Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies: = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis/Dissertation Book |
| Language: | English |
| Published: |
2012
|
| Subjects: | |
| Links: | https://open.fau.de/handle/openfau/3528 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-48913 http://d-nb.info/1038871204/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026809789&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Physical Description: | VII, 129 S. Ill., graph. Darst. 21 cm |
Staff View
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| 100 | 1 | |a Karbach, Astrid |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies |b = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper |c vorgelegt von Astrid Karbach |
| 246 | 1 | 1 | |a Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper |
| 264 | 1 | |c 2012 | |
| 300 | |a VII, 129 S. |b Ill., graph. Darst. |c 21 cm | ||
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| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2012 | ||
| 650 | 0 | 7 | |a Cytomegalie-Virus |0 (DE-588)4238363-8 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Antikörper |0 (DE-588)4002290-0 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Impfung |0 (DE-588)4026656-4 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Antikörper |0 (DE-588)4002290-0 |D s |
| 689 | 0 | 1 | |a Cytomegalie-Virus |0 (DE-588)4238363-8 |D s |
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| 912 | |a ebook | ||
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-026809789 | |
Record in the Search Index
| DE-BY-OTHR_katkey | 5266005 |
|---|---|
| DE-BY-TUM_katkey | 1957849 |
| _version_ | 1831256893670031360 |
| adam_text | TABLE OF CONTENTS
1 ZUSAMMENFASSUNG 1
2 SUMMARY 2
3 INTRODUCTION 3
3.1 THE HUMAN CYTOMEGALOVIRUS 3
3.1.1 STRUCTURE AND
MORPHOLOGY 3
3.1.2 EPIDEMIOLOGY, PATHOGENESIS,
AND CLINICAL
MANIFESTATIONS 4
3.2 THE HUMORAL IMMUNE RESPONSE
TO ANTIGENS 5
3.2.1 GENERATION AND
STRUCTURE OF ANTIBODIES 6
3.2.2 FUNCTION OF ANTIBODIES 7
3.2.3 MONOCLONAL ANTIBODIES 8
3.2.4 FEY RECEPTORS 9
3.2.5 VACCINATION 9
3.3 THE IMMUNE RESPONSE
TO CYTOMEGALOVIRUS
INFECTION 10
3.3.1 THE CELLULAR IMMUNE
RESPONSE TO CYTOMEGALOVIRUS
INFECTION 10
3.3.2 THE HUMORAL
IMMUNE RESPONSE TO CYTOMEGALOVIRUS INFECTION 11
3.4 GLYCOPROTEIN B OF THE HUMAN CYTOMEGALOVIRUS 11
3.4.1 CHARACTERIZATION OF GLYCOPROTEIN B 11
3.4.2 THE IMMUNE RESPONSE
TO GLYCOPROTEIN B 12
3.5 THE MURINE CYTOMEGALOVIRUS AND
ITS GLYCOPROTEIN
B 13
3.6 THERAPY FOR AND PROPHYLAXIS AGAINST HCMV 14
3.6.1 ANTIVIRAL CHEMOTHERAPY 14
3.6.2 PASSIVE IMMUNIZATION 14
3.6.3 ACTIVE IMMUNIZATION 15
4 PROJECT RATIONALE 18
5 RESULTS 19
5.1 CLONING OF GLYCOPROTEIN B OF THE MURINE CYTOMEGALOVIRUS 19
5.2 GENERATION OF HYBRIDOMA CELLS 19
5.3 SCREENING OF HYBRIDOMA CELLS 20
5.3.1 SCREENING FOR RECOGNITION
OF MCMV PARTICLES 20
5.3.2 SCREENING FOR GLYCOPROTEIN
B RECOGNITION
AND FOR NEUTRALIZATION OF FREE VIRUS 20
5.4 DETERMINATION OF ANTIBODY ISOTYPE 22
5.5 RECOGNITION OF VIRIONS 23
HTTP://D-NB.INFO/1042313687
5.5.1 RECOGNITION OF VIRIONS BY MONOCLONAL ANTIBODIES BY ELISA 23
5.5.2 RECOGNITION OF VIRIONS BY MONOCLONAL ANTIBODIES BY WESTERN
BLOTTING 24
5.6 LOCALIZATION OF EPITOPES
WITHIN MCMV GLYCOPROTEIN B 25
5.6.1 STRUCTURAL PREDICTIONS
AND BIOINFORMATIC
MODEL FOR MCMV GLYCOPROTEIN
B 25
5.6.2 CLONING OF GB
SUBSTRUCTURES IN EUKARYOTIC AND PROKARYOTIC
EXPRESSION VECTORS 26
5.6.3 EXPRESSION AND
DETECTION OF EUKARYOTICALLY EXPRESSED
PROTEIN DOMAINS 27
5.6.4 PURIFICATION AND
DETECTION
OF GST-TAGGED
PROTEIN FRAGMENTS 29
5.6.5 SEROPOSITIVITY RATE
AGAINST ANTIGENIC DOMAINS ON MCMV GB 33
5.7 NEUTRALIZATION CAPACITY OF GB-SPEDFIC ANTIBODIES 34
5.7.1 NEUTRALIZATION CAPACITY OF GB-SPECIFIC ANTIBODIES ON DIFFERENT
CELL
TYPES 34
5.8 A GN-SPECIFIC
ANTIBODY WITH EXTRAORDINARY NEUTRALIZATION CAPACITY 38
5.8.1 MONOCLONAL 32.22 RECOGNIZES
A MCMV PROTEIN 38
5.8.2 32.22 SPECIFICALLY BINDS
MCMV GN 39
5.8.3 NEUTRALIZATION CAPACITY
OF THE GN-SPECIFIC
ANTIBODY 41
5.8.4 SEROPOSITIVITY RATE
AGAINST GN 42
5.9 LARGE SCALE PURIFICATION AND
CONCENTRATION OF ANTIBODIES 43
5.9.1 BINDING OF ANTIBODIES
TO PROTEIN A AND
ELUTION 43
5.10 THERAPY OF INFECTED RAG MICE
WITH MCMV-SPECIFIC ANTIBODIES 45
5.10.1 REDUCTION OF VIAL
LOAD FOLLOWING
THERAPY WITH
MCMV-SPECIFIC ANTIBODIES 45
5.10.2 IN VIVO KINETICS
OF MONOCLONAL ANTIBODIES 47
5.10.3 SURVIVAL FOLLOWING
THERAPY WITH
MCMV-SPECIFIC ANTIBODIES 48
5.10.4 THERAPY OF RAG
MICE WITH FCY-RECEPTOR
KNOCK OUT 49
5.11 PROPHYLACTIC TREATMENT OF RAG MICE WITH
MCMV-SPECIFIC ANTIBODIES 50
5.11.1 REDUCED VIRAL REPLICATION
AFTER PROPHYLACTIC TREATMENT
WITH ANTI-GB
ANTIBODIES 50
5.11.2 REDUCED VIRAL REPLICATION
AFTER PROPHYLACTIC
TREATMENT WITH 32.22 51
5.12 ANALYSIS OF STERILIZING IMMUNITY FOLLOWING PASSIVE
IMMUNIZATION 53
5.12.1 DETERMINATION OF AN APPROPRIATE VIRUS
INOCULUM 53
5.12.2 SURVIVAL FOLLOWING
PASSIVE IMMUNIZATION S3
5.12.3 DETECTION OF VIRAL DNA
IN ORGANS 54
5.13 IMMUNIZATION WITH UV-INACTIVATED VIRIONS OR DONTLL
OF MCMV GB 56
5.13.1 EXPERIMENTAL SETUP 56
5.13.2 ANALYSIS OF IMMUNIZATION 57
5.13.3 PROPHYLACTIC TRANSFER OF MCMV-SPECIFIC AND
DOMLL-SPECIFIC B-CELLS 59
5.13.4 PROPHYLACTIC TRANSFER OF DOMLL-SPECIFIC SERA 62
5.13.5 ADDITION OF ISCOM AND CPG
TO THE VACCINE AND BOOST
OF INFECTED MICE 64
5.13.6 IMMUNOFLUORESCENCE ANALYSIS WITH
SERA OF IMMUNIZED
MICE 65
5.13.7 COMPETITION OF DOMLL SPECIFIC SERA
AND A DOMLL
SPECIFIC MONOCLONAL
(1F11) 67
5.13.8 DOMLL PEPTIDE
SCANNING AND
RECOGNITION
OF PEPTIDES
BY DOMLL
SPECIFIC SERA 68
5.13.9 GLYCOSYLATION PATTERN
OF DOMLL 69
5.14 IMMUNIZATION WITH MCMV GB AD-1 70
5.14.1 ANALYSIS OF IMMUNIZATION WITH AD-1
GST. 70
5.14.2 NEUTRALIZATION CAPACITY OF AD-L-SPECIFIC SERA 71
6 DISCUSSION 73
6.1 GENERATION OF MONOCLONAL ANTIBODIES PROTECTING
AGAINST MCMV 73
6.1.1 MOST NEUTRALIZING ANTIBODIES INDUCED
BY MCMV INFECTION
RECOGNIZE GB 74
6.1.2 PROTECTION FROM MCMV
INFECTION BY GLYCOPROTEIN-SPECIFIC ANTIBODIES 74
6.1.3 CORRELATION OF IN
VITRO NEUTRALIZATION CAPACITY WITH
IN VIVO
PROTECTION 75
6.1.4 TRANSFER OF STERILIZING
IMMUNITY BY CMV-SPECIFIC
ANTIBODIES 77
6.1.5 INFLUENCE OF FC RECEPTORS
ON ANTIVIRAL ACTIVITY 78
6.2 VACCINATION AGAINST
CMV-INFECTION 79
6.2.1 SEROPOSITIVE RATE AGAINST VIRAL GLYCOPROTEINS
AND PROTEIN
SUBDOMAINS IN MICE 80
6.2.2 IMMUNIZATION WITH DIFFERENT GB-DERIVED ANTIGENS
OR UV-LNACTIVATED
VIRIONS 80
6.2.3 ANALYSIS OF THE
IMPACT OF ALTERNATIVE ADJUVANTS ON ANTIVIRAL RESPONSES 82
6.3 CONCLUSIONS AND FUTURE
PROSPECTS 83
7 METHODS 84
7.1 CELL CULTURE
TECHNIQUES 84
7.1.1 MAINTENANCE OF CELL
CULTURES 84
7.1.2 CRYO-CONSERVING OF EUKARYOTIC CELLS 84
7.1.3 HYBRIDOMA TECHNIQUE 84
7.1.4 TRANSFECTION OF CULTURED CELLS 85
7.2 IMMUNOLOGICAL METHODS 85
7.2.1 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 85
7.2.2 IMMUNOFLUORESCENCE ANALYSIS 87
7.2.3 WESTERN BLOTTING 87
7.2.4 FLOW CYTOMETRIC ANALYSIS AND
SINGLE CELL SORTING 87
7.3 VIROLOGICAL METHODS 88
7.3.1 IN VITRO AMPLIFICATION AND
PURIFICATION OF MCMV PARTICLES 88
7.3.2 DETERMINATION OF VIRAL TITERS
OF MCMV
PREPARATIONS 88
7.3.3 LUCIFERASE-BASED IN VITRO
NEUTRALIZATION ASSAY 88
7.4 MOLECULARBIOLOGICAL METHODS 89
7.4.1 POLYMERASE CHAIN REACTION 89
7.4.2 CLONING 90
7.4.3 REMOVING N-LINKED
OLIGOSACCHARIDES BY PNGASE F CLEAVAGE 91
7.5 PROTEINBIOCHEMLCAL METHODS 91
7.5.1 DETERMINING PROTEIN
CONCENTRATIONS 91
7.5.2 SDS POLYACRYLAMIDE GEL
ELECTROPHORESIS (PAGE) 92
7.5
J PURIFICATION OF MONOCLONAL ANTIBODIES
UTILIZING PROTEIN A COLUMNS 92
7.5.4 PURIFICATION OF PROKARYOTICALLY EXPRESSED
PROTEINS 92
7.5.5 BIOTINYLATION OF ANTIBODIES 93
7.6
IN
VIVO AND
EX VIVO
EXPERIMENTS 93
7.6.1 INFECTION, ANTIBODY APPLICATION,
AND EUTHANASIA 93
7.6.2 COLLECTION OF BLOOD 93
7.6.3 BIOLUMINESCENCE IMAGING 94
7.6.4 PURIFICATION OF B-CELLS FROM
MURINE SPLEENS FOR
ADOPTIVE TRANSFER 94
7.6.5 DETERMINATION OF THE VIRAL LOAD
IN ORGAN
SAMPLES 95
7.6.6 ADOPTIVE B-CELL- OR SERUM TRANSFER 95
7.6.7 IMMUNIZATION 96
8 MATERIAL 97
8.1 CELL LINES AND
CULTURE MEDIA 97
8.2 ANTIBODIES 98
8.3 OLIGONUCLEOTIDES 98
8.4 PEPTIDES 99
8.5 BUFFERS AND SOLUTIONS 99
8.6 KITS 100
8.7 STANDARDS 101
8.8 VIRUS 101
8.9 ADJUVANS 101
8.10 PROTEIN A COLUMN 101
8.11 CONSUMABLES AND GLASSWARE 101
8.12 CHEMICALS AND REAGENTS 101
8.13 SOFTWARE 102
8.14 INSTRUMENTS 102
9 BIBLIOGRAPHY 104
10 APPENDIX 121
10.1 LIST OF ABBREVIATIONS 121
10.2 LIST OF TABLES 123
10.3 LIST OF FIGURES 123
10.4 AMINOACID ALIGNMENT OF HCMV AND MCMV GLYCOPROTEIN B 125
10.5 VECTOR MAPS 127
10.5.1 PCDNA3-BASED VECTORS CONTAINING
GB OR
FRAGMENTS OF GB 127
10.5.2 PCGEX6PL-BASED VECTORS
CONTAINING FRAGMENTS
OF GB 128
10.5.3 VECTORS CONTAINING
A FRAGMENT OF GN 128
10.6 ALIGNMENT OF M60, M73, AND M73.5 PROTEIN SEQUENCES 129
|
| any_adam_object | 1 |
| author | Karbach, Astrid |
| author_facet | Karbach, Astrid |
| author_role | aut |
| author_sort | Karbach, Astrid |
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| building | Verbundindex |
| bvnumber | BV041361450 |
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| ctrlnum | (OCoLC)862823004 (DE-599)DNB1042313687 |
| dewey-full | 570 610 |
| dewey-hundreds | 500 - Natural sciences and mathematics 600 - Technology (Applied sciences) |
| dewey-ones | 570 - Biology 610 - Medicine and health |
| dewey-raw | 570 610 |
| dewey-search | 570 610 |
| dewey-sort | 3570 |
| dewey-tens | 570 - Biology 610 - Medicine and health |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| indexdate | 2024-12-20T16:45:42Z |
| institution | BVB |
| language | English |
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| physical | VII, 129 S. Ill., graph. Darst. 21 cm |
| psigel | ebook |
| publishDate | 2012 |
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| spellingShingle | Karbach, Astrid Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper Cytomegalie-Virus (DE-588)4238363-8 gnd Antikörper (DE-588)4002290-0 gnd Impfung (DE-588)4026656-4 gnd |
| subject_GND | (DE-588)4238363-8 (DE-588)4002290-0 (DE-588)4026656-4 (DE-588)4113937-9 |
| title | Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper |
| title_alt | Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper |
| title_auth | Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper |
| title_exact_search | Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper |
| title_full | Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper vorgelegt von Astrid Karbach |
| title_fullStr | Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper vorgelegt von Astrid Karbach |
| title_full_unstemmed | Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper vorgelegt von Astrid Karbach |
| title_short | Protection from cytomegalovirus infection by glycoprotein-specific monoclonal antibodies |
| title_sort | protection from cytomegalovirus infection by glycoprotein specific monoclonal antibodies schutz vor infektion mit dem cytomegalievirus durch glykoproteinspezifische monoklonale antikorper |
| title_sub | = Schutz vor Infektion mit dem Cytomegalievirus durch glykoproteinspezifische monoklonale Antikörper |
| topic | Cytomegalie-Virus (DE-588)4238363-8 gnd Antikörper (DE-588)4002290-0 gnd Impfung (DE-588)4026656-4 gnd |
| topic_facet | Cytomegalie-Virus Antikörper Impfung Hochschulschrift |
| url | https://open.fau.de/handle/openfau/3528 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-48913 http://d-nb.info/1038871204/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026809789&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT karbachastrid protectionfromcytomegalovirusinfectionbyglycoproteinspecificmonoclonalantibodiesschutzvorinfektionmitdemcytomegalievirusdurchglykoproteinspezifischemonoklonaleantikorper AT karbachastrid schutzvorinfektionmitdemcytomegalievirusdurchglykoproteinspezifischemonoklonaleantikorper |