Development and characterisation of an immunochemical test system for the determination of bacterial signal molecules (N-acylated homoserine lactones) [Elektronische Ressource] / Xiao Chen. Betreuer: Anton Hartmann

Chen Chen, Jing Xiao

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Doctoral thesis, Xiao Chen, 2011




Development and characterisation of an immunochemical test
system for the determination of bacterial signal molecules (N-
acylated homoserine lactones)




Dissertation der Fakultät für Biologie der Ludwig-Maximilians-
Universität München

vorgelegt von
Xiao Chen

November 2010, München
1
Doctoral thesis, Xiao Chen, 2011


Ehrenwörtliche Erklärung
Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte
Hilfe angefertigt habe. Ich habe weder anderweitig versucht, eine Dissertation einzureichen
oder eine Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile
derselben einer anderen Prüfungskommission vorgelegt.







Begutachter 1: Herr Prof. Dr. Anton Hartmann
Begutachter 2: Frau Prof. Dr. Elisabeth Weiss
Datum der Abgabe der Dissertation: 04 November 2010
Datum der mündlichen Prüfung: 10 Mai 2011
2
Doctoral thesis, Xiao Chen, 2011


Lernen ist wie Rudern gegen den Strom. Hört man damit auf,
treibt man zurück.

----Laozi
3
Doctoral thesis, Xiao Chen, 2011


INDEX OF CONTENTS
INDEX OF FIGURES .................................................................................................. 8
INDEX OF TABLES . 10
INDEX OF ATTACHMENTS .................................................................................... 11
ABBREVIATION ...................................................................................................... 12
1 INTRODUCTION ................. 14
1.1 Quorum sensing ...................................................................................................................... 14
1.1.1 Discovery of quorum sensing ................................... 14
1.1.2 Mechanism of quorum sensing . 15
1.1.3 Gram-negative bacteria and AHL (HSL) quorum sensing molecules ....... 18
1.1.3.1 Burkholderia cepacia and quorum sensing ................................................................................ 20
1.1.3.2 Pseudomonas aeruginosa and quorum sensing ......... 21
1.1.3.3 Interspecies communication between P. aeruginosa and B. cepacia ......... 22
1.1.3.4 Pseudomonas putida and quorum sensing ................. 22
1.1.3.5 Quorum sensing in biofilm communities and 1,2,4-trichlorobenzene (TCB) biomineralisation24
1.2 Quorum quenching ................................................................................................................. 25
1.2.1 Inhibition of AHL signal sensing .............................. 25
1.2.2 Limitation of signal accumulation-HSL degradation ................................................................ 26
1.2.2.1 Abiotic degradation .................... 26
1.2.2.2 Enzymatic degradation............................................................................................................... 27
1.2.3 The biological functions of the AHLases.................. 28
1.3 Current HSL analytical methods ........................................................................................... 28
1.3.1 Conventional chemical analysis ................................ 28
1.3.2 Bioreporters .............................................................. 30
1.4 Immunochemistry ................................................................................... 31
1.4.1 Introduction of immunochemical techniques ............ 31
1.4.2 Production of antibody .............................................. 31
1.4.2.1 Antibody introduction ................ 31
1.4.2.2 Target molecule selection .......................................................................... 32
1.4.2.3 Hapten design ............................................................................................................................ 33
1.4.2.4 Hapten synthesis and conjugation to carrier molecules ............................. 33
1.4.2.5 Immunisation, fusion and hybridoma ........................ 34
1.4.3 Immunoassay and different formats .......................... 36
1.4.3.1 Enzyme-tracer format ELISA .................................................................................................... 37
1.4.3.2 Coating antigen format ELISA .. 37
1.4.3.3 Sandwich ELISA ....................................................... 37
1.4.4 Optimisation of immunoassays . 37
1.4.4.1 Test sensitivity ........................................................................................... 37
1.4.4.2 Reagents ..................................................................... 38
1.4.4.3 Hapten conjugates ...................... 38
1.4.4.4 Buffers and blocking systems .................................................................... 39
1.4.4.5 Immobilisation on the surface of microtiter plate ...... 39
1.4.5 Immunoassay evaluation ........................................... 39
1.4.6 Fluorescence in immunochemistry ........................... 40
4
Doctoral thesis, Xiao Chen, 2011

1.4.6.1 Fluorescence .............................................................................................................................. 40
1.4.6.2 Fluorescent dye conjugation to antibody ................... 41
1.4.6.3 Tyramide Signal Amplification (TSA) ...................... 42
1.4.7 Aqua-Optosensor (AOS) ........................................................................................................... 42
1.5 Outline of goal and tasks for this work ................. 44
2 MATERIALS AND METHODS ............................................................ 45
2.1 Chemicals, standards and proteins ........................................................ 45
2.2 Instruments for hapten syntheses .......................... 46
2.3 Synthesis of HSL haptens ....................................................................................................... 47
2.3.1 Synthesis of sebacic acid monobenzyl ester (1) ........ 47
2.3.2 Synthesis of N-(9-benzylcarboxyl-1-hydroxy)-Meldrum’s acid (2) ......... 48
2.3.3 Synthesis of N-(11-benzylcarboxy-3-oxoundecanoyl)-L-homoserine lactone (3) .................... 48
2.3.4 Synthesis of N-(11-carboxy-3-oxoundecanoyl)-L-homoserine lactone (HSL1) (4) ................. 49
2.3.5 Synthesis of N-(5-carboxypentanoyl)-L-homoserine lactone (HSL2) (5) ................................. 50
2.3.6 Synthesis of N-(11-benzylcarboxy-3-hydroxyundecanoyl)-L-homoserine lactone (6) ............. 51
2.3.7 Synthesis of N-(11-carboxy-3-hydroxyundecanoyl)-L-homoserine lactone (HSL3) (7) .......... 51
2.3.8 Synthesis of N-(9-benzylcarboxy nonanoyl)-L-homoserine lactone (8) ... 52
2.3.9 Synthesis of N-(9-carboxynonanoyl)-L-homoserine lactone 8 (HSL 4) ................................... 53
2.4 Materials and instruments for ELISA .................................................................................. 54
2.5 Preparation of hapten-protein conjugates for immunogens and coating antigens............ 54
2.6 Characterisation of hapten protein conjugates with UV absorbance spectra ................... 57
2.7 Preparation of enzyme-tracers .............................................................................................. 57
2.8 Production of anti-HSL monoclonal antibodies ................................... 58
2.9 Determination of immunoglobulin type ................................................................................ 59
2.10 Purification of monoclonal antibodies and determination of protein concentration ........ 59
2.11 Characterisation of HSL antibodies (anti-HSL mAbs) with enzyme-linked
immunosorbent assay (ELISA) .............................................................................................. 59
2.11.1 Standard procedure of coating antigen format ELISA 60
2.11.2 ELISA in the enzyme-tracer format .......................... 61
2.11.3 Titration and screening against HSL substances ....................................................................... 62
2.11.4 Binding test to a few possible HSL degradation substances ..................... 63
2.11.5 Standard curves ......................................................................................................................... 64
2.11.6 Determination of cross reactivity .............................. 65
2.12 Detection of HSLs in biological samples with ELISA .......................... 65
2.12.1 Standard curves in matrix: determination of matrix effects ...................................................... 66
2.12.2 Standard curves in ABC Medium: determination of HSL in B. cepacia LA3 supernatants ..... 66
2.13 Preparation of biological samples .......................................................... 66
2.13.1 Preparation of Burkholderia cepacia culture supernatants ....................................................... 67
2.13.2 Preparation of Pseudomonas putida culture supernatants ......................... 67
2.13.3 Preparation of 1,2,4-TCB-mineralising bacterial biofilm community samples ........................ 68
2.14 HSL antibody characterisation with Aqua-Optosensor (AOS)........................................... 68
2.15 Optimisation of fluorescent labelling .......................................