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Publié par | ludwig-maximilians-universitat_munchen |
Publié le | 01 janvier 2007 |
Nombre de lectures | 19 |
Extrait
AusdemGastrointestinalLabor
derTierärztlichenFakultätderTexasA&MUniversität,CollegeStation,Texas,USA
Vorstand:SandeeHartsfield,DVM,MS,Dipl.ACVA
AngefertigtunterderLeitungvon
JörgM.Steiner,Dr.med.vet.,PhD,Dipl.ACVIM,Dipl.ECVIM.CA
Vorgelegtüber
Prof.Dr.Dr.habil.JohannesHirschberger
MedizinischeKleintierklinik
derTierärztlichenFakultätderLudwig.Maximilians. UniversitätMünchen
Vorstand:Prof.Dr.Dr.habil.KatrinHartmann
ReinigungundTeilcharakterisierungvoncaniner
neutrophilerElastaseunddieEntwicklungeines
ImmunoassayszurMessungderKonzentrationder
neutrophilenElastaseimSerum
PurificationandPartialCharacterizationofCanine
NeutrophilElastaseandtheDevelopmentofan
ImmunoassayfortheMeasurementofNeutrophilElastase
ConcentrationinSerum
Inaugural.Dissertation
zurErlangungdertiermedizinischenDoktorwürde
derTierärztlichenFakultät
derLudwig.Maximilians.UniversitätMünchen
von
AnjaStoll
aus
Hamburg
München2007
I
GedrucktmitGenehmigungderTierärztlichenFakultät
derLudwig'Maximilians'UniversitätMünchen
Dekan: Univ..Prof.Dr.E.P.Märtlbauer
Referent: Prof.Dr.Hirschberger
Korreferent: Prof.Dr.Potschka
TagderPromotion:9.Februar2007
IITABLEOFCONTENTS
DEDICATION............................................................................................................ VI
ACKNOWLEDGEMENTS.......................................................................................VII
LISTOFABBREVIATIONS..................................................................................... IX
1.INTRODUCTION..........................................................................................1
1.1Hypothesisandobjectives.....................................................................................1
2.LITERATUREREVIEW............................................................................2
2.1.NeutrophilElastase(NE) .....................................................................................2
2.1.1.Nomenclature............................................................................................2
2.1.2.Structure....................................................................................................5
2.1.3.Function ....................................................................................................6
2.1.4.Inhibitionofneutrophilelastase................................................................7
2.2.Neutrophilelastaseanddisease ...........................................................................8
2.2.1.Alpha .proteinaseinhibitordeficiency .....................................................81
2.2.2.Hereditaryneutropenia..............................................................................9
2.2.3.Neutrophilelastaseasamarkerofdiseaseseverityofinflammatory
boweldiseaseinhumans........................................................................10
2.3.Diagnosisofidiopathicinflammatoryboweldiseaseindogs ...........................12
2.3.1SerologicmarkersforcanineIBD ...........................................................13
3.MATERIALSANDMETHODS.............................................................14
3.1.Materials ...........................................................................................................14
3.1.1.ChemicalsandReagents .........................................................................14
3.1.2.Instruments..............................................................................................17
3.1.3.Disposables .............................................................................................19
3.2.Methods.............................................................................................................20
III3.2.1.Purificationofcanineneutrophilelastase ...............................................20
3.2.1.1.Assayforproteolyticactivity..................................................20
3.2.1.2.Preparationofneutrophils.......................................................20
3.2.1.3.Extractionofneutrophilelastase.............................................21
3.2.1.4.Columnchromatography.strongcation.exc hange
chromatography ......................................................................................21
3.2.1.5.Continuous.elutionelectrophoresis ........................................22
3.2.1.6.Sodium.dodecyl.sulfategelelectrophore se(SDS.PAGE) .....22
3.2.2.Partialcharacterizationofcanineneutrophilelastase.............................23
3.2.2.1.Estimationofthemolecularweight ........................................23
3.2.2.2.Estimationofisoelectricpoint ................................................24
3.2.2.3.DeterminationoftheextinctioncoefficientforNE ................25
3.2.2.4.MaterialpreparationforN.terminalaminoacidsequencing..26
3.2.3.Productionofanti.canineneutrophilelastaseantiserum ........................26
3.2.3.1.Determinationofantibodytiter...............................................26
3.2.3.2.Ouchterlonydoubleimmunodiffusionassay ..........................27
3.2.4.Purificationofanti.canineneutrophilelastaseantibodies ......................27
3.2.4.1.Preparationofaffinitychromatographycolumn.....................27
3.2.4.2.Affinitychromatographyofanti.canineNEantibodies..........28
3.2.4.3.Biotinylationofpurifiedantibodies........................................29
3.2.5. Set.upofanenzymelinkedimmunoassay(ELISA)forthemeasurement
ofcanineneutrophilelastaseconentrationinserum...............................29
3.2.5.1.ValidationoftheELISA .........................................................30
3.2.6. Serumcanineneutrophilelastaseconcentrationinhealthydogs............31
3.2.7. Serumcanineneutrophilelastaseconcentrationindogswith
gastrointestinaldisease............................................................................31
4.RESULTS ........................................................................................................33
4.1.PurificationofNeutrophilElastase(NE)...........................................................33
4.2.PartialcharacterizationofcNE ..........................................................................41
4.3.Productionofanti.canineneutrophilelastaseantibodies...................................47
IV4.4.DevelopmentandvalidationofanELISAforthemeasurmentofcNE
concentrationinserum........................................................................................50
4.5.Serumcanineneutrophilelastaseconcentrationsinhealthydogs.....................56
4.6.Serumcanineneutrophilelastaseconcentrationsindogswithgastrointestinal
diseaseorpancreatitis .........................................................................................59
5.DISCUSSION.................................................................................................66
5.1.Purificationofcanineneutrophilelastase..........................................................66
5.2.Partialcharacterizationofcanineneutrophilelastase........................................67
5.3.Productionofanti.canineneutrophilelastaseantibodies...................................68
5.4.DevelopmentandvalidationofanELISAforthemeasurementofcanine
neutrophilelastase...............................................................................................69
5.5.SerumcNEconcentrationsinclinicallyhealthydogs .......................................70
5.6.SerumcNEconcentrationsindiseaseddogs .....................................................70
6.SUMMARY ....................................................................................................73
7.ZUSAMMENFASSUNG............................................................................75
8.REFERENCES..............................................................................................77
APPENDIX..................................................................................................................84
A.1.LegendsforFigures .........................................................................................84
A.2.LegendsforTables...........................................................................................86
A.3.ListofBuffer....................................................................................................87
CURRICULUMVITAE..............................................................................................89
VDEDICATION
Dedicated to my parents,
Karin & Fritz Stoll.
VIACKNOWLEDGEMENTS
First of all, I would like to thank Prof. Dr. Johannes Hirschberger for providing a
contactwiththeGastrointestinalLaboratoryatTexasA&MUniversity.
MyspecialthankstoDr.Jörg Steinerforallowingmetoworkonthisstudyinhis
laboratory, for his mentorship, and his guidance through the completion of this
project.
Further,IwouldliketoexpressmygratitudetoDr.JanSuchodolskiandDr.Craig
Ruaux.Theirexpertiseandencouragementwereinvaluablefortheaccomplishmentof
thisproject.
Special thanks to Kathrin Burke and Nora Berghoff for teaching me some of the
laboratory skills needed to complete this project. I also would like do thank Panos
Xenoulisforhishelp.