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Introduction: When a new disease occurs, one of the most affordable remedies is drugs containing specific antibodies to this infectious agent. The use of such drugs is aimed at reducing the amount of the pathogen in the macroorganism and the associated reduction in the severity of the symptoms of the disease or recovery. The purpose of this review is to analyze the experience of using immunoglobulins and monoclonal antibodies in the treatment of COVID-19 patients during the pandemic., Results and Conclusion: The two main groups of medical protective agents that block the penetration of the SARS-CoV-2 virus into permissive cells are drugs obtained from blood plasma of convalescents (immunoglobulin) and human monoclonal antibodies. The first group of drugs in the treatment of COVID-19 includes blood plasma of convalescents, which can be successfully used for emergency prevention. The main disadvantage of using blood plasma convalescents is the difficulty of standardization due to the different content of specific antibodies in donors. Another disadvantage is the undesirable side effects in recipients that occur after plasma administration. An alternative approach to COVID-19 therapy is the use of humanized and genetically engineered human monoclonal antibodies against certain epitopes of the SARS-CoV-2 virus. For example, monoclonal antibodies against receptor-binding domain of the S-protein, which prevents the virus from entering permissive cells and interrupts the development of infection. The advantages of these drugs are their safety, high specific activity, and the possibility of standardization. However, the complexity of their production and high cost make them inaccessible for mass use in practical medicine.

Previously, we have reported the crystal structures of Fab fragment of Infliximab in complex with TNFα. The structurally identified epitope on TNFα revealed the mechanism of TNFα inhibition by partially overlapping with the TNFα-receptor interface and the possibility to optimize the binding affinity. In this study, we launched a screen of a phage display library to isolate novel anti-TNFα antibodies based on the infliximab epitope. To develop novel anti-TNFα antibodies, structural analysis, the phage display antibody isolation, step by step antibody optimization, CDR residues random mutagenesis, and binding affinity characterization were performed. One of the novel antibodies generated on the backbone of infliximab, Inf3D6, has the superior binding affinity to TNFα, thus, demonstrating the potential for structure guided optimization for improvement of existing antibody-based therapeutics.

The immunochromatographic test-system was developed for detecting protein of pathogenicity of CagA Helicobacter pylori in various biological samples (feces, content of dento-gingival recesses) and also in culture of H.pylori. The test-system represents multi-membrane composite on the basis of membranes manufactured by "MDI" (India). The main immunochemical components of test-system are conjugate of nanoparticles of colloid gold with size of 30 nm with monoclonal antibodies (clone HP-387), applied to membrane for conjugate; monoclonal antibodies (clone HP-1811) and anti-species antibodies of goat against Ig of mouse, applied to nitrocellulose membrane correspondingly in test and control zones. All antibodies are produced by the firm "Bialeksa" (Russia)., Competing Interests: The authors declare no conflict of interest.

Some drugs candidates for treatment of Ebola virus disease (EVD), have been studied, monoclonal antibody (mAb) cocktails have shown great potential as EVD therapeutics. The advantages of mAb therapy include low toxicity, high specifcity and versatility, with the range of biological effects being dependent upon the Fc region. Functions of mAbs include pathogen opsonisation, complement activation, antibody-dependent cell cytotoxicity and virus neutralization characteristics. The most known mAb cocktail, used as therapeutic, is ZMapр, manufactured by «Leaf Biopharmaceutical» from 2004. The elaborated mAb cocktails, structures and properties s of mAbs, the protective characteristics of mAbs and development of new pan-ebolavirus mAbs are reviewed in this article., Competing Interests: The authors declare no conflict of interest.

Co-circulation of two evolutionary distinct lineages of influenza virus in one epidemic season has led to development specifc reagents for rapid identifcation and typing of new isolates. Panel of MAbs to hemagglutinin of influenza virus B/Brisbane/46/15 belonging to Victoria evolutionary lineage was developed. All MAbs reacted in ELISA with B/Victoria-like strains only. There were no interactions with heterologous influenza viruses of B/Yamagata lineage, seasonal and potentially pandemic influenza A viruses. All MAbs reacted in hemagglutination inhibition and virus neutralization. MAbs interacted in hemagglutination inhibition only with B/Victoria-like viruses, but did not interacted B/Yamagata-like strains. Neutralization and hemagglutination inhibition studies of viruses isolated before 1983 with MAbs revealed that MAbs 6E11, 9G5, 9B5 and 6A4 had the ability to interact with the virus B/ Russia/69 which may evidence that B strains of early isolation period (before lineage separation) have common epitope with recent Victoria lineage viruses. MAbs 7C8, 7G9, 7H8 and 8D11 were directed to a conserved epitope (or epitopes) specifc for influenza hemagglutinin viruses of B/Victoria group. The presence of differences in the effectiveness of the interaction of MAbs 6A9, 7G9 and 8A8 in hemagglutination inhibition test allows the identifcation and differentiation of strains isolated in chicken embryos and MDCK cell culture. Thus, the developed MAbs can be successfully used for identifcation and antigenic analysis of B/Victoria-like strains., Competing Interests: The authors declare no conflict of interest.

The technology for the generating of single-domain recombinant monoclonal antibodies (nanoantibodies) based on the immunization of a camel, cloning of induced sequences encoding single-domain antigen-recognizing fragments of non-canonical camel antibodies, as well as functional selection of clones of nanoantibodies by the phage display method, was used to obtain new effective tools for more efficient diagnostics of Chlamydia infection and to develop new approaches for effective therapy. Two promising nanoantibodies were obtained. They showed effective binding to extracellular and intracellular forms of C. trachomatis, and also had activity that inhibited the development of chlamydial infection in vitro.

In the literature review covered issues opening protein-proprotein convertase, subtilisin/kexin-type9 (PCSK9), its modern terminology, the results of its biochemical, molecular and genetic studies, metabolic regulation, functions and clinical findings in the blood content ofPCSK9 in lipid disorders and clinical pharmacological studies of monoclonal antibodies to this protein for the correction of lipid metabolism of major interest for cardiology and lipidology.

The comparative evaluation was carried out concerning systems of detection using conjugates on the basis of peroxidase, alkaline phosphatase, colloid gold and system of amplification “Super-CARD" for multiplex dot-immune assay of antibodies. It is established that sensitivity of system of detection with colloid gold is approximately 8 times higher than with amplification “SuperCARD"; 30 times higher than with conjugate of alkaline phosphatase and 250 times higher than with peroxidase conjugate. The immunesols of gold maintain limit of detection of human IgG 10 pg with dynamic range of signal alteration from 5 ng to10 pg overlapping the range of concentrations of specific IgG in case of typical natural immune response. The most specific results provides conjugate of colloid gold with monoclonal antibodies to human IgG.

The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.

The change in the concentration and antigen-binding activity of 28 monoclonal antibodies was studied after their adsorption on the surface of polystyrene microplates in buffers with different pH values (1.0, 2.8, 7.5, 9.6, and 11.9). We used 16 clones to the HIV p24 protein and 12 clones to the surface antigen of Hepatitis B Virus. The binding efficiency of adsorbed antibodies to the labeled antigen was evaluated by the slope of the linear region of the binding curve to the concentration axis. It was shown that the antigen-binding activity of six antibodies (21.5%) statistically significantly increased after adsorption at pH 2.8 and 11.9 as compared to pH 7.5 and 9.5. The maximum amount of antibodies was found to be adsorbed on the solid surface at pH 7.5. The analysis of the binding of 125I-HBs-antigen to adsorbed antibodies made it possible to evaluate the concentration of active antibodies on the polystyrene surface. It was shown that the increase in the antigen-binding activity was due to an increase in the proportion of antibodies with retained activity after adsorption at pH 2.8 and 11.9. Under these conditions, about 20% of the antibodies retained their antigen-binding activity, and 6% did so after immobilization at pH 7.5.

Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

A hybridoma panel producing monoclonal antibodies to immunochemically non-identical antigenic epitopes of the protein nature located in outer membrane of Yersinia pseudotuberculosis was obtained. It was revealed that the previously identified B-antigen protecting laboratory animals from experimental plague was detected using both monoclonal antibodies against mentioned protein determinants and the determinants of lipopolysaccharide O-side chains. The B-antigen is a component of the Y. pseudotuberculosis outer membrane, egested in the form of the vesicles (OMVs) by bacteria cultivated in fluid nutrient medium.

The goal of this work was to analyze the antigenic structure of the hemagglutinin (HA) of the pandemic influenza virus A(H1N1)pdm09 using monoclonal antibodies (MAbs) and to develop a sandwich ELISA for identification of pandemic strains. Competitive ELISA demonstrated that 6 MAbs against HA of the pandemic influenza A/ IIV-Moscow/01/2009 (H1N1)pdm09 virus identified six epitopes. Binding of MAbs with 22 strains circulating in Russian Federation during 2009-2012 was analyzed in the hemagglutination-inhibition test (HI). The MAbs differed considerably in their ability to decrease the HI activity of these strains. MAb 5F7 identified all examined strains; MAbs 3A3 and 10G2 reacted with the majority of them. A highly sensitive sandwich ELISA was constructed based on these three MAbs that can differentiate the pandemic influenza strains from the seasonal influenza virus. The constancy of the HA epitope that reacts with MAb 5F7 provides its use for identification of the pandemic influenza strains in HI test. MAbs 3D9, 6A3 and 1E7 are directed against the variable HA epitopes, being sensitive to several amino acid changes in Sa, Sb, and Ca2 antigenic sites and in receptor binding site. These MAbs can be used to detect differences in HA structure and to study the antigenic drift of the pandemic influenza virus A(H1N1)pdm09.

A study of the influence of exogenous factors on the immunochemical activity of the bacterium Yersinia pseudotuberculosis and lipopolysaccharide preparations isolated from bacteria was performed using monoclonal antibodies. It was shown that the hybridomas that were obtained in this work produce antibodies against different and, most likely, species-specific epitopes associated with lipopolysaccharide O side chains. The antibody concentrations produced increased with a decrease in the temperature, at which the bacteria were cultivated. An inhibitory effect of proteinase K, pepsin, and trypsin on the immunochemical activity of bacterial cells, determined using a solid-phase enzyme immunoassay, was demonstrated. Treatment with sodium periodate showed no uniform effect on the reactions between monoclonal antibodies and antigens (lipopolysaccharides and microbial cells), as adjudged by an immunoassay, which is most likely a consequence of the different localization of lipopolysaccharide epitopes recognized by the antibodies from four hybridomas.

An enzyme immunoassay-based test system for Y. pestis V antigen detection was developed. The specificity and sensitivity of this system met the requirements for medical immunobiological preparations for the identification of causative agents of highly fatal diseases. The sensitivity of the test system was assessed, and its high specificity was also demonstrated: the test system did not detect bacterial cells of closely related (four Y. pseudotuberculosis strains) and heterologous microorganism strains. The test system developed was able to detect the V antigen at concentrations as low as 2.0 ng/mL in cells of nine experimental Y. pestis cultures. The obtained preparation can be recommended for use in laboratory diagnostics of plaque.

A panel of five monoclonal antibodies (MAbs) to the HA1 molecule of the influenza B virus of the Victorian lineage with high virus-neutralizing activity was developed. For identification of the virus neutralizing epitopes in HA1 escape mutants (EM) of the influenza BIShandong/07/97 and B/Malaysia/2506/04 virus were selected using virus- neutralizing antibodies (MAbs). Three EMs had single, two--double and one--triple amino acid substitutions (AAS) in HA1 (H122N, A202E, K203T, K2031, K203N or A317V). In addition, AAS N197S was detected in three EMs. A correlation of AAS identified with peculiarities of interaction of EMs with Mabs was discussed.

Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate.

The source of monoclonal antibodies was chosen the cultural fluid of hybridoma-producers deposited in the specialized collection of cell cultures of vertebrates (St. Petersburg) with numbers RKKK(P) 386D and RKKK(P) 674D. The specific immunoglobulin (Ig) from cultural fluid was concentrated by precipitation with saturated solution of ammonium sulfate. The scheme of obtaining monoclonal antibodies included activation of peroxidase, conjugation of activated peroxidase with Ig, removal of unbounded proteins, storage and control. The preservation of activity of conjugates was supported with BSA (10%) or glycerin (50%). The last on is preferable to be applied for this purpose. The test of monoclonal antibody-01 and monoclonal antibody-0139 of peroxidase conjugates with kit of strains of comma bacillus 01 and 0139 demonstrated their strict specificity because they interacted only with corresponding serum groups under absence of crossed reactions with representatives of geterologic microorganisms. The direct dot-immune analysis is carried out during 1.5 hour and its sensitivity is within the limits 105-106. The application of diagnostic monoclonal peroxidase conjugates 01, 0139 in laboratory practice can promote the increase of specificity of serologic analysis of cholera and saving time-frame of its application.

A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 10(6) cells/mL (5 x 10(4) cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.

The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013-0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.

A new construction of a test-system for free thyroid hormone 3,3',5-triiodothyronine determination (free T3) in human blood serum in ELISA and IRMA variants was described. For this purpose a low-molecular weight bifunctional conjugate containing T3 and vitamin H (biotin, Bt) residues was synthesized. The conjugate (T3-Bt) can be bound via its biotin function to biotin-binding proteins on a solid phase whereas its T3-portion can interact with monoclonal antibody against T3 (anti-T3-MAb) labeled with horseradish peroxidase or iodine-125 in an enzyme immunoassay or an immunoradiometric assay system (ELISA or IRMA), respectively. The immunochemical interaction is hindered if the T3 residue takes part as a component of a preformed solid phase complex between T3-Bt and avidin. Computer modeling shows that in this complex glycan component of avidin shields iodothyronine residue. The binding of T3-Bt both with T3-free sites on anti-T3-MAb and the solid phase avidin achieves with help of a delayed T3-Bt addition into avidin-covered containers with preliminary co-incubated labeled anti-T3-MAb and an assayed T3 sample.

Brain-specific anion transporter BSAT1 extracellular fragment (451-557) cDNA was cloned in a vector for prokaryotic expression, a producer E. coli strain was obtained, and recombinant extracellular fragment BSAT1(451-557)was purified and used for immunization of BALB/c mice. Splenic cells from mice with verified immune response were used for hybridoma generation. Several hybridoma clones producing monoclonal antibodies to BSAT1 extracellular fragment were selected. Antibody specificity was confirmed by ELISA, immunoblotting with recombinant BSAT1(451-557), and immunofluorescent BSAT1 assay on rat brain sections and cultured HEK293 cells. It was demonstrated that the obtained antibodies specifically bind native rat and human BSAT1 and can be used in both fundamental studies of structures forming the blood-brain barrier and development of targeted transport of diagnostic preparations and drugs across the blood-brain barrier.

The impact of monoclonal antibodies (mAb) biotinylation on the output and the repertoire of selected peptides in the biopanning procedure were tested. A comparative analysis of the peptides selected from phage library using the biotinylated and non-biotinylated mAb 2F5 was performed. It was shown that the output of peptides homologous to the native epitope was 1.7-fold higher for biotinylated antibodies, whereas the binding capacity of the selected phages with mAb 2F5 in ELISA was higher in the case of using non-biotinylated antibodies. It should be noted that the phages exposing peptides, which have 4-5 amino acid sequence similarity with the native epitope, demonstrate the highest binding affinity. The phages that expose peptides with 3 amino acid sequence similarity demonstrate different binding affinity: from the smallest to the largest. Based on the obtained data, it is safe to suggest that the rational biopanning may proceed in accordance with the task.

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

On the basis of synthesized negatively charged hydrosol hexacianferrate (II) ferrum (III) (HCFF) by diameter 10-20 HM are received stable conjugates with antibodies and antigens glycoprotein and lipopolysaccharide (LPS) nature. Synthesized hydrosol (HCFF) is new type of a disperse phase in the lateral flow assay. Conjugates mentioned above were applied for construction lateral flow tests-systems for revealing cholera toxin, the rabbit antibodies to recombinant glycoproteine complex from Micobacterium tuberculosis H37 Rv, human immunoglobulin, LPS antigens S. typhimurium, S. enteritidis. Developed lateral flow tests-systems had high analysis speed (5-7 min), good specificity and sensitivity: on cholera toxin of 2.0 ug/ml, on LPS antigens S. typhimurium, S. enteritidis 0.5 ug/ml.

Anthrax is the widespread acute infection disease, affecting animals and humans, refers to the bioterrorist threat agents of category A, because of the high resistance of Bacillus anthracis spores to adverse environmental factors and the ease of receiving them. We obtain a representative panel of 20 monoclonal antibodies against the key component of pathogenic exotoxins, anthrax protective antigen. Quantitative sandwich-ELISA for protective antigen with antibody obtained was developed. Six pairs of monoclonal antibodies showed the detection limit up to 1 ng/ml concentration of the protective antigen in blood serum.

One-step rapid immunochromatographic method for detection of diphtheria toxin in different water samples (phosphate buffer, milk, human nasopharyngeal swab) with the conjugate of monoclonal antibodies labeled with colloidal gold was developed. The limit of visible detection of the diphtheria toxin is 10 ng/ml and 15 min time analysis. The use of silver sensitivity enhancement and scanning equipment decreased the detection limit to 1.25 ng/ml.

The authors have obtained a panel of 7 monoclonal antibodies (MAbs) against pandemic influenza virus A/IIV-Moscow/01/2009 (HIN1)swl isolated in Russia. One MAb is directed to a NP protein linear epitope and interacts with all the influenza A viruses under study. Six other MAbs are directed to H1 hemagglutinin conformation-dependent determinants and detect homologous virus in the hemagglutination-inhibition test, enzyme immunoassay, immunofluorescence and virus neutralization tests. MAbs differentiate pandemic influenza viruses A(H1N1)swl from seasonal influenza A(H1N1), A(H3N2), and B viruses. The high neutralizing activity of MAbs permits their use to study the fine antigen structure of influenza virus hemagglutinin and to differentiate the A(H1N1) pandemic influenza viruses and offers promise for obtaining humanized antibodies in order to make specific prevention and treatment of influenza.

Aim: Development of new method of C4B isotype functional activity evaluation in enzyme immunoassay by using pharmaceutical preparation derinat as a classical pathway complement activator and its use for blood sera isotyping in confirmed urogenital tract chlamydia infection., Materials and Methods: Enzyme immunoassay was used to detect C4A and C4B isotype functional deficiency in blood sera of patients. Chlamydia etiology urogenital infection diagnosis was based on results of standard clinical-instrumental examination methods: vaginal clinical smear analysis, scrape sample light microscopy with consequent treatment by fluorescent monoclonal antibodies against Chlamydia trachomatis and PCR., Results: In acute form of the disease C4A deficiency frequency of occurrence was 0.36, and C4B deficiency - 0.55. In chronic form of the disease deficiency frequency of occurrence was 0.38 for both isotypes. In the group of healthy people isotype deficiency was 0.08 and 0.25, respectively., Conclusion: Innate masked C4 deficiency interfere with the normal immune defense of organism against chlamydia infection, and antigen carbohydrate pathogenicity may possibly be more significant for the development of immune response to which C4B isotype activity is necessary.

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.

Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.

Twenty unique phage antibodies to human tumor necrosis factor alpha were selected from a naive combinatorial library of human single chain fragment variable. Analysis of gene segments encoding selected antibodies shown that repertoire of variable domains of heavy and light chains included variable domains of both naive autoantibodies and antibodies produced as a result of somatic hypermutagenesis.

Despite combination therapy with immunosuppressive agents, 32.5% of patients with IBD showed the formation of antibodies to infliximab. Simultaneous study of the concentration of the drug (infliximab), TNF-alpha and antibodies to it in the blood serum allows to judge not only on the effectiveness of anticytokine therapy, but also on the advisability of further conducting therapy. Elevated levels of AINF may lead to infusion reactions, reducing the effectiveness and duration of response to this therapy. Transplantation of MSCs reduces the level of antibodies to infliximab, but in 2 (5%) patients noticed a gradual increase of these antibodies. After infliximab infusion from 4 to 8 weeks the level in serum increased up to 45 mg/ml and higher, further serological concentration of infliximab is gradually reduced and then falls below the-horn. High concentrations of infliximab (> 45 mkg/ml) in blood samples at combined immunosuppressive therapy (infliximab + glucocorticoids + cytotoxic agents) should be considered as a sign of potential complications. The absence of antibodies to antigens of HLA I and class II after systemic transplantation of allogeneic mesenchymal stromal cells (MSCs) of bone marrow demonstrates not only the effectiveness but also the safety of transplantation of allogeneic MSCs, in relation with that the special selection of donors for transplantation of allogeneic MSCs is not required.

The splenocytes isolated from the mice immunized with wild-type or guinea pig-adapted Ebola virus strains were used to obtain hybridoma collections. Investigation of the monoclonal antibodies (mAb) obtained to one of the strains to another revealed antigenic interstrain differences in nucleoprotein and VP40. It is interesting that the differences were found in the hydridoma collection obtained against the wild-type strain. The mAbs produced by hydridomas to the adapted strain were found to equally well the antigens of both strains.

Aim: Comparative study of sensitivity and specificity of immunochromatographic (IC) assay kit and dot-immunoanalysis for assessment of feasibility of their use for laboratory diagnostics of cholera., Materials and Methods: Experimental lots of IC assay kit and dot-immunoassay (DIA) for detection of Vibrio cholerae serogroup 01 serovars Ogava and Inaba were constructed on the basis of species-specific monoclonal antibodies (MCA) conjugated with colloid gold (IC) and peroxidase (DIA). Hybridoma-producer of MCAwas obtained and stored in liquid nitrogen in Rostov-on-Don Research Institute for Plague Control. It was deposited in specialized collection of cell cultures of vertebrates in Institute of Cytology (Saint Petersburg)., Results: Strong specificity of IC assay kit and DIA relative to cholera vibrios 01 and absence of crossreactivity with closely related and heterologous microorganisms were shown. Minimal quantity of vibrios, which could be detected using IC assay kit and DIA, was 107 and 105-106 microbial cells respectively., Conclusion: Performance of IC assay takes 5-15 min, DIA--1.5 hour, they allow to visually assess the reaction, do not require instrumentation and in perspective both methods could be used on defined stages of scheme for laboratory analysis of cholera.

An indirect enzyme immunoassay (EIA) using the purified fraction of surface viral glycoproteins (GP) as an antigen for solid phase sensitization was not shown to be a specific method for the differential detection of influenza A(HS) (HS-Ab) virus antibodies (Abs) due to total conservative epitopes in the structure of GPs of influenza A(H5) and A(H1NI) viruses. The cross activity of some monoclonal Abs (MAbs) to influenza A(H5) and A(HIN1) viruses, which had been obtained at the Research Institute of Influenza, was proof of the presence of total immunodominant determinants in the structure of influenza H1 and H5 virus hemagglutinin (HA). In this connection, an EIA, which was based on the competition of influenza A(H5) H5-Ab virus HA-specific MAbs in the test sera for an association with influenza A(H5) virus, was proposed for the subtype-specific detection of H5 Ab. Comparison of the results of competitive EIA (cEIA), microneutralization (MN) test and HA inhibition test (HAIT) (using equine red blood cells) in the examination of sera obtained from 44 volunteers immunized with inactivated vaccine containing influenza A/Indonesia/5/2005 (H5N1) virus showed the high sensitivity and specificity of cEIA in detecting H5-specific Abs. The effectiveness of cEIA for the sera strictly positive for the content of H5 Abs was close to that of MN test and was 9-34% higher than HAIT (depending on those used in the analysis of H5 virus antigens). cEIA may be proposed to assess new influenza vaccines as an additional laboratory test. Since the infectious virus is not used during cEIA, it may be recommended for the serodiagnosis of influenza A(H5) at practical virological laboratories.

To define of possible mechanism of monoclonal antibody B2 interaction with haptens and peptide-mimotope of benzo[a]pyrene Fab-fragment structure was modeled. Antibody active centre structure was defined using docking with a number of different polyclic aromatic hydrocarbons. It was shown that active center of monoclonal antibody B2 composed of two binding pockets. Correlation was revealed between experimental and calculated data on relative free energies of monoclonal antibody B2 binding with different ligands. We found that synthetic peptide-mimotope of benzo[a]pyrene weakly competeswith benzo[a]pyrene conjugate in monoclonal antibody B2 binding. Immunization of mice with peptide-mimotope conjugate did not revealed antibodies to benzo[a]pyrene. To define structural features of peptide-mimotope of benzo[a]pyrene preliminary model of peptide-mimotope in the frame of pIII protein was calculated. It was shown that tryptophan located inside of peptide can be exhibited on a surface and accessible to antibody. Obtained modeling results could be applied for following optimizations ofbenzo[a]pyrene peptide-mimotope and active center of monoclonal antibody B2.

Aim: To develop and characterize by immunochemical methods the panel of monoclonal antibodies (MAbs) recognizing different antigenic determinants ofhuman secretory component (SC) molecule., Materials and Methods: sIgA and SC were obtained from colostrum by combination of ion-exchange chromatography and gel filtration. Recombinant SC was expressed in Escherichia coli cells transformed by construction that contained fragment of gene coding extracellular domain of receptor for polymeric Ig. MAbs were produced and studied using hybridoma technologies and different methods of immunoenzyme analysis respectively., Results: Panel comprising 10 MAbs against human SC of which 4 types of antibodies recognize cryptic epitopes of free SC and other 6 types recognize epitopes exposed both on SC and sIgA. MAbs panel contains antibodies interacting with conformational and linear epitopes of antigen. Three from obtained MAbs bind to SC epitopes which structure is determined by presence of carbohydrate residues in the molecule of antigen. Immunometric systems were developed which allow to differentially detect free SC and sIgA., Conclusion: Developed and characterized MAbs panel recognizing different epitopes of SC molecule opens new opportunities for laboratory and basic research of human secretory immunity.

Aim: To obtain and study of immunochemical characteristics of monoclonal antibodies (MAbs) to CagA cytotoxin of Helicobacter pylori employing recombinant fragments of CagA protein., Materials and Methods: Standard methods of construction and selection of hybridomas, different variants of immunoenzyme analysis and immunoblotting were used. Molecular genotyping of H. pylori cultures by amplification of cagA gene fragments was performed., Results: Panel of MAbs recognizing 4 different linear epitopes on the CagA molecule, three of which are localized in conservative parts of cytotoxin and one--in variable region of CagA, was developed. On the basis of two obtained antibodies, system of two-center immunoenzyme assay for quantitative detection of CagA protein which is characterized by high sensitivity and specificity, was developed. Obtained MAbs allow to differentiate CagA-positive and CagA-negative strains of H. pylori by immunochemical methods., Conclusion: Employing pure recombinant fragments of CagA protein, first panel of MAbs to CagA cytotoxin of H. pylori was developed and characterized. Obtained MAbs open perspectives for study of the H. pylori cytotoxin molecule and construction of immunodiagnostic assays aimed on detection of CagA antigen.

Aim: To develop highly sensitive sandwich technique for identification of surface hepatitis B virus antigen (HBsAg) in serum and analyse of possible improvement of solid phase for immunoenzyme sandwich technique of HBsAg identification through variation of pH-dependent sorption of monoclonal antibodies on the surface of immune plates., Materials and Methods: Calibration curves for identification of HBsAg in sandwich techniques using 36 possible binary combinations of monoclonal antibodies of our panel (including high affinity antibodies to HBsAg produced by 6 hybridomas) were compared. Immobilization of antibodies on solid phase (by passive sorption) was performed at different pH values (2.8, 7.5, and 9.5)., Results: Analysis of panel of antibodies to HBsAg produced by 6 hybridomas revealed pH-dependent monoclonal antibodies (18C8), which immobilization at low pH values together with detecting antibodies F4F3 allowed to greatly improve sensitivity of the sandwich technique. Minimal credibly detectable concentration of HBsAg in sera of persons infected with hepatitis B virus was 0.013 - 0.017 ng/ml. Validation of sandwich technique was performed on certified panel of serum samples with various concentrations of HBsAg (different serotypes)., Conclusion: Highly sensitive sandwich technique for detection of HBsAg was developed. It was shown that analysis of panel of monoclonal antibodies on pH-dependence could be used as simple methodical approach for optimization of immunoenzyme sandwich techniques for detection of different antigens.

The electrooptical abilities of the microbial suspensions during a cells interaction with antibodies (ABs) of a different specificity have been studied on the example of the Azospirillum brasilense Sp245 cells and their interaction with the polyclonal monospecific and polyspecific antibodies. Measuring of the orientational spectra of the cells has been performed using the ELUS electrooptical analyzer. A discrete frequency set of an orienting electric field (740, 1000, 1450, 2000, and 2800 kHz) was used. It has been shown that an interaction of the polyspecific AB with the investigated cells redoubles the value of an electrooptical signal of the cells' suspension as compared with the monospecific antibodies. These findings can be used for a development a new method of microorganism detection.

The presence of polyreactive immunoglobulins in sera may substantially influence on the accuracy of antibody affinity determination. In order to obtain precise values of antibody affinity one should apply one of two following ways. First, one should block polyreactive immunoglobulins with high concentration of Twin 20 and high concentration of any antigen which does not interact with studying antibody. After this antibody affinity may be determined by traditional methods. Another way is the application of the method suggested by us earlier, which allow determining affinity of two antibodies in a mixture and the relation of their concentrations.

Antiinflammatory and analgesic effects of artrofoon in osteoarthritis and the absence of side effects were demonstrated. The maximum antiinflammatory effect was attained by the end of the 3rd month of artrofoon treatment. The effect persisted from 6 months to 2 years against the background of maintaining therapy with artroofoon.

Analysis of membrane topology of connexin-43 (Cx-43) made it possible to determine one of its extracellular fragments (E2): Q173-1208. The nucleotide sequence of this fragment was cloned into pCBDQ and pHPML vectors containing the sequences of calmodulin-binding domain (CBD) and HPML-domain of Ca-ATPase of human hPMCA4b cells plasma membrane. This yields two chimeric proteins with N-terminal 6-histidine motif containing the extracellular fragment Cx43 E2 and one of hPMCA4b domains (Cx43-CBD and Cx43-HPML). The latter were inserted into the recombinant polypeptide to improve solubility and enhance immunogenicity of the product. Affinity-purified on Ni-NTA agarose recombinant Cx43-CBD was used for immunization of mice and obtaining of monoclonal antibodies. Primary selection of clones was carried out by solid-phase IEA with immobilized Cx43-HPML and by immunoblotting with Cx43-HPML. The positive clones were tested immunohistochemically on rat brain sections. This two-stage testing made it possible to select two hybridomas, which produced monoclonal antibodies to Cx43 in native conformation. The resultant antibodies can be used in vitro and in vivo for immunophenotyping of various Cx43-positive cells.

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

In patients with ankylosing spondyloarthritis, artrofoon produced a more potent antiinflammatory effect than nonsteroidal antiinflammatory drugs. The study demonstrated a positive effect of artrofoon on clinical and laboratory parameters, good tolerability of the preparation, and the absence of negative effects on the hepatic and renal functions. Being added to complex therapy, artrofoon led to a decrease in the dose of nonsteroidal antiinflammatory drugs.

A panel of hybridomas producing monoclonal antibodies (MAbs) to nucleocapsid protein (NP) of avian influenza A virus was obtained. On the basis of 2 MAbs, the authors designed an antigen-bound ELISA (sandwich ELISA), in which NP3 MAbs were used as antigen-bound antibodies and NP MAbs conjugated with horse radish peroxidase as antigen detection antibodies. The specificity of the test system to avian influenza virus was determined. The developed test system was ascertained to specifically detect influenza A virus of all study subtypes and to yield no cross reactions with other tested virus pathogens. The sensitivity of the sandwich ELISA was 30 ng/ml of NP in the urine-treated virus preparations. The assay was tested on experimental H5N1-infected mice. The findings positively correlated with the results of postmortem studies and with the virus isolation method in the chick embryos. The developed test system may be used to detect avian influenza A virus as an alternative or supplement to other diagnostic techniques.

The use of two monoclonal antibody types specific to different epitopes of diphtheria toxin systems have been developed to reveal diphtheria corynebacteria toxigenicity rapidly based on immunochromatographic and latex-agglutination detection of the diphtheria toxin. The methods have been tested on a sample of 36 clinical isolates. The possibility of significant detection of the toxigenic properties of the Corynebacterium strain, grown for 1 day, has been demonstrated. The developed methods allow for the detection of diphtheria toxin in concentrations of 3-4 ng/ml. The developed test systems are a perspective tool for diphtheria diagnostics because of significant time shortening as compared to traditional microbiological methods.

It was shown that application of the traditional method for antibody affinity determination may give not exact and non complete information about the characteristics of studied antibodies especially if studying specimens contain two or more species of antibodies with different affinity. In order to avoid this problem it is recommended to use another approach, suggested by us earlier. Our approach allows elucidating that studying specimens contain more than one kind of antibodies and this allows applying a proper method for an- tibody affinity evaluation. Such method, which allows determining the affinity of two antibodies in a mixture that have different affinity, is also described.

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.