Therapeutic Drug Monitoring Antibody Biosensors

104 Pages·2016·13.23 MB·English

Therapeutic Drug Monitoring Antibody
Biosensors
A study of monoclonal antibodies for the detection of levels of ciprofloxacin in vitro
Authors
AndersSøbye
AsgerKolding
Marie-LouiseKnopLund
SchoolofEngineeringandScience
AalborgUniversity2016
Group4.205

SixthSemester,Schoolof
EngineeringandScience
Nanotechnology
Skjernvej4A
9220AalborgØ
http://www.nano.aau.dk
Title: Abstract:
TherapeuticDrugMonitoring
Infectiousdiseasesareestimatedtocausenearly12
AntibodyBiosensors
million deaths a year, but most infectious diseases
aretreatableorpreventablewithappropriatephar-
Project:
maceuticals, leading to an enormous demand for
P6
antibiotics. Prescription doses are fixed, based on
bioavailabilitytrialsdoneonhealthyhumansubjects.
ProjectPeriod:
Individualbiologicalfactorsandexposuretomulti-
January2016-May2016 ple doses of multiple drugs can have major effects
on the bioavailability of drugs, which can lead to
Projectgroup: reduceddrugeffectivenessandadversedrugeffects.
4.205 It is therefore necessary to develop a precise ana-
lyticalassayfordetectionofcertainantibioticsina
Participants:
solution,inordertodetermineifthefreeconcentra-
AndersSøbye tionissafeandensuresappropriatedrugefficacy. In
AsgerKolding thisprojectabiosensorhasbeendeveloped,where
Marie-LouiseK.Lund surface plasmon resonance is used with different
antibodiesprovidedbyBioPortoA/S,todetermine
Supervisors: the binding kinetics of the antibiotic ciprofloxacin
(CPFX)tothespecificantibodies. Differentconcen-
EvamariaPetersen
trationsofhumanserumalbuminwaspresentdur-
LeonidGurevich
ing the experiments as a model protein. Two anti-
bodiesweresuccessfullyimmobilizedusingcovalent
aminecoupling,andsubjectedtoextensiveexperi-
PrintedCopies: 6
ments. Theexperimentsyieldedinconclusiveresults,
TotalPageNumber: 104
astowhichantibodywasmostsuitableforfurther
Appendix: 0
testing and assay development. While they both
Finished: May,2016
showed specific binding, inconsistent kinetic and
equilibriumconstantswereobtained. Furtherstud-
ies, including thermodynamic measurements and
epitope mapping are needed in order to verify the
validity behind using these specific antibodies for
thedetectionofCPFXinclinicaldiagnostics.
Thecontentofthisreportisfreelyavailable,butpublication(withcitation)mayonlytakeplaceinagreement
withtheauthors.

Preface
Thisprojectiswrittenbygroup4.205consistingofsixthsemesterNanotechnologystudentsofAalborg
UniversityfromJanuary2nd toMay30th. LectorEvamariaPetersenandLectorLeonidGurevichare
theprimarysupervisors. Thetopicofthisprojectisbiosensors,whichwillbefabricated,relyingon
monoclonalantibodiesforthedetectionoftheantibiotic,ciprofloxacin. Furthermore,thefabricated
biosensorswillrelyonsurfaceplasmonresonanceprinciples.
This project will introduce the topic and importance of biosensors in treatments with antibiotics,
along with the theoretical elucidation of biosensor principles. The project includes theoretical
chaptersdescribingantibodies,humanserumalbumin,ciprofloxacin,andsurfaceplasmonresonance
spectroscopy. Adescriptionoftheexperimentsandresultswillfollow,leadingtoadiscussionand
conclusionoftheexperimentsaccordingtorelevanttheory.
Throughoutthisproject,eachchapterandsectionwillhavenumberedtitles. Furthermore,allfigures,
significantequations,andtableswillbenumbered. Thereferencesystemappliedisthenumerical
systemandeveryreferenceisrepresentedbya[number],whichrefersdirectlytoaspecificsourcein
thebibliography. Figureswithnocitationarecomposedbythegroupitself.
AndersSøbye AsgerKolding
Marie-LouiseKnopLund
Group4.205 5of104

List of Abbreviations
CPFX: Ciprofloxacin
DMSO: Dimethylsulphoxide
EDC: N’(3-Dimethylaminopropyl)-N-Ethylcarbodiimide Hydrochlroic
Acid
HSA: Human Serum Albumin
Ig: Immunoglobulin
LMW: Low-Molecular-Weight
MES: 4-Morpholine Ethanesulfonic Acid
MTL: Mass Transport Limitation
NHS: N-Hydroxysuccinimide
ff
PBS: Phosphate-Bu ered Saline
SDS: Sodium Dodecyl Sulphate
SPR: Surface Plasmon Resonance
SP: Surface Plasmon
Tris: Tris(hydroxymethyl)aminomethane
Group4.205 7of104

Table of Contents
1 Introduction 11
2 Biosensors 13
3 Antibodies 17
3.1 BindingofAntigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.2 ProductionofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4 HumanSerumAlbumin 23
5 Ciprofloxacin 25
6 SurfacePlasmonResonanceBiosensors 27
6.1 ImmobilizationofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
6.2 ReactionKinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
6.3 DetectionofLow-Molecular-WeightAnalytes . . . . . . . . . . . . . . . . . . . . . . . . 34
7 MaterialsandMethods 37
7.1 StockSolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
7.1.1 RunningBufferSolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
7.1.2 FlowSystemRinsingSolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
7.1.3 CouplingBufferSolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
7.1.4 ActivationSolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
7.1.5 QuenchingSolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
7.1.6 RegenerationSolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
7.1.7 AntibodySolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
7.1.8 AntigenSolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
7.1.9 DMSOCalibrationSolutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
7.2 SensorChipInstallationandRinsing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
7.3 pHandConcentrationScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7.4 ImmobilizationofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
7.5 RegenerationConditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
7.6 DataCollection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.7 DataProcessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
8 Results 45
8.1 SensorChip1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
8.1.1 SensorChipInstallationandRinsing . . . . . . . . . . . . . . . . . . . . . . . . . 45
8.1.2 ImmobilizationofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
8.2 SensorChip2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
8.2.1 SensorChipInstallationandRinsing . . . . . . . . . . . . . . . . . . . . . . . . . 47
8.2.2 pHScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
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TableofContents
8.2.3 ImmobilizationofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.2.4 DiverterValveTest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.3 SensorChip3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.3.1 SensorChipInstallationandRinsing . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.3.2 pHScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.3.3 ImmobilizationofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
8.4 SensorChip4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
8.4.1 SensorChipInstallationandRinsing . . . . . . . . . . . . . . . . . . . . . . . . . 53
8.4.2 pHandConcentrationScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
8.4.3 ImmobilizationofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
8.4.4 RegenerationConditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
8.4.5 DataProcessingof0µMHSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
8.4.6 DataProcessingof0.38µMHSA . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
8.4.7 DataProcessingof0.75µMHSA . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
8.5 SensorChip5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
8.5.1 SensorChipInstallationandRinsing . . . . . . . . . . . . . . . . . . . . . . . . . 67
8.5.2 pHandConcentrationScouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
8.5.3 ImmobilizationofAntibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
8.5.4 RegenerationConditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
8.5.5 DataProcessingof0µMHSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
8.5.6 DataProcessingof0.38µMHSA . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
8.5.7 DataProcessingof0.75µMHSA . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
9 Discussion 81
9.1 ImprovementsandFurtherStudies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
10 Conclusion 93
Bibliography 95
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