Characterisation of Anti-glucagon-like Peptide-1 Antibodies Utilising Surface Plasmon Resonance

114 Pages·2015·2.69 MB·English

Nanotechnology
InstituteofPhysicsandNanotechnology
Skjernvej4A,9220AalborgOest
Phone[phone],Fax[phone]
www.nano.aau.dk
Title:
Characterisation of Anti-glucagon-like
Peptide-1 Antibodies Utilising Surface
PlasmonResonance
Synopsis:
ProjectPeriod: Thethesisexploredtheversatilityofsurfaceplas-
Master thesis, September 2014 to mon resonance to characterise anti-GLP-1 anti-
September2015 bodiesthroughkinetic,thermodynamicandsand-
wichanalysis. Theantibodiesdisplayeda(cid:14)nities
in the range of 2.93*10(cid:0)8-3.56*10(cid:0)7 M. The in-
teractionswerefoundenthalpydriven,describing
Author:
theformationandstabilisationofhydrogenbonds
LasseThomsen
at lower temperatures and consequently higher
a(cid:14)nities. Furthermore, antibodies displayed en-
hanceda(cid:14)nitiesof1.21*10(cid:0)9-4.87*10(cid:0)9Mwhen
servingassandwichantibodies.
ProjectSupervisor: In addition, pair-wise epitope mapping was per-
formed to investigate sandwich combination po-
LeonidGurevich
tential and specificity of the antibodies. The re-
sults suggested a specific epitope recognition or-
der starting from the N-terminal of ABS 033-
04,HYB147-13,HYB147-12,HYB147-08and
ABS047-03.
CompanyContactPerson: In comparison with enzyme-linked immunosor-
KristianBangert bent assay, surface plasmon resonance o(cid:11)ers
several advantageous but require considerable
amount of optimisation to produce valid results.
AmountPublished: 3
NumberofPages: 104
Published: 15-09-2015

Nanoteknologi
InstitutforFysikogNanoteknologi
Skjernvej4A,9220AalborgOest
Telefonnummer+4599409215,Fax[phone]
www.nano.aau.dk
Titel:
Karakterisering af anti-glucagon-like
peptide-1 antisto(cid:11)er ved brug af over-
Synopsis:
fladeplasmonresonans
I dette speciale blev alsidigheden af overflade
ProjektPeriode: plasmonresonansbenyttettilatkarakterisereanti-
Speciale,september2014tilseptember GLP-1antisto(cid:11)erkinetisk,termodynamiskognår
2015 antisto(cid:11)etbenyttessom’sandwich’antistof.Anti-
sto(cid:11)erneudvistebindingsa(cid:14)niteteraf2.93*10(cid:0)8-
3.56*10(cid:0)7 M. Interaktionen var fundet entalpy-
dreven, hvilketbeskriverformationenogstabilis-
Forfatter
eringen af hydrogen bindinger ved lavere tem-
LasseThomsen perature, som yderligere medfører højere bind-
ings a(cid:14)niteter. Antisto(cid:11)er benyttet som sand-
wichantisto(cid:11)erudvisteøgetbindingsa(cid:14)niteteraf
1.21*10(cid:0)9-4.87*10(cid:0)9M.
Parvis epitope kortlægning var udført for at un-
Vejleder:
dersøge sandwich potentiale af forskellige kom-
LeonidGurevich
binationer og specificiteten af antisto(cid:11)erne. Re-
sultaternevisteenspecifikrækkefølgehvorpåan-
tisto(cid:11)erne bandt sig, startende fra N-terminalen:
ABS 033-04, HYB 147-13, HYB 147-12, HYB
VirksomhedsKontaktPerson: 147-08andABS047-03.
KristianBangert Overflade plasmon resonans tilbyder en lang
række fordele over ELISA, men kræver
tilgengæld betydelig optimisering for at pro-
AntalPrintet: 3 ducerebrugbareresultater.
AntalSider: 104
AfleveringsDato: 15-09-2015

Preface
This report is the conclusion of a master thesis performed by a nanobiotechnology student at Aalborg
University in the period of September 1st, 2014 to September 15th, 2015. The work was performed in
collaborationwithKristianBangert,headofresearchanddevelopmentatBioPortoDiagnosticsA/S,who
suppliedallproteinsusedthroughouttheprojectandtowhomaspecialthankisgiven.
Reading Guide Throughout the report, there will be references to various sources. These will be listed
in the form [#] where the number in the angular brackets refers to a specific source in the bibliography
at the end of the report. In the bibliography the sources will be listed with its title, author, and other
relevantinformationdependingonwhetherthesourceisabook,article,orwebpage. Thebibliographic
referenceswillbelistedafterthespecificsectioninwhichtheyareused;thisindicatesthatthereference
appliestoalloftheaboveifnothingelseisstated.
Tablesandfiguresarelistedafterthenumberofthechapterinwhichtheyaredisplayed. Hencethefirst
figureinchapter4wouldbenamed’figure4.1’whereasthesecondonewouldbe’figure4.2’etc. Since
tables are numbered according to the same system both ’table 4.1’ and ’figure 4.1’ are possible in the
samechapter. Toeachfigure/tableashortdescriptivecaptionwillbemadetogetherwithabibliographic
referencewherenecessary. AllfigurescanbefoundontheattachedDVD.
AalborgUniversity,15/09-2015
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iv
Contents
Contents v
1 Introduction 1
1.1 Glucagon-likePeptide-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3 SurfacePlasmonResonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2 MaterialsandMethods 43
2.1 Chemicals,ProteinsandEquipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.2 SolutionMixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.3 SensorchipInstallation&Rinsing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.4 pHScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.5 AntibodyImmobilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3 Results 53
3.1 SensorchipInstallation&Rinsing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.2 pHScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3 AntibodyImmobilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.4 RegenerationCondition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.5 KineticAnalysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.6 ThermodynamicAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.7 SandwichAntibodyKinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.8 Pair-wiseEpitopeMapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4 Discussion 77
4.1 SensorchipComparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.2 pHScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4.3 AntibodyImmobilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.4 RegenerationConditionEstablishment . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.5 KineticAnalysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.6 ThermodynamicAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.7 SandwichKineticAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.8 Pair-wiseEpitopeMapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.9 SPR-ELISAComparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
v
CONTENTS
5 Conclusion 87
Bibliography 89
A Appendix 99
A.1 CalibrationProcedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
A.2 ExcessiveTreatedFigures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
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CONTENTS
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