Antibody Molecules Making Antibodies 10 Talking About Antibodies 13 Finding and Getting Antibodies 14 Choice of Primary 1 Antibodies 15 Antibodies Handling and Storing 16 Recommended Storage Freezer 20 16 Reco ID: 35911
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Chapter2 Antibodies Keywords Immunohistochemistry · Antibodylabeling · Fluorescencemicro- scopy · Fluorescentimmunocytochemistry · Fluorescentimmunohistochem- istry · Indirectimmunocytochemistry · Immunostaining Contents Introduction ................................... 7 AntibodyMolecules ............................... 8 MakingAntibodies ................................ 10 TalkingAboutAntibodies ............................. 13 FindingandGettingAntibodies .......................... 14 ChoiceofPrimary(1 )Antibodies ......................... 15 AntibodiesHandlingandStoring ......................... 16 RecommendedStorageFreezer,Ð20 C ..................... 16 RecommendedStorageRefrigerator,4 C .................... 16 Introduction Anantibody(Ab)isthekeyreagentofimmunocytochemistry.Touseantibodies effectively,considertheirstructure,function,andgeneration.Suchbasicknowledge aboutantibodiesisessentialtosucceedinidentifyingsuitableexperimentaldesign, Þndingantibodies,andtrouble-shootingproblems. Immunocytochemistrytakesadvantageofthreepropertiesofantibodies: 1.Antibodiesuniquelybindtoaproteinorothermolecule. 2.Antibodybindingtomoleculesisessentiallypermanentatphysiological conditions. 3.Newantibodiescanbemadetailoredtonewinterestingmolecules. 7 R.W.Burry, Immunocytochemistry ,DOI10.1007/978-1-4419-1304-3_2, C SpringerScience+BusinessMedia,LLC2010 8 2Antibodies AntibodyMolecules Animmuneresponsegeneratesantibodiesorproteinscalled immunoglobulins(Igs) . AntibodiesarefurtherclassiÞedintomultiple isotypesorclasses (Table2.1).In immunocytochemistry,the IgG isotypeispreferredbecauseitsgenerationandbind- ingismoreconsistent.IgMantibodiescanbeusedifnootherisotypeisavailable. TheIgGmoleculescanbebrokendownintofour subclasses ,IgG1,IgG2,IgG3, andIgG4.Inimmunocytochemistryexperiments,thesesubclassesdonotmatterfor mostspeciesofanimals,buttheyareimportantforantibodiesgeneratedinmouse monoclonalantibodies(IgG1,IgG2a,IgG2b,andIgG3),aswewillseeinlater chapters. Table2.1 Igisotypes AntibodyisotypeAction IgAGut,respiratory,urinaryresponse IgDInitialimmunesystemresponse IgEResponsetoallergensÐhistaminerelease IgGImmuneresponsetoinvadingpathogens IgMEarlyimmuneresponseforpathogens Inusingantibodies,knowledgeoftheIgGstructureisimportant(Fig.2.1). IgG hasaconstantregionandavariableregion .The constantregion containsspecies- speciÞcsequencesandthe Fcportion thatbindsanFcreceptor(Fig.2.1,clearend), whichisfoundoncirculatingwhitecells,macrophages,andnaturalkillercells. TheFcportionalsohasspecies-speciÞcsitesthatareuniquetotheanimalspecies Fig.2.1 Theantibody.An IgGantibodyhasasingle constantregion( white )with theFcportionandthe species-speciÞcantigens.The variableregion( gray ) containstheFabportionthat bindstheepitopeportionof theantigen.Thesmall protein,onlyinthevariable region,isknownasthelight chain;thelargeproteinthatis partoftheconstantand variableregionistheheavy chain.TheIgGcanbe digestedbytheprotease enzyme,papain,intoanFc end(constantend)andaFab end(variableend)