Prof. Dr. Vladka Čurin Šerbec

PP10
Prof. Dr. Vladka Čurin Šerbec
Zavod Republike Slovenije za transfuzijsko medicino, Center za razvoj in za izdelavo diagnostičnih
reagentov & raziskovalna skupina Biomedicina
Blood Transfusion Centre of Slovenia, Centre for the production of diagnostic reagents and for
research & research group Biomedicine
Šlajmerjeva 6, 1000 Ljubljana, Slovenia
Blood Transfusion Centre of Slovenia:
(Anja Colja Venturini, PhD , student )
Maja Černilec, MSc
(Mascia Ghielmetti, PhD, postdoc)
Melita Gracar, BSc
(Simon Koren, PhD , student)
Miha Kosmač, PhD, student
(Jelka Kutić)
Anja Lukan, PhD student
(Sendi Montanič, MSc)
(Katrina Pretnar Hartman, PhD)
Boštjan Smrekar, BSc
Marjana Šprohar
Tanja Vranac, PhD
ANTIBODIES
MONOCLONAL ANTIBODIES
Georges Koehler
Cesar Milstein
“antibodies on demand”, Nobel Prize in Medicine, 1984
MONOCLONAL ANTIBODY
PRODUCTION
BLOOD TYPING
BLOOD TYPING
DIAGNOSTIC REAGENTS
PRION DISEASES
transmissible spongiform encephalopathies (TSE)
BSE
neurodegenerative
central nervous system
PrPC PrPSc
no cure
scrapie (Sc)
sporadic, inherrited, iatrogenic (vCJD, kuru)
“proteinaceous”
“infectious”
PRION
“protein-only “ hypothesis
Nobel Prize in Medicine, 1997
S. B. Prusiner, Science 1982
“for the discovery of Prions – a new biological principle of infection”
PRION PROTEIN (PrP)
conformational change
PrPC
PrPSc
?
Govaerts et al.; 2004
DeMarco et al.; 2004
Kobb, Surewicz; 2009
Cobb et al.; 2007
- 1996, vCJD
- relation with BSE
Will, R. G., et al. Lancet. 1996; 347 : 921-52.
- 2004, possible transmission by blood
Llewelyn, C. A., et al. Lancet 2004; 363: 417-21.
Peden, A. H., et al. Lancet 2004; 364: 527-29.
Wroe, S. J., et al. Lancet 2006; 368: 2061-67.
Collins S. J., et al. Lancet 2004; 363:51-61.
DIAGNOSTICS
monoclonal antibodies
PrP, resistant to proteinase K (PrPres)
the importance of conformation specific mAbs
TREATMENT OF PRION DISEASES
•
molecular mechanisms of neurotoxicity???
PrPC
• target:
PrPC
PrPSc
• immunotherapy: active or passive
PrPSc
mAb v5B2 was used as the basis for the development of a new
generation of postmortem enzyme immunoassay for BSE diagnostics
without PK digestion, which was in the process of validation by
European Comission.
Sequencing of mAb variable regions is important to identify the cell line.
Chatterjee R., Cell biology. Science. 2007; 315(5814):928-931.
EPITOPE MAPPING STUDIES
In further studies, crucial amino acids in peptide P1 sequence (and
consequently in PrP), important for binding of V5B2, were determined
by using a panel of synthetic peptides with substituted amino acids
predominantly at N- (cysteine) and C-terminal part (double tyrosine) as
well as phage-displayed peptides. Our results led us to the conclusion
about the importancy of tyrosine (s) for binding of V5B2 as well as to
the identification of C-terminally cleaved PrPSc (PrP226*) in amyloid
aggregates.
(in collaboration with SISSA , prof. G. Legname, and University of
Trieste, prof. R. Gennaro)
Kosmač et al., Mol. Immunol., 2011
Figure 6: Immunohistochemistry of the PrPSc deposits in the cerebellum of a sCJD patient (upper three figures). Immunolabeling was performed
with whole mAb V5B2, murine scFv (moScFv) and 24 humanized scFv (huScFv) of V5B2. The arrow marks PrPSc plaques, while the triangle marks
the diffuse, synaptic PrPSc deposition. On the lower three figures immunolabeling was performed on the cerebellum of the CJD negative patient.
Škrlj N. et al., PlosOne, 2011
Immunisation of Prnp 0/0 mice with oxidised recombinant
human PrP lead to a panel of N-terminal anti-PrP mAbs
(collaboration with SISSA, prof. G. Legname)
Monoclonal antibodies against BTL –
collaboration with the University of
Trieste (prof. S. Passamonti)
Cell line
2G2
3G7
5C1
5E11
5G11
5H11
6D5
6E4
6F4
6H6
1B4
2F2
6B4
2H11
1A10
1E8
1H6
2G3
4E2
5A3
6E7
7H2
ELISA test
(peptide A)
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ELISA test
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ELISA test
(Microsomes)
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Figure 5: Western blot analyses of rat liver denaturated microsome (M),
performed with purified mAb 6E4/1F2/1E2 (10 mg/ml).
Figure 6: ICC staining of
HepG2 cells with mAb
6E4/1F2/1E2 (10 mg/ml).
Figure 7: Reactivity of mAb 6E4/1F2/1E2 with native BTL,
expressed on HepG2 cells, as determined by flow cytometry.
Antibodies can DETECT and could be DETECTED.
ANTI-IDIOTYPIC PRINCIPLE
Anti-idiotypic antibodies (Ab2) are a part of the immune response.
Ab2 α idiotopes apart from the binding site
for the Ag on Ab1
Ab2 β
Ab2 γ
internal image of Ag
epitopes in the Ag binding site,
structurally different from Ag
Towards a network theory of the immune
Niels Jerne
system, Ann. Immunol., 1974
Nobel Prize in Medicine, 1984
ANTIBODIES AGAINST
BIOLOGICAL DRUGS
HEMOCHROMATOSIS
• Cukjati, M., Vaupotič, T., Rupreht, R., Čurin Šerbec, V. (2007) Prevalence
of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in
Slovenian population by an improved high-throughput genotyping assay.
BMC Medical Genetics, 2007, 8, 1-9.
• Cukjati, M., Koren, S., Čurin Šerbec, V., Vidan Jeras, B., Rupreht, R. (2007)
A novel homozygous frameshift deletion c.471del of HFE associated with
hemochromatosis. Clin. Genet. 71, 1-4.
• Ramagopalan, S.V., Cukjati, M., Černilec, M., DeLuca, G.C., Čurin Šerbec,
V. Mutations in the hemochromatosis gene and the clinical outcome of
multiple sclerosis. J.Neuroimmunol., 2008, 203, 1, 104-107.
TEACHING
Undergraduate study:
Molecular immunology (FCCE UL)
Molecular immunology with immunochemistry (FCCE UL)
Monoclonal antibodies (FCCE UL)
Postgraduate study:
Immunology and clinical immunology (Biomedicine, MF UL)
Monoclonal antibodies: an overview and their use in research, diagnostics and
in therapy (Biomedicine, MF UL)
Animal and human cell cultures and hybridoma technology (Biotechnology, BF
UL)
Immunological techniques (Biotechnology, BF UL)
MENTORSHIPS
Diploma work
Natalija Povodnik, 2001
Blanka Premrov, 2001
Anja Venturini, 2004
Simon Koren, 2004
Tomaž Vaupotič, 2004
Katja Škerget, 2004
Sendi Montanič, 2005
Boštjan Smrekar, 2005
Aleš Volgemut, 2005
Dejan Verhovšek, 2006
Miha Kosmač, 2006
Luka Omahen, 2006
Melita Gracar, 2006
Primož Treven, 2009
Adrijana Oblak, 2009
Marija Kokalj, 2010
MENTORSHIPS
MSc Thesis
Maja Černilec, 1995
Petra Žunec, 1996
Vesna Bojanič, 1998
Andreja Kamin, 2005
Katja Trontelj, 2005
Sendi Montanič, 2008
MENTORSHIPS
PhD Thesis
Ruth Rupreht, 2000
Vesna Galvani, 2001
Tanja Vranac, 2005
Katrina Pretnar Hartman, 2005
Simon Koren, 2009
Anja Venturini, 2010
Katja Trontelj, 2010
Miha Kosmač, 2011
MENTORSHIPS - POSTDOC
Dr. Mascia Ghielmetti, University in Bern (2007-2008)
Dr. Michela Terdoslavich, University in Trieste (2007-2008)
MENTORSHIPS - STUDENT’S AWARDS
Maja Alscher, Krkina nagrada za študente, 1996
Gregor Kosec, Prešernova nagrada za študente, 2000
Natalija Povodnik, Prešernova nagrada za študente, 2001
Vesna Galvani, Krkina nagrada za študente, 2001
Senta Frol, Martin Hren, Prešernova nagrada za študente, 2001
Blanka Premrov, Krkina nagrada za študente, 2002
Tomaž Vaupotič, Krkina nagrada za študente, 2003
Anja Venturini, Krkina nagrada za študente, 2004
Simon Koren, Prešernova nagrada Univerze v Ljubljani, 2004
Miha Kosmač, Prešernova nagrada Univerze v Ljubljani, 2006
Marija Kokalj, Prešernova nagrada za študente, 2010
Katja Trontelj, Krkina nagrada za študente, 2010