Monoclonal human antibody against alpha-toxine from s. aureus and its application in treatment or prevention of abscess formation

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biochemistry, in particular to a monoclonal human antibody, specific to alpha-toxin of S. aureus. The claimed invention additionally relates to pharmaceutical compositions for treatment of prevention of the abscess formation in an organ, which contains at least one antibody or one nucleic acid, which codes the said antibody.

EFFECT: invention makes it possible to extend an assortment of antibodies, specific to alpha-toxin of S aureus.

23 cl, 7 dwg, 4 tbl, 6 ex

 

The present invention relates to monoclonal human antibody specific against the alpha-toxin of S. aureus, hybridoma producing the specified antibody, nucleic acid encoding it, and also to the cells of the host, transfected with the indicated nucleic acids. In addition, the present invention relates to methods for the indicated monoclonal antibodies. Additionally, the present invention relates to pharmaceutical compositions containing at least one antibody or at least one nucleic acid encoding the indicated antibody. The present invention also relates to the use of this monoclonal antibody for the treatment or prevention of abscess formation.

Staphylococcus aureus (S. aureus) is a facultative anaerobic gram-positive spherical bacterium, which is considered an opportunistic pathogen. S. aureus is usually colonize the nose, the skin and the mucosa of the gastric part-intestinal tract of healthy people. At any point in time, roughly 20-30% of the population infected with S. aureus. In healthy people, these bacteria often cause secondary infections, such as pimples and boils. Typically, the mucous membranes and epidermal barriers (skin) protects against infection with S. aureus. Violation of the integrity of these natural barriers in rez is ltate damage such as burns, trauma or surgery, significantly increases the risk of infection and can cause severe and/or systemic diseases. Diseases that suppress the immune system (eg, diabetes, late-stage renal disease, cancer, AIDS and other viral infections), and immunosuppressive therapies, such as radiation, chemotherapy and transplant therapy, increase the risk of infection. Opportunistic infections caused by S. aureus can become quite serious and to induce the development of endocarditis, bacteremia, osteomyelitis, and the formation of abscesses, which, in turn, can cause serious illness and death. Staphylococcal infections can be divided into localized infections, such as pneumonia, and clinically more complicated staphylococcal infections, such as sepsis and the formation of abscesses caused by dissemination of pathogenic a distant organ.

Worldwide S. aureus is a major cause of sepsis, infections of skin, soft tissue, and lower respiratory tract. For many years the number of cases as nosocomial and community-acquired infections has been steadily increasing. In addition, treatment of these infections is complicated by the emergence of strains with multidrug resistant what ivascu. In developed countries such as the USA, resistance methicillin-resistant Staphylococcus aureus (MRSA) antibiotic β-lactam series is a serious problem in hospitals and other medical institutions. It is important that the incidence of all invasive infections caused by MRSA, including community-acquired infection is high compared with other bacterial pathogens, and 20% of these infections lead to death. Additionally, the emergence of acquired resistance to vancomycin were further limited the possibilities for treatment of severe staphylococcal infections.

S. aureus has a diverse Arsenal of virulence factors that contribute to the pathogenesis of the disease. They can roughly be divided into surface proteins and proteins that are excreted into the environment. Surface proteins include structural components of the cell wall of bacteria, such as peptidoglycan and lipoteichoic acid, and surface proteins predominantly expressed during the exponential phase of growth, including protein a, fibronectin-binding protein a and clumping factor. Secreted proteins, as a rule, differ from bacterial cells during the stationary phase of bacterial growth and include several toxins such as alpha-toxin (also known as alpha-hemolysin), enterotoxin B, lakeside is s (including leukocidin Panton-Valentine, PVL), a lipase and protease V8. However, despite the large amount of knowledge about the biochemical and molecular properties of these toxins, their exact role in the pathogenesis of infections caused by S. aureus, is not clear until the end.

Experimental confirmation and epidemiological data suggest that alpha-toxin may be involved in the pathogenesis of pneumonia along with other cytotoxins (Me Eiroy et al, 1999). Assume that alpha-toxin binds to surface receptors on susceptible host cells and thus is attached to the cell surface. This event promotes oligomerization of the toxin in heptaminol structure, forming the early days, followed by injection of β-cylindrical structure with a pore diameter of 2 nm in the plasma membrane. The formation of pores leads to loss of membrane integrity, destabilizes the cell and, ultimately triggers apoptosis and lysis of cells. To the formation of pores induced alpha-toxin, especially such sensitive cells like lymphocytes, macrophages, alveolar epithelial cells, pulmonary endothelium and erythrocytes. At the same time, granulocytes and fibroblasts, apparently resistant to lysis (Me Eiroy et al, 1999).

The exact role of alpha-toxin in the inflammatory response and induction of the innate immune response to bacterial infection is not fully understood. S. aureus ex is reserued a number of other virulence factors, and today, the contribution of each virulence factor in manifestation of the disease is not fully understood and represents a challenge for the development of prevention and treatment of clinically complex infections caused by Staphylococcus aureus.

As you know, the alpha-toxin is one of the virulence factors in the development of host infection induced S. aureus, and several studies have highlighted the importance of alpha-toxin in the course of the disease, for example, instillation of purified alpha-toxin rabbit or in the lung tissue of the rat causes vascular leakage and pulmonary hypertension, which is associated with the release of various signaling molecules (e.g., phosphatidylinositol, nitric oxide, prostaglandins and thromboxane A2). In the literature it has been shown that immunity to alpha toxin protects against the harmful effects of toxins, however, the development of vaccines against alpha-toxin remains a serious problem.

Wardenburg and Schneewind (2008) showed that the severity of lung disease in mice correlates with the levels of alpha-toxin, produced by certain isolates of Staphylococcus aureus. In addition, the authors showed that immunization against the variant alpha-toxin, incapable of forming pores, induces the development of immunity to pneumonia caused by S. aureus. These results are consistent with research which ia the same group, demonstrating that alpha-toxin are important for the pathogenesis of community-acquired pneumonia caused by (methicillin-resistant Staphylococcus aureus). In another experiment, the authors showed that antibodies to alpha-toxin also defended the epithelial cells of the human lung from S. aureus-induced lysis (Wardenburg and Schneewind (2008)).

Although these results indicate that alpha-toxin is involved in the destruction of lung tissues, it remains unclear whether the death of the animals in the above-described experiments, the result of direct destruction of cells of the lung toxin or excessive inflammatory response, or both reasons. Passive transfer of antibodies to alpha-toxin significantly reduced circulating levels of interleukin-1β is a cytokine, which is known to be accompanied by acute lung injury. Thus, it is logical to conclude that the inflammatory response may contribute to lung damage, mediated by alpha-toxin.

When a localized infection, such as pneumonia in humans, approximately 40% of patients with pneumonia caused by S. aureus, develop infections of the blood stream and disseminated lesions. The spread of bacterial infection can lead to a blood infection and colonization of the remote body. Infection of the blood stream can lead to septicemia, rapidly progressive and often summer is gnome complication of infections, due to S. aureus.

The spread of infection S.aureus also often observed in model animals with pneumonia caused by S. aureus, and also have approximately 40% of the animals developed disseminated bacteremia due to tissue damage and infection through the layers of the epithelium into the blood stream and lymphatic tissue. However, dissemination is largely dependent on the genetic background used lines of animals, as well as the potential of the innate immune system, such as activation of neutrophils to control growth, for example, animals line C57B/L, characterized by depletion of neutrophils, are very susceptible to infection of the kidneys of zolotisty Staphylococcus, whereas immunocompetent animals resistant to infection. In contrast, animals line a/J is very sensitive to infections, mainly due to the delayed receipt of neutrophils in the kidney (von Kockritz-Blickwede, 2008).

Although data on the structure and function of proteins of S.aureus were more than full, the development of an effective vaccine remains a difficult task.

An attempt was made to secure immunity to alpha-toxin and bacteria S.aureus using compositions containing a combination of antibodies that are specific contact with antigen - alpha-toxin S.aureus, and antibodies that are specific associated with other bacterial Antiga is ω (WO 2007/145689). These songs though, and include the number of antibodies, which by themselves are not effective, neutralize infection and/or provide protection against infection in the synergistic activity of the combination antibodies.

It was shown that the protective efficacy of this combination antibodies, neutralizing the toxin S.aureus, and opsonic antibodies in 72 hours after infection with S.aureus isolate higher than the protective effect of immunization only neutralizing or only opsonic antibodies. The combination of opsonic and neutralizing the toxin antibodies demonstrated a protective effect, preventing infection of the skin and soft tissues, as well as the dissemination of bodies. However, disclosed in the patent application of neutralizing anti-alpha-toxin antibodies alone are not enough to prevent dissemination of the body/abscess formation, or neutralize the infection.

Another attempt was made Heveker et al.(A, 1994b), which describes the neutralizing monoclonal antibodies to human and mouse directed against the alpha-toxin S.aureus. Monoclonal antibody human IgG subtype/lambda characterized through the sequence and demonstrate neutralization.

Described Heveker (a) line cells hybridoma person producing antibodies to alpha-toxin was isolated using peri is eticheskikh of blood leukocytes in healthy volunteers, previously immunized test vaccine containing alpha-toxoid S.aureus. Because the alpha toxoid used in the study Heveker, is a chemically modified alpha-toxin, it can be assumed that the change really makes antigenic determinants are less immunogenic or even non-immunogenic. This approach, therefore, as such, may not lead to the formation of the same effective immunity, as it was shown for other bacterial toxins such as cholera toxin: toxoid vaccine stimulated the development of anti-toxin antibodies, which did not cause the development of immunity to infections (Levine(1983)).

In the literature identified various factors that are important virulence factors for abscess formation, such as toxins, composition, extracellular factors and enzymes. The assumption about the possible role of alpha-toxin in the formation of abscesses was expressed Kapral et al. (1980). It was reported that alpha-toxin accumulates dramatically in the abscess after puberty, when this has not been demonstrated whether alpha-toxin is required for the formation of an abscess. The second publication made Adiam et al. (1977), denied a key role of alpha-toxin in the formation of abscess. The authors showed that alpha-toxin plays a key rol is in the spread of hemorrhagic forms of infectious mastitis rabbits (blue-breast), observed in natural outbreaks. They have reproduced in the laboratory of clinical picture, using two strains of staphylococci, non-relatives. High titers of circulating anti-alpha-toxin antibodies led to the formation of protect against this lethal form of mastitis. Thus, the neutralizing antibody titer can prevent death, changing clinical picture in the direction of the state, characterized by a less severe abscesses. However, neutralization of alpha-toxin had no effect / not prevented the formation of abscesses in rabbits. In a later publication Kielian et al. (2001) investigated the role of alpha-toxin in the formation of abscess in the brain of mice. In the experiment, the authors implanted strain wild type S. aureus and its mutants in the brain tissue of the frontal lobes and assessed the ability of each strain to cause brain abscesses. The authors used the strains mutated in genes associated with the expression of known virulence factors, for example, mutants of the locus sarA and agr, each of which is involved in the overall regulation of important virulence factors. Because alpha-toxin is under control of the regulatory system sarA/agr, the authors have also included in their experiments, the strain mutant for alpha toxin. Experimental data showed that the replication of bacterial strains, mutant by the alpha-Tox is well or locus sarA/agr, reduced virulence of bacterial cells when injecting into the skull compared to the isogenic control strain RN6390, which has resulted in the reduction of bacteria and reduce detectable foci of inflammation in the brain of animals compared with large, well-formed abscess in mice treated isogenic strain.

However, in experimental models of brain abscess mutant strains were not completely avirulent and therefore cannot exclude the possibility that in addition to alpha-toxin, an additional factor or factors may play an important role/role in the formation of abscess of the brain.

The role of alpha-toxin in the formation of abscess was assessed in the other experimental conditions as set forth Schwan et al. (2003) analysis of local, systemic and abscess-forming infectious staphylococcal model. The authors noticed that, over time, non-haemolytic strains of Staphylococcus aureus have become more common in mouse models of abscess and models of RAS, but not in the tissues of the organs associated with systemic infections. For example, in a mixed infection using all variants of Staphylococcus aureus RN6390 (hyperhemolytic, haemolytic and non-haemolytic) in the model of abscess, hyperhemolytic group decreased significantly on day 7 after infection, and megemliteni the population has increased significantly. Sequencing of some of the mutants obtained by site-directed mutagenesis revealed the presence of mutations in agrC gene or intergenic region agrA-agrC, which led to decreased activity as alpha-toxin, Delta-toxin. In the analysis of certain mutant strains on the activity agr (agr-) or alpha-toxin (hla) abscess, wound and systemic infection model, agr-mutant strain and hla-mutant strain did not show any differences in the total number of bacteria in the abscesses of mice on day 4 compared to the original strains of wild-type (RN6390). The same is true for local infections (wound model), while in the model of systemic infection was a significant purification of hla - and agr-mutant strain. The result clearly indicates the importance of alpha-toxin in systemic infections, but not in the local infections or abscess formation. Indeed, mixed infections caused by strains of hla-mutant and wild-type showed in the model abscess low prevalence populations hla-mutant over the wild-type strain. Further, the authors concluded that mutations agr cause a decrease in the expression of alpha - and Delta-toxins, which, in turn, help predominant growth of this agr-mutant group in a mixed population of cells of S. aureus present in abscesses and wounds. The results clearly contradict the results described Kielin et al., where the absence of alpha-toxin was reduced bacterial virulence. Therefore, the role of alpha-toxin in the formation of abscess is not clear.

In General, there is no evidence pointing to a single virulence factor as a major force in the formation of abscess. Therefore, the study was focused on the detection of additional factors that are not fully controlled by Staphylococcus aureus, such as environmental factors or certain structural areas, as a General key factors in the formation of an abscess. For example, recent data on the virulence factors influencing the formation of abscesses, indicate the influence of unrelated divalent metal ions, such as N+and Ca2+, in the formation of abscess and multiplication of bacteria in it. The chelation of metal ions in animals inhibits the formation of abscesses in the liver and inhibits the growth of S. aureus in the abscess (Corbin 2008). On the other hand, Tzianabos et al. (2001) suggested that the formation of pathological structures, such as abscesses in the tissues, the organism as S. Aureus needs virulence factors present on the surface of bacterial cells. The authors showed that the strains that are closely associated with clinical cases, the formation of abscesses or may have one or more polysaccharides associated with the cell wall is coy and containing zwitterion sites in the molecule (chemical compound, the total charge is equal to zero, but electrically neutral molecule carries formal positive and negative charges on different atoms). In the absence of zwitterionic site abscess is not formed. The authors concluded that these polysaccharide polymers can modulate the formation of abscess in the body. In addition, they presented evidence of participation in this process, not only the main polysaccharides SR and Vietnam, but lipoteichoic acid (LTA), an additional well-known virulence factor present in the cell wall. They also identified zwitterionic site in the molecule of lipoteichoic acid and, thus, has expanded its hypothesis abscess formation include the presence of zwitterionic plot in any virulence factor, which is key in the formation of an abscess.

Based on the results, indicating that different factors contribute to the formation of abscess induced S.aureus, a specialist in this area cannot be expected that the neutralization of only one factor will prevent the formation of abscess.

Thus, the object of the invention is to provide means and methods for the prevention and treatment of clinically complex infectious diseases caused by S.aureus, such as the formation of abscess.

Accordingly, one technical problem underlying the present invention is to provide a monoclonal antibody specific to alpha-toxin of S. aureus, and the antibody has an in vivo protective capacity against clinically complex infectious diseases caused by S.aureus, such as the formation of abscess.

The mentioned technical problem is solved by using monoclonal antiteloobrazovania below.

According to the present invention proposed a monoclonal antibody, designated 243-4, and specific alpha-toxin of S. aureus, and the variable region of the light chain of the antibody comprises at least one of SEQ ID NO: 1 in the region CDR1, SEQ ID NO: 2 in the region CDR2, and SEQ ID NO: 3 in the region CDR3, and a variable region light chain antibody contains at least one of SEQ ID NO:1 in the region CDR1, SEQ ID NO:2 in the region CDR2, and SEQ ID NO:3 in the region CDR3, and variable region of the heavy chain of the antibody comprises at least one of SEQ ID NO:4 in the region CDR1, SEQ ID NO:5 in the region CDR2, and SEQ ID NO:6 in the region CDR3, or a fragment or mutein specified antibodies, capable of binding to alpha-toxin of S. aureus, and mutein monoclonal antibodies carries at least one conservative substitution at any of the sites CDR of the heavy or light chain of the antibody.

In accordance with the preferred implementation according to the present izobreteny the proposed human monoclonal antibody, specific for alpha-toxin of S. aureus, where the variable region of the light chain of the antibody comprises SEQ ID NO:1 in the region CDR1, SEQ ID:2 in the region CDR2, and SEQ ID NO:3 in the region CDR3, and where the variable region of the heavy chain of the antibody contains SEQ ID NO:4 in the region CDR1, SEQ ID NO:5 in the region CDR2, and SEQ ID NO:6 in the region CDR3, or a fragment or mutein, capable of binding to alpha-toxin of S. aureus, where mutein monoclonal antibodies carries at least one a conservative substitution at any of the sites CDR (hypervariable sites) heavy or light chain of the antibody.

Unexpectedly it was found that the monoclonal antibodies according to the present invention show a high ability to prevent the formation of abscess. Preventing the development of abscess was demonstrated in a model using the kidneys of mice by injecting monoclonal human antibodies specific for alpha-toxin, according to the present invention. Judging by the structure of the toxin, which is rather secretively protein, and not the component (a polysaccharide cell wall, any direct bactericidal effect, such as the destruction of bacterial cells, or indirect effector function mediated by the immune system, for example, opsona-phagocytic destruction of the pathogen, due to the activation of the complement system, can be excluded and do not cause otsutstvie abscess formation.

In the present description, the term "monoclonal antibody" includes any monoclonal antibody human or part thereof, regardless of the source of the receipt. Preferred is a fully human monoclonal antibody. It is preferable for obtaining monoclonal antibodies using hybridoma. Hybridoma can be hybridomas of mammalian cells, such as murine, bovine or human. Preferred is hybridoma of human origin. To obtain monoclonal human antibodies suitable hybridoma obtained by the merger of B-cells with cell myeloma or heteromyinae.

B-cells can be obtained by immunization of a healthy subject or by selecting convalescent patients with subsequent selections they have samples Krai of which can be selected b cells. the selection of B-cells from blood samples can be carried out by known methods (see, for example. Current Protocols in Immunology. Chapter 7.1. Isolation of whole mononuclear cells from peripheral blood and cord blood. Ed. Wiley & sons, Eds. J.C. Coligan et al.). The merger of B-cells with cell myeloma or heteromyinae getting hybridoma carried out in accordance with standard methods according to the classical approach Koler and Milstein. To fit the myeloma cells are derived P3X63, yet is how P3X63Ag8. 653 (ATCC CRL-1580) or SP2/Q (ATSS CRL-1646). Suitable cells heteromyinae include, for example, F3B6 (ATCC HB-8785). Received hybridoma can be subjected to selection in accordance with known procedures. Hybridoma cultured in an appropriate culture medium and secrete antibody from the supernatant.

Monoclonal antibody can also be obtained by the methods of genetic engineering and, in particular, the grafting of CDR segments defined in the claims, the available monoclonal antibodies

by replacing section CDR primary antibodies to specific segments CDR as defined by the claims.

The term "plot CDR" refers to the hypervariable area antibodies, that is, the region that defines the specificity of antibodies to a particular antigen. Three sites CDR (CDR1-CDR3) as light and heavy chains, responsible for binding to the antigen.

Plot CDR has the following localization in the heavy chain:

Plot CDR1: the amino acids 26 to 33 in the VH exon,

Plot CDR2: amino acids 51 to 58 in the VH exon,

CDR3 section: amino acids 97 to 110 in the VH exon.

The location of a parcel CDR does not depend on the class of antibodies, that is, IgM, IgA or IgG.

Plot CDR has the following location in the light chain lambda type:

Plot CDR1: the amino acids 26 to 33 in exon Vλ,

Plot CDR2: amino acids 51 to 58 in exon Vλ,

CDR3 plot: the amino acid is you from 90 to 110 in exon Vλ.

Mapping (alignment) aminokisloty the sequence th VH exons, Vχ and Vλ can be obtained from the database V Base (http://imqt.cines.fr/IMGT_vquest/share/textes/.

The term "fragment" refers to any fragment of an antibody able to bind with alpha-toxin of S. aureus. The fragment has a length of at least 10 amino acids, preferably 20, most preferably 50 amino acids. In addition, preferably, if the fragment includes the binding site of the antibody. In the preferred case, the fragment javljaetsja Fab, F(ab')2, single-stranded or a domain antibody. Most preferred is a Fab fragment or F(ab')2, or their mixture. Fragments of antibodies may be glycosylated, e.g contain carbohydrate residues in the variable regions of antibodies.

Accordingly, the present invention further provides a monoclonal antibody as defined in the present description, the antibody presents Fab, F(ab')2, single chain or a domain antibody fragment.

The term "mutein" includes any mutiny monoclonal antibodies, characterized by adding, removing and/or replacing at least one amino acid. The preferred implementation option is available in mateine monoclonal antibodies at least one conservative substitution at any of the sites CDR of the heavy or light chain of the antibody, as Asano in the claims. The preferred implementation option is available in mateine not more than 5, not more than 4, preferably not more than 3 and especially preferably not more than 2 conservative substitutions. The ability of the fragment or mutein antibodies to contact the alpha-toxin of S. aureus determine direct enzyme linked immunosorbent assay, as described in the "Examples"section: purified alpha-toxin immobilized on the solid phase tablets for ELISA. Fragments of antibodies or mutiny antibodies incubated with immobilized alpha-toxin, then the bound antibodies or their mutiny visualize using the appropriate secondary antibody conjugated with an enzyme.

The term "conservative substitution" means the replacement of one amino acid belonging to a specific physical-chemical group to an amino acid belonging to the same physico-chemical group. Physico-chemical groups are defined as follows:

Physico-chemical group of nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, Proline, phenylalanine and tryptophan. The group of amino acids having uncharged polar side chains consisting of asparagine, glutamine, tyrosine, cysteine and cystine. Physico-chemical group of amino acids with positively charged polar side chain consists of lysine, arginine and histidinemia chemical group of amino acids with negatively charged polar side chain consists of aspartic and glutamic acids and their carboxylate anions, also known as aspartate and glutamate.

According to another implementation variant, the invention provides a monoclonal antibody specific to alpha-toxin of S. aureus, in which the variable region of the light chain of the antibody has the amino acid sequence of SEQ ID NO:7, and the variable region of the heavy chain has the amino acid sequence of SEQ ID NO:8, or fragments of the indicated antibodies which are capable of contacting the alpha-toxin of S. aureus, or a variant of the specified antibodies are able to bind with alpha-toxin of S. aureus, in which the amino acid sequence of the variable region of the light chain of the antibody of at least 85% identical to SEQ ID NO:7 and amino acid sequence variable regions of the heavy chain of the antibody of at least 85% identical to SEQ ID NO:8. In the present description, the term "variant" refers to a polypeptide in which amino acid sequence shows a certain degree of identity with the amino acid sequence shown in the sequence listing.

The term "% identity", well-known experts in this field, indicates the degree of similarity between two or more polypeptide molecules, which is determined by the matching sequences. The percentage of identity is calculated based on the percentage of identical sites in dahili more sequences with accounting omissions, or other features of the sequences.

The percentage identity close polypeptides may be determined using known techniques. As a rule, use special computer programs with algorithms tailored to the specific needs. Preferred methods to determine identity first generate the greatest similarity between the studied sequences. Computer programs for determining identity between two sequences include, but are not limited to, the GCG software package, including GAP (Devereux J et al., (1984); Genetics Computer Group, University of Wisconsin, Madison (Wisconsin); BLASTP, BLASTN and FASTA (Altschul S, et al., (1990)). The BLAST program X can be obtained from the National center for biotechnology information (NCBI) and other sources (Reference BLAST, Altschul S, et al., NCB NLM NIH Bethesda, Maryland, 20894; Altschul S, et al., 1990). To determine the percentage identity can be used a well-known algorithm of Smith-Waterman.

Below are the preferred options for comparing sequences:

Algorithm: Needleman S.. C.D. Wunsch(1970),

Matrix comparison: BLOSUM62 from Henikoff C. and J.G. Henikoff (1992),

The penalty for pass in sequence: 12,

The penalty for the length of a gap in the sequence: 2.

The GAP program is also suitable for the use of the Finance with the specified parameters. The above settings are standard (default settings) for comparison of amino acid sequences in which the gaps at the ends do not decrease the value of identity. When a very short sequence is compared with the base sequence may additionally require larger values of mathematical expectation to 100,000, and in some cases reducing the word length (word size) to 2.

Can be used for additional model algorithms, the penalty for creating a gap, the penalty for extending a gap and matrix comparisons, including those mentioned in the "Handbook of the programs, the Wisconsin package, version 9, September 1997. The choice will depend on the comparisons that must be made, and further, whether the comparison between pairs of sequences in this case are preferred GAP or Best Fit, or between one sequence and a large database of sequences, in this case, preferred are FASTA and BLAST. The similarity of 85%, determined by means of these algorithms, described as 85% identity. The same applies to a higher degree of identity.

In a preferred implementation, the variants according to the present invention have the identity, 85% or more, preferably 90% or more, and most the e preferably 95% or more.

In accordance with a preferred implementation of the present invention, the monoclonal antibody is a human antibody. In the present description, the term "human" means that the monoclonal antibody man practically does not contain the amino acid sequences of alien species, in a preferred embodiment, monoclonal antibody man (human monoclonal antibody) entirely consists of the amino acid sequence of a human.

Light chain monoclonal antibodies according to the present invention may be Kappa or lambda type.

In a preferred implementation of the present invention light chain belongs to the lambda type. Light chain can be represented by a natural chain, including natural realignment, genetically modified or synthetic light chain.

Heavy chain of the monoclonal antibody according to the present invention can be selected from the isotypes IgM, IgA, or IgG, preferably IgG.

In accordance with another preferred implementation of the invention, the heavy chain of monoclonal antibodies belong to the IgG.

In the present description, the term "ability to communicate" refers to the binding that occurs between the antibody and detected by the antigen to which directed this antibody is. This type of binding is specific, in contrast, is not specific, which occurs in the absence of antigen.

Antibodies are able to bind with alpha-toxin, obtained using hybridoma technology, in which b-cells are of mammalian cells, for example, mouse, bovine or human. Preferred option b cells are b-cells. In addition, a monoclonal antibody able to bind with alpha-toxin, can be obtained by CDR-grafting sites CDR, as specified in the claims, the available monoclonal antibodies with obtaining monoclonal antibodies specific against the alpha-toxin of S. aureus according to the present invention.

According to the following implementation variant of the invention proposed a monoclonal antibody able to bind with alpha-toxin of S. aureus, which can be derived from b cells of mammals, such as mice, cattle or humans, the preferred option is a human b-cells or hybridoma obtained by merging these In-human cells with myeloma cells or heteromyinae.

According to the following implementation variant of the invention the proposed hybridoma, capable of producing monoclonal antibodies that bind to the alpha-toxin of S. aureus, is definitely in this application.

In the present description, the term "alpha-toxin" refers to a bacterial protein produced by S. aureus. After binding to the surface of the host cell alpha-toxin undergoes oligomerization to heptamer. The formation of pores is the main cause of apoptosis and cell lysis. For potential protection from pathogen fundamental importance is the ability of monoclonal antibodies to contact with both Monomeric and oligomeric form of alpha-toxin produced by S. aureus.

In accordance with another preferred implementation of the invention the monoclonal antibody according to the present invention is capable of specific bind Monomeric and oligomeric forms of alpha-toxin of S. aureus. In accordance with another variant of realization monoclonal antibody according to the present invention or its fragment or mutein capable of specific contact with both Monomeric and oligomeric form of alpha-toxin of S. aureus or both.

In the present description, the term "oligomeric form includes a form different from Monomeric forms of alpha-toxin, such as dimeric, trimeric, tetramer, pentamers, laksamana, hatamura etc., or polymeric forms such as hatamura form of alpha-toxin, prior to the formation of pores.

In accordance with another preferred option is realizatsii monoclonal antibody according to the present invention modified at the N end, the inner region or the C-end. Modifications include di-, oligo - or polymerization of the Monomeric form, for example by Poperechnaya using dicyclohexylcarbodiimide. Thus obtained di-, oligo-or polymers can be separated by gel filtration. Further modifications include modification of the side chain, such as change in stocks e-amino of lysine or amino - and carboxy-terminal modifications, respectively. Further modifications include post-translational modifications, such as glycosylation and/or partial or complete deglycosylation proteins and the formation of disulfide bonds. Antibodies can also be conjugated with a label, such as an enzymatic, fluorescent or radioactive labels. The preferred modification is chosen from at least one oligomerization, glycosylation or conjugation of a drug or label.

In addition, in the present invention proposed a nucleic acid, encoding, respectively, the heavy and the light chain of the monoclonal antibodies of the present invention. Nucleic acids may be represented by natural nucleic acid or derived from the germ line or rearrangements that occur in b cells, also nucleic acids may be synthetic. Synthetic nucleic what iSlate also include nucleic acids, having modified vnutrikletochnye, including phosphothioate to increase the stability of nucleic acids to destruction. Nucleic acids can be obtained by genetic engineering methods or completely through the synthesis of nucleotides.

The present invention additionally proposed vectors containing at least one nucleic acid encoding a light chain of the monoclonal antibody according to the present invention and/or at least one nucleic acid encoding a heavy chain of a monoclonal antibody according to the present invention. Nucleic acids can be installed in the same vector or may be present in the form of binary vectors. The vector preferably contains a promoter functionally linked to the nucleic acid to facilitate the expression of a nucleic acid encoding the light and/or heavy chain.

In a preferred embodiment, the vector further comprises the origin for replication and maintenance in the cell host. The vector may also include a nucleotide sequence encoding a signal sequence located at the 5'end of the nucleic acid encoding the light or heavy chain. The signal sequence may promote secretion of the encoded circuit on Wednesday.

In a preferred embodiment, the sector derived from adenoviruses, the vaccine viruses, baculoviruses, SV 40 virus, retroviruses, viruses, plants or bacteriophages such as lambda derivatives, or M13. Most preferred is a vector containing the constant region of the heavy and light chain human Ig, such as an integrated vector system for the expression of antibodies in eukaryotic cells, described Persic et al., 1987.

In addition, in the present invention proposed cell host containing the vector and/or nucleic acid and suitable for the expression vector. In the field of technology to which this invention relates, famous for numerous prokaryotic and eukaryotic expression systems, and eukaryotic cell hosts, such as yeast cells, insect cells, plant cells and mammalian cells, such as lines NEC, Regs, Cho, COS or HeLa and their derivatives, are preferred. Especially preferred are producing a line of human cells. In the preferred case, the transfected cells are the owners allocate produced antibodies into the culture medium. If achieved intracellular expression, are renaturation in accordance with standard techniques, such as described Benetti et al., 1998.

Monoclonal antibodies according to the present invention extracted from blood lymphocytes withdraw iwaisako patient, and the result of antibodies that have been purified and selected natural way with a high affinity, neutralizing and effective protection against infections.

The present invention also provides methods for obtaining monoclonal antibodies. In one implementation options of the monoclonal antibody produced by culturing the above-described hybridoma. Produced monoclonal antibody secreted in the supernatant and can be purified from it by conventional chromatography methods.

Alternatively, the monoclonal antibody is produced by cells of the host containing the vector according to the present invention by culturing host cells under conditions suitable for recombinant expression of the encoded chain antibodies. In a preferred embodiment, a host cell contains at least one nucleic acid encoding a light chain and at least one nucleic acid encoding a heavy chain, and provides the Assembly of monoclonal antibodies with obtaining three-dimensional spatial patterns corresponding to the three-dimensional structure of a monoclonal antibody produced by a mammal, preferably a human cell. If the light chain is produced separately from the heavy chain, both chains can be cleaned and then assembled with the teachings of monoclonal antibodies, having a three-dimensional structure of monoclonal antibodies, essentially similar to the structure of antibodies produced by a mammal, preferably a human cell.

Monoclonal antibody can also be obtained by recombinant expression of the encoded light and/or heavy chains, where the nucleic acid is obtained by separating the nucleic acid encoding the monoclonal antibody, in a known manner and then insert nucleic acid sequence that encodes a CDR plots in the selected nucleic acid, as defined in the formula of the present invention.

The present invention also proposed a pharmaceutical composition comprising at least one monoclonal antibody and/or at least one nucleic acid encoding a light and/or heavy chain of the monoclonal antibody.

The pharmaceutical composition may additionally contain antibiotics such as streptomycin, penicillin and vancomycin, etc., preferably associated with a monoclonal antibody according to the present invention.

The pharmaceutical compositions contain monoclonal antibody at doses of 0.1-100 mg/kg of body weight.

The pharmaceutical compositions can be entered by any known method, for example intravenously, intramuscularly, intracutaneously, subcutaneously, NR is trebusina, topically, intranasally, or in the form of inhaled spray.

In a preferred embodiment of the invention the pharmaceutical composition is used for the prevention or treatment of abscess formation in the body of the patient is a mammal, such as cattle, pig, cat, dog, horse, man. In a preferred embodiment of the invention the pharmaceutical composition is used for people. In another embodiment of the invention the formation of abscess caused by staphylococcal infection.

In addition, S. aureus-induced infection, which is treated using the pharmaceutical compositions according to the present invention, may be infection of the mammary gland, such as mastitis.

Accordingly, the present invention proposed the use of monoclonal antibodies or nucleic acid that encodes a variable region light chain and/or heavy chain according to the present invention, for manufacturing a pharmaceutical composition for the prevention or treatment of obrazovaniya abscess in the body of a mammal, preferably human.

In a preferred embodiment of the invention the pharmaceutical composition, a monoclonal antibody or a nucleic acid encoding a variable region of a light or heavy chain, according to the present invention is used for PR is the prevention or treatment of an abscess in the body, such as the kidneys, heart, liver, gallbladder, pancreas, small intestine, large intestine, lungs, brain, skin, eyes, lymphatic tissue and spleen. In a preferred embodiment of the invention, the treatment is directed to an abdominal abscess. Accordingly, the organ of the abdominal cavity, which will be subjected to treatment is a liver, gallbladder, spleen, pancreas, kidneys, thin or large intestine.

In the present description, the term "education abscess" refers to the formation of abscess in the body, such as kidney, heart, liver, gall bladder, pancreas, thin or large intestine, lung, brain, skin, eyes, lymphatic tissue and spleen. In the present description, the term "access" means a collection of pus that accumulates due to infectious process (usually caused by bacteria or parasites) formed in a tissue cavity. Toxins secreted by proliferating bacteria that destroy cells and cause an inflammatory response, which are attracted to this area a large number of lymphocytes and increases the area of the local blood flow. Leukocytes destroy dead tissue and absorb bacteria by phagocytosis. From the destroyed tissue, dead bacteria and white blood cells, as well as the accumulated extracellular fluid formed a cloudy green or yellow is awaty pus. For abscess characterized by encapsulating wall, which is formed adjacent healthy cells, trying to isolate the pus from nearby structures. This is a protective reaction of tissue to pause the spread of infectious materials to other parts of the body. Abscesses may develop in any type of dense tissue, but most often on the skin surface (where they can manifest as superficial pustules (boils) or deep skin abscesses), in the lungs, brain, kidneys and tonsils. The main complication is the spread of abscess contents, such as the sowing of the body, adjacent or distant tissues and extensive local necrosis (gangrene). The formation of abscess determined by assessing the bacterial load in the body.

In the present description, the term "abdominal abscess" refers to the body abscess of the abdominal cavity. The abdominal cavity is the body cavity that contains most of the internal organs, it is located below (or inferior) thoracic cavity and above the pelvic cavity. It is part of the abdominal-pelvic cavity. The organs of the abdominal cavity include the stomach, liver, gall bladder, spleen, pancreas, intestine, kidneys.

In the present description, the term "colonization of the authority" means the distribution of live bacteria from your local hearth in which eccii in remote tissues and organs. The sowing of the body characterized by the presence of living infectious bacterial cells in healthy tissue without the formation of macroscopic encapsulated colonies of bacteria.

In the present description, the term "bacterial load" is defined as the number of live bacterial cells within anatomically well-defined tissue, and is expressed as the number of bacterial cells that form colonies on solid medium such as agar. To assess bacterial load in the body is extracted surgically from the surrounding tissue, and then in sterile tissue of the body is crushed in sterile saline for destruction of tissue structure and separation of bacterial cells from the tissues of mammals. A certain amount of cell suspension (or its serial dilution in sterile saline) is applied on the solid nutrient medium for bacteria. Bacterial load is expressed as the number of colony forming units in the kidney (e.g., CFU/kidney).

The present invention also proposed a test kit for the diagnosis of infections caused by S. aureus containing at least one monoclonal antibody according to the present invention, and optionally additional components suitable for carrying out a diagnostic test.

Ing eventy, suitable for carrying out a diagnostic test, for example, a buffer solution with osmollnosti, ranging from 280 to 320 mOsm/l, and pH value in the range from 6 to 8; buffer solution containing chelators; buffer solution containing monovalent or divalent cations with a total concentration of cations in the buffer from 0.02 M to 2.0 M and/or buffer solution containing human serum or serum of animal origin with a concentration of from 0.01% to 20%.

The test set is applicable for specific reliable diagnosis of infections caused by S.aureus. Quantitative determination can be based on standard ELISA in the liquid phase or solid-phase ELISA. Detection can be premim or indirect, as is well known in the art to which this invention relates, moreover, antibodies can be optional conjugated with an enzyme, fluorescent or radioactive label.

Accordingly, the present invention also provides the use of at least one monoclonal antibody according to the present invention for detecting binding of alpha-toxin in the sample. The binding of an antibody according to the present izobreteniye alpha-toxin can be detected using, for example, secondary antibodies goat to human IgG, conjugated with horseradish peroxidase.

BRIEF DESCRIPTION of GRAPHIC MATERIALS

FIGURE 1

The figure 1 shows the DNA sequence and amino acid sequence of the variable region of the heavy chain of monoclonal human antibodies 243-4. Plot CDR1 of the antibody 243-4 is located in clauses 26 to 33, Plot CDR2 antibody 243-4 is located at positions 51 through 58, and the plot CDR3 antibody 243-4 is located in provisions 97 through 110.

FIGURE 2

The figure 2 shows the DNA sequence and amino acid sequence of the variable region of the light chain of monoclonal human antibodies 243-4. Plot CDR1 of the antibody 243-4 is located in clauses 26 to 33, Plot CDR2 antibody 243-4 is located in clauses 51 to 53, and the plot CDR3 antibody 243-4 is located at positions 90 to 101.

FIGURE 3

The figure 3 shows the antigenic specificity of monoclonal antibodies person 243-4. Antigenic specificity of antibodies 243-4 was evaluated by binding with the panel of bacterial toxins in enzyme-linked immunosorbent ELISA analysis. The ELISA was carried out in tablets for micrometrology coated with purified toxins. After 24-hour incubation at room temperature tablets for micrometrology were blocked by bovine serum albumin. Then, using secondary antibodies goat to human IgG, conjugated with horseradish peroxidase was determined by the binding of monoclonal antitle-4 with immobilized toxins. Monoclonal antibody human 243-4 was associated with alpha-toxin of S. aureus significantly stronger compared to other examinees toxins.

FIGURE 4

The figure 4 shows the results of the analysis of the binding of monoclonal antibodies person 243-4 with alpha-toxin of epidemic strains of S. aureus by the method of Western blot. Monitored the allocation of alpha-toxin twelve epidemic strains of S. aureus in the stationary growth phase of the bacterial culture. After cultivation normalized bacterial supernatant and purified alpha-toxin downloaded LTO-SDS page gel and made electroblotting. After blocking the nitrocellulose membrane was incubated with purified monoclonal antibody human 243-4.

The production and recognition of Monomeric and/or heptameric form of alpha-toxin from each investigated epidemic strain was confirmed by binding of monoclonal antibodies person 243-4.

On the figure 4 M denotes the size of the marker, numbers 1-12 represent epidemic strains of S. aureus, and Utah denotes purified alpha-toxin.

FIGURE 5

The figure 5 shows the determination of the affinity of monoclonal antibodies person 243-4 in BIAcore analysis. The kinetics of binding of monoclonal antibodies person 243-4 analyzed using the BIAcore 2000. Different concentrations of al the f-toxin was loaded into the flow cell with immobilized on the chip monoclonal antibody human 243-4. To calculate the dissociation constants of the antibodies was detected phase Association and dissociation. Kinetic data were evaluated by a General approximation using the software BIAevaluation 4.1.

FIGURE

The figure 6 shows the neutralization of alpha-toxin a monoclonal antibody human 243-4 model alveolar damage in human cells in tissue culture. Epithelial human cells A were cultured with alpha-toxin for 16 hours in the presence of either antibodies of isotype control or monoclonal antibodies 243-4. After that, the cells were examined using quantitative analysis of lactate dehydrogenase (LDH), which detects the presence of cells, the damaged alpha-toxin and indicates the degree of protection that can be achieved with the use of antibodies. To interpret the results for 100% took the degree of lysis of the cells during incubation with the highest concentration of alpha-toxin. The degree of lysis of the cells treated only alpha-toxin, depended on its concentration. Similar results were obtained for cells treated with alpha-toxin and labeled antibody. These data indicate that the protective effect of antibody isotype control. Incubation of alveolar cells of the lung of a person with a monoclonal antibody human 243-4, on the contrary, he defended them from the of ISIS, induced by alpha-toxin. There have been three independent experiments, each of which confirmed the protective properties of the antibodies 243-4.

FIGURE 7

The figure 7 shows the protective effect of monoclonal antibodies person 243-4, introduced through the catheter into the upper Vena cava, the model mice with multi-organ infection. 24 hours after the catheter was inserted mice using a catheter was introduced 1x107SOME strains of S. aureus 300 and 7.5 mg/kg monoclonal antibodies person 243-4 or saline. Two days later, the mice in the treatment group was administered a second dose of antibody (5 mg/kg), and mouse from the control group received only saline. Five days after surgery, mice were subjected to euthanasia to control bacterial load of the kidneys and the formation of abscess. In mice immunized with a monoclonal antibody 243-4, observed a significantly lower bacterial load and the absence of abscesses in the kidneys, and the control mice was observed by high bacterial load of the kidneys and expressed the formation of abscesses.

The following examples illustrate the invention but do not limit the scope of the present invention. Further variations will be apparent to the person skilled in the art upon study of the description and given known data.

EXAMPLES

Example 1

Succession is alnost DNA and amino acid sequence of the antibody 243-4

The antibody specificity is caused by the DNA sequences and amino acids, respectively. Were defined sequence of DNA nucleotides variable regions of heavy and light chains. For RNA extraction, the suspension of cells hybridoma (5x10) besieged by centrifugation and homogenized in a Qiashredder columns (#79654, Qiagen). Then mRNA was isolated from homogenised sediment cells hybridoma using the RNeasy-Kit (#74124, Qiagen) according to the manufacturer's instructions. cDNA was synthesized on the basis of isolated mRNA by reverse transcription using reverse transcriptase Superscript II (# 18064-022, Invitrogen). Genes 243-4 antibodies amplified from the synthesized cDNA using a set of Advantage 2 PCR Kit (#639206, Clontech) according to the manufacturer's instructions. Specific amplification of genes of antibodies provided by applying a combination of primers designed to amplify the variable region of the modified human IgG (Welschof et al., 1995). For amplification of variable regions of heavy (UN) and light chain (vl) used a set of straight chain-specific primers in combination with one reverse primer which is specific joins in the constant domain of the heavy or light chain (vh amplification of the heavy chain of IgG in combination with vhi, Vm, Us; Amplification of vl light chain λ-type in combination with V a I Vu 2/5, Vu 3 a, 4a, Vu 4b and VU 6; see Table 1). Amplificate obtained by PCR were cloned in plasmid pCR4-MAINTENANCE kit for sequencing TORO TA Cloning Kit (# K457540, Invitrogen), and purified plasmid DNA was sent for sequencing (Microsynth, Balgach, Switzerland) using plasmid-specific primers from TORO Cloning Kit (T3 and T7, see table 1). The obtained DNA sequences were processed and investigated their primary structure using the software package for the analysis of the results of clone (# 875-501-1787, Scientific and Educational Software). The analysis of the primary structure was obtained consensus sequence, which is then analyzed using V Base - database of all germline sequences of human variable regions (http://imqt.cines.fr/IMGT_vquest/share/textes/). Based on the results of the analysis of the initial sequencing were designed additional primers specific for the heavy and light chain (Vb-atox as and University as atoc, see table 1). These primers were used to confirm the identified sequence of the antibody in the hybridization sites previously used combinations of primers.

Obtained in this way genes of antibodies used for sequencing as described above and as shown in Figures 1 and 2.

Table 1
Sequences of primers used for amplification and sequencing of variable regions of antibodies 243-4
PrimerSourceRoom SEQIDSequence (5'-3')Application
Heavy chain IgGWelschof et al.,O Immunol Met, 175, 19959GAC C(C50)GA TGG GCC CTT GGA GOT*PCR, sequencing
VH110C(G50)AG GTG CAG CTG GTG CAG TCT*PCR, sequencing
VH211CAG GTG(A50) CFG CTG CFG G(C50)AG TC*PCR, sequencing
VH312GAG GTG CAG CTG G(C50)TG GAG TCT*PCR, sequencing
Light chain λ13AGA GGA G(C50)GG GAA CAG AGTGAC* PCR, sequencing
VLλ 114CAG TCT GTC T(C50)ACG TG(T50) CAG CCG CCC TCA*PCR, sequencing
VLλ 2/515CAG TCT GCG CTG ACT CAA(G50) CCG G(C50)TCT CC*PCR, sequencing
VLλ 316TCC TAT GAA CTG ACT CAG CCA CCC(T50) TPCR, sequencing
VLλ 4a17TCT GAA CTG ACT CAG CCC(A33T33) C(G50)CC TC*PCR, sequencing
VLλ 4b18TCT GAA CTG ACT CAG GAC CCT GC(T50T*PCR, sequencing
VLλ 619A(G50AT TTT ATG HUNDRED and ACT CAG CCC CAC TCT*PCR, sequencing
T3#K457540 InvitrogenATT AAC CCT CAC TAA AGGGAsequencing
T721TAA TAC GAC TCA HUNDRED TAGGGsequencing
VL-atox asSelf-designed primers22AGG CTG TCA TCC CAT GCA CAGPCR, sequencing
VH-atox as23CTG CTG CTC CCA GAT CGT CTC GCPCR, sequencing

The reasons indicated in parentheses indicate replacement in the preceding provision, including the percentage of substituted nucleotides.

Example 2:

Antigenic specificity of monoclonal antibodies person 243-4 (ELISA)

Antigenic specificity of antibodies 243-4 was evaluated by binding with the panel of bacterial toxins (alpha-toxin: No. 120, List biological Laboratories, all other toxins: own production, Kenta Biotech AG). Analysis was performed by the method of enzyme-linked immunosorbent assay (ELISA) plates for micrometrology (#439454, Nunc MaxiSorp)coated with purified toxins at a concentration of 1 µg/ml After 24 h incubation at room t is mperature tablets for micrometrology blocked for 2 h with 0.5% bovine serum albumin. Binding of monoclonal antibodies person 243-4 (1 μg/ml) to immobilized toxins was determined using secondary goat to human IgG, conjugated with horseradish peroxidase (HRP)in a dilution of 1:2000 (# 62-8420, Zymed Laboratories, Invitrogen). The reaction was stopped by model HC1.

The optical density was recorded on the device ELISA reader at 490 nm using Softmax Pro®, as shown in figure 3.

Example 3:

The binding of alpha-toxin of epidemic strains of S. aureus in the analysis Western blot

Select the alpha toxin of the twelve epidemic S. aureus was controlled after 16 hours of growth in the medium BHI (# 255003, Becton Dickinson) at 37°C.

The strains obtained from the German reference centre for Golden staph (Robert Koch Institute, Wernigerode) and represent the most widespread epidemic strains of S. aureus that cause disease. Some of these strains produce less of alpha-toxin in comparison with others, which leads to different signal intensity.

Different genotypes of the investigated strains are shown in table 2. After culturing the bacteria besieged by centrifugation, and then the optical density of the supernatant of the culture was normalized to the value D600=0,6 original bacterial culture. 25 µl of each supernatant was loaded on 4-20% LTOs-SDS page gel (#ES, Invitrogen) and subjected to electroblotting for 1 hour. One microgram of purified alpha-toxin (#120, List biological Laboratories) were downloaded and examined in parallel with the control. After blocking in 5% dry milk for 1 h nitrocellulose membrane (#LC2000, Invitrogen) were incubated with 50 μg/ml of purified monoclonal human antibodies 243-4. The binding of an antibody 243-4 with alpha-toxin were detected using secondary antibodies goat to human IgG, conjugated with horseradish peroxidase at a dilution of 1:2000 (# 62-8420, Zymed Laboratories, Invitrogen), as shown in figure 4.

TABLE 2
Panel examples of clonal lines of strains of S. aureus (MRSA), received in hospital and non-hospital settings
The designation of the Western blotThe number of isolates from the Robert Koch InstituteMLST*1SS*2spa-type sequence*3
193-00134ST2478t051
206-00842ST88 t008
306-02222ST98t008
406-01579ST2398t031
506-00219ST55t002
606-00409ST2255t003
706-01019ST4545tl384
806-02182ST2222t965
903-02773ST11tl75
1006-00373ST88t008
1105-0189 ST2222t310
1206-00300ST8080t044

*1) Multiloci typing sequence.

*2) Clonal complex.

*3) analysis of the variable repeat region of the gene of the protein A (spa).

Example 4:

Determination of affinity (using BIAcore)

Range surface plasmon resonance was measured using the system BIAcore 2000 (BIAcore). All experiments were performed in 20 mm MOPS buffer, pH 7.0, 150 mm Nad, 0.1 mg/ml BSA. First, antibodies goat to human IgG (# 81-7100, Zymed Laboratories, Invitrogen) was immobilized on the chip SM (BIAcore) using binding assays amines to about 13 200 Rel. units, as described in the manual BIAapplications. In addition to the primary covalently linked coating antibody 243-4 was immobilized on the sensor chip via interaction with pre-immobilized antibody to human IgG, resulting formed an additional layer immobilization of approximately 240 Rel. unit To determine the reaction kinetics of antigen-antibody cell was perfesional solutions of alpha-toxin with increasing concentrations (3,9 nm and 7.8 nm, nm 15.62 wide, of 31.25 nm, at 62.5 nm, 125 nm, 250 nm and 500 nm, #10, List biological Laboratories) at a flow rate of 50 μl/min After each measurement cycle (5 min associations, then 30 min of dissociation of the complex of antibody-antigen was restored by regeneration of the surface of 10 mm glycine-Hcl at pH of 1.7. To calculate the dissociation constants of the phase Association and dissociation of antibodies 243-4 registered, evaluated by means of a common approximation using the software BIAevaluation 4.1 (BIAcore AB, as shown in figure 5). For in the analysis by the overall approximation takes into account only the indicated concentration of antigen, which allowed to perform the analysis using model binding Langmuir, gave a 1:1 ratio (<125 nm, table 2), as described in the BIAcore.

TABLE 3
Kinetic constants quantitative interaction of alpha-toxin antibody 243-4
The concentration of antigenkass(I mol-1c-1)kdiss(c-1)ToD(M)
3,9 nm5,7*1048,2* Yu51,4* j-9
7,8 nm 6,1*1041,0* Yu51.6 x 10-'
15,nm6,2*1048,0* Yu51,3* n-9
31,25nm6,3* 1049,7* Yu51,5* Yu9
at 62.5 nm6,6*1049,0*10'51,4*10'9
125 nm6,2*1048,9* Yu51,4* j-9
Average6,2*104±0,3*1048,9*10-5±0,7*10-51,4*10-9±0,1*10-9

Example 5:

Model of acute powiedzenia light on the culture of cells of the alveolar epithelium person

Culture of cells of the alveolar epithelium person A were sown in medium RPMI (#R0883, Sigma-Aldrich) at a density of 3x105cells per well. Simultaneously, a series of solutions with increasing concentration of alpha-toxin (5 μg/ml - 50 μg/ml, #120, List biological Laboratories) advanced the satisfactory incubated only with the environment, 20 μg/ml antibody for control isotype (IgG1 λ person, purified myeloma protein; #1 5029, Sigma-Aldrich) or 20 μg/ml of purified monoclonal antibodies 243-4. After 4 h incubation at 37°C alpha-toxin or a solution of alpha-toxin-antibody was added to the cells and incubation was continued for another 16 hours. After that, the cells were analyzed using a kit for the quantitative determination of lactate dehydrogenase (LDH) (#04744934001, Roche), which allows us to estimate the release of LDH from the cells into the culture medium, as shown in figure 6.

Example 6:

The model of multi-organ infection in mice

Female mice of BALB/C (Charles River, Sulzfeld, Germany) weighing 27-31 g acclimatized for 14 days before surgery. Mice were received from the supplier indicated that the supplied mouse free from pathogens. For placement of the catheter mice were anestesiology, introducing xylazine intraperitoneally (8 mg/kg body mass)/ketamine (100 mg/kg body weight). For one odnovremennogo polyethylene catheter in the upper Vena cava on the left side shaved neck was made of the minimum horizontal incision of the skin (outer diameter of 0.6 mm, F6hr Medical Instruments, Seeheim, Germany). Twenty-four hours after installation mice through the catheter was injected 1×107SOME strains of S. aureus US 300 (100 μl) and 7.5 mg/kg purified antibodies 243-4 or physiological rest the p (50 μl). Two days later, the mice receiving the treatment group was administered a second dose of antibody (5 mg/kg), whereas mice from the control group received only saline. Five days after surgery, mice were euthanized to control bacterial load and abscess formation in the kidneys. For these purposes, the kidneys were aseptically removed from the bodies of animals, euthanized, and homogenized in saline solution. Before removing the authority has examined the location of the catheter in the upper Vena cava. Before homogenization body macroscopically examined for the presence of an abscess. After serial dilution of the homogenate body were cultured on cups RTOS for 48 h at 37°C. the Number of colony forming units (CFU) rasschityvala and recorded as CFU/kidney, as shown in figure 7.

TABLE 4
The different nature of the formation of an abscess of the kidney in the control group of animals and the group treated
The group treated with monoclonal antibodyMouse 1The abscess is missing in both kidneys
Mouse 2Abscess away is there in both kidneys
Mouse 3The abscess is missing in both kidneys
The control groupMouse 4The pronounced formation of abscess in both kidneys
Mouse 5The pronounced formation of abscess in both kidneys
Mouse 6Was not determined, because the mouse died before the end of the experiment

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WO 2007/145689 Use of Alpha-Toxin for treating and preventing Staphylococcus infections.

1. Monoclonal antibody specific to alpha-toxin of S. aureus, harakterizuyu is that the variable region of the light chain of the antibody contains SEQ ID NO:l in the region CDR1, SEQ ID NO:2 in the region CDR2, and SEQ ID NO:3 in the region CDR3, and the variable region of the heavy chain of the antibody contains SEQ ID NO:4 in the region CDR1, SEQ ID NO:5 in the region CDR2, and SEQ ID NO:6 in the region CDR3, or a fragment, or mutein specified antibodies, capable of binding to alpha-toxin of S. aureus, and mutein monoclonal antibodies carries at least one conservative substitution in one part of the CDRs in the heavy or light chain.

2. Monoclonal antibody according to claim 1, in which the variable region of the light chain of the antibody has the amino acid sequence of SEQ ID NO:7 and the variable region of the heavy chain has the amino acid sequence of SEQ ID NO:8, or a fragment of the indicated antibodies capable of binding to alpha-toxin of S. aureus, or variant specified antibodies, capable of binding to alpha-toxin of S. aureus, and the amino acid sequence of the variable region of the light chain of the antibody of at least 85% identical to SEQ ID NO:7 and amino acid sequence of the variable region of the heavy chain of the antibody of at least 85% identical to SEQ ID NO:8.

3. Monoclonal antibody according to claim 1, the antibody fragment is a Fab, F(ab')2, single-stranded, or a domain antibody.

4. Monoclonal antibody according to claim 1, the antibody is a human antibody.

5. Monoclonal antibody according to claim 1, and light chain type of the lambda.

6. Monoclonal antibody according to claim 1, characterized in that the heavy chain belongs to the IgG type.

7. Monoclonal antibody according to claim 1, and an antibody capable of specific contact Monomeric and oligomeric forms of alpha-toxin of S. aureus.

8. Monoclonal antibody according to claim 1, the antibody contains N-terminal, internal or C-terminal modification.

9. Monoclonal antibody of claim 8, the modification is selected from at least one of oligomerization, glycosylation or conjugation with drug or label.

10. Monoclonal antibody according to any one of claims 1 to 9, derived from b-cells of a mammal or hybridoma obtained by merging these b-cells of the mammal with myeloma cells or heteromyinae.

11. Nucleic acid encoding a light chain of the monoclonal antibody according to any one of claims 1 to 10.

12. Nucleic acid encoding a heavy chain of a monoclonal antibody according to any one of claims 1 to 10.

13. The vector for expression of the antibody according to any one of claims 1 to 10 or a fragment of the indicated antibodies containing at least one nucleic acid encoding a light chain according to claim 11, or at least one nucleic acid encoding a heavy chain indicated in paragraph 12, or at least one nucleic acid encoding a light chain according to claim 11, and at least one nucleic acid coding for the total heavy chain according to item 12.

14. The vector according to item 13, and the specified vector further comprises a promoter functionally linked to the nucleic acid and amplifying her expression.

15. A host cell to obtain the antibody according to any one of claims 1 to 10 or a fragment of the indicated antibodies containing vector for 14 or the nucleic acid according to claim 11 or 12.

16. Pharmaceutical composition for treatment or prevention of abscess formation in the body, containing at least one monoclonal antibody according to claims 1-10, or at least one nucleic acid according to claim 11 and at least one nucleic acid according to item 12, and a pharmaceutically acceptable carrier or ingredient.

17. The use of monoclonal antibodies according to any one of claims 1 to 9 for the prevention and treatment of abscess formation in the body.

18. The use of a nucleic acid according to claim 11 and nucleic acids 12 to obtain a pharmaceutical composition for the prevention and treatment of abscess formation in the body.

19. Use 17 or 18, and an abscess in the body is an abdominal abscess.

20. The use according to any one of p or 18, and the organ is a kidney, heart, liver, lung, brain, skin and spleen.

21. The use according to any one of p or 18, with the formation of an abscess caused by S. aureus infection.

22. Test kit for the diagnosis of infection with S.aureus in the sample, VK is uchumi at least one monoclonal antibody according to any one of claims 1 to 10 or a nucleic acid according to claim 11 and nucleic acid according to item 12.

23. The use of at least one monoclonal antibody according to any one of claims 1 to 10 for detecting the binding of alpha-toxin in the sample.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology and biotechnology. Presented are isolated artificial or recombinant antibody or its functional part, capable of specific binding F-antigen of respiratory-syncytial virus, which include: variable sequence of heavy chain, containing CDR1 NYIIN (SEQ ID NO:1), CDR2 GIIPVLGTVHYAPKFQG (SEQ ID NO:2), CDR3 ETALVVSTTYLPHYFDN (SEQ ID NO:3), and variable sequence of light chain, containing CDR1 QASQDIVNYLN (SEQ ID NO:4), CDR2 VASNLET (SEQ ID NO:5), CDR3 QQYDNLP (SEQ ID NO:6); as well as nucleotide sequence coding said antibody. Described is isolated mammalian cell, including said nucleic acid, where said cell expresses said nucleic sequence. Disclosed is method of obtaining antibody or its functional part, including: cultivation of said cell in vitro; and obtaining antibody or its functional fragment, produced by said cell. Described is composition for treatment or prevention of RSV-associated disorder, or preventing or counteracting unfavourable effect of RSV infection on humans, including therapeutically efficient quantity of said antibody or its functional part and pharmaceutically acceptable carrier, solvent or excipient. Claimed is application of said antibody or coding it nucleic sequence for obtaining medication for treatment or prevention of RSV-associated disorder, or preventing or counteracting unfavourable effect of RSV infection on humans.

EFFECT: invention makes it possible to extend arsenal of means for treatment or prevention of RSV-associated disorders.

22 cl, 16 dwg, 3 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology and immunology. Described are versions of antibodies, binding the GRM molecule, as well as their antigen-binding fragments, amino acid sequences of variable parts of which are presented in the claim materials. Nucleic acid, coding the said antibodies, is presented. Claimed is a method of obtaining the RGM-binding protein, which includes cultivation of a host cell in a culture medium under conditions suitable for obtaining the binding protein, capable of binding with RGM, where the host cell contains an expression vector, containing the separated nucleic acid, coding the said antibody. Described is a pharmaceutical composition for treating a disease, in which the SGM A activity produces a negative impact, which contains a therapeutically efficient quantity of the said antibody and a pharmaceutically acceptable carrier. Claimed is an application of the said antibody for obtaining a medication, used for a) reduction of hRGM A binding with a patient's Neogenin receptor; or b) for reduction of hRGM A binding with BMP-2 and BMP-4 in the patient.

EFFECT: invention makes it possible to obtain antibodies against GRM, which are used for treating diseases, associated with excessive interaction of RGM with the Neogenin receptor, BMP-2 and BMP-4.

13 cl, 16 dwg, 10 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention relates to biochemistry. A method of immunoassay of human protein CXCL1 is described. Human CXCL1 or its fragment is measured in a sample with application of two or more types of monoclonal antibodies to human CXCL1 or their fragments. Each of two or more types of the monoclonal antibodies to human CXCL1 or their fragments specifically identifies any of regions of a sequence of amino acid sequences, represented in SEQ ID NO:1-3, which represent partial sequences of an amino acid sequence, constituting human protein CXCL1. Two or more types of the monoclonal antibodies to human CXCL1 or their fragments specifically identify regions of the sequence, different from each other. Claimed are the monoclonal antibodies or their fragments, each of which specifically identifies any region of the amino acid sequence, represented in SEQ ID NO:1-3, and has a new amino acid sequence.

EFFECT: invention makes it possible to determine human protein CXCL1 with high sensitivity.

15 cl, 9 dwg, 1 tbl, 21 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: compounds can be applied for treatment of oncologic and autoimmune diseases. Invention also characterises method of obtaining conjugates, pharmaceutical composition and medication, which contains modified proteins. In general formulae 1 or 2 , R1 is selected from the group representing (CH3)2N-,

R2 is selected from the group representing where R3 as terminal substituent represents -NH2, or and R4 represents H or C1-C3alkyl.

EFFECT: novel compounds possess affinity for CD16a receptor.

18 cl, 20 dwg, 3 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

Antibodies to her // 2504553

FIELD: biotechnologies.

SUBSTANCE: invention describes versions of bispecific antibodies specifically bound to EGFR and HER3, which contain amino-acid sequences of variable regions of heavy and light chains respectively, SEQ ID NO: 30 and 29; or SEQ ID NO: 28 and 27; or SEQ ID NO: 28 and 29; or contain complementary regions CDR of heavy and light chains of the above sequences of variable regions. The invention describes nucleic acid coding a versions antibody, and a host cell containing the above nucleic acid and used for expression of the anitbody. Immunoconjugate containing antibody versions and cytotoxic agent used for treatment of cancer containing cells that express EGFR and HER3 are presented. A method for obtaining a bispecific antibody, which involves cultivation of a host cell so that an antibody is generated, is described. The invention describes a pharmaceutical composition for treatment of cancer containing EGFR- and HER3-expressing cells, which contains effective amount of bispecific antibody and pharmaceutically acceptable carrier. The invention proposes a treatment method of cancer containing EGFR- and HER3-expressing cells and an inhibition method of biological activity of EGFR and/or HER3 of a specimen, which involve introduction of effective amount of a bispecific antibody. Use of the above antibody in production of a remedy for treatment of cancer, the cells of which express EGFR and HER3, is described.

EFFECT: invention allows obtaining bispecific antibodies binding EGFR and HER3, which are not conjugates of two antibodies.

22 cl, 33 dwg, 4 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.

EFFECT: invention allows obtaining antibodies to IL-21 of a human being.

48 cl, 4 dwg, 16 tbl, 23 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.

EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.

3 cl, 5 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: invention is a strain of cultured hybrid cells of animal Mus musculus L. CCHFV Vd-3 deposited in the State collection of pathogenic microorganisms and cell cultures "SCPM-OBOLENSK" under number H-27, which is a producer of monoclonal antibody 3H6/F2 to virus of Crimean Congo haemorrhagic fever (CCHF), is suitable for manufacture on its base of one of the components of a set of reagents for immunoenzymometric detection of antigens of CCHF virus in samples of biological material.

EFFECT: invention enables to obtain highly specific monoclonal antibodies suitable for use as one of the components of the set of reagents for immunoenzymometric detection of antigens of CCHF virus.

1 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: invention is a strain of cultured hybrid cells of animal Mus musculus L. CCHFV Vd-2 deposited in the State collection of pathogenic microorganisms and cell cultures "SCPM-OBOLENSK" under number H-26, which is a producer of monoclonal antibody 1E2/E5 to virus of Crimean Congo haemorrhagic fever (CCHF), suitable for the manufacture on its base of one of the components of the set of reagents for immunoenzymometric detection of antigens of CCHF virus in samples of biological material.

EFFECT: invention enables to obtain highly specific monoclonal antibodies suitable for use as one of the components of the set of reagents for immunoenzymometric detection of antigens of CCHF virus.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. What is presented is a method for generating an antibody and its functional fragments against a tumour antigen expressed on the tumour surface resistant to at least one anti-tumour compound by applying ground homogenate, and/or suspension, and/or cell lysate originated from the same tumour for immunisation. There are also disclosed using the method according to the invention for producing the monoclonal antibodies and their functional fragments, the monoclonal antibodies produced by the method, nucleic acids coding them, an expression vector, a host cell, and a method for preparing the antibody with using them, as well as hybridomes secreting these antibodies and their functional fragments for preparing a drug, the anti-tumour composition and using it as a drug.

EFFECT: invention can find further application in therapy of resistant tumours.

42 cl, 7 dwg, 4 ex, 6 tbl

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

FIELD: biotechnologies.

SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.

EFFECT: invention allows obtaining antibodies to IL-21 of a human being.

48 cl, 4 dwg, 16 tbl, 23 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: this invention relates to biotechnology and immunology. One proposes: JAM-A protein antibody or functional fragment thereof, hybridoma secreting such antibody, nucleic acid, expression vector and host cell as well as a method for the antibody and composition production. One considers application of the JAM-A protein antibody or functional fragment thereof.

EFFECT: invention usage ensures creation of new JAM-A protein antibodies which may be further applied in treatment or prevention of diseases related to proliferation of tumour cells extracting JAM-A protein.

34 cl, 31 dwg, 5 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention presents cultivated hybrid cell strains of Mus. musculus animals Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-1C6, Sp2/0-BC/rhPC-3H6, producers of monoclonal antibodies specific to human protein C. The strains are deposited in the Russian National Collection of Industrial Microorganisms of Federal Unitary Enterprise State Research Institute 'Genetics', No. (VKPM H-111), (VKPM H-112), respectively. The antibodies belong to hPROC-specific murine immunoglobulin G possessing cross-responsiveness, are selectively bound with human protein C and form a stable complex.

EFFECT: antibodies under the invention may be used for purposes of pharmaceutical and biomedical analytical studies, particularly for quantitative detection of the human recombinant factor C.

1 dwg, 4 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to infectious diseases, and can be used for treating whooping cough and/or for Bordetella infection protection. Polypeptide under the invention represents a fragment of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain from an amino acid sequence extended from position 1166 to position 1281 SEQ ID NO:1. Said invention also concerns the specific fragments of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain, and to their application, particularly for targeting of molecules of interest to CD11b expressing cells.

EFFECT: use of the inventions allows extending the range of products for treatment and prevention of the Bordetella infection.

26 cl, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: in invention described is hybridoma, producing monoclonal antibodies, which bind with protein 161P2F10B in a specific way. Invention describes versions of monoclonal antibodies, one of which includes amino acid sequence of heavy and light chain of antibody, produced by said hybridoma, and the other includes amono acid sequence of variable part of heavy and light chain of antibody, produced by said hybridoma, or amino acid sequence Fab, F(ab')2, Fv or Sfv of antibody fragment, which binds with protein 161P2F10B, 161P2F10B demonstrates tissue-specific expression in healthy, mature tissue and is abnormally expressed in case of oncologic diseases.

EFFECT: antibodies can be used as anti-cancer medication and in composition of pharmaceutical composition for treating cancer, in particular, kidney cancer, reducing tumour growth, as well as for detection of protein 161P2F10B in biological sample.

11 cl, 212 dwg, 8 tbl, 20 ex

FIELD: medicine.

SUBSTANCE: essence of invention includes contact of liquid sample, taken from mammal organism, with one or several monoclonal antibodies to Lawsonia intracellularis antigen, secreted by cell lines of ECACC hybridomes, which have registration numbers. Invention also includes diagnostic test-kit, containing antibodies specific for Lawsonia intracellularis.

EFFECT: increased specificity of antigen detection.

11 cl, 5 ex, 5 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.

EFFECT: invention enables treating or preventing autoimmune disorders of skin, inflammatory diseases of skin or mucous membrane, or injured skin in an animal effectively.

16 cl, 19 dwg, 3 tbl, 12 ex

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