[Neutralizing single-chain antibodies against the tick-borne encephalitis virus].

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA 13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the sc13D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified sc13D6 was (3.0 +/- 0.2) x 10(7) M(-1) for the equilibrium state and (2.8 +/- 0.3) x 10(7) M(-1) in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC50 value for sc13D6 was 16.7 microg/ml.

[Interrelations between Ixodes persulcatus ticks and the tick-borne encephalitis virus of the red vole (Clethrionomys rutilis) in western Siberia]. / Vzaimootnosheniia kleshchei Ixodes persulcatus i virusa kleshchevogo éntsefalita s krasnoi polevkoi (Clethrionomys rutilus) v zapadnoi ibiri.

We present the data of 12-year survey (1989-2001) of the red vole population in southeastern West Siberia, including estimation of host relative numbers, abundance of immature taiga ticks, and percentage of animals with antigemagglutitnins against tick-borne encephalitis (TBE) virus. We discuss the role of demographic groups of voles as tick's hosts and their participation in the maintenance of TBE causative agent population. The estimation of spontaneous TBE infection rate in summer as well as in winter and early spring seasons, which have been made using a set of molecular-biological, serological and virological methods, demonstrates that a high proportion of red voles maintain non-pathogenic TBE causative agent over a long time, presumably, in the form of persistent infection.

[Preparation and selection of monoclonal antibodies for detecting hepatitis B surface antigen in blood sera]. / Poluchenie i otbor monoklonal'nykh antitel dlia vyiavleniia poverkhnostnogo antigena virusa gepatita B v syvorotkakh krovi.

HV monoclonal antibodies (MAb) were produced in order to improve the quality of HBsAg detection and their specific characteristics were compared with those of other MAbs. MAbs were characterized by asymmetric interactions with the antigen when used as first or second antibodies. The reactivity of a panel of HV and X MAb to ad and ay subtypes was studied by enzyme immunoassay. Mutual blocking (epitope mapping) of MAb helped select antibody couples for the creation of highly effective test system for the diagnosis of the major HBsAg subtypes. The sensitivity and specificity of MAbs were evaluated on reference and control panels of HBsAg sera and on serum specimens from a random sampling of 300 blood donors. The sensitivity of the most specific MAb pairs was 0.1 ng/ml for HBsAg subtype ay and 0.25 ng/ml for subtype ad. The specificity of attested MAb was 98.5% in incubation with stirring and 97% in static incubation. The optimal combinations of attested MAbs were used in the manufacture of Recomnathep B test system in the sandwich format.

[Monoclonal antibody study of antigens E and NS1 of Western Siberian tick borne encephalitis virus strains]. / Issledovanie s pomoshch'iu monoklonal'nykh antitel antigenov E i NS1 shtammov virusa kleshchevogo éntsefalita Zapadnoi Sibiri.

Antigenic structure of tick-borne encephalitis virus proteins was studied by ELISA with monoclonal antibodies (MAb) to E and NS1 glycoproteins of strain Sofyin. Envelope proteins appeared to be conservative which corresponded to previously determined nucleotide sequences of E gene fragments and deduced primary structures of the corresponding E protein. Five of six studied MAb to NS1 nonstructural glycoprotein of strain Sofyin reacted with this protein of all studied strains. The only exception was MAb 17C3 which discriminates West Siberian strains from Far Eastern strain Sofyin.

Tick-borne encephalitis virus strains of Western Siberia.

Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89-98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations.

[Preparation and properties of monoclonal antibodies to tick-borne encephalitis viral nonstructural proteins]. / Poluchenie i svoistva monoklonal'nykh antitel k nestrukturnym belkam virusa kleshchevogo entsefalita.

Hybridomas secreting monoclonal antibodies (Mab) to tick-borne encephalitis (TBE) virus are obtained. Immunodiffusion showed that 3 Mabs to TBE protein NS3 belong to class IgM and the rest to IgG1. Mabs to TBE protein NS1 were tested in hemagglutination inhibition, complement fixation, neutralization, and protection tests. Only 1 hybridoma produced Mab specific for protein NS1 of TBE strain Sofyin, the rest reacted with the common antigenic determinants of nonstructural TBE complex.

Comparative analysis of serological activity of non-structural protein (NS1) from tick-borne encephalitis virus and its analog expressed in bacterial cells.

By means of immunoaffinity chromatography and expression of the gene in Escherichia coli, non-structural glycoprotein NS1 of tick-borne encephalitis virus (TBEV) and its recombinant analog were prepared. Antisera against these proteins were obtained by hyperimmunisation of rabbits. The antisera were tested by means of complement fixation, agar diffusion, hemagglutination inhibition and virus neutralization. Although both antisera are reacted with natural antigen, the recombinant analog of NS1 did not bind antibodies against natural protein in complement fixation and immunoprecipitation. Nevertheless the NS1 analog was rather active in ELISA. Neither the natural nor the recombinant protein protected experimental animals from lethal virus infection. A contamination of natural NS1 antigen with small amounts of structural glycoprotein E may be responsible for both antibody formation and virus neutralization. This can be relevant for the design of a subunit vaccine.

Epitope analysis of tick-borne encephalitis (TBE) complex viruses using monoclonal antibodies to envelope glycoprotein of TBE virus (persulcatus subtype).

The arrangement of envelope protein epitopes of tick-borne encephalitis viruses (TBEV) (persulcatus or eastern subtype, Sofjin strain and ricinus or western subtype, Minsk-256 strain) and Kyasanur Forest disease virus (KFDV) was investigated using competitive binding of monoclonal antibodies against the Sofjin E protein. The E protein of TBEV Sofjin strain forms three antigenic domains: E1, E2 and E3, represented by 12, 9 and 2 epitopes respectively; two additional epitopes stand alone. Domains E1 and E2 are heterogeneous. On the epitope map of the Minsk-256 strain domain E3 remains intact, domains E1 and E2 overlap and the relative arrangement of virus-neutralizing epitopes from E1 and E2 domains is changed. The epitope map of KFDV is significantly dissimilar to TBEV. The viruses can be distinguished by epitopes with identical serological reactivity. A satisfactory agreement between our epitope maps and previously published antigenic models of flavivirus envelope protein (Guirakhoo et al., 1989; Mandl et al., 1989a) was observed. The main difference of our map is that domains corresponding to domains B and C (Sofjin strain) and A, B and C (Minsk-256 strain) in Heinz's model are overlapping. The results of competition analysis depend on the nature of the antigen (virion or purified protein) and the immunoassay technique.

[Expression of the gene for tick-borne viral encephalitis virus NS3 protein in Escherichia coli cells]. / Ekspressiia v kletkakh Escherichia coli gena belka NS3 virusa kleshchevogo éntsefalita.

On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E. coli cells. It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues. A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained. The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated. According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.

[Localization of the antigenic segment of the tick-borne encephalitis virus envelope protein using monoclonal antibodies]. / Lokalizatsiia antigennogo uchstka belka obolochki virusa kleshchevogo éntsefalita s ispol'zovaniem monoklonal'nykh antitel.

The largest cyanogen bromide fragment (GP-14,5; coordinates 78-176) of E protein belonging to the envelope of the tick-borne encephalitis (TBE) virus (Far Eastern subtype, strain Sofjin) interacted with five out of twelve E-specific monoclonal antibodies (MAbs). Having compared; efficiencies of some MAbs binding to the antigens of TBE viruses of Far Eastern and West European subtypes and primary structures of analogous peptides of these viruses, we suggested the epitopes of these MAbs to be located in the vicinity of 89 and/or 116-th amino acid residues of E protein. Effect of denaturing agents and reduction followed by carboxymethylation on the protein E antigenic properties was studied.

[Identification of individual antigenic epitopes of the envelope protein in the tick-borne encephalitis virus using monoclonal antibodies]. / Identifikatsiia otdel'nykh antigennykh épitopov belka obolochki virusa kleshchevogo éntsefalita s primeneniem monoklonal'nykh antitel.

A variant analysis of virus antigens of tick-borne encephalitis complex was carried out by enzyme immunoassays and topographic mapping of this protein using a panel of monoclonal antibodies to the structural glycoprotein of tick-borne encephalitis (TBE) virus of the Far East subtype. The results of the study confirmed the existence on the structural protein of both identical determinants typical of all the antigens of the complex and of subgroup-specific determinants. The topological analysis of the epitopes binding monoclonal antibodies revealed 3 separate domains possessing different functional properties. The results of topological mapping and immune typing of TBE virus antigen were compared.

[Monoclonal antibodies against tick-borne encephalitis virus glycoprotein: preliminary characterization]. / Monoklonal'nye antitela k glikoproteidu virusa kleshchevogo éntsefalita: predvaritel'naia kharakteristika.

Characterization of 12 clones of monoclonal antibodies (MAb) generated for the main immunogen of tick-borne encephalitis virus, glycoprotein E, is presented. The following MAb parameters have been determined: constants of binding with antigen, classes and subclasses of immunoglobulins, the activity in two variants of solid-phase enzyme-immunoassay, binding with protein A, and MAb behavior in serologic tests: hemagglutination-inhibition, diffuse precipitation in agar, and virus neutralization. The preliminary studies revealed the presence in these MAb of at least three groups of antibody complementary to various nonoverlapping sites on the structural virus glycoprotein. Further employment of these MAb in practical and research work is discussed.

[Use of protein A in the immunosorbent analysis of antibodies to the tick-borne encephalitis virus]. / Primenenie belka A v immunosorbentnom analize antitel k virusu kleshchevogo éntsefalita.

The results of the studies made with a view to developing the method for the determination of specific antibodies to the antigen of tick-borne encephalitis virus in human blood serum and liquor are presented. The method is based on the capacity of Staphylococcus aureus protein A to bind with Fc-region of immunoglobulins, which makes it possible to use this protein as the "second" system of antibodies. The conditions for the sorption of the antigen on polystyrene test tubes and for binding 125I-or horse radish peroxidase-labeled protein A preparations with antibodies have been determined, and the method has been approved in tests made on sera and liquor obtained from donors and tick-borne encephalitis patients.

[Development of a quick method of radioimmunoanalysis of myoglobin for the rapid diagnosis of myocardial infarction]. / Razrabotka bystrogo metoda radioimmunoanaliza na mioglobin dlia ékspress-diagnostiki infarkta miokarda.

Rapid radioimmunoassay of myoglobin is developed. The complete procedure requires about 60 min. The procedure might be used in routine laboratory practice. Content of myoglobin constituted from 14 to 56 ng/ml in blood serum preparations of 50 donors. In blood sera of 120 patients with acute myocardial infarction concentration of myoglobin was increased up to 90--6,000 ng/ml within 2-6 hrs, returning to the normal values within 38-48 hrs after the heart attack.