Russian Research Center for Molecular Diagnostics and Therapy

Moscow, Russia

Russian Research Center for Molecular Diagnostics and Therapy

Moscow, Russia
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Yabbarov N.G.,Russian Academy of Sciences | Posypanova G.A.,RAS Research Center Kurchatov Institute | Vorontsov E.A.,RAS Research Center Kurchatov Institute | Obydenny S.I.,Moscow State University | Severin E.S.,Russian Research Center for Molecular Diagnostics and Therapy
Journal of Controlled Release | Year: 2013

The use of vector molecules for the targeted delivery of antitumor drugs provides their selectivity for cancer cells. The recombinant receptor-binding fragment of alpha-fetoprotein (rAFP3D) was used as a vector molecule. The specific receptor of alpha-fetoprotein is a universal tumor marker, being expressed on the surface of many tumor cells, but not in normal human tissues. And rAFP3D includes the receptor binding cite of AFP. A three-component delivery system including vector protein rAFP3D, PAMAM G2 dendrimer and antitumor antibiotic doxorubicin (Dox) was synthesized. The attachment of two dendrimer molecules to the vector protein did not affect the effectiveness of rAFP3D binding to AFP receptor on the surface of tumor cells nor the effectiveness of receptor-mediated endocytosis. Dox was conjugated with G2 via cis-aconitic anhydride (acid labile linker). The in vitro Dox release study showed that the conjugate was stable at neutral pH but was labile at pH < 6. The Dox release was correlated with the intracellular distribution of conjugate in tumor cells. The rAFP3D-G2-Dox conjugate demonstrated a high cytotoxic activity against human ovarian adenocarcinoma cell lines: Dox-sensitive SKOV3 cells and Dox-resistant SKVLB cells and was low-toxic against human peripheral blood lymphocytes. Based on our findings, we may conclude that it is possible to significantly increase the effectiveness of Dox delivery to tumor cells by using the targeted delivery system comprising the recombinant third domain rAFP3D as a vector molecule. © 2013 Elsevier B.V.


PubMed | Moscow State University, Russian Research Center for Molecular Diagnostics and Therapy and RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry
Type: Journal Article | Journal: Doklady. Biochemistry and biophysics | Year: 2016

We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained.


Yabbarov N.G.,Russian Academy of Sciences | Posypanova G.A.,RAS Research Center Kurchatov Institute | Vorontsov E.A.,Russian Research Center for Molecular Diagnostics and Therapy | Popova O.N.,RAS Research Center Kurchatov Institute | Severin E.S.,Russian Research Center for Molecular Diagnostics and Therapy
Biochemistry (Moscow) | Year: 2013

Polyamidoamine (PAMAM) dendrimers of the second generation (G2) are branched polymers containing 16 surface amino groups that allow them to be used as universal carriers on creating systems for drug delivery. G2 labeled with fluorescein isothiocyanate (FITC) efficiently bound with the surface of tumor cells at 4 C and was absorbed by the cells at 37 C. The covalent binding to G2-FITC of a vector protein, a recombinant fragment of the human alpha-fetoprotein receptor-binding domain (rAFP3D), increased the binding and endocytosis efficiency more than threefold. Covalent conjugates of G2 with doxorubicin (Dox) obtained by acid-labile linking of cis-aconitic anhydride (CAA) without the vector protein (G2-Dox) and with the vector protein rAFP3D (rAFP3D-G2-Dox) were accumulated by the tumor cells with high efficiency. However, a selective effect was observed only in rAFP3D-G2-Dox, which also demonstrated high cytotoxic activity against the human ovarian adenocarcinoma SKOV3 cells and low cytotoxicity against human peripheral blood lymphocytes. Based on these results, rAFP3D-G2 conjugate is promising for selective delivery of antitumor drugs. © 2013 Pleiades Publishing, Ltd.


Severin E.S.,Russian Research Center for Molecular Diagnostics and Therapy
Russian Chemical Reviews | Year: 2015

Basic approaches to the design of targeted drugs for the treatment of human malignant tumours have been considered. The stages of the development of these approaches have been described in detail and theoretically substantiated, and basic experimental results have been reported. Considerable attention is paid to the general characteristic of nanopharmacological drugs and to the description of mechanisms of cellular interactions with nanodrugs. The potentialities and limitations of application of nanodrugs for cancer therapy and treatment of other diseases have been considered. The use of nanodrugs conjugated with vector molecules seems to be the most promising trend of targeted therapy of malignant tumours. © 2015 Russian Academy of Sciences and Turpion Ltd


Urusov A.E.,Russian Academy of Sciences | Kostenko S.N.,Russian Academy of Sciences | Sveshnikov P.G.,Russian Research Center for Molecular Diagnostics and Therapy | Zherdev A.V.,Russian Academy of Sciences | Dzantiev B.B.,Russian Academy of Sciences
Sensors and Actuators, B: Chemical | Year: 2011

An immunoanalytical system was developed for the determination of ochratoxin A with the use of a surface plasmon resonance (SPR) sensor amplified by the anti-species antibody-colloidal gold particle (CGP) conjugate. The use of the binding of immune complexes to the CGP-anti-species antibody conjugate leads to the SPR signal amplification by a factor of more than 10 and results in the 60 pg/mL limit of detection of ochratoxin A with an assay time of 30 min. These characteristics are superior to those obtained both in the conventional enzyme immunoassay with the use of the same reagents and the SPR assay with unmodified antibodies and specific antibodies conjugated to colloidal gold. © 2011 Elsevier B.V.


Sukoyan G.V.,Russian Research Center for Molecular Diagnostics and Therapy | Gongadze N.V.,Russian Research Center for Molecular Diagnostics and Therapy
Bulletin of Experimental Biology and Medicine | Year: 2011

The therapeutic action of adenocine during cardiac insufficiency (heart failure) caused by ischemic (stenosis) or reperfusion (removal of ligature) injury to the myocardium prevents depletion of ATP, the major energy source for myocytes in the right and left ventricles, and a drop in NAD/NADH ratio. The development of energy shortage during heart failure cannot be eliminated by β-acetyldigoxin, levosimendan, or milrinone: the content of ATP in the right and left ventricular myocardium remained below the normal level by 28 and 29%, 37 and 33%, 32 and 28%, respectively; the NAD/NADH ratio of the energy supply system in cardiomyocytes did not return to normal. Adenocine increased the content of NAD to the normal level in both the right and left ventricles, while it remained below the normal level after administration of β-acetyldigoxin (by 24 and 19.5%, respectively), levosimendan (by 27 and 29%), and milrinone (by 26 and 24%). In contrast to β-acetyldigoxin, levosimendan, and milrinone, adenocine inhibited activity of poly(ADP-ribose) polymerase in both ventricles. It is concluded that adenocine directly inhibits the key enzyme triggering apoptosis; we also hypothesized that this drug activates the regulatory and signal mechanisms arresting apoptotic alterations in the myocardium during heart failure. © 2011 Springer Science+Business Media, Inc.


Urusov A.E.,Russian Academy of Sciences | Kostenko S.N.,Russian Academy of Sciences | Sveshnikov P.G.,Russian Research Center for Molecular Diagnostics and Therapy | Zherdev A.V.,Russian Academy of Sciences | Dzantiev B.B.,Russian Academy of Sciences
Journal of Analytical Chemistry | Year: 2011

The detection of mycotoxins-toxic contaminants of fungal origin-is an important problem in the food and feed quality control. An immunochromatographic system was developed for the detection of ochratoxin A (OTA), which is one of the priority contaminants in grain. Monoclonal antibodies against OTA and their conjugates with colloidal gold nanoparticles were prepared. The detection is based on the competition of OTA in a sample and an OTA-protein conjugate immobilized on a test strip for the binding to anti-bodies on the colloidal particle surface. The method was tested in the analysis of plant extracts (maize and barley extracts). It was shown that OTA can be detected in a medium with a high content of an organic solvent (up to 35% of methanol). The disappearance of the line in the test zone is visually detected at OTA concentrations starting from 50 ng/mL. In the case of the video-digital detection of changes in the color intensity of the test zone, the limit of detection of OTA is 5 ng/mL. The duration of the assay is 10 min. © 2011 Pleiades Publishing, Ltd.


Sharapova O.A.,Moscow Research Institute of Medical Ecology | Yurkova M.S.,Russian Research Center for Molecular Diagnostics and Therapy | Laurinavichyute D.K.,Russian Research Center for Molecular Diagnostics and Therapy | Andronova S.M.,Russian Research Center for Molecular Diagnostics and Therapy | And 3 more authors.
Journal of Chromatography A | Year: 2011

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26. kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins. © 2011 Elsevier B.V.


Hendrickson O.,Russian Academy of Sciences | Fedyunina N.,Russian Academy of Sciences | Zherdev A.,Russian Academy of Sciences | Solopova O.,Russian Research Center for Molecular Diagnostics and Therapy | And 2 more authors.
Analyst | Year: 2012

The aim of the present study was to produce monoclonal anti-fullerene C 60 antibodies and to develop the enzyme immunoassay for the detection in the first use of free fullerene C 60 both in solutions and in multicomponent biological probes. The immunization of mice with the conjugate of fullerene C 60 carboxylic derivative with thyroglobulin synthesized by carbodiimide activation led to the production of eight clones of anti-fullerene antibodies. The specificity of the antibody-fullerene binding was confirmed. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of water-soluble protein-conjugated fullerene, the fullerene aminocaproic acid, fullerenol and for pristine fullerene in solution. To solubilize extremely hydrophobic free fullerene C 60 a specially selected water-organic mixture compatible with immunoassay was proposed. The detection limit of free fullerene C 60 in solution was 2 μg L -1. Fullerene C 60 was also detected by ELISA in organ homogenates of rats intraperitoneally or intragastrically administered with fullerene. To reduce the influence of biomatrices on the assay results a technique was developed for the biological sample pretreatment by the extraction of C 60 from bioprobe by toluene followed by the evaporation of toluene and dissolution of the fullerene-containing extract in the selected water-organic media. The ELISA procedure in the first use allowed the detection of fullerene C 60 in different tissues.


Byzova N.A.,Russian Academy of Sciences | Zvereva E.A.,Russian Academy of Sciences | Zherdev A.V.,Russian Academy of Sciences | Eremin S.A.,Moscow State University | And 2 more authors.
Analytica Chimica Acta | Year: 2011

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR-protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500ngmL -1) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16-250ngmL -1 its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR-protein conjugate. The duration of the assay is 10min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested). © 2011 Elsevier B.V.

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