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ΑΝΟΣΟΠΟΙΗΣΗ ΠΟΝΤΙΚΩΝ ΜΕ ΤΟ ΠΕΠΤΙΔΙΟ TAX1BP1 ΚΑΙ ΕΛΕΓΧΟΣ ΓΙΑ ΔΗΜΙΟΥΡΓΙΑ ΑΝΤΙΣΩΜΑΤΩΝ ΕΝΑΝΤΙ ΤΗΣ ΥΔΑΤΟΠΟΡΙΝΗΣ-4.
Board 1 / Thu 10:20, 11 Dec 2014

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Introduction
Antibodies against aquaporin-4 are specific and pathogenetic
for Neuromyelitis Optica. In a previous study, 3 linear
intracellular B- cell epitopes were found. One epitope
presented a high molecular similarity with a fraction of the
human TAX1BP1 protein, which is necessary for the
replication of HTLV-1 virus, the etiological agent of HAM/TSP.
Aim of the Study
The aim of the study was to investigate whether
immunization of mice with the TAX1BP1 peptide could
produce epitope spreading against linear or conformational
aquaporin-4 epitopes.
Methods
Peptide Synthesis
13 peptides (MAP) – 2 homologous peps & 11 AQP4 epitopes
Immunizations
16 female C57Bl/6 mice, 6-8 weeks old.
 8 immunized with 100λ ΤΑΧ1ΒΡ1pep 1mg/ml in Complete
Freud’s Adjuvant (CFA)
 8 received equal volumes of CFA in PBS 1x (control group)

ELISA & Inhibition Assays
All sera were evaluated by ELISA assays for antibodies against the peptide
TAX1BP1pep, the homologous AQP4 peptide AQPpep8’ and all linear AQP4
epitopes. Homologous and cross-inhibition assays were performed to evaluate
binding specificity.
Cell Based Assays
The existence of antibodies against conformational AQP4 epitopes was evaluated
by a cell based assay, using M23-transfected HEK293 cells.
Results
 All immunized animals showed high reactivity against the immunization peptide,
its’ homologous AQPpep8’ and the full linear AQP4 epitope AQPpep8 [252-275].
 Reactivity gradually increased after each immunization boos
 No antibodies were produced against any other linear epitopes.

 Sera obtained from control animals presented no reactivity against the peptides.
 No antibodies were produced against conformational epitopes.

 Inhibition assays confirmed binding specificity.

Conclusions
Our study demonstrated the production of highly specific
antibodies against the linear intracellular AQP4 epitope
AQPpep8, after immunization. Epitope spreading is a very
important mechanism towards enhancement of the
immune response. The lack of epitope spreading in our
experiments implies that the cross-reactivity of the anti-
TAX1BP1pep and anti-AQPpep8 antibodies may be an
epiphenomenon. However, studies have described
relapses of autoimmune disorders in the absence of
epitope spreading, therefore the pathogenetic activity of
the anti-TAX1BP1pep antibodies could be confined to
cases of HTLV-1 latent infection.
References
Alexopoulos, H., E. I. Kampylafka, I. Chatzi, et al (2013). "Reactivity to
AQP4 epitopes in relapsing-remitting multiple sclerosis." J Neuroimmunol
260(1-2): 117-120.
Kampylafka, E. I., J. G. Routsias, H. Alexopoulos, et al (2011). "Fine
specificity of antibodies against AQP4: epitope mapping reveals
intracellular epitopes." J Autoimmun 36(3-4): 221-227.