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Papasarantos I.,Institute of Radioisotopes and Radiodiagnostic Products | Klimentzou P.,Institute of Radioisotopes and Radiodiagnostic Products | Koutrafouri V.,Institute of Radioisotopes and Radiodiagnostic Products | Anagnostouli M.,National and Kapodistrian University of Athens | And 3 more authors.
Applied Biochemistry and Biotechnology | Year: 2010

A biotin derivative, namely biotin-aminocaproic acid-lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid-lysine "long-arm" was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its Nε-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic "lysine-dendrimer" was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten. © 2009 Springer Science+Business Media, LLC.


Tsoutsou E.,National and Kapodistrian University of Athens | Tzetis M.,National and Kapodistrian University of Athens | Giannikou K.,National and Kapodistrian University of Athens | Syrmou A.,National and Kapodistrian University of Athens | And 5 more authors.
European Journal of Paediatric Neurology | Year: 2013

A 28-month-old girl with dysmorphic craniofacial features, microcephaly, hypotonia, psychomotor retardation, failure to thrive and gastrointestinal problems was referred for clinical evaluation. Array-CGH analysis revealed one of the smallest de novo microdeletions on chromosome 16q21q22.1, 2.03 Mb in size. Advanced molecular analysis contributes to more precise genotype-phenotype correlation and accurate definition of the breakpoints in the deleted/duplicated regions. © 2012 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.


Ioannou K.,National and Kapodistrian University of Athens | Samara P.,National and Kapodistrian University of Athens | Livaniou E.,Institute of Radioisotopes and Radiodiagnostic Products | Derhovanessian E.,University of Tübingen | Tsitsilonis O.E.,National and Kapodistrian University of Athens
Cancer Immunology, Immunotherapy | Year: 2012

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as "thymic hormones," are produced by this gland. Although the majority of them have not been proven to be thymus-specific, thymic peptides comprise an effective group of regulators, mediating important immune functions. Thymosin fraction five (TFV) was the first thymic extract shown to stimulate lymphocyte proliferation and differentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin α (proTα) and thymosin α1 (Tα1) showed that they are of clinical significance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeficiencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their effect are yet not fully elucidated, proTα and Tα1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proTα, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of Tα1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proTα into the clinical setting. © Springer-Verlag 2012.


Niotis A.E.,Institute of Radioisotopes and Radiodiagnostic Products | Mastichiadis C.,Institute of Radioisotopes and Radiodiagnostic Products | Petrou P.S.,Institute of Radioisotopes and Radiodiagnostic Products | Christofidis I.,Institute of Radioisotopes and Radiodiagnostic Products | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2010

The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin- horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction. © 2009 Springer-Verlag.


Misiakos K.,Institute of Microelectronics, Greece | Petrou P.S.,Institute of Radioisotopes and Radiodiagnostic Products | Kakabakos S.E.,Institute of Radioisotopes and Radiodiagnostic Products | Yannoukakos D.,Institute of Radioisotopes and Radiodiagnostic Products | And 8 more authors.
Biosensors and Bioelectronics | Year: 2010

The development and testing of a portable bioanalytical device which was capable for real-time monitoring of binding assays was demonstrated. The device was based on arrays of nine optoelectronic transducers monolithically integrated on silicon chips. The optocouplers consisted of nine silicon avalanche diodes self-aligned to nine silicon nitride waveguides all converging to a single silicon detector. The waveguides were biofunctionalized by appropriate recognition molecules. Integrated thick polymer microchannels provided the necessary fluidic functions to the chip. A single sided direct contact scheme through a board-to-board receptacle was developed and combined with a portable customized readout and control instrument. Real-time detection of deleterious mutations in BRCA1 gene related to predisposition to hereditary breast/ovarian cancer was performed with the instrument developed using PCR products. Detection was based on waveguided photons elimination through interaction with fluorescently labeled PCR products. Detection of single biomolecular binding events was also demonstrated using nanoparticles as labels. In addition, label-free monitoring of bioreactions in real time was achieved by exploiting wavelength filtering on photonic crystal engineered waveguides. The proposed miniaturized sensing device with proper packaging and accompanied by a portable instrument can find wide application as a platform for reliable and cost effective point-of-care diagnosis. © 2010 Elsevier B.V.


Georgiadou D.,Institute of Radioisotopes and Radiodiagnostic Products | Hearn A.,University of Massachusetts Medical School | Evnouchidou I.,Institute of Radioisotopes and Radiodiagnostic Products | Chroni A.,Greek National Center For Scientific Research | And 4 more authors.
Journal of Immunology | Year: 2010

All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8-9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing. Copyright © 2010 by The American Association of Immunologists, Inc.


Psarouli A.,Institute of Radioisotopes and Radiodiagnostic Products | Psarouli A.,University of Crete | Bourkoula A.,Institute of Radioisotopes and Radiodiagnostic Products | Petrou P.,Institute of Radioisotopes and Radiodiagnostic Products | And 3 more authors.
Procedia Engineering | Year: 2011

Effective immobilization of biomolecules at transducer interfaces plays a crucial role in biosensors development. In this work, we compared several silicon nitride (Si 3N 4) surface silanization methods in terms of efficient biomolecule immobilization either through physical adsorption or covalent binding. Two silanizing agents, namely 3-aminopropyltriethoxysilane (APTES) and 3-glycidyloxypropyltrimethoxysilane (GOPTS), were employed. It was found that silanization with GOPTS provided slightly higher protein immobilization capacity and homogeneity and lower non-specific binding as compared to APTES modification. Therefore, it could be employed for the effective functionalization of Si 3N 4 waveguides for biosensing applications. © 2011 Published by Elsevier Ltd.


PubMed | Institute of Radioisotopes and Radiodiagnostic Products
Type: Journal Article | Journal: Analytical and bioanalytical chemistry | Year: 2010

The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin-horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction.


Fotiadou K.,Institute of Physical Chemistry | Thanassoulas A.,Institute of Radioisotopes and Radiodiagnostic Products | Nounesis G.,Institute of Radioisotopes and Radiodiagnostic Products | Yannakopoulou K.,Institute of Physical Chemistry
Supramolecular Chemistry | Year: 2011

The anionic cyclodextrins (CDs), heptakis[6-(3-thiopropionate)-6-deoxy]- β-CD, heptakis[(6-(thio-ethanoate)-6-deoxy]-β-CD and partially phosphorylated 6-(aminoethylphosphate)-6-deoxy-β-CD as triethylammonium salts, bpsp.NHEt3, bpse.NHEt3 and bphos.NHEt3, respectively, interacted with the positively charged picrate salt of heptakis[6-(guanidino)-6-deoxy] - CD, bguan.picrate, to form heterodimers as shown by NMR spectroscopic and microcalorimetric isothermal titration calorimetry (ITC) titrations in solution. Association constants, Ka (M-1) in DMSO-d6 and in DMSO-d6-H2O (80/20, v/v) were: bguan.picrate/bpsp.NHEt3, 6.4×105 and 5.9×104; bguan.picrate/bpse.NHEt3, 4.2×104 and 1.2×104; ITC data in DMSO agreed with those above: bguan.picrate/bpsp.NHEt3, Ka=4. 7±0.1×105M-1, H=-83.97kJmol-1, S=-173.19Jmol-1K-1; bguan.picrate/bpse.NHEt3, Ka=2.4±0.2×105M-1, H=-88.49kJmol-1 and S=-182.27Jmol-1K-1. Heterodimers are highly stable in DMSO and less stable in DMSO-H2O. Multivalency in the interactions is manifested by positive cooperativity, negative enthalpy of formation (δH) and sizeable negative entropy (δS), in support of the development of well-ordered supramolecular structures in solution. © 2011 Taylor & Francis.


Sarantopoulou E.,National Hellenic Research Foundation | Petrou P.S.,Institute of Radioisotopes and Radiodiagnostic Products | Kollia Z.,National Hellenic Research Foundation | Palles D.,National Hellenic Research Foundation | And 3 more authors.
Journal of Applied Physics | Year: 2011

The bovine serum albumin (BSA)-polystyrene (PS) interface layer is laser photo activated at 157 nm for site selective multiple target-protein immobilization. The 5-15 nm photon induced interface layer has different chemical, wetting, and stiffness properties than the PS photon processed surface. The irradiated areas exhibit target-protein binding, followed by localized probe-target protein detection. The photon induced chemical modification of the BSA-PS interface layer is identified by: (1) Morphological, imaging, and analysis of surface parameters with atomic force microscopy, (2) spectroscopic shift (4 cm-1), of the amide I group and formation of new C=N, NH2, C-O, C=O, and O-C=O groups following irradiation, identified with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and (3) the different hydrophilichydrophobic and force-distance response of the bare PS and BSA-PS surfaces. Near field edge diffraction (Fresnel) fluorescence imaging specifies the threshold photon energy and the fluence required to optically detect the protein binding on the photon induced BSA-PS interface layer. By approximating the Fresnel integrals with analytical functions, the threshold photon energy and the fluence are expressed as the sum of zero, first, and second order harmonic terms of two characteristic diffracted modes and they are specified to be 8. 73 × 10-9 J and 623 J m-2, respectively. Furthermore, a bioarray of three probe-target proteins is fabricated with 1.5 m spatial resolution using a 157 nm laser microstepper. The methodology eliminates the use of intermediate polymer layers between the blocking BSA protein and the PS substrate in bioarray fabrication. © 2011 American Institute of Physics.

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