Comparison of different serological test-systems to analyse specific IgM and IgA immuneresponses against measles virus [Elektronische Ressource] / vorgelegt von Dana Christina Wenz

Ein Geehrter Microsoft-Kunde

Sujets

• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2004
• Nombre de lectures: 59
• Langue: English

Extrait

Aus dem Laboratoire National de Santé Luxembourg
Abteilung Virologie/Immunologie
Direktor: Professor Dr. C. P. Muller




Comparison of different serological test-systems to
analyse specific IgM and IgA immunresponses against
measles virus


INAUGURAL-DISSERTATION
zur Erlangung des Doktorgrades
der Medizin

der
MEDIZINISCHEN FAKULTÄT
der Eberhard-Karls-Universität
zu Tübingen



vorgelegt von
DANA CHRISTINA WENZ
aus
Germersheim
2004




























Dekan: Professor Dr. C. D. Claussen
1. Berichterstatter: Professor Dr. C. P. Muller
2. Berichterstatter: Professor Dr. B. Flehmig

gedruckt 2004 bei Hans-Joachim Köhler, Druck & Kopie, Tübingen
Note of thanks
I would like to thank all the people, which have involved their knowledge, skills
and patience for the successful outcome of this doctoral thesis. All colleagues at
the laboratory were very open minded and helpful and became, next to work,
also good friends.
My special thanks go to my supervisor Prof. Dr. C. P. Muller. He introduced me
to the way of scientific laboratory work. He taught me how to arrange my data
and to present the results in a professional way. I am also very grateful for all
the caring organization and correspondence done by his wife Ulla Muller and for
the fellowship from the Ministère de la Santé, Luxembourg.
A lot of thanks go to Wim Ammerlaan for teaching me very patiently how to
work sufficiently with cells and for the excellent technical guidance at the
fluorescence-activated cell scanner.
I am very thankful for all the valuable tips from Heike Hartter, Oliver Ertl, Dr.
Fabienne Bouche, Dr. Stephanie Kreis and Dr. Fred Fack.
For thorough proof-reading I would like to thank Sonja Krauß.
Thank you, Thomas Kieber for your help, the scarifies and your love.
“You see, Momo,” he told her one day, “it’s like this. Sometimes, when you’ve a
very long street ahead of you, you think how terribly long it is and feel sure you’ll
never get it swept.”
He gazed silently into space before continuing. “And then you start to hurry,” he
went on. “You work faster and faster, and every time you look up there seems
to be just as much left to sweep as before, and you try even harder, and you
panic, and in the end you’re out of breath and have to stop - and still the street
stretches away in front of you. That’s not the way to do it.”
He pondered a while. Then he said, “You must never think of the whole street at
once, understand? You must only concentrate on the next step, the next breath,
the next stroke of the broom, and the next, and the next. Nothing else.”
Again he paused for thought before adding, “That way you enjoy work, which is
important, because then you make a good job of it. An that’s how it ought to be.”

Monologue, “Beppo Roadsweeper”
Michael Ende, “Momo”, K. Thienemanns Verlag, Stuttgart, 1973

















To my parents










List of contents
List of contents I
Table of abbreviations IV
1Introduction1
1.1 The measles virus2
1.1.1 Classification and morphology
1.1.2 Isolation of MV5
1.1.3 Virus replication6
1.1.4 Molecular epidemiology7
1.2 Measles disease 8
1.2.1 Clinical features
1.2.2 Complications 10
1.3 Immunity against measles virus 12
1.3.1 Immune response
1.3.2 Specific immune response against MV 13
1.3.3 Surveillance of measles immunity 16
1.3.4 Alternatives 18
1.4 Measles eradication
1.4.1 Measles vaccines and vaccination strategies 18
1.4.2 Side effects of measles vaccination 20
1.4.3 Eradication efforts 21
1.5 Goals of this thesis 24
1.5.1 Comparison of 5 different serological measles IgM assays 24
1.5.2 Evaluation of IgA response against MV-H 25
I2 Materials and Methods 27
2.1 Test panels
2.1.1 Panel A for comparison of MV-specific IgM assays 27
2.1.2 Panel B for evaluating IgA response against MV-H 29
2.1.3 Specimen handling 30
2.1.4 Assays performed on different panels 31
2.2 Cell culture 31
2.2.1 Transfected human melanoma cells (Mel-JuSo)
2.2.2 Materials 32
2.2.3 Procedure 33
2.2.4 Transfected baby hamster kidney cells (BHK-21 cells) 34
2.3 Serological methods 35
2.3.1 Indirect whole measles virus ELISA (IgM-/IgG-Enzygnost®) 35
2.3.2 Capture measles-IgM ELISA (IgM-Comfort EIA) 39
2.3.3 Recombinant H-ELISA (H-ELISA) 42
2.3.4 FACS-measured immunofluorescence assay (FACScan) 45
2.4 Statistical evaluation 47
3 Results 51
3.1 Comparison of different serological measles IgM assays 51
3.1.1 Panel used
3.1.2 Test standardization
3.1.3 Gold standard 52
3.1.4 Characteristics of the different assays 52
3.1.5 Receiver operating characteristic curves (ROC) 58
3.2 Evaluation of IgA response against MV-H 62
3.2.1 Panel used 62
3.2.2 Test standardization
II3.2.3 Serum IgA titers 64
3.2.4 Measles IgA in saliva 68
4 Discussion 72
4.1 Comparison of different serological measles IgM assays 73
4.1.1 Demand for new serological assays for measles diagnosis 73
4.1.2 Evaluation of different assays 75
4.1.3 User-friendliness of tests 77
4.2 Evaluation of IgA response against MV-H 78
4.2.1 Observations on the panel, reagents and the system
4.2.2 Evaluation of MV-IgA in different specimens 79
5 Summary 83
6 References 84
7 Curriculum Vitae 112

IIITable of abbreviations
Aa amino acid
AFU arbitrary fluorescence unit
aq. bidi aqua bidestillata
BSA bovine serum albumin
CDC Centers of Disease Control and Prevention
C.U. Capture Units
EBVEpstein-BarrVirus
EDTA ethylene diamin tetraacetic acid
Efefficiency
ELISA enzyme linked immunosorbent assay
ER endoplasmatic reticulum
FACS flourescence activated cell scanner
F-FACS Mel-JuSo/MV-F/wt tested in FACS
FCS fetal calf serum
F fusion protein
FITCfluoresceinisothiocyanate
hhour
Hhemagglutinationprotein
H-FACS Mel-JuSo/MV-H/wt tested in FACS
HI hemagglutination inhibition
HIV Human Immunodeficiency Virus
IgAimmunoglobulinA
IgGimmunoglobulinG
IgG-Enzygnost Enzygnost® Anti-Measles-Virus/IgG
IgM immunoglobulinM
IgM-EnzygnostEnzygnost®Anti-Measles-Virus/IgM
IUinternational units
kDakilodalton
L largeprotein
IVmM milli Mol
Mmatrixprotein
mAb monoclonal antibody
MWRST Mann-Whitney Rank Sum Test
MCV measles containing vaccine
MHC major histocompatibility complex
min minutes
MMR Measles- Mumps- Rubella
MVmeaslesvirus
MV-F measles virus fusion protein
MV-H measles virus H protein
MV-NP measles virus nucleoprotein protein
N nucleoprotein
N nitrogen 2
NPV negative predictive value
NTneutralizationassay
O.D. optical density
PBS phosphate buffered saline
PCR polymerase chain reaction
PO/PODPeroxidase
PPMC Pearson Product Moment Correlation
PPV positive predictive value
PSG penicillin/streptomycin/glutamin
RFrheumatoid factor
RNAribonucleicacid
rpm rounds per minute
Se sensitivity
SFV Semliki Forest virus
Sp specificity
SROC Spearman Rank Order Correlation
SSPE subacute sclerosing panencephalitis
T T- cytotoxic lymphocyte C
V