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E Kontseková

Publications and source records attributed to E Kontseková.

At least 19 recordsLinked to original sources
PubMed2000

Radioiodination of human interferon-alpha2 interferes with binding of C-terminal specific antibodies.

Radioimmunoassays based on reactivity between a monoclonal antibody (mAb) and human 125I-interferon (IFN)-alpha2 are frequently exploited in interferon research. In general, epitopes of antibodies specific for human IFN-alpha2 are located on the two immunodominant structures formed in the N- and C-terminal domains, respectively. We found that labelling of IFN-alpha2 with Na(125)I by the chloramine-T method did not affect the binding of antibodies recognising the N-terminal region 30-53. In contrast, radioiodination of IFN was associated with a dramatic decrease in IFN reactivity with mAbs specific for the C-terminus (residues approximately 120-145 approximately ). We suggest that steric hindrance araising from the incorporation of 125I into the tyrosine residues at positions 123, 130 and 136 may be responsible for the change in immunoreactivity. The adverse effect of radioiodination of IFN-alpha2 on the binding potency of C-terminal specific mAbs must be taken into consideration in experiments based on the interaction of such antibodies (i.e. NK2) with the labelled antigen.

Antibodies, Monoclonal↗
PubMed2000

Engineered acid-stabile human interferon gamma.

Loss of anti-viral potency upon pH2-treatment is an inherent feature of interferon (IFN)-gamma. The phenomenon seems to be caused by dissociation of IFN-gamma homodimer into subunits upon acidification and subsequent self-association of monomers into aggregates with reduced activity after neutralization. We demonstrated that acid-stability could be engineered into human IFN-gamma without affecting its specific activity. An artificial intra-monomer disulphide bond E7C/S69C stabilizes the dimeric form of the cytokine, which retained its full bioactivity after exposure to pH2. Acidification did not modify the antigenic structure of IFN-gamma as proved by a panel of mouse anti-human IFNgamma antibodies.

Amino Acid Substitution↗
PubMed2000

The carboxyterminal domains of human IFN-alpha2 and IFN-alpha8 are antigenically homologous.

The antigenic properties of human hybrid IFN-alpha8(60)/alpha1(92)/alpha8 were compared with those of human IFN-alpha1 and IFN-alpha2 using monoclonal antibodies (mAb). Hybrid IFN demonstrated a significantly closer antigenic relationship to the subtype alpha2 than to the subtype alpha1. In particular, high homology was observed between antigenic structures located in the C-terminal domains (93-166) of IFN-alpha8 and IFN-alpha2, whereas the corresponding N-terminal receptor-binding domains (30-53) showed distinct antigenic characteristics. The 100% homology between IFN-alpha8 and IFN-alpha2 in the region 114-131 (helix D) indicated the role of this region in formation of the common antigenic structure. In IFN-alpha8/1/8, this shared antigenic structure was important for antiviral activity and exhibited immunodominant properties, consistent with functional and antigenic properties of the corresponding structure in IFN-alpha2. Based on this antigenic homology, we suggest that IFN-alpha8 and IFN-alpha2 are evolutionarily more closely related to each other than to IFN-alpha1. This study will contribute to a better understanding of evolutionary events in the human IFN-alpha family.

Amino Acid Sequence↗
PubMed1999

Structural and functional heterogeneity of the amino-terminal receptor-binding domain of human interferon-alpha 2.

Structural immunoanalysis of human interferon (IFN)-alpha 2c revealed antigenic and functional heterogeneity in its N-terminal receptor-binding domain (loop AB). Monoclonal antibodies (mAbs) mapped to the region 30-53 of IFN-alpha 2 defined three partially overlapping antigenic sites designated here as 'a', 'b' and 'c'. For the high-affinity binding of IFN-alpha 2c to the cellular receptor, site b located in segment 34-41 and site c (residues 43-53) appeared to be most important. Only the part of site a (amino acids 30-33) seemed to be involved in the interaction with receptor. The segment of residues 30-46 forms a relatively straight structure on the protein surface, according to the three-dimensional model of human IFN-alpha 2.

Antibodies, Monoclonal↗
PubMed1999

Immunogenicity of interferon-alpha 2 in therapy: structural and physiological aspects.

Recombinant human interferon (rIFN)-alpha 2 has been approved for therapeutic application in a range of human oncological and viral diseases. However, some patients can develop strictly specific antibody response to rIFN-alpha 2, which may diminish its therapeutic potential. Such humoral response appears to be quite complex and obviously depends on multiple parameters. Our review is aimed primarily to factors associated with structural modifications of rIFN-alpha 2 that we consider crucial for formation of therapy-induced antibodies. These factors are either related to inherent conformational differences between three IFN-alpha 2 subvariants or to immunogenically active contaminating derivatives resulting from production, purification and storage of this recombinant protein. In addition, the role of treatment regimen and physiological variables modulating the immune response to rIFN-alpha 2 in the challenged organism are mentioned.

Antibody Formation↗
PubMed1998

Therapy-induced antibodies to interferon-alpha 2a recognise its receptor-binding site.

Fifty-eight patients with chronic hepatitis B (HB) or C (HC) were treated with recombinant human interferon (rIFN)-alpha 2 and their sera were assayed for antibodies to rIFN-alpha 2c. Twelve of these patients produced low titres and two high titres of the antibodies. We localized the region which was recognised by the high-titre therapy-induced antibodies on the IFN molecule by testing the antibodies with a set of murine monoclonal antibodies (MoAbs) to IFN-alpha 2 in a competitive radioimmune assay (RIA). Only MoAbs with epitopes located in the amino-terminal portion of IFN-alpha 2 could inhibit the binding of radiolabelled IFN-alpha 2 by patients' sera. Our data indicate that the therapy-induced antibodies were directed to the receptor-binding domain of IFN-alpha 2 formed by amino acids (aa) 30-53. In accordance with this observation, human anti-IFN sera inhibited the binding of rIFN-alpha 2 to human cells.

Adult↗
PubMed1998

[Present views on interferons].

The nature of interferons and mechanisms of their action have been studied for more than four decades. The review article brings the current view of interferon, putting emphasis on historical transitions in its definition and changes in the understanding of the physiological role of interferon in organism. The recent classification of interferons together with the more detailed characterization of human interferons are presented. (Tab. 1, Ref. 40.)

Humans↗
PubMed1997

Analysis of an interaction between the soluble vaccinia virus-coded type I interferon (IFN)-receptor and human IFN-alpha1 and IFN-alpha2.

The soluble B18R protein coded by vaccinia virus exerts properties of a type I interferon (IFN)-receptor with broad species specificity. We analyzed neutralizing and binding activity of the B18R protein against several recombinant human type I IFNs. The B18R protein inhibited the antiviral potency of IFN-alpha1, IFN-alpha2, IFN-alpha8/1/8, and IFN-omega on human cells. The N-terminal domain of human type I IFN is involved in the high affinity binding to its cellular receptor. To localize the binding domain(s) of IFN with the B18R protein, competition experiments between B18R, and mapped monoclonal antibodies to IFN-alpha1 and IFN-alpha2 were performed. Surprisingly, our data indicated that the contact area between the B18R protein and IFN comprised in addition to the N-terminal region of IFN-molecule also its C-terminal portion. We suggest that this different pattern of interaction with a ligand might determine the ability of B18R protein to bind type I IFNs of different species.

Antiviral Agents↗
PubMed1997

Structural immuno-analysis of human and porcine interferon gamma: identification of shared antigenic domain.

We examined the antigenic resemblance between human (h) and porcine (p) interferon (IFN)-gamma by binding (ELISA) and neutralization assays. The murine polyclonal antisera and sets of murine monoclonal antibodies (mAbs) raised against either IFN were tested in confrontation with recombinant IFNs of either species, and with site-specific mutants of hIFN-gamma. Several of the mAbs raised against pIFN-gamma cross-reacted in ELISA with hIFN-gamma. In contrast, none of the anti-hIFN-gamma mAbs cross-reacted. By employing site-specific mutants of recombinant hIFN-gamma as antigens in ELISA we succeeded in identifying the C-terminal portion 97-111 as the antigenic site in hIFN-gamma recognized by the cross-reactive anti-pIFn-gamma mAbs. None of the mAbs recognizing the common antigenic structure had neutralizing potency, although His111 was determined by others as the residue important for bioactivity of hIFN-gamma. Mutations in the domain 97-111 had no or little influence on homospecific reactivity of anti-hIFN-gamma mAbs, indicating that this domain, while being mouse-immunodominant in the case of pIFn-gamma was poorly immunogenic in the case of hIFN-gamma. The epitopes of three out of five anti-hIFN-gamma mAbs mapped in the N-terminal region 1-23, indicating immunodominance of this region in hIFN-gamma. Another mAb (D9D10), also directed to the N-terminus of hIFN-gamma, apparently recognized a conformational epitope. This antibody lacked ELISA-reactivity with the wild-type hIFN-gamma but strongly bound mutant protein with an engineered disulfide bridge Cys7-Cys69. Surprisingly, D9D10 showed high reactivity also with the wild type hIFN-gamma produced by baculovirus construct coding for the mature protein with signal sequence or with wild type protein possessing residues Cys-Tyr-Cys from the signal sequence.

Animals↗
PubMed1997

Different stabilities of the N- and C- terminal domains of human interferon alpha.

The present results are consistent with the hypothesis predicting two structurally independent polypeptide domains in the regions 1-92 and 111-166 of the type I IFNs. However, we observed differences in molecular unfolding between the N- and C-terminal portions of human IFN-alpha during denaturation in SDS solutions. Monoclonal antibodies (mAbs) detected changes in the N-terminal region (residues 1-85) of denaturated IFN-alpha 1 or IFN-alpha 2. In contrast, SDS-denaturation of antigens did not affect the reactivity of mAbs with epitopes located in the C-terminal portion (residues 105-166) of both IFNs. The N-terminal domain is known to be involved in the high affinity receptor binding of IFN-alpha. Therefore a theory that the active sites may be conformationally more flexible than the rest of the polypeptide might explain the lower conformational stability of this domain.

Animals↗
PubMed1997

Forty years of interferon.

The nature and mode of action of interferon (IFN) have been intensively studied for more than 40 years. In this review, we summarize the current knowledge of IFN putting emphasize on transitions in its definition, understanding of its physiological role and nomenclature, and on brief characterization of individual IFN families. Finally, the evolution of IFN and relations between the IFN families are discussed.

Animals↗
PubMed1996

Conformational changes in pH2-treated human interferon-alpha 2 detected with monoclonal antibodies.

Monoclonal antibodies allowed to demonstrate the existence of alternative antigenic forms of the same molecule as of human interferon (IFN)-alpha 2. Exposure of recombinant IFN to pH 2, although not affecting its bioactivity, induced structural modulation of molecular surface. The antigenic structure of IFN-alpha 2 appeared to be built of the acid-stable and acid-labile epitopes. In general, the acid-stable sites determined subtype-specific antigenic properties of the protein, whereas the acid-labile determinants were responsible for antigenic characteristics shared by some other human IFNs. Acidification of IFN-alpha 2 to pH 2 for at least 1-2 h resulted in simultaneous structural rearrangement of all acid-labile sites.

Antibodies, Monoclonal↗
PubMed1995

Relativity of an antigenic homology between human interferon-alpha 1 and interferon-alpha 2c.

Analysis of an antigenic relatedness between human interferon (IFN)-alpha 1 and IFN-alpha 2 was performed with mapped monoclonal antibodies raised to the respective subtypes. Antigenic properties of immunoreactive domains located in the N-terminal segments 30-67 of IFN-alpha 1 and IFN-alpha 2 were found distinct when compared by neutralization bioassay or ELISA. On the other hand, corresponding domains exhibited an unexpectedly high antigenic homology when tested by Western blot. We suppose that this relativity in antigenic relation lies in the various extent of denaturation of IFN-molecules in bioassay, ELISA and immunoblot. Structural differences of tested antigens may be responsible for a conformation-determined access of antibodies to the shared epitopes.

Amino Acid Sequence↗
PubMed1992

Common and different antigenic properties of the rabies virus glycoprotein of strains SAD-Vnukovo and Pitman-Moore.

Two fixed rabies virus strains, SAD-Vnukovo and Pitman-Moore (PM) were used as combined immunogens for the generation of hybridomas secreting specific monoclonal antibodies (MoAbs). The obtained hybridomas were primarily screened by an ELISA for production of MoAbs to antigen of SAD-Vnukovo strain. Six positive clones were established. A panel of MoAbs has been characterized according to reactivity in immunofluorescence, immunoblot, ELISA and neutralization tests. All MoAbs were positive in immunofluorescence when cells infected with the SAD-Vnukovo strain were used. By immunoblot, four MoAbs showed specificity for the viral glycoprotein of both SAD-Vnukovo and PM rabies strains. This pattern of reactivity indicated the existence of shared conformation-independent epitopes located on the related antigens. However, in ELISA, the tested MoAbs did not recognize viral glycoproteins of the PM strain. This indicates, that the different strain-specific conformations of the native glycoprotein determine the accessibility of the common linear determinants for respective antibodies. Only one antibody, with conformation-dependent glycoprotein specificity, was capable to neutralize the CVS strain of rabies virus.

Animals↗
PubMed1992

[Bispecific antibodies: present status and possibile applications].

The review article information on the current status in the research regarding bispecific antibodies. The theoretical and methodical aspects of preparation of heterohybridomas secreting bifunctional monoclonal antibodies are discussed in details. The question of purification of the antibody fraction with dual specificity is also addressed. The possible application of bispecific antibodies in human medicine for immunodiagnostics and aimed immunotherapy is described. (Fig. 7, Ref. 22.).

Antibody Specificity↗
PubMed1991

Distinct effect of pH 2 on a common antigenic structure found in human interferons-alpha 1 and -alpha 2 in the region 30-35.

The antigenic similarity between molecules of recombinant human interferon-alpha 1 (IFN-alpha 1) and recombinant human IFN-alpha 2 was demonstrated with neutralizing monoclonal antibody (mAb) 1-46. The common epitope for the mAb 1-46 was localized into amino-terminal region of IFN-alpha molecule around residues 30-35. Following pH 2 treatment, the biological activity of both IFN-alpha 1 and IFN-alpha 2 was retained but the antigenic relatedness between corresponding sequences 30-35 was diminished. The common structure on the IFN-alpha 1 molecule proved acid stable and the mAb 1-46 retained the ability to neutralize the pH 2 treated IFN-alpha 1. However, the neutralization of pH 2-treated IFN-alpha 2 by specific antibody was completely suppressed. These results complemented our earlier finding of the dramatic effect of acidic pH on the antigenic structure of region 132-137 of the IFN-alpha 2 molecule. We conclude that pH 2 may induce a conformational rearrangement of the IFN-alpha 2 molecule, resulting in an altered tertiary structure with deviating antigenic characteristics.

Acids↗
PubMed1991

Enhancement of neutralizing efficacy by combining three monoclonal antibodies to human interferon-alpha.

Three murine monoclonal antibodies (mAb) directed to distinct epitopes on recombinant human interferon (IFN)-alpha 1, and three mAb recognizing distinct epitopes on recombinant human interferon (IFN) alpha 1, and three mAb recognizing distinct epitopes on recombinant human IFN-alpha sc, were studied by IFN-neutralizing assays. The efficacy of neutralization of the anti-viral and the anti-proliferative activities of IFN-alpha 1, or IFN-alpha 2c, by the specific antibodies used, individually or in combination, were evaluated. In comparison with single mAb, the mixtures of three mAb against IFN-alpha 1 or three mAb against IFN-alpha 2c were capable of neutralizing more than 10-times larger amounts of IFN-alpha 1 and alpha 2c, respectively. The strong potentiation of the neutralization efficacy resulting from mixing different mAb was demonstrated by neutralization of the anti-viral as well as the anti-proliferative activities of both recombinant IFN. The neutralization experiments support the interpretation that the observed potentiation results from simultaneous interaction of anti-IFN mAb with different epitope specificity.

Antibodies, Monoclonal↗