestion
andactprincipallyasneutralisingantibodies.
•BreastmilkandcolostrumhavehighlevelsofIgA
whichcoattheaerodigestivetracts;protecting
againstinfectionsinbreast-fedbabies.
Classification

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Majorimmunoglobulins
IgM
•IgMantibodiesareexpressedonthesurfaceofB-
cellsasmonomersbutsecretedaspentameters.
•Apentameterhasfiveantibodiesconnectedbya
joiningchain,withtenantigen-bindingsitesintotal.
•Itisthefirstimmunoglobulinproducedduringfoetal
developmentandthefirsttobeproducedbyB-cells
againstanewinfection.
•IgMhashighavidity,meaningtheantibody-antigen
complexisstrong,butlowaffinity,sothestrengthofa
singleepitope-antibodyinteractionisweak.
Classification

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Minorimmunoglobulins
IgD
•IgDispresentonthesurfaceofB-cells.Ithasarole
inB-cellandantibodyproduction.AllnaiveBcells
expressIgDandIgM.
IgE
•IgEismainlyfoundonmastcellsbutisalsopresent
atlowlevelsinthebloodandextracellularfluid.Itis
associatedwithallergy,particularlytype
Ihypersensitivityreactions,includingatopicdisease
(e.g.asthmaanddermatitis)andanaphylaxis.
•Ittriggershistaminereleasefrommastcellsand
basophils.IgEisalsopartofthebody’sresponseto
parasiticinfections.
Classification

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Classification

15. Antigens
•Antigensaremoleculesormolecularstructuresthat
areforeigntothebodyandgenerallyinducean
immunereactionintheformoftheproductionof
antibodiesagainstthem.
•Insimplewords,antigenscanbeanythingthat
doesn’tbelongtothebodyandareforeign.
•Eventhoughantigensareusuallydefinedbythe
inductionofanimmuneresponse,allantigensmight
notinduceanimmuneresponse.Theantigensthat
inducearesponsearetermedimmunogens.
•Theabilityofantigenstoelicitanimmuneresponse
dependsonthepresenceofspecificregionsonthe
antigenscalledantigenicdeterminants.
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16. Antigens
•Thedeterminantsbindtoreceptormoleculeswiththe
complementarystructureonimmunecellstoelicita
response.
•Mostantigensinhumansareproteins,peptides,or
polysaccharides;however,lipidandnucleicacidscan
alsoactasantigenswhencombinedwithproteinsor
polysaccharides.
•Inaddition,antigensmightalsobeintentionally
introducedintothebodyintheformofvaccinesin
ordertoinducetheadaptiveimmunesystemofthe
bodyagainsttheantigen.
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17. Properties of Antigens
•Antigenshavedifferentpropertieswhichdetermine
theimmunogenicityoftheantigensandthusare
essentialinordertounderstandtheimmunereaction
againstthem.
•Since these propertiesdetermine the
immunogenicity,theseareconsideredproperties
requiredtoformagoodantigen.
•Thefollowingaresomeofthepropertiesofantigens;
1.ForeignNature
2.ChemicalNature
3.MolecularSize
4.MolecularRigidityandComplexity
5.AntigenicDeterminantsandCross-reactivity
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18. 1. Foreign Nature
•Allantigensthatinduceanimmuneresponseinthehost
areforeigntothebodyoftherecipient.
•Thehostbodyrecognizestheantigentobedifferent
fromthenormalbodycomponents.
•Theimmunogenicityoftheantigenincreaseswiththe
increaseinthedegreeofforeignness.Inthecaseof
biologicalantigens,theforeignnessincreaseswiththe
increaseinthephylogeneticgapbetweenthetwo
species.
•However,therearesomeexceptionsinthatsome
proteinsoccurringwithinthehostmightalsoinducean
immuneresponse,asinthecaseofautoantigens.
•Similarly,proteinsandothermoleculesfromother
speciesmightalsonotinduceanimmuneresponseif
theylackantigenicdeterminantsorepitopes.
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19. 2. Chemical Nature
•Themostpotentandcommonlyencountered
antigensareproteinsfollowedbypolysaccharides.
•However,othermoleculeslikelipidsandnucleic
acidscanalsoactasantigenswhencomplexwith
proteinsandpolysaccharides.
•Inthecaseofproteins,theantigenshouldcontain
immunogenicregionswithatleast30%ofamino
acidslikelysine,glutamine,arginine,glutamicacids,
aspargine,andasparticacid,alongwithahigh
numberofhydrophilicorchargedgroups.
•Thelevelofimmunogenicityalsoincreaseswiththe
heterogenicityofthemolecules.Homopolymersare
usuallylessimmunogenicthanheteropolymers.
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20. 3. Molecular Size
•Themolecularsizeoftheantigensisalsocrucialin
theimmunogenicityofthemolecules.
•Ithasbeenestablishedthatantigensshouldhavea
minimumsizeofgreaterthan5000Dabeforethey
canbeconsideredimmunogenic.
•However,lowmolecularweightsubstancescan
demonstrateimmunogenicitywhencoupledwith
large-sizedcarriers.
•Thelowmolecularweightsubstancesaretermed
haptensthatareconsidered‘partialantigens’withat
leastoneantigenicdeterminant.
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21. 4. Molecular Rigidity and Complexity
•Therigidityandcomplexityofmoleculesareessential
factorsthatdetermineimmunogenicity.
•Ingeneral,rigidmoleculesaregoodantigensasthey
canraiseantibodiestocertainstructureswhen
comparedtothelessrigidones.
•Thecomplexityofthestructureisalsoanessential
factorasapeptideantigenwitharepeatingunitofa
singleaminoacidislessimmunogenicthana
moleculewithtwoormorerepeatingaminoacids
units.
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22. 5. Antigenic Determinants and Cross-reactivity
•Antigenicdeterminantsareregionsinanantigen
moleculethatisinvolvedinthereactionwith
antibodies.
•Usually,antigenswithtwoormoreantigenic
determinantscaninduceantibodyproduction.Thus,
asmallerantigenusuallydoesn’tinduceantibody
productionasitisnotpossibleforasmallmoleculeto
havemorethanoneantigenicdeterminant.
•Cross-reactivityofantigensisalsoanessentialfactor
whereantibodiesinducedbyadifferentantigencan
interactwithanotherantigen.
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23. Types of Antigens
•Antigenscanbegroupedintodifferenttypesbased
ondifferentfactors.Someofthecommon
classificationsarebasedontheoriginofthe
antigenanditsimmunogenicity.
•A.TypesofAntigenontheBasisofOrigin:
•ExogenousAntigens:antigenwhichareproduced
outsidethebodyandstartcirculatinginthebodyfluid
andtrappedbyantigenprocessingcellssuchas
macrophages,dendriticcellsandthisprocessis
phagocyticinnature.Examples:bacteria,viruses,
fungietc.Buttherearesomeantigenswhoareearlier
exogenousinnaturebutlaterbecomeendogenousin
nature,thesetypesofantigenknownasintracellular
antigen.
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24. Types of Antigens
•EndogenousAntigen:Theyareproducedinsidethe
cellasaresultofnormalcellmetabolismorbecause
ofanybacterialcellinfection.Thefragmentsarethen
presentonthesurfaceoftheinfectedcellsinthe
complexwithMHCclass1molecules.
•Autoantigens:Theyareselfproteinornucleicacid
thatisattackedbythehostimmunesystem,andthey
causeautoimmunedisease.Theyareformedmainly
duetogeneticandenvironmentalfactors.
•TumorAntigens:ThesearepresentedbyMHC
moleculesonthesurfaceoftumorcells.Theyresult
intumor-specificmutationduringtransformationof
normalcellsintocancercells.
•
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25. Types of Antigens
•NativeAntigens:Itisanantigenthatisnot
processedbyanAPCtosmallerparts.Tcellscannot
bindtonativeantigen,whereasBcellscanbe
activatedbynativeones.
•B.OntheBasisofImmuneResponse:
1.Immunogen:Theyareproteinorpolysaccharides
andcangenerateanimmuneresponseontheirown.
2.Hapten:Thesearenon-protein,foreignsubstances
thatrequirecarriermoleculestoinduceanimmune
response.
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26. Structure of Antigens:
•Themolecularstructureofanantigenis
characterizedbyitsabilitytobindtotheantigen-
bindingsiteofanantibody.
•Antibodiesdifferentiatebetweendifferentantigenson
thebasisofthespecificmolecularstructurespresent
onthesurfaceoftheantigen.
•Mostantigensareproteinsorpolysaccharides.These
canincludecoats,capsules,flagella,toxins,and
fimbriaeofbacteria,viruses,orother
microorganisms.Besides,secretionsandother
chemicalsofthesamenaturecanalsoactas
antigens.
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27. Structure of Antigens:
•Lipidsandnucleicacidsofthesemicroorganismsare
onlyantigenicwhenthesearecombinedwithproteins
orpolysaccharides.
•Thestructureofantigensmightbedifferent
dependingonthenatureoftheantigen,theirsize,
andimmunogenicity.
•Allimmunogenicantigenshaveaspecificstructural
componentcalledepitopeorantigenicdeterminant.
•Thenumberofepitopesdiffersindifferentantigens
anddeterminesthenumberofantibodiesasingle
antigencanboundto.
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28. Structure of Antigens:
•Thestructuralcomponentsofinteractioninantigens
aredifferent,whichdeterminestheclassesof
antibodiestheyboundto.
•Theregiononantibodiesthatinteractswithantigens
iscalledaparatope.Ithasbeenestablishedthatthe
structureofepitopeandparatopecanbedefinedwith
alockandkeymetaphorasthestructuresare
specificandfitwithoneanother.
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29. Difference Between Antigen and Antibody
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30. Antigen-Antibody Interaction
•Antigen-Antibody(Ag-Ab)Interactionisa
biochemicalreactionbetweenantibodiesandspecific
antigens.
•Itisthebindingofparatopesofantibodiestospecific
antigensontheirepitopesthatinitiatesaseriesof
immunologicalresponsestoactagainstthe
respectiveantigensfortheirremovalordestruction.
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31. Antigen-Antibody Interaction
•ThereactionbetweenAgandAboccurinthree
stages.
Infirststage,therectioninvolvesformationofAg-
Abcomplex.
Thesecondstageleadstovisibleeventslike
precipitation,agglutinationetc.
ThethirdstageincludesdestructionofAgorits
neutralization.
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32. Salient features of Ag-Ab reaction:
•SpecificityofAg-Abreaction
•Immunecomplex
•BindingsiteofAg-Abreaction
•BindingforceofAg-Abreaction
•SpecificityofAg-Abreaction
•ThereactionbetweenAgandAbishighlyspecific.
•Specificityreferstotheabilityofanindividual
antibodycombiningsitetoreactwithonlyone
antigenicdeterminantortheabilityofapopulationof
antibodymoleculestoreactwithonlyoneantigen.
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33. Salient features of Ag-Ab reaction:
•Forexample,theantibodyproducedagainstlens
antigenwillreactonlywithlens-antigen.Similarly,the
antibodyproducedagainstkidneyantigenwillreact
withonlykidney-antigen.Astandardlockcanbe
openedbyitsownkeyonlyasoneantibodycanreact
withitsownantigen.
•Immunecomplex
•Animmunecomplexisformedfromtheintegral
bindingofanantibodytoasolubleantigen.
•Theboundantigenactingasaspecificepitope,
boundtoanantibodyisreferredtoasasingular
immunecomplex.
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34. Salient features of Ag-Ab reaction:
•BindingsiteofAg-Abreaction
•Inantigen-antibodyreaction,theantibodyattaches
withtheantigen.
•Thepartofantigenwhichcombineswithantibodyis
calledEpitope.
•Anepitope,alsoknownasantigenicdeterminant,is
thepartofanantigenthatisrecognizedbythe
immunesystem,specificallybyantibodies,Bcells,or
Tcells.
•Thepartofanantibodythatrecognizestheepitopeis
calledaparatope.
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35. Salient features of Ag-Ab reaction:
•BindingforceofAg-Abreaction
•ThebindingbetweenantigenandantibodyinAg-Ab
reactionisduetothreefactorsnamely:
Closenessbetweenantigenandantibody.
Non-covalentbondsorintermolecularforces.
Affinityofantibody.
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36. Salient features of Ag-Ab reaction:
Closenessbetweenantigenandantibody:when
antigenandantibodyarecloselyfit,thestrengthof
bindingisgreat.Whentheyareapartbinding
strengthislow.
Non-covalentbondsorintermolecularforces:the
bondsthatholdtheantigentotheantibodycombining
siteareallnon-covalentinnature.Theseinclude
hydrogenbonds,electrostaticbond,vanderwaals
forcesandhydrophobicbonds.
Affinityofantibody:antibodyaffinityisthestrength
ofthereactionbetweenasingleantigenic
determinantandasinglecombiningsiteonthe
antibody.
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37. Salient features of Ag-Ab reaction:
StrengthofAg-Abreaction
•Thenon-covalentinteractionthatformthebasisof
antigen-antibodybindingincludehydrogenbond,
ionicbond,hydrophobicinteractionandVanderwalls
interaction.
•Astrongantigen-antibodyinteractiondependsona
veryclosefitbetweentheantigenandantibodywhich
requireshighdegreeofspecificity.
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38. Salient features of Ag-Ab reaction:
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39. Properties of Ag-Ab reaction:
•Affinityofantibody:
•Interactionsbetweenantigenandantibodyinvolve
non-covalentbindingofanantigenicdeterminant
(epitope)tothevariableregion(complementarity
determiningregion,CDR)ofboththeheavyandlight
immunoglobulinchains.Theseinteractionsare
analogoustothoseobservedinenzyme-substrate
interactionsandtheycanbedefinedsimilarly.
•Avidityofantibody:
•ItisthestrengthofthebondaftertheformationofAg-
Abcomplexes.
•ItisusedtodenotetheoverallcapacityofAntibodies
tocombinewiththemultivalentantigen.
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40. Properties of Ag-Ab reaction:
•AmultivalentAghasmanytypesofantigenic
determinants.
•Wheninjectedintotheblood,eachantigenic
determinantstimulatestheproductionofaparticular
antibody.
•ThevariousantibodiesproducedbyasingleAg
combinewiththedifferentantigenicdeterminantsof
theAg.
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41. Types of Ag-Ab reaction:
•ThetypesofAg-Abreactionsare:
•Precipitationreaction
•Agglutinationreaction
•Complementfixation
•ELISA-EnzymeLinkedImmunoSorbentAssay
•Immunofluorescence
•Precipitationreaction
•WhenasolubleAgcombineswithitsAbinthe
presenceofanelectrolyte(NaCl)ataparticular
temperatureandpH,itformsaninsolubleprecipitate
ofAg-Abcomplex.TheAbcausingprecipitationis
calledprecipitinandthereactioniscalled
precipitationreaction.
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42. Types of Ag-Ab reaction:
Functionofprecipitationreaction:precipitation
occursintwomedia:
Liquid
Gel
Precipitationinliquid:
•Ag-Abreactionperformedbyplacingaconstant
amountofAbinaseriesoftubesandadding
increasedamountofAg.Ag-Abreactstogether
resultinginprecipitation.Plottingtheamountof
precipitateagainstincreasingantigenconcentration
yieldsaprecipitationcurve.
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44. Types of Ag-Ab reaction:
Precipitationingel:
•RadialImmunodiffusion(Mancini):
•Inthesemethod,agargelorsimilargelsareusedon
platesorpetriplates.BothAgandAbdiffusefreelyin
thegelsysteminalldirections.
•Atacertainpointdependingontherateofdiffusion
andconcentrationofthereactants,azoneof
equivalencewillbeformed,whichisseenasavisible
precipitation.
•IfAgorAbpreparationsarecomplex,multiplebands
form.Theseareagainof2types-Singlediffusion
methodsanddoublediffusionmethods
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46. Types of Ag-Ab reaction:
Agglutinationreaction:
•WhenaparticularAgismixedwithitsAb’sinthe
presenceofelectrolytesatasuitabletemperature
andpH,theparticlesareclumpedoragglutinated.
•TheAboftheserumcausesthecellularAg’stoform
clumpsandthesearecalledAgglutinins.
•Theparticulateantigensthatareaggregatedare
termedasAgglutinogens.
•Slideagglutination:thisisarapidmethodto
determinethepresenceofagglutinatingantibodies.
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47. Types of Ag-Ab reaction:
Complementfixation:
•LysisofRBCorbacteriarequiressomenon-specific
unstablecomponentsoffreshserumwhicharecalled
complement.
•Thiscomplementsystemcomprisesof11proteins
andarepresentineverindividual.TheybindtoFc
componentofAbinvolvedinAg-Abcomplex.This
abilityoftheAg-Abcomplextofixcomplementis
usedincomplementfixationtest.
•Inthefirststage,thetestAgandtheantiserumare
mixedinthepresenceofknownamountof
complement.
•IfAbspecificfortheAGispresentintheserum,Ag-
Abcomplexwillbeformedthatwillfixthe
complement.
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48. Types of Ag-Ab reaction:
ELISA-EnzymeLinkedImmunoSorbentAssay:
•In1971,enzymelabelledAg’sandAb’swere
developedasserologicalreagentsfortheassayof
Ab’sandAg’s.
•Theseareverysimple,sensitive,economicandless
hazardwhencomparedtoRIA.
•Theligandusedhereisamoleculewhichcandetect
theAbandiscovalentlycoupledtoanenzymesuch
asperoxidase,betagalactosidase,alkaline
phosphataseetc.
•ELISAisof3types:
•IndirectELISA
•SandwichELISA
•CompetitiveELISA
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49. Types of Ag-Ab reaction:
IndirectELISA:
•ThistechniqueisusedforthedetectionofHIV.The
envelopproteinsaredevelopedbyrecombinant
technologyandcoatedonthesurfaceofthemicrotire
plates.Suspectsserumisadded,andunbound
proteinsarewashedoff.
•SandwichELISA:
•UsedtodetectthepresenceofAginasample.The
welliscoatedwithAbspecifictotheAgandthen
suspectserumisaddedandallowedtoreact.The
wellsarewashedtoremoveunboundAg’s.
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50. Types of Ag-Ab reaction:
•ThenalabelledAbagainstadifferentepitopeofthe
Agisadded.UnboundAb’sareremovedbywashing
andthisisfollowedbyadditionofcoloredsubstrate
anddevelopmentofcolor.Theintensityofthecoloris
directlyproportionaltotheconcentrationoftheAgin
theserum.
•CompetitiveELISA:
•Anothervariationformeasuringamountsofantigenis
competitiveELISA.Inthistechnique,antibodyisforst
incubatedinsolutionwithasamplecontaining
antigen.
•TheAg-AbmixtureisthenaddedtoanAgcoated
microtiterwell.
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51. Types of Ag-Ab reaction:
•Themoreantigenpresentinthesample,thelessfree
antibodywillbeavailabletobindtotheAg-coated
well.additionofanenzyme-conjugatedsecondary
antibody(Ab2)specificfortheisotypeoftheprimary
antibodycanbeusedtodeterminetheamountof
primaryantibodyboundtothewellasinanindirect
ELISA.
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52. Types of Ag-Ab reaction:
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53. Application of Ag-Ab reaction:
•ThechiefuseofAg-Abreactionsare:
•Determinationofbloodgroupsfortransfusion.
•Serologicalascertainmentofexposuretoinfectious
agents.
•Developmentofimmunoassaysforthequantification
ofvarioussubstances.
•Todetectthepresenceorabsenceofproteinin
serum.
•Determiningthecharacteristicsofcertain
immunodeficiencydisease.
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54. Primary and Secondary Immunoresponses
•Whenanantigenisinjected,antibodiesinblood
appearwithinabout10days,reachapeaklevel
within20daysandresponsedeclinesbyabout30
days.ThelgMmoleculeswillbepredominantinthis
primaryresponse.
•Whenthesameantigenisreinjectedintothesame
animalafterafewmonths,theantibodyresponseis
quicker(within3days),stronger(100to1000times
morequantityofantibody),moreavid(lgGtype)and
moreprolonged(responselastsformonths).Thisis
thesecondaryimmunoresponse,whichisdueto
thememorycellsproducedintheprimaryresponse.
Thisisthebasisofimmunization.
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55. Primary and Secondary Immunoresponses
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56. Primary and Secondary Immunoresponses
•Activeimmunityisinducedbyimmunizationwith
toxoid,orkilledorattenuatedorganisms.Examples
arediphtheria,pertussis,tetanus(DPT)vaccine,oral
poliovaccineandhepatitisBvaccine.
•Inpassiveimmunity,protectionisgivenby
preformedantibodies.Thisisusedinimmuno-
therapyagainstdiphtheria,tetanus,snake-bites,etc.
7/16/2024 56FKBIWOTT/MBM125/2023-2024

57. 7/16/2024 57FKBIWOTT/MBM125/2023-2024
THANK YOU