Manymonoclonalantibodiesofmouseoriginarevaluablediagnosticagents.TheirproductionbyclassicalhybridomatechniquesisfrequentlylimitedbytheinstABIlityofcelllines,lowantibodyyieldsandthelimitationsofimmunizingmicewithtoxicantigens.Apromisingalternativetothehybridomatechnologyistheproductionofrecombinantantibodies.PioneeringworkofthelastdecadeshowedthatitispossIBLetoamplifyrearrangedimmunoglobulingenesfromB-lymphocytes,toinsertthemintodifferentvectors,andtoexpresstheminbacteria,yeast,insect,mammalianorplantcells.Moreover,therandomizedcombinationofclonedheavyandlightchainimmunoglobulingenefragmentsallowedtheconstructionofmouseantibodylibraries.Theselibrariesenabletheisolationofspecificantibodiesagainstparticularantigensbyphagedisplaytechniques.Oneprerequisiteforgeneratinghighlydiversifiedmouseantibodylibraries,however,isthedevelopmentofPCRprimerscapableofamplifyingallrearrangedimmunoglobulingenes.Inimmunoglobulinrepertoirelibrarycloning,thehomologybetweenaparticularprimersequenceanditstargettemplate,aswellasthediversityofaprimerpoolarethetwomostimportantparameterswhichdeterminethecloningefficiencyandthesizeofaresultingrepertoirelibrary.ThisscreeningstrategyallowstheamplificationofrearrangedmouseimmunoglobulingenesofindividualBcellclonesaswellasoflargerBcellpopulationsfortheconstructionofmousescFv-antibodylibraries.
KitComponents
PrimerSet1forPCRAmplificationI1904-10A1:HeavyChainVariablePrimer(Set1)11x20ulI1904-10A2:HeavyChainConstantPrimer(Set1)1x220ulI1904-10A3:LightChainVariable(kappa)Primer(Set1)10x20ulI1904-10A4:LightChainConstant(kappa)Primer(Set1)1x220ulI1904-10A5:LightChainVariable(lam
BDa)Primer(Set1)1x20ulI1904-10A6:LightChainConstant(lambda)Primer(Set1)1x20ulPrimerSet2forCloningI1904-10B7:HeavyChainVariablePrimer(Set2)11x20ulI1904-10B8:HeavyChainConstantPrimer(Set2)1x220ulI1904-10B9:LightChainVariable(kappa)Primer(Set2)10x20ulI1904-10B10:LightChainConstant(kappa)Primer(Set2)1x20ulI1904-10B11:LightChainConstant(lambda)Primer(Set2)1x220ulI1904-10B12:LightChainConstant(lambda)Primer(Set2)1x20ul
PCRamplification(Set1)andcloning(Set2)ofmouseIgGheavyandlightchainvariabledomaincodingregions.
Applications
GenerationoflargerepertoiresofrearrangedimmunoglobulinvariabledomaincodingregionsfortheconstructionofmouseIgGscFv-antibodylibraries.AmplificationofimmunoglobulinvariablegenefragmentsfromsingleBcellclones.
RecommendedDilution
Workingdilution10pmol/ul(Ready-to-useforcommonPCR).DilutionbufferH2O.CategoryPCRoligonucleotideprimer.
StorageandStability
Storeat-20°C.Formaximumrecoveryofproduct,centrifugetheoriginalvialafterthawingandpriortoremovingthecap.Aliquotsarestablefor6months.
References
1.Zhou,H.,etal.,NucleicAcidsResearch22:888-889(1994).2.Breitling,F.,etal.,Gene104:147-153(1991).3.Dübel,S.,etal.,Gene128:97-101(1993).
