RU2420588C2 - MURINE MONOCLONAL ANTIBODIES BOUND WITH ANTIGEN F1 OF Yersinia pestis, METHOD FOR PRODUCING THEREOF WITH USING YEAST, METHOD AND KIT FOR Yersinia pestis DETECTION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there are described isolated mouse antibody selectively bound with antigen F1 of Yersinia pestis, and an antigen-binding fragment (Fab). There is presented yeast cell producing the described antigen-binding fragment (Fab). There is offered method for producing described antigen-binding fragment. Also, there is presented method for Yersinia pestis detection and a kit for detection of antigen F1 of Yersinia pestis.

EFFECT: invention presents a Yersinia pestis detection reagent.

9 cl, 12 dwg, 7 ex

Description

Technical field

The present invention relates to antibodies, in particular to murine monoclonal antibodies that bind to F1 protein from Yersinia pestis, a method for producing said antibodies using yeast, the use of said antibodies to detect Yersinia pestis, and to a kit for said use containing said antibodies.

State of the art

Antibodies usually consist of two heavy chains linked together by disulfide bonds, and light chains associated with the N-terminus of each of the heavy chains. Each heavy chain contains at the N-terminus a variable domain with a constant domain at the other end. Each light chain also contains at the N-terminus a variable domain with a constant domain. The variable domains of each pair of light and heavy chains form an antigen-binding site. The variable domains of light and heavy chains have a similar overall structure, and each domain includes a framework of four regions, the sequences of which are relatively conservative, linked through three regions that define complementarity (complementarity determining regions, CDRs). Four frame sections form a beta-fold type conformation, and CDRs form loops connecting the beta-fold structure. Plots CDRs are located in close proximity to each other due to the frame sections and contribute to the formation of antigennegative plot. Plots of CDRs and frame sections of antibodies can be determined by reference to the Kabat numbering system, Kabat et al., (1987) "Sequences of Proteins of Immunological Interest", US Dept. of Health and Human Services, US Government Printing Office ) in combination in conjunction with x-ray crystallography, as set forth in WO 91/09967.

Kohler and Milstein (Kohler et al., (1976) Nature 256: 495-497) are usually used to produce antibodies that can bind to any specific antigen. It mainly includes immunization of a mouse with an antigen, fusion of spleen cells from an immunized mouse with mouse myeloma cells, and selection among one or more hybridomas thus obtained that secrete a monoclonal antibody specific for the target antigen.

The capsule antigen F1 is the main immunochemical component of the antigen located on the surface of Y. pestis. The F1 antigen is attached to the outer membrane of the microbe Y. pestis in the form of an oligomeric protein that forms a granular layer and gradually diffuses into the environment. The capsule polymer consists of many similar hydrophobic protein subunits aggregated under physiological conditions. The F1 antigen subunit is initially synthesized from 170 amino acids and has a molecular weight of 17.6 kDa, then, after removal of the signal peptide during secretion to the surface, a protein with a molecular mass of 15.6 kDa and an isoelectric point of 4.1 is formed (Karlyshev et al., FEBS Lett. 1992 Feb 3 ; 297 (1-2): 77-80).

SUMMARY OF THE INVENTION

The aim of the present invention was to obtain a reagent for the detection of Yersinia pestis.

This goal was achieved by isolating a murine antibody capable of binding F1 antigen from Yersinia pestis, determining its sequence and demonstrating the high specificity of the indicated antibody in binding of F1 antigen from Yersinia pestis. Also, this goal was achieved by creating a method of obtaining the specified functional antibodies using yeast cells.

The present invention provides a novel monoclonal murine antibody comprising the amino acid sequence of SEQ ID NO: 1 as the variable region of the heavy chain (VH) of the antibody, the amino acid sequence of SEQ ID NO: 2 as the variable region of the light chain (VL) of the antibody and conserved regions of both chains necessary for the functioning of the specified antibodies. Such a new monoclonal mouse antibody selectively binds to the F1 antigen from Yersinia pestis.

The present invention also provides an isolated DNA fragment encoding the antibody described above.

The present invention also provides an isolated fragment with a fragment antigen binding (Fab) site of a monoclonal mouse antibody that selectively binds to the F1 antigen from Yersinia pestis and includes the variable region of the heavy chain (VH) with the amino acid sequence of SEQ ID NO: 1 and the variable region of the light chain (VL) with the amino acid sequence of SEQ ID NO: 2.

The present invention also provides an isolated DNA fragment encoding a fragment with an antigen binding site (fragment antigen binding, Fab) described above.

The present invention also provides a yeast cell transformed with the DNA fragment described above having the ability to produce said Fab.

The present invention also provides a yeast cell as described above, wherein said yeast cell is Pichia pastoris.

The present invention also provides a method for producing the Fab described above, comprising growing said yeast in a culture medium and isolating said Fab from the culture fluid.

The present invention also provides a method for detecting Yersinia pestis, comprising: (a) obtaining a sample from an animal or human; (b) the interaction of the specified sample with the antibody or Fab described above; (c) detecting a complex formed by said antibody or Fab and F1 antigen; and, optionally, (d) determining the amount of F1 antigen present in said sample based on the detected amount of the complex formed by the antibody and the F1 antigen, or the complex formed by the Fab and the F1 antigen.

The present invention also provides a kit for the detection of F1 antigen from Yersinia pestis, containing isolated monoclonal antibodies that selectively bind to the F1 antigen from Yersinia pestis, and means for detecting said F1 antigen using said antibody.

Brief Description of the Drawings

Figure 1 shows Western blotting (lanes 1-2) and electrophoresis in 12% SDS-PAGE (reducing conditions) of F1 antigen from Y. pestis with murine antibodies. Tracks; 1 - conjugate anti-IgG-POD (control), 2 - mouse antibodies (20 μg / ml), 3 - F1 antigen from Y. pestis, 4 - molecular weight standard (97, 69, 45, 30, 20 kDa).

Figure 2 shows the immunoreactivity of an antibody using the F1 antigen from Y. pestis, determined using the ELISA method.

Figure 3 shows the calibration curves for determining the dissociation constant of antibodies and F1 antigen from Y. pestis, obtained using the ELISA method determined by ELISA method.

Figure 4 shows the mortality curves of mice in both the experimental and control groups.

Figure 5 shows the nucleotide and amino acid sequences of the VH region. The 5'-primer used to clone the gene encoding the Fab fragment of the mouse monoclonal anti-F1 antigen from Y. pestis is shown in blue, the frame regions are shown in yellow, the hypervariable loops (CDRs) are shown in green, the constant domain of the heavy (IgG1) chain of the mouse antibody is shown in red.

Figure 6 shows the nucleotide and amino acid sequences of the VL region. The 5′-primer used to clone the gene encoding the Fab fragment of the mouse monoclonal anti-F1 antigen from Y. pestis is shown in blue, the frame regions are shown in yellow, the hypervariable loops (CDRs) are shown in green, the constant domain of the light chain (kappa) of the mouse antibody is shown in red.

7 shows the construction of plasmids pPICZαA-F1-mouse-H and pPICZαA-F1-mouse-L.

On Fig shows a schematic map of the plasmid pPICZαA-F1-mouse-H-L.

Figure 9 shows Western blotting of the culture fluid of strain GS115 / AOX-F1-mouse-H-L producing the Fab fragment with anti-mouse antibodies. Lanes with β-mercaptoethanol: 1 - weight markers; 2 - GS115 / AOX-F1-mouse-H-L, 3-GS115.

Figure 10 shows the protein analysis data (12% SDS-PAGE) for 4 fractions of the Fab preparation after purification by gel filtration on a column with a Sephadex G50. Lanes: 1 - standard, 2-5 - various fractions of the antibody preparation.

11 shows a calibration curve for Fab against F1 antigen from Y. pestis, obtained by ELISA.

12 shows the binding curve for natural (a) and recombinant (b) Fab against the F1 antigen from Y. pestis in Klotz coordinates.

Detailed description of the present invention

A suitable method for isolating antibodies effective for use in the framework of the present invention includes (a) administering to the animal an effective amount of a protein, peptide or mimotope thereof to produce antibodies, and (b) isolating said antibodies. Antibodies obtained against certain proteins or mimotopes may have some advantages, since such antibodies are not heavily contaminated with antibodies against other compounds, which could otherwise affect the accuracy of the diagnostic method. Methods for producing such antibodies are known in the art and are described in detail by Harlow et al. (Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press, 1988) and include immunizing animals to produce antibody preparations that are isolated from, for example, plasma or ascites fluid and is purified by methods known in the art to obtain preparations that give a reaction with an antigen. Many species have proteins with close sequences and therefore difficulties may arise when using standard immunization protocols to produce antibodies that recognize a protein of only one species. Therefore, it is also worth considering modifications to standard methods for producing antibodies, such as, for example, difference (subtractive) hybridization techniques. Such modifications may be known to those skilled in the art, and are also described herein. In other methods, antibodies that can be used in the framework of the present invention, obtained using recombinant methods described by Sambrook and others (Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Labs Press, 1989)).

Briefly, monoclonal antibodies are obtained by fusion of spleen cells from an immunized animal and myeloma cells to produce a hybridoma. Hybridomas can be tested for the ability to produce the desired antibody, then hybridomas can be grown, and these antibodies can be isolated. The term "grown cells", as used here, means hybridomas or other cell lines that produce antibodies. Methods for obtaining and testing such grown cells are described by Harlow et al. (See above). The preparation of material used as an antigen for injection of animals includes methods well known in the art, for example, the use of a full-sized protein, the use of a peptide selected from immunogenic regions of the protein, modification of the antigen by methods such as, for example, binding to dinitrophenol, binding with arsanilic acid, antigen denaturation, antigen binding to a carrier protein, such as, for example, keyhole limpet hemacyanin, with peptides containing T-glue receptor binding sites Class II current, with beads, as well as any other methods known in the art. See Harlow et al. (See above).

In particular, the antibodies of the present invention are murine monoclonal antibodies having the ability to bind F1 antigen from Yersinia pestis (Y. pestis). These antibodies are murine monoclonal antibodies containing the amino acid sequence of SEQ ID NO: 1 as the variable region of the antibody heavy chain (VH), the amino acid sequence of SEQ ID NO: 2 as the variable region of the antibody light chain (VL) of the conserved regions of both chains necessary for the functioning of the indicated antibodies. Conservative sites of both chains necessary for the functioning of the indicated antibodies are, but are not limited to, the amino acid sequences represented by SEQ ID NO: 54 and SEQ ID NO: 55. These new murine monoclonal antibodies selectively bind to the F1 antigen from Y. pestis.

Further, the fragment antigen binding (Fab) site of the present invention is an isolated Fab that selectively binds to F1 antigen from Yersinia pestis and includes a heavy chain variable region (VH) with the amino acid sequence of SEQ ID NO: 1 and a variable region light chain (VL) with the amino acid sequence of SEQ ID NO: 2.

In the present invention, the phrase "antibody or Fab capable of binding to the F1 antigen from Yersinia pestis" means a molecule that binds to the F1 antigen and forms a stable complex. A stable complex is a complex in which binding between partners occurs for a period of time sufficient to detect the specified complex using the methods described here. The term “selectively binds F1 antigen from Yersinia pestis” means the ability of said molecule to preferably bind to the F1 antigen, as opposed to binding to proteins unrelated to the F1 antigen or binding to non-protein components present in the sample. An antibody or Fab that preferably binds to the F1 antigen is an antibody or Fab that binds to the F1 antigen but does not bind substantially to other molecules or components that may be present in the sample. Significant binding involves, for example, the binding of an antibody that binds to the F1 antigen with a molecule that is not an F1 antigen, with an affinity or strength sufficient to interfere with the ability of the antibody that binds to the F1 antigen to determine the level of F1 antigen from Y. pestis in sample. Examples of such molecules and components that may be present in a sample are, but are not limited to, non-F1 antigen proteins, lipids and carbohydrates.

The ability of an antibody or Fab to bind to an antigen can be determined by a person skilled in the art using methods including, but not limited to, ELISA and equilibrium dialysis. Methods for determining affinity and binding strength are well known to those skilled in the art, described in detail by Janeway et al. (Immunobiology: The Immune System in Health and Disease (Garland Publishing Company, 1996)).

A Fab suitable for practicing the present invention is a Fab having the ability to bind F1 antigen from Y. pestis when the concentration of F1 antigen is between about 10 ng / ml and about 10 pg / ml. In particular, antibodies useful in the framework of the present invention bind F1 antigen from Y. pestis when the concentration of F1 antigen is 10 ng / ml, about 1 ng / lm or less, preferably 100 pg / ml. Such antibodies and Fab are described in the accompanying Examples.

An isolated DNA fragment encoding an antibody of the present invention is an expressed DNA fragment containing a promoter, a signal sequence, a nucleotide sequence encoding the structural parts of the antibody heavy and light chains, and a transcription termination site.

In particular, the nucleotide sequences encoding the structural parts of the heavy and light chains of an antibody of the present invention comprise a DNA fragment encoding a heavy chain variable region (VH) (SEQ ID NO: 3) and a light chain variable region (VL) (SEQ ID NO: 4) associated with a DNA fragment encoding the conserved regions of both chains necessary for the functioning of the specified antibodies. The nucleotide sequences encoding the conserved regions of both strands necessary for the functioning of this antibody are presented, but are not limited to the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57.

Also, the nucleotide sequences encoding the Fab of the present invention comprise a DNA fragment encoding a heavy chain variable region (VH) (SEQ ID NO: 3) and a light chain variable region (VL) (SEQ ID NO: 4).

Due to the degeneracy of the translation code, there may be differences in the DNA sequence. The DNA fragments of the present invention are not limited to the fragments shown in SEQ ID NO: 3 or 4, provided that they encode antibody chain sections with the amino acid sequence shown in SEQ ID NO: 1 or 2.

The yeast cells of the present invention are yeast cells transformed with the DNA fragment described above and having the ability to produce Fab against the F1 antigen from Y. pestis.

The phrase “yeast cells transformed with a DNA fragment” means that the desired DNA fragment has been introduced into the yeast cell using methods known to one skilled in the art. Transformation of a yeast cell with a DNA fragment leads to an increase in the expression of the DNA fragment encoding the antibody of the present invention. The presence of an α-factor signal sequence results in secretion of the produced antibody into the culture fluid. Methods for transforming yeast cells include all known methods, for example, a modified version of the procedure described for S. cerevisiae (Gietz and Schiesti, 1996).

The method according to the present invention is a method for producing a Fab against F1 antigen from Y. pestis, comprising growing yeast in a nutrient medium and isolating the obtained Fab from the culture fluid.

An example of yeast cells suitable for producing Fab according to the present invention are, but are not limited to, Pichia pastoris yeast cells. The phrase "Pichia pastoris yeast" means that said yeast is classified as Pichia pastoris (P. pastoris) in accordance with a classification known to one skilled in the art of microbiology. Examples of P. pastoris yeast useful in the framework of the present invention include, but are not limited to, P. pastoris GS115 yeast (Invitrogen).

In the present invention, the cultivation, accumulation and purification of Fab from the culture fluid and other fluids can be carried out by a method similar to traditional fermentation methods, when a certain protein is produced using a microorganism.

The growth medium can be either synthetic or natural, provided that the medium contains a carbon source, a nitrogen source, minerals and, if necessary, a suitable amount of the nutrients that yeast needs to grow. Carbon sources include carbohydrates such as glucose and sucrose, and various organic acids. Depending on the method of assimilation, the indicated microorganism can use alcohol, including methanol, ethanol, glycerin. Various ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, and digested fermented microorganisms, can be used as a nitrogen source. As a source of minerals, potassium monophosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. As vitamins, thiamine, yeast extract and the like can be used.

The cultivation is preferably carried out under aerobic conditions, such as cultivation with stirring, shaking with aeration, at a temperature of from 20 to 40 ° C, preferably from 28 to 30 ° C. The pH of the medium is usually maintained in the range from 2 to 9, preferably in the range from 6 to 7.5. The pH of the medium can be adjusted using ammonia, calcium carbonate, various bases and buffers. Typically, growing for 1 to 7 days leads to the accumulation of Fab in the culture fluid.

After growth, solid components, such as cells, can be removed from the culture fluid by centrifugation or filtration using a membrane, and then the antibodies or Fab can be isolated and purified by salt precipitation using sodium sulfate or ammonium sulfate, affinity chromatography, ion exchange chromatography, etc.

Monoclonal murine antibodies of the invention that selectively bind to the F1 antigen from Y. pestis can also be prepared using a suitable hybridoma. Hybridomas can be grown in culture medium and accumulated antibodies can be isolated. Hereinafter, the term "grown cells" refers to hybridomas or other cell lines that produce antibodies. Methods for the preparation and verification of such cultured cells are described by Harlow et al. (See above). Methods for producing antigenic material for injection into an animal include methods known in the art, for example, the use of a full-sized protein, peptides selected from the immunogenic region of the protein, modification of the antigen by methods such as, for example, binding to dinitrophenol, binding to arsanilic acid, denaturation of antigen, binding of antigen to a carrier protein, such as, for example, keyhole limpet hemacyanin, with peptides containing binding sites for class II T cell receptors, beads, as well as bubbled other methods known in the art. See Harlow et al. (See above).

Another method according to the present invention is a method for detecting Yersinia pestis. The specified method includes: (a) obtaining a sample from an animal or human; (b) the interaction of the specified sample with the antibody or Fab described above; (c) detecting a complex formed by said antibody or Fab and F1 antigen; and, optionally, (d) determining the amount of F1 antigen present in said sample based on the detected amount of the complex formed by the antibody and the F1 antigen, or the complex formed by the Fab and the F1 antigen. The presence of any amount of F1 antigen from H. pestis in the sample is evidence of a disease.

In one embodiment of the present invention, a sample is obtained or isolated from an animal or human in order to test for the presence of Y. pestis. An animal may or may not be suspected of having an early stage of the disease. As a sample that can be used to determine the presence of Y. pestis, a sample of any animal tissue can be used. Blood, sputum, bubo content, tissue samples are suitable samples. The preferred sample is blood. A person skilled in the art can easily select suitable samples.

Although not necessary in the framework of the present invention, the sample may be subjected to the desired pre-treatment. For example, a sample can be normalized to achieve the desired degree of specific activity. Rationing the sample using a suitable dilution method known in the art allows quantification of the F1 antigen from Y. pestis, regardless of the initial concentration (e.g., specific gravity) in the sample.

After receiving the sample, determine the level of F1 antigen from Y. pestis in this sample. The terms “define”, “determine the level of F1 antigen from Y. pestis”, “determine the amount of F1 antigen from Y. pestis” and the like used here are used to outline a range of methods that can be used to determine or measure the amount of F1 antigen from Y. pestis in the sample. Such methods can give qualitative or quantitative results. The level of F1 antigen from Y. pestis can be determined by the content of the whole protein of the F1 antigen from Y. pestis protein or by the content of fragments, degradation products or reaction products of the F1 antigen from Y. pestis.

Antibodies that bind to the F1 antigen from Y. pestis useful in the methods of the present invention may include multifunctional antibodies, for example bifunctional antibodies, containing at least one functional part that specifically binds to the F1 antigen. Such multifunctional antibodies can include, for example, chimeric molecules, including a molecule that binds to the F1 antigen, and a second part that allows the chimeric molecule to bind to the substrate or allows it to be detected in a way in which binding to the F1 antigen does not worsens. Examples of such second parts are, but are not limited to, fragments of an immunoglobulin molecule, a fluorescent protein, or an enzyme.

Hybrids or antibody fusion proteins that retain the ability to bind to F1 antigen from Y. pestis can also be used. In such hybrids, the F1 antigen binding portion can be linked to a second portion that can bind to the substrate or can be detected. Examples of such second parts include, but are not limited to, fragments of immunoglobulin molecules, epitopes, fluorescent proteins or enzymes.

An antibody having the ability to bind to the F1 antigen from Y. pestis, used in the framework of the present invention, may be contained in the composition for detection. For example, an antibody may be contained in a buffer in which the antibody is dissolved and / or combined with a carrier. Suitable buffers and carriers are known to those skilled in the art. Examples of suitable buffers include any buffer in which the antibody can function with selective binding of the F1 antigen, such as, but not limited to, phosphate buffered saline, water, HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic buffer salt acid), TES buffer (Tris-EDTA salt buffer), Tris buffer and TAE buffer (Tris-acetate-EDTA). Examples of carriers include, but are not limited to, polymer matrices, toxoids, serum albumin, such as bovine serum albumin. Carriers can be combined with the antibody or conjugated (attached) to the antibody in a manner that does not substantially alter the ability of the antibody to bind F1 antigen.

The term “interaction” as used herein means the introduction, addition of a sample, presumably containing the F1 antigen from Y. pestis, to the antibody that binds the F1 antigen, for example, by combining or mixing the sample with the antibody. If the F1 antigen from Y. pestis is present in the sample, a complex of F1 antigen with an antibody is formed; the formation of such a complex means the ability of an antibody to selectively bind to the F1 antigen to form a stable complex that can be detected. Detection can be qualitative, quantitative or semi-quantitative. The binding of F1 antigen from the sample to the antibody occurs under conditions suitable for complex formation. Such conditions (e.g., suitable concentrations, buffers, temperature, reaction time), as well as methods for optimizing such conditions, are well known to those skilled in the art. Binding can be detected and measured using a variety of methods that are standard in the art, including, but not limited to, enzymatic immunochemistry methods (e.g., ELISA), immunoprecipitation, immunoblot methods, and other immunochemistry methods described, for example, Sambrook et al. . (see above) and Harlow et al. (see above). These links also describe examples of conditions in which the complex is formed.

In the framework of one of the embodiments of the present invention, the complex of antigen F1 and antibodies can form in solution. In another embodiment of the present invention, an antigen F1 complex with an antibody can be formed in which the F1 antigen or antibody is immobilized on a support (eg, supported on a support). Immobilization techniques are well known to those skilled in the art. Suitable substrate materials include, but are not limited to, plastic, glass, gel, celluloid, fabrics, paper, and other materials. Examples of support materials include, but are not limited to latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF (polyvinylidene fluoride), and magnetic resins. Suitable forms of the substrate material include, but are not limited to recesses (e.g., microtiter plates), microtiter plates, rods, strips, beads, branch flow apparatuses, membranes, filters, tubes, cups, celluloid matrix, magnetic particles, others particulate matter. Particularly preferred substrates are, for example, ELISA plates, rods, immunoassay strips, radio immunological plates, agarose beads, plastic beads, latex beads, bending, cotton threads, plastic chips, immunoblot membranes, immunoblot paper and flow membranes . In one of the embodiments of the invention, the substrate indicated above may contain a marker for detection. For a description of examples of substrate materials, see, for example, Kemeny, D.M. (1991) A Practical Guide to ELISA, Pergamon Press, Elmsford, NY pp 33-44, and Price, C.) and Newman, D. eds. (Principles and Practice of Immunoassay, 2 ^nd edition (1997) Stockton Press, NY, NY).

The antibody can be immobilized on a substrate, such as a microtiter plate, a rod, an immunoassay strip, branch flow devices. A sample obtained from an animal or human is applied to a substrate and incubated under conditions suitable (i.e., sufficient) to form the F1 antigen complex with the antibody bound to the substrate.

In accordance with the present invention, an antigen F1 complex with an antibody is detected. The term "complex formation detection" as used herein means the identification of the presence of an antibody bound to the F1 antigen. If a complex is formed, then the amount of the complex formed can be quantified, but this is not necessary. Complex formation or selective binding between the putative F1 antigen with an antibody can be measured (i.e., detected, determined) using a variety of methods that are standard in the art (see, for example, Sambrook et al., Supra), examples of which are described here. The complex can be detected by a variety of methods, including but not limited to the use of one or several methods: an enzyme-like immuno-method, a competitive enzyme-like immuno-method, a radioimmune, fluorescent immuno-method, a chemiluminescent immuno-method, a method using apparatuses with a branching method, a branching flow method, using particles (for example, using particles such as but not limited to magnetic particles or plastic polymers such as latex or polystyrene beads), immunoprecipitation, BioCore ™ (e.g. using colloidal gold), immunodot (e.g. Heska AG's Immunodot System, Friborg, Switzerland) and immunoblotting (e.g. Western blotting), phosphorescence method, flow method, chromatography method, polyacrylamide gel method, plasmon surface resonance method, spectrophotometry method, particle method, electronic sensor method. These methods are well known to those skilled in the art.

These methods can be used to obtain quantitative or qualitative results, depending on how these methods are used. The results of the methods can be based on the detection of a native F1 antigen molecule or its fragments, their degradation products or reaction products of F1 antigen. Some methods, such as agglutination, particle separation and immunoprecipitation, can give visual results (for example, observation with the naked eye or using devices such as a densitometer or spectrophotometer) that do not require the use of a detection marker.

In other methods, conjugation (i.e., coupling) of an antibody with a detection marker or with a reagent that selectively binds to the antibody (e.g., antibody to antibody that binds F1 antigen) helps in detecting complex formation. Such compounds and reagents are examples of molecules for detection (i.e., molecules capable of detecting an antibody complex with F1 antigen). A marker for detection can be attached to the part of the molecule that does not affect the ability of this molecule to bind to the F1 antigen. Conjugation methods are well known to those skilled in the art. Examples of markers for detection include, but are not limited to, radioactive, fluorescent, chemiluminescent, chromophore, enzymatic, phosphorescent, electronic markers; tags from metal sols, non-ferrous beads, physical tags or ligands. Beads are macroparticles consisting of a matrix material, such as latex or polystyrene, which can be covalently or non-covalently linked to a molecule for detection. Ligands include molecules that selectively bind to another molecule. Preferred detection markers include, but are not limited to fluorescein, radioactive isotopes, phosphatase (e.g., alkaline phosphatase), biotin, avidin, peroxidase (e.g., horseradish peroxidase), β-galactosidase, compounds like biotin or avididin (e.g., strep or ImmunoPure® NeutrAvidin). For example, in the most preferred embodiment of the invention, antibodies binding to the F1 antigen are conjugated to their latex beads.

The present invention may also include one or more layers and / or types of secondary molecules or other binding molecules capable of detecting the presence of an antibody complex with F1 antigen. For example, unmodified (i.e., unrelated to a detection molecule) secondary antibodies that selectively bind to an F1 antigen binding antibody can bind to a modified (i.e. bound to a detection molecule) tertiary antibody that selectively binds secondary antibodies, which allows detection F1 antigen. Suitable secondary antibodies, tertiary antibodies and other secondary and tertiary molecules can be easily selected by a person skilled in the art. Preferred tertiary molecules can also be selected by a person skilled in the art based on the characteristics of the secondary molecule. A similar strategy can be applied to successive layers.

Non-immunological methods may also be used. In order to detect F1 antigen, methods can be used, such as pre-concentration of the sample, to increase the concentration of F1 antigen in order to increase sensitivity to the protein. Such non-immunological methods also include, for example, electrophoresis, where the detection of F1 antigen can be performed by methods known in the art, for example, protein staining. In another embodiment, a protein-based F1 antigen test can be used to determine the F1 antigen in a pre-concentrated sample from an animal or human.

Once the level of F1 antigen is measured, a conclusion can be made about the presence of the disease. The conclusion about the presence of the disease means comparing the level of F1 antigen in the test sample with the level found in a healthy animal or person. In the framework of the present invention, a conclusion about the presence of a disease is made when any amount of F1 antigen is detected.

The present invention also includes a kit suitable for detecting (detecting) F1 antigen from Y. pestis using the method described above. The term “suitable” means that detection includes the methods described herein using antibodies that bind the F1 antigen in accordance with the present invention. Such antibodies can be conjugated to a marker for detection. In another embodiment, the kit may contain unlabeled antibodies in accordance with the present invention, as well as labeled antibodies with or without detection markers having the ability to detect F1 antigen. The kit may also contain related components, such as, but not limited to, buffers, tags, containers, inserts, tubing, vials, syringes, and the like.

Methods for obtaining plasmid DNA, DNA cleavage and ligation, transformation, selection of oligonucleotides as primers and similar methods can be standard methods well known to those skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, E.F., and Maniatis, T., ("Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)).

The following examples are provided for purposes of explanation and do not in any way limit the scope of the present invention.

Example 1. Purification of F1 antigen.

Cultivation.

The cultivation of the microorganism Y. pestis EB76 (strain with a live vaccine) was carried out in a dense nutrient medium, on Hottinger's agar (Hottinger's agar) with the addition of magnesium sulfate to 0.25%, calcium chloride to 0.025%, hemolyzed blood to 0.1% at 37 ° C for 48 hours.

Purification of F1 antigen.

The culture was washed off the surface of the agar with sodium phosphate buffer pH 8.5 (PBS 8.5). Cells were pelleted by centrifugation at 5000 g for 15 minutes. The supernatant was collected, the pellet was carefully resuspended in PBS pH 8.5 and washed twice (at this stage, the capsular antigen was washed into the solution from the cell surface). Wash supernatants were combined and used as a source of F1 antigen (F1 coarse extract). The presence of F1 antigen in solution was monitored by collecting ELISA data using self-prepared conjugates of rabbit antibodies against peroxidase F1 antigen and murine monoclonal antibodies against F1 antigen.

Protein precipitation from coarse F1 antigen extracts was carried out using ammonium sulfate with 30% saturation. After incubation for 12 hours at 4 ° С, the precipitate formed was centrifuged at 10,000 g for 20 minutes, the obtained precipitate (precipitate SA30) was dissolved in PBS buffer and again precipitated at the isoelectric point at pH 4.05 ± 0.05 by titration using a dilute HCl solution. The resulting aggregates were separated by centrifugation at 10,000 g for 20 minutes, dissolved in phosphate buffer, and then, to prepare for storage, the obtained proteins were dialyzed against deionized water and lyophilized.

Determination of the amount of protein in samples with F1 antigen

Protein quantification was performed by the Lowry method. A solution of albumin 1 mg / ml was used as a standard.

Electrophoresis of samples with F1 antigen

Capsular protein electrophoresis was performed in 12.0% SDS-PAGE by the Laemmly method. The F1 protein was used as a standard (State Research Center for Applied Microbiology, Obolensk, Russia). Electrophoresis analysis confirmed that the purified F1 sample had the same gel mobility as the internal F1 standard. Preparative amounts of F1 antigen from Y. pestis were obtained.

Example 2. The accumulation and characterization of a murine monoclonal antibody to the F1 antigen from Y. pestis

Immunization

Eight-week-old Balb / c mice were immunized three times with an interval of 2 weeks with 50 μg of antigen. For the first immunization, a 1: 1 mixture of antigen and complete Freund's adjuvant in the form of a homogeneous emulsion was used. Antigen for the second immunization was prepared in a similar way, but only with incomplete Freund's adjuvant. Third, the final immunization was carried out using 50 μg of antigen in PBS buffer.

Getting a hybrid

Mice were terminated on the 4th day after the third immunization, blood was extracted from them and serum was obtained, which was used as a positive control. The spleen was homogenized, half of the obtained lymphocytes were frozen in liquid nitrogen using 10% DMSO, using the remaining lymphocytes, cells fused with SP2 / 0 myeloma were obtained in accordance with the standard method using 50% PEG 4000.

These cells were plated in 96-well plates containing DMEM growth medium (Gibco, cat.No. 52100-039) supplemented with L-glutamine, sodium pyruvate, gentamicin, HAT (100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine) and 8% fetal calf serum. First, a suspension of top dressing cells, perinatal macrophages obtained by washing the abdominal cavity of a healthy mouse, was introduced into the wells. Primary culture growth was determined visually using a microscope. After 10-12 days after hybridization, the supernatants from the wells were checked for the content of specific antibodies using ELISA.

Primary antibody culture wells were cloned twice using a limited dilution method in DMEM growth medium with the same additives, with the exception of HAT, onto a layer of top dressing cells. Supernatant testing was performed on day 10-12. If desired, positive clones were cloned 1-3 more times, and then their isotypes were determined using EIA.

Ascites

Selected clones were inoculated in mice in which Pristane was previously introduced. Ascites were isolated 7-10 days after inoculation with cells.

Purification of monoclonal antibodies against F1 antigen from Y. pestis

Ascites (20 ml) were diluted in MES buffer (150 mM sodium chloride, 50 mM 2- [N-morpholino] ethanesulfonic acid, pH 5.7) in a 1: 4 ratio and applied to a Protein G-column (Protein G-Sepharose 4B Fast Flow (Pharmacia, 17-0618-03)). The mouse IgG1 isotype yield was 3.8 mg / ml ascites. After purification, the purity of the antibodies was checked in SDS-PAGE and using immunoreactivity (indirect ELISA). Antibody purity was about 95%.

In accordance with the results of immunoblotting (Figure 1), F19 antibodies specifically stained one band in sample F1, a band with a high molecular weight corresponds to F1 dimer.

The cross-reactivity test with antigens F. tularensis, B. anthracis PA and SP, T. gondii, Salmonella O Ag, Legionella pn., VAg Y. pestis, SEB, E. coli was negative.

Immunoreactivity of purified antibodies

Antibody immunoreactivity was tested by indirect ELISA using F1 antigen from Y. pestis (F1 coating - 5 μg / ml in 0.1 M carbonate buffer, antibodies - 1 μg / ml, conjugate - mouse anti-IgG, substrate - 3.3 ', 5 , 5'-tetramethyl- [1,1'-biphenyl] -4,4'-diamine (TMB). Data were obtained on a Multiscan multichannel photometer at a wavelength of 450 nm. The results are shown in Figure 2. These monoclonal antibodies showed high specific activity and sensitivity up to 0.5 ng / ml.

Determination of the dissociation constant for antibodies against the F1 antigen from Y. pestis The dissociation constant was determined by the Klotz method (Klotz IM, The Proteins, Vol. 1, eds H. Neurath and K. Bailey, Academic Press, New York, 1953, p.727) in modified version of B. Friguet, AFChaffotte, L. Djavadi-Ohaniance and ME Goldberg (Journal of Immunological Methods, 77 (1985), 305-319).

To restore equilibrium in the antibody-antigen system, the plates that were previously blocked with 1% bovine serum albumin (BSA) were coated with 50 μl of F1 antigen solution in PBS-AT buffer at concentrations of 10 ^-8 and 10 ^-9 M, 3 rows for each, and titrated with serial dilutions of 1/2. Then all wells from these 6 rows were coated with 50 μl of antibodies at a selected concentration. Standard antibody solutions 1; 0.8; 0.5; 0.2; 0.1 was introduced into 3 rows of the same plate, where 1 corresponded to the concentration of antibodies to cover with a layer of titrated antigen, several wells were covered with 50 μl of antibody to determine Ao (optical density without antigen). All wells containing antibodies were also coated with 50 μl PBS-AT buffer (0.05 M phosphate buffer (pH 7.5) with 0.2% BSA and 0.1% Tween 20 added). Incubation was carried out for 2 hours at room temperature in a stirrer. After incubation, the contents of all wells were transferred to a dish pre-coated with a 5 μg / ml antigen layer in PBS buffer. The incubation was carried out for 1 hour, the plates were washed with PBS-T buffer (0.05 M phosphate buffer (pH 7.5) with the addition of 0.1% Tween 20) and covered with a layer of anti-mouse IgG conjugate. After 1 hour of incubation, the plate was washed and TMB was applied; after 20 minutes, the results were obtained by measuring with a Multiscan multichannel photometer at 450 nm. If antibody standards were linear, experiments were considered valid. Ao was calculated as the average of all wells in which antibodies were applied without antigen. The results were presented in figure 2. Dissociation constants were determined based on a calibration curve.

Thus, the Kd for monoclonal antibodies to the F1 antigen from Y. pestis is 4.5 × 10 ^-9 M.

Example 3. In vivo study of the neutralizing activity of murine monoclonal antibodies against F1 antigen from Y. pestis by the ability to protect animals from the virulent strain of Y. pestis F1 (+).

The ability of mouse antibodies against F1 antigen from Y. pestis to protect mice from the virulent strain of Y. pestis F1 (+) was studied in vivo. Antibodies were injected into mice intravenously before and after infection. Protectability of antibodies was evaluated by comparing LD ₅₀ (with antibodies) and LD ₅₀ (without antibodies). The parameter "average life expectancy" was evaluated in the case of a minimum difference in the values of LD ₅₀ .

M. musculus Balb / c mice (18-20 g, ten in each experimental group) were subcutaneously inoculated with Y. pestis 231 (F1 +) strain (State Research Center for Applied Microbiology, Obolensk, Russia) with a dose of 30 cells in 0.2 ml saline per mouse. Antibody treatment was performed 2 hours before infection. Antibodies in a dosage of 1 mg (in 0.2 ml of physiological saline) per mouse were injected into the abdominal region. The effectiveness of the treatment was evaluated by the survival time of the animals for 21 days and by the number of dead animals. The specificity of death was determined by isolating the units of colony formation (cfu) of Y. pestis from organs after preparation. The results are presented in the Table.

Table Antibodies for treatment Dead mouse / total number of mice Surviving mice,% Life expectancy, days Mab anti F1 C1 F19 2/10 80 fourteen Mab va53 10/10 0 9.2 Mab va68 10/10 0 9.2 Mab va67 10/10 0 6.0 Mab va49 8/10 twenty 6.5 Mab Va224 10/10 0 6.0 Mab va52 10/10 0 8.0 Mab va48 8/10 twenty 6.5 The control 10/10 0 4.6

Surviving animals on the 21st day after infection were divided into three groups who received immunotherapy with antibodies Mab anti F1 F1 F19, anti VAg C1 Va48 and Va49. In the first case, the therapeutic effect was high: 80% of the mice survived, in other cases, only 20%. It should be noted that bacteriological studies of the organs and blood of surviving mice for the presence of Y. pestis 231 culture yielded negative results. All dead animals contained a specific specific culture of the plague pathogen. The mortality curves of mice in both the experimental and control groups are shown in FIG. 4.

Thus, the test results showed that the Mab "anti F1 F1 F19" was highly effective in protecting mice against infection with a virulent strain of Y. pestis F1 (+).

Example 4. Cloning of genes encoding Fab fragments of mouse monoclonal antibodies against F1 antigen from Y. pestis, using mRNA from suitable cell lines as templates.

Total RNA was purified from 1 × 10 ⁷ cells of suitable hybridomas containing antibodies of the present invention Mab anti F1 F1 F19. Cells (10 ⁷ cells) were washed twice with a single PBS buffer. 400 μl of a GTB solution (4 M guanidine thiocinate, 30 mM sodium citrate, 1% N-lauryl sarcosyl sodium, 120 mM β-merthoethanol) was added to a test tube with 100 μl of cells. Cells were suspended by pipetting for 2-3 minutes and placed on ice. Then, 50 μl of 3 M sodium acetate (pH 5.2) and 500 μl of phenol saturated with water were added, and the resulting suspension was thoroughly mixed on a Vortex mixer. Then, 100 μl of a mixture of chloroform - isoamyl alcohol was added and the suspension was thoroughly mixed on a Vortex mixer for 1 min with 1 minute interruptions. The suspension was unscrewed for 10 minutes and the supernatant was transferred to a new tube. The procedure was repeated. An equal volume of isopropanol (500 μl) was added and stored at -20 ° C. The sample was untwisted for 10 minutes, the precipitate was washed with ethanol (70%) and dissolved in water (50 μl). A total of 7.5 μg RNA (0.15 μg / μl) was isolated, which was used for further cDNA synthesis.

cDNA was synthesized using the RevertAid ™ H Minus First Strand cDNA Synthesis (Fermentas) kit according to the “PCR-suitable cDNA synthesis protocol” recommended by the manufacturer.

Design primers for cloning a gene encoding a heavy chain.

In order to identify a pair of primers suitable for cloning a gene encoding a heavy chain, PCR was performed for cloning using Tag DNA polymerase (the reaction mixture contained 10X Tag for DNA polymerase Tag (67 mM Tris-HCl (pH 8.8 at 25 ° C), 166 mM (NH ₄ ) ₂ SO ₄ , 100 mM 2-mercaptoethanol) +20 mM MgCl ₂ , 1.25 mM dNTP, 5'-terminal primer (25 pmol), 3'-terminal primer (25 pmol), Taq DNA polymerase ( 2.5 U), cDNA 1 ml, bidistilled water up to 50 μl). Total cDNA was used as template. The PCR program was as follows: 95 ° C for 3 minutes; 35 cycles of 94 ° C for 1 minute, 45 ° C for 1 minute and 72 ° C for 1 minute; at the end of 72 ° C for 10 minutes.

The 5'-terminal primer corresponded to a set of 16 degenerative primers (SEQ ID NO: 5-20) recognizing 95% of the signal peptide sequences in the heavy chain encoding genes laid out in the Kabat database (Kabat, E., Wu, T., Perry, N. 1991. Sequences of proteins of lmmunological Interest, 5th edn. US Departament of Health and Human Services, Public Health Service, NIH). Primer A26 (SEQ ID NO: 21) was used as the 3'-terminal primer in PCR corresponding to the 3'-end of the heavy chain of the Fab fragment (IgG1) with the XhoI site inserted at the 5'-end. Using electrophoresis of PCR products, it was shown that the use of a single pair of primers (SEQ ID NO: 16 and 21) led to the appearance of a PCR fragment. The size of the obtained PCR product (≈700 base pairs) was in good agreement with the predicted cDNA sequence of the Mus musculus IgG heavy chain gene from the Gene-Bank and IMGT-GENE-DB databases.

Design primers for cloning a gene encoding a light chain.

It was previously shown that the antibody under study contains a kappa-type light chain. Based on these data, a set of 23 degenerative primers (SEQ ID NOS: 22-44) was used as the 5'-terminal primer. This kit recognizes 95% of the sequences corresponding to signal peptides in the kappa chain coding genes published in the Kabat database (Kabat, E., Wu, T., Perry, N. 1991. Sequences of proteins of Immunological Interest, 5th edn. US Departament of Health and Human Services, Public Health Service, NIH). A31 (SEQ ID NO: 45) was used as the 3'-terminal primer in PCR corresponding to the 3'-end of the heavy chain of the Fab fragment (IgG1) with the XhoI site inserted at the 5'-end. Using electrophoresis of PCR products, it was shown that using a single pair of primers (SEQ ID NOs: 39 and 45) led to the appearance of a PCR fragment. The size of the obtained PCR product (≈700 base pairs) was in good agreement with the predicted cDNA sequence of the Mus musculus IgG light chain gene from the Gene-Bank and IMGT-GENE-DB databases.

Cloning of genes encoding Fab fragments of antibodies against the F1 antigen from Y. pestis

Pfu DNA polymerase (Fermentas) was used to clone genes encoding antibody chains. PCR conditions were optimized by selecting annealing temperature and Mg ²⁺ concentration (the reaction mixture contained 10X buffer for Pfu DNA polymerase (200 mM Tris-HCl (pH 8.8 at 25 ° С), 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCl, 1% Triton X-100, 1 mg / ml BSA) +20 mM MgCl ₂ , 1.25 mM dNTP, 5'-terminal primer (25 pmol), 3'-terminal primer (25 pmol), Pfu DNA polymerase (1.25 U) , cDNA 1 μl, bidistilled water up to 50 μl). Each of the two independent PCR products was eluted from the agarose gel and phosphorylated using T4 polynucleotide kinase DNA. The program for PCR was as follows: 95 ° C for 3 minutes; 35 cycles of 94 ° C for 1 minute, 45 ° C for 1 minute and 72 ° C for 2 minutes; at the end of 72 ° C for 10 minutes. The resulting fragments were cloned into the plasmid vector pUC18 linearized with SmaI restrictase. Recombinant plasmids containing the Fab heavy and light chain genes of the indicated antibody were isolated and named pUC18-F1-H and pUC18-F1-L, respectively.

Example 5. Sequencing of gene fragments encoding the variable parts of the Fab fragment of a murine monoclonal antibody against F1 antigen from Y. pestis.

Recombinant plasmid pUC18-F1-H containing an independent PCR fragment encoding an anti-F1 antigen heavy chain from Y. pestis, and plasmid pUC18-F1-L containing an independent PCR fragment encoding an antibody light chain was isolated and PCR fragments were sequenced into both directions. Used standard sequencing primers SEQ ID NO: 46 and 47. According to the results of WAMPredict (https://antibody.bath.ac.uk/WAMpredict.html), the canonical classes of hypervariable loops are as follows: CDR-L1 - 1, CDR- L2 - 1, CDR-L3 - 1, CDR-H1 - 1, CDR-H2 - 2, CDR-H3, Length: 6, Bend status: Curved.

Example 6. Construction of an expression system for the production of a Fab fragment of a mouse monoclonal antibody against the F1 antigen from Y. pestis and obtaining the specified antibodies in yeast.

Construction of expression cassettes.

Genes encoding Fab chains of a mouse anti-F1 antigen F1 fragment from Y. pestis were amplified by PCR to introduce them into expression cassettes. Plasmids pUC18-F1-L and pUC18-F1-H were used as matrices.

To amplify the DNA fragment encoding the variable domain of the light chain, we used the 5'-primer Lf (SEQ ID NO: 48) and the 3'-primer Lr (SEQ ID NO: 50. The 5'-primer Lf contains a restriction site at the 5'-end XhoI, necessary for further attachment to the α-factor signal sequence, the Lr 3′-primer contains the XbaI restriction site at the 5 ′ end.The XhoI DNA fragment — Light chain XbaI was ligated into the modified plasmid pPICZαA (Invitrogen), in which the PmeI restriction site was destroyed by site directed mutagenesis.

For amplification of the DNA fragment encoding the variable domain of the heavy chain, 5'-primer Hf (SEQ ID NO: 51) and 3'-primer Hr (SEQ ID NO: 53) were used. The 5'-primer Hf contains at the 5'-end the XhoI restriction site necessary for further attachment to the signal sequence of the α-factor, the 3'-primer Hr contains the XbaI restriction site at the 5'end. The XhoI DNA fragment - heavy chain XbaI was ligated into the original version of the plasmid pPICZαA.

Gene fusion

Combining the genes of both chains of the Fab fragment on one plasmid was carried out by double cleavage of the light chain vector with BglII and BamHI restriction enzymes, followed by the introduction of a heavy chain vector into the unique BamHI site.

Thus, the expression vector pPICZαA-F1-mouse-HL was constructed containing sequences encoding both strands of the Fab fragment of the mouse anti-F1 F1 antigen from Y. pestis, each of which is directly connected in the same reading frame to the α-signal signal sequence from Saccharomyces cerevisiae under the control of the AOX1 promoter (Fig. 8).

Obtaining yeast transformants and analysis of Fab products.

The expression plasmid pPICZαA-F1-mouse-H-L, linearized with the restriction enzyme PmeI, was introduced into GS115 cells according to the INVITROGEN protocol using a technique using L1C1. The resulting transformants were designated as GS115 / AOX-F1-mouse-H-L.

12 independent transformants of the GS115 / AOX-F1-mouse-H-L clones were grown in test tubes (V = 4 ml) for 45 hours at 30 ° C and pH 6.0 using BMMY growth medium (Invitrogen) (final optical density OD 40). The number of Fab fragments in the culture fluid was measured by ELISA. In accordance with the results of this method, strains producing the Fab fragment of the mouse anti-F1 antigen from Y. pestis were selected. Selected clones of producer strains GS115 / AOX-F1-mouse-H-L were grown in flasks (V = 100 ml) for 45 hours at 30 ° C and pH 6.0 on YPM medium.

The culture fluids of the producer strains were analyzed using Western blotting. The results are presented in Fig.9. From these data, the specialist can conclude that some producer strains GS115 / AOX-F1-mouse-H-L secrete proteins into the culture fluid that can be detected by interaction with anti-mouse antibodies using Western blotting. These proteins are believed to be separate strands of the Fab fragment of the mouse anti-F1 antigen from Y. pestis.

ELISA method for analysis of antibody fragments in the culture fluid:

- Sorption of protein culture fluid 100 μl per well for 2 hours at room temperature;

- Washing the wells three times with PBS;

- Block 3% BSA in PBS (200 μl per well) for 2 hours at room temperature;

- Washing the wells three times with PBS buffer with 0.05% Tween 20;

- Incubation with goat anti-mouse antibodies;

- Rinse 5 times with PBS buffer with 0.05% Tween 20;

- Addition of o-phenylenediamine 100 μl (4 mg / 10 ml) with H ₂ O ₂ in pH 5.0 buffer;

- Stop the reaction by adding 100 μl of 10% H ₂ SO ₄ .

Example 7. Characterization of the affinity of Fab fragments of murine monoclonal antibodies against the F1 antigen from Y. pestis produced by yeast.

Production and characterization of a natural mouse Fab fragment from MoAb F19. Proteolysis of MoAb F19 was performed with pepsin at a ratio of 1: 100 to total IgG in the presence of 10 mM cysteine for 20 hours at 37 ° C, which would result in a Fab fragment. As follows from the electrophoresis data, the obtained preparation contained a certain amount of other hydrolysis products, which made it difficult to determine the amount of Fab fragment, therefore, the purification was successfully carried out by gel filtration on a column with a Sephadex G50. Analysis of 4 obtained fractions is shown in Fig.10. All 4 fractions contained both Fab fragments and Fab 'fragments. Fab fragments prevailing in the fourth fraction were selected for subsequent experiments.

Development of an ELISA method for estimating the amount of a recombinant Fab fragment.

To develop an ELISA method for detecting a recombinant Fab fragment, an attempt was made to use the anti-His ₆ tail conjugate, which was unsuccessful due to the incomplete accessibility of all 6 histidines for binding. A horseradish peroxidase and MoAb conjugate was prepared against murine Ig Kappa light chains. For this, 0.5 ml of MoAb ascites against mouse Kappa-light chains, clone EM-34.1 (Sigma, cat. No K-2132), was purified by three-fold precipitation in sodium sulfate and conjugated with horseradish peroxidase. Natural murine Fab MoAb F19 fragments were used as standard. The results are shown in FIG. 11.

Determination of the dissociation constants of recombinant and natural Fab fragments.

Dissociation constants (Kd) were determined during the two-step ELISA method, as described in Example 2. The results are presented in Fig.12.

Thus, the Kd for the natural monoclonal Fab fragment of the naturally occurring murine antibody MoAb F19 against the F1 antigen from Y. pestis is

Kd for the recombinant Fab fragment against the F1 antigen from Y. pestis is

Thus, a recombinant Fab fragment was obtained that has high specificity against the F1 antigen from Y. pestis and a high dissociation constant.

Although the present invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various substitutions can be made and equivalents applied that are not outside the scope of the present invention. All documents cited here are part of this application, incorporated by reference.

Claims (9)

1. An isolated mouse antibody that selectively binds to the F1 antigen from Yersinia pestis, characterized in that the variable domain of the heavy chain (VH) of the indicated antibody includes the amino acid sequence of SEQ ID NO: 1, and the variable domain of the light chain (VL) of the indicated antibody includes the amino acid sequence of SEQ ID NO: 2. 2. An isolated DNA fragment encoding the antibody of claim 1. 3. Antigen-binding fragment (Fab), selectively binding to the F1 antigen from Yersinia pestis, which includes the variable domain of the heavy chain (VH) with the amino acid sequence of SEQ ID NO: 1 and the variable domain of the light chain (VL) with the amino acid sequence of SEQ ID NO : 2. 4. An isolated DNA fragment encoding an antigen binding fragment (Fab) according to claim 3. 5. A yeast cell transformed with a DNA fragment according to claim 4 and having the ability to produce Fab according to claim 3. 6. The yeast cell according to claim 5, characterized in that said yeast is Pichia pastoris yeast. 7. The method of producing Fab according to claim 3, comprising growing the yeast cells according to claim 5 in a nutrient medium and isolating said Fab from the culture fluid. 8. A method for detecting Yersinia pestis, comprising: (a) obtaining a sample from an animal or human; (b) the interaction of the specified sample with the antibody according to claim 1 or Fab according to claim 3; (c) detecting a complex formed by said antibody or Fab and antigen F1; and, optionally, (d) determining the amount of F1 antigen present in said sample based on the detected amount of the complex formed by the antibody and the F1 antigen, or the complex formed by the Fab and the F1 antigen. 9. The kit for the detection of F1 antigen from Yersinia pestis, containing the isolated monoclonal antibodies according to claim 1 or Fab according to claim 3, which selectively bind to the F1 antigen from Yersinia pestis, and means for detecting the specified F1 antigen using the specified antibody or Fab .

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