KR102196550B1 - Monoclonal Antibodies Specifically Binding to Glycoprotein 51 of Bovine Leukemia Virus and Its Use - Google Patents

Abstract

The present invention provides a monoclonal antibody against the glycoprotein 51 of the Sorrhosis virus, and a hybridoma cell line producing the antibody. The monoclonal antibody against the glycoprotein 51 of the Sorucocosis virus of the present invention has a high affinity specific binding ability for the glycoprotein 51 of the Sorheukosis virus, and thus can be usefully used for diagnosing Sor. The monoclonal antibody against the soleukosis virus of the present invention is also very useful in the manufacture of a kit for diagnosis of soleukosis.

Description

Monoclonal Antibodies Specifically Binding to Glycoprotein 51 of Bovine Leukemia Virus and Its Use}

The present invention relates to a monoclonal antibody that specifically binds to Sor. In more detail, a monoclonal antibody against Sorococcus virus glycoprotein 51, a hybridoma cell line that produces the antibody, a composition for diagnosis of Sorucosis and a kit for diagnosis of Sorucosis comprising the monoclonal antibody as an active ingredient About.

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Soleukemia is a type 2 legal livestock infectious disease in cattle that is infected by the bovine leukemia virus (BLV). It can be infected at all ages, including the fetus, and most of them are unexplained infections or some (~30%) PL ( persistent lymphocytosis), lymphosarcoma in various internal organs, and clinical symptoms depending on the affected organ. Cows with lymphosarcoma die immediately or after several weeks to several months, and once infected, they exist in the blood of cattle for life and are difficult to eradicate.

BLV is a virus of the genus Deltaretrovirus belonging to the Retroviridae family in viral taxonomics. BLV consists of a single-stranded RNA genome, several enzymes and structural proteins, including reverse transcriptase. Structural proteins constituting BLV include internal proteins (p24, p15, p12, p10) and envelope glycoproteins (gp51, gp30). Among these, the gp51 protein, one of the envelope glycoproteins, recognizes cellular viral receptors and plays a direct role in viral infection, and induces a strong immune response. Therefore, BLV gp51 protein is regarded as an important antigenic site of antibody testing for disease diagnosis or monitoring, and it is important to secure monoclonal antibodies that recognize epitopes in BLV gp51 protein for antibody testing to achieve this purpose.

To this end, a hybridoma cell line producing a monoclonal antibody against the BLV gp51 protein was constructed, and the monoclonal antibody produced by the hybridoma cell line specifically recognized the epitope region of the BLV gp51 protein, and was subjected to blocking ELISA. When applied, it was confirmed that BLV antibodies can be effectively detected in outdoor infected individuals.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

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The present inventors made intensive research efforts to develop a monoclonal antibody capable of specifically detecting a bovine leukemia virus in a biological sample. As a result, the production of monoclonal antibodies that specifically bind to glycoprotein 51 by overexpressing and purifying a recombinant protein against glycoprotein 51 of Sor. The present invention was completed by successfully preparing a bridoma cell line.

Accordingly, an object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to the recombinant protein of Sor.

Another object of the present invention is to provide a monoclonal antibody against Sor.

Another object of the present invention is to provide a hybridoma cell that produces a monoclonal antibody against Sor.

Another object of the present invention is to provide a composition for diagnosing Soleukosis comprising the monoclonal antibody as an active ingredient.

Another object of the present invention is to provide a diagnostic kit for Soleukosis comprising the monoclonal antibody as an active ingredient.

Another object of the present invention is to provide a method of detecting an antibody against a soleukosis virus using the monoclonal antibody.

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Other objects and advantages of the present invention will become more apparent by the following detailed description and claims.

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According to one aspect of the present invention, the present invention is an antibody that specifically binds to a recombinant protein of bovine leukemia virus glycoprotein 51 represented by the amino acid sequence of SEQ ID NO: 3 or its Antigen binding fragments are provided.

According to another aspect of the present invention, the present invention provides a monoclonal antibody against Soryochosis virus glycoprotein 51 produced by hybridoma cells with accession numbers KCTC 18355P and KCTC 18356P.

According to another aspect of the present invention, the present invention provides hybridoma cells with accession numbers KCTC 18355P and KCTC 18356P, which produce monoclonal antibodies against Sor.

The monoclonal antibody of the present invention specifically binds to bovine leukemia virus glycoprotein 51 while having high affinity.

According to a preferred embodiment of the present invention, the monoclonal antibody of the present invention specifically binds to the 51 site of the Soleucosis virus glycoprotein of SEQ ID NO: 4.

The monoclonal antibody of the present invention is an antibody produced by a hybridoma cell line deposited with the accession numbers KCTC 18355P and KCTC 18356P on February 17, 2015 with the Korea Research Institute of Bioscience and Biotechnology KCTC (Korean Collection for Type Cultures).

In the present specification, the term "antibody" refers to a specific antibody against Soreocyssis virus glycoprotein 51, and is meant to include not only the complete antibody form but also the antigen-binding fragment of the antibody molecule.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma4(γ4), alpha1(α1) and alpha2(α2). The constant region of the light chain has kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, MA (1984)).

The antigen-binding fragment of an antibody molecule refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, and the like. Among the antibody fragments, Fab has a structure having a light chain and a heavy chain variable region, a light chain constant region, and a heavy chain first constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are generated by disulfide bonds between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and the recombinant technology for generating the Fv fragment is PCT International Patent Application WO88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. The double-chain Fv (twochain Fv) is a non-covalent bond where the heavy chain variable region and the light chain variable region are connected, and the single-chain Fv (single-chain Fv) is generally covalently bonded to the variable region of the heavy chain and the variable region of the single chain through a peptide linker. It may be linked or linked directly at the C-terminus to form a dimer-like structure such as a double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the whole antibody with papain yields Fab, and digestion with pepsin yields F(ab')2 fragments), preferably It can be produced through genetic recombination technology.

In the present invention, the antibody is preferably in the form of Fab or in the form of a complete antibody.

In the present specification, the term “heavy chain” refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3, and its It means all short stories.

In addition, in the present specification, the term "light chain" refers to both a full-length light chain including a variable region domain VL and a constant region domain CL including an amino acid sequence having a sufficient variable region sequence to impart specificity to an antigen, and a fragment thereof.

As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.

The monoclonal antibody of the present invention can be obtained from hybridoma cells produced by a cell fusion method known in the art. In general, hybridoma cells that secrete monoclonal antibodies are produced by fusing immune cells from an immunologically suitable host animal such as a mouse injected with an antigenic protein and a cancer cell line. The fusion of these two cells is fused through a method using polyethyleneglycol known in the art, and the antibody-producing cells are proliferated by a standard culture method. After subcloning by limited dilution is performed to obtain a uniform cell population, hybridoma cells capable of producing antigen-specific antibodies are cultured in large quantities in vitro or in vivo.

Myeloma cells used for cell fusion include various cell lines such as mouse-derived p3/x63-Ag8, p3-U1, NS-1, MPC-11, SP2/0, F0, P3x63 Ag8, V653, S194, rat-derived R210, etc. You can use The cell line used in a specific example of the present invention is myeloma cell SP2/0.

The monoclonal antibody produced by the hybridoma cells may be used without purification, and various conventional methods, such as dialysis, salt precipitation, ion exchange chromatography, size exclusion chromatography, and affinity chromatography. It can be used after being purified with high purity using the same.

Various methods commonly used to select monoclonals that selectively recognize Sor. glycoprotein 51, for example, radioimmunoassay (RIA), enzyme immunosorbent method (ELISA), immunofluorescence method (Immunofluorescence), western black Ratting (Western blotting) and flow cytometry may be used, but are not limited thereto. According to a specific embodiment of the present invention, monoclonals are selected by enzyme immunosorbent method (ELISA).

According to another aspect of the present invention, the present invention provides a composition for diagnosing soleukosis comprising the antibody of the present invention against the above-described soleukosis virus.

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The monoclonal antibody that specifically binds to the glycoprotein 51 of the soleukosis virus of the present invention can be applied to a biological sample and used to diagnose soleukosis.

The term “biological sample” as used herein includes tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells and bacterial expression systems, etc. Can, but is not limited thereto. By detecting the formation of an antigen-antibody complex using the antibody of the present invention with or without the manipulation of these biological samples, it is possible to confirm the presence of S.
The formation of the above-described antigen-antibody complex is a colormetric method, an electrochemical method, a fluorescence method, a luminometry, a particle counting method, a visual assessment, or It can be detected by scintillation counting method.

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"Detection" in the present specification is for detecting the antigen-antibody complex, and can be performed using various markers. Specific examples of the label include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, or radioactive isotopes.
Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase, and fluorescent substances include fluorescein. Phosphorus, Eu ³⁺ , Eu ³⁺ chelate or cryptate, etc., as a ligand, a biotin derivative, etc., as a luminescent material, an acridinium ester and an isoluminol derivative, etc., and as a microparticle, a colloid It includes gold and colored latex, and the like, and radioactive isotopes include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bonton Hunter's reagent.

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Preferably, the antigen-antibody complex can be detected using an enzyme immunosorbent assay (ELISA). In the enzyme immunosorbent method (ELISA), a blocking ELISA, labeled antibody and biological sample using the principle that antibodies against various disease agents present in a biological sample react with an antigen attached to a solid support to interfere with the reaction of the labeled antibody with the antigen. Competitive ELISA using the principle that antibodies against disease agents present in the body compete at the same time to react with antigens attached to a solid support, direct ELISA using a labeled antibody that recognizes antigens attached to a solid support, and attached to a solid support. Indirect ELISA using a labeled secondary antibody that recognizes the capture antibody in a complex of antigen-recognizing antibodies, direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of an antibody attached to a solid support and an antigen, a solid support It includes various ELISA methods such as indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antibody attached to the antigen and the antigen. The antibody of the present invention may have a detection label, and if it does not have a detection label, the antibody of the present invention can be captured and confirmed by treatment with another antibody having a detection label.

According to another aspect of the present invention, the present invention provides a kit for diagnosing soleukosis comprising the antibody of the present invention against the above-described soleukosis virus.

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The antibody of the present invention, which uses the glycoprotein 51 of the Sorheococcus virus as an antigen, can be applied to a biological sample to diagnose whether it is infected with the Sor.

The diagnostic kit for Soleukosis of the present invention may include a substrate, an appropriate buffer solution, an antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like for immunological detection of an antibody. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a slide glass made of glass, and the color developing enzyme is peroxidase, alkaline Phosphatase (alkaline phosphatase) can be used, fluorescent materials FITC, RITC, etc. can be used, and the color developing substrate solution is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) )) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

According to another aspect of the present invention, in order to provide information on the diagnosis of Sorucocosis, the present invention uses a monoclonal antibody against Sorucosis virus of the present invention to treat Sorucocosis virus in a biological sample isolated from cattle. It provides a method of detecting an antibody against.

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The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a monoclonal antibody against the glycoprotein 51 of the Sorheukosis virus, and a hybridoma cell line producing the antibody.

(Ii) The monoclonal antibody against the glycoprotein 51 of the Sorucosis virus of the present invention has a high affinity specific binding ability for the glycoprotein 51 of the Sorucosis virus, so it can be usefully used in the diagnosis of Sorryucosis. have.

(Iii) The monoclonal antibody against the soleukosis virus of the present invention is also very useful in the manufacture of a kit for diagnosis of soleukosis.

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1 is a schematic diagram of BLV gp51 recombinant protein production.
2 is a result of confirming the expression and purification of the BLV gp51 recombinant protein. (a) BLV gp51 P1, (b) BLV gp51 P2, (c) BLV gp51 P3, (d) BLV gp51 P4
3 is a graph showing the reactivity of BLV gp51 P3 recombinant protein with monoclonal antibodies and four types of BLV gp51 recombinant protein.
Figure 4 is a result of confirming the blocking ability of the BLV gp51 P3 recombinant protein monoclonal antibodies and BLV gp51 recombinant protein. (a) BLV gp51 P1, (b) BLV gp51 P2, (c) BLV gp51 P3, (d) BLV gp51 P4
5A is a result of confirming the blocking ability of BLV gp51 P1, P2, P4 recombinant proteins with monoclonal antibodies and BLV virus.
5B is a result of confirming the blocking ability of BLV gp51 P3 recombinant protein with monoclonal antibodies and BLV virus.
6 is a photograph of the result of Western blot for monoclonal antibody 9D72.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example One: BLV gp51 Recombinant protein Antigen development

As a BLV gp51 recombinant protein, a total of four proteins named BLV gp51 P1, P2, P3, and P4 were produced.

1-1: BLV gp51 recombinant protein

The BLV gp51 P1 and P2 recombinant proteins are regions containing the BLV gp51 and gp30 genes, and the BLV gp51 P3 and P4 recombinant proteins are E. coli recombinant proteins produced by PCR cloning the BLV gp51 gene region into the pET21a vector (Fig. BLV gp51 P1, P2, P3 and P4 recombinant proteins were expressed in large amounts as insoluble proteins, and were purified by denaturation. As a result, proteins of 57.2 KDa, 43.5 KDa, 27.5 KDa and 17.5 KDa were obtained, respectively.

Genetic information of BLV gp51 recombinant protein Gene Origin Recombinant protein Gene Region Primers Size
(kDa) Fusion
protein BLV gp51 P1 1446 bp
(gp51+gp30) Forward:
5'-CGCGAATTCTGGAGATGCTCCCTGTCCCTAGGAAA-3`
Reverse:
5'-CCCGCGGCCGCCCAGGTTAGA GTGAAAATTCC-3` 57.2 C-terminal
6X histidine BLV gp51 P2 1089 bp
(gp51+gp30) Forward:
5'-CGCGAATTCTGGAGATGCTCCCTGTCCCTAGGAAA-3`
Reverse:
5'-CGCCTCGAGAATGCGCAGGAAACAGCAAGGCTCATTA -3` 43.5 C-terminal
6X histidine BLV gp51 P3 648 bp
(gp51) Forward:
5'-CGCGAATTCTGGAGATGCTCCCTGTCCCTAGGAAA-3`
Reverse:
5'-CGCCTCGAGCCAGAAGATTTGGGCGTCC-3` 27.5 C-terminal
6X histidine BLV gp51 P4 381 bp
(gp51) Forward:
5'-CGCGAATTCTGGAGATGCTCCCTGTCCCTAGGAAA-3`
Reverse:
5'-CGCCTCGAGCCCAGAAGATTTGGGCGTCC-3` 17.5 C-terminal
6X histidine

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Step 1: gene amplification

For gene amplification, PCR was performed using the primer pairs shown in Table 1. PCR conditions are as follows:

Denaturation: 94°C for 3 minutes, 3-step cycling (35 cycles): 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, last extension: 72°C for 10 minutes.

The PCR product was cloned into the pET21a vector, and then colonies were selected and sequenced to confirm the nucleotide sequence.

Step 2: Induction of expression of recombinant protein

The cloned vector was introduced into E. coli and cultured to induce expression. SDS-PAGE was performed to confirm the size and protein expression characteristics of the expressed protein (lysate pellet, insoluble protein). The expressed protein was purified using 6XHis Tag, and the degree of purification and amount were measured by performing SDS-PAGE (FIGS. 2a-2d).

Example 2: BLV gp51 Antibody development

Recombinant proteins (BLV gp51 P1, P2, P3, P4) were developed to develop antibodies that react specifically to BLV gp51, and mouse immunization was performed to produce monoclonal antibodies using the developed recombinant proteins.

2-1: monoclonal antibody against recombinant protein

Step 1: Immunity

In order to produce a monoclonal antibody against the recombinant protein prepared above, immunization was performed on the experimental animals. As antigens, each recombinant protein (BLV gp51 P1, P2, P3, P4) was prepared at a concentration of 0.5 mg/ml, and 200 μl of the recombinant protein and 200 μl of Freund's Adjuvant (FA) were mixed in the abdominal cavity of Balb/C mice every two weeks. 1-3 immunizations were performed. For the fusion immunity test, one week after the third immunization, an indirect ELISA test was performed using mouse serum. For final boosting, 100 μl of the recombinant protein was immunized intraperitoneally and used for cell fusion after 4 days.

Step 2: cell fusion and Hybridoma Produce

The spleen located on the left side of the body of the immunized mouse is excised and suspended in DMEM medium. The extracted spleen was ground with a mesh to separate cells and mixed with culture medium DMEM to prepare a splenocyte suspension. The suspension was centrifuged to remove the supernatant, and the splenocytes and bone marrow cell line (SP2/0 Ag14 (ATCC, USA)) were mixed at a ratio of 1:5, followed by centrifugation to precipitate the cells to remove the supernatant. After slowly dispersing the centrifuged cells, 1 ml of polyethylene glycol 1500 (PEG1500, Roche) was treated, and maintained at 37°C for 1 minute, and 1 ml of DMEM was added. Thereafter, 10 ml of DMEM was added for 1 minute, reacted in water at 37° C. for 5 minutes, adjusted to 50 ml, and centrifuged again. The cell sediment was resuspended in a separation medium (HAT medium) at a concentration of 1 to 2×10 ⁵ cells/ml, dispensed into a 96-well plate by 0.1 ml, and cultured in a carbon dioxide incubator at 37°C.

Step 3: Selection of hybridoma cells

An ELISA assay was used to select hybridoma cells that specifically react to BLV gp51 P1, P2, P3, and P4 from the hybridoma cell group prepared in step 2 above.

To a 96-well microplate, 100 μl (0.5 μg/ml) of target antigen BLV gp51 P1, P2, P3, P4 and control antigen normal cell lysate were added to each well and adhered to the plate surface. Was removed by washing. BLV virus culture supernatant and culture medium containing only 10% FBS were added to the plate surface by adding 100 μl to each well, and unreacted antigens were washed away. 100 μl of the hybridoma cell culture solution was added to each well and reacted at room temperature for 1 hour, followed by washing three times with a phosphate buffer solution-Tween 20 (PBS-T) solution to remove the unreacted culture solution. Goat anti-mouse IgG-HRP was added thereto, reacted at room temperature for 1 hour, and then washed three times with PBS-T. After washing, a substrate solution (TMB) of peroxidase was added to react, and 0.5 M of sulfuric acid was dispensed into each well of the plate by 50 μl, and the absorbance was measured at 450 nm using a Sunrise ELISA reader (Tecan). Hybridoma cell lines that secrete antibodies that are highly reactive to the antigen and not reactive to the control antigen were selected. The selected hybridoma cell line was subjected to limited dilution and cloning to be monocloned, and then hybridoma cell lines secreting the target antibody were selected in the same manner as above.

As described above, 10 monoclonal antibodies to BLV gp51 P1 recombinant protein, 22 monoclonal antibodies to BLV gp51 P2 recombinant protein, 18 monoclonal antibodies to BLV gp51 P3 recombinant protein, and 11 monoclonal antibodies to BLV gp51 P4 recombinant protein Antibodies were selected. The results of the selected antibodies are shown in Table 2-5.

Example 3: BLV gp51 monoclonal antibody selection having blocking ability

3-1: Confirmation of blocking ability with 4 kinds of BLV gp51 recombinant protein

The reactivity and blocking ability of the monoclonal antibody selected by the above method with 4 kinds of BLV gp51 recombinant proteins were confirmed using indirect ELISA and blocking ELISA.

Indirect ELISA

Step 1: reaction of mAb with antigen

mAb was diluted 1/10 in PBS buffer, and then 100 μl was dispensed onto the plate coated with BLV gp51 recombinant protein and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 2: reaction with secondary Ab

The secondary antibody, Peroxidase labeled anti-mouse IgG (H+L) antibody (KPL), was diluted to 50 ng, and then 100 μl was dispensed onto the plate and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 3: Substrate reaction

Substrate (TMB; Surmodics) was dispensed into the plate by 100 μl, and reacted for 10 minutes at room temperature.

Step 4: Stop and read the substrate reaction

After dispensing 50 μl of 0.5 M sulfuric acid into the plate, the results were analyzed after reading at 450 nm using a Sunrise ELISA reader (Tecan).

Blocking ELISA

Step 1: Serum and antigen reaction

Serum was diluted 1/2 in 0.1% PBST buffer, and then 100 μl was dispensed into a plate coated with BLV gp51 recombinant protein, and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 2: reaction of mAb with antigen

The mAb was diluted 1/10 in PBS buffer, and then 100 μl was dispensed onto the plate and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 3: reaction with secondary Ab

The secondary antibody, Peroxidase labeled anti-mouse IgG (H+L) antibody (KPL), was diluted to 50 ng, and then 100 μl was dispensed onto the plate and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 4: Substrate reaction

Substrate (TMB; Surmodics) was dispensed into the plate by 100 μl, and reacted for 10 minutes at room temperature.

Step 5: Stop and read the substrate reaction

After dispensing 50 μl of 0.5 M sulfuric acid into the plate, the results were analyzed after reading at 450 nm using a Sunrise ELISA reader (Tecan).

As a result of the experiment, all of the monoclonal antibodies made from the four BLV gp51 recombinant proteins were all reactive with the recombinant proteins of P1, P2, P3, and P4, respectively, but had no blocking ability (the results of BLV gp51 P1, P2, P4 monoclonal antibodies were omitted). Among the experimental results, the results of 9 monoclonal antibodies made from BLV gp51 P3 recombinant protein are shown in Figs. 4a-4d.

3-2: Confirmation of blocking ability with BLV virus

The blocking ability between the selected monoclonal antibody and BLV virus was confirmed using a blocking ELISA method.

Blocking ELISA

Step 1: Serum and antigen reaction

Serum was diluted 1/2 in 0.1% PBST buffer, and then 100 μl was dispensed into the plate coated with BLV virus, and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 2: reaction of mAb with antigen

The mAb was diluted 1/10 in PBS buffer, and then 100 μl was dispensed onto the plate and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 3: reaction with secondary Ab

The secondary antibody, Peroxidase labeled anti-mouse IgG (H+L) antibody (KPL), was diluted to 50 ng, and then 100 μl was dispensed onto the plate and reacted at 37° C. for 1 hour. After the reaction, it was washed 3 times with 0.05% PBST buffer.

Step 4: Substrate reaction

Substrate (TMB; Surmodics) was dispensed into the plate by 100 μl, and reacted for 10 minutes at room temperature.

Step 5: Stop and read the substrate reaction

After dispensing 50 μl of 0.5 M sulfuric acid into the plate, the results were analyzed after reading at 450 nm using a Sunrise ELISA reader (Tecan).

As a result of the experiment, among the four monoclonal antibodies made from BLV gp51 recombinant proteins, among the monoclonal antibodies made from BLV gp51 P1, P2, P4 recombinant proteins, there were no antibodies with blocking ability, and all antibodies with blocking ability were BLV gp51. It was found in antibodies made from P3 recombinant protein. Among the monoclonal antibodies made from BLV gp51 P3 recombinant protein, only 8A8, 10B43, 9D72, 2E42, 8F76, 12F12, and 10H37 antibodies (total of 7) showed excellent blocking ability. Among the experimental results, the results of some of the monoclonal antibodies made from BLV gp51 P1, P2, P4 recombinant protein and the monoclonal antibodies made from BLV gp51 P3 recombinant protein are shown in FIGS. 5A and 5B (BLV gp51 P1, P2, P4 monoclonal antibodies. Omit some results).

Example 4: Determination of the region and type of monoclonal antibody epitope recognition made of BLV gp51 P3 recombinant protein

Among the monoclonal antibodies made from BLV gp51 P3 recombinant protein, 8A8, 10B43, 9D72, 2E42, 8F76, 12F12, and 10H37 antibodies (7 in total) were found in BLV gp51 P1, P2, P3, and P4 recombinant proteins as shown in the above ELISA results. Everyone reacts. Therefore, they appear to be antibodies that recognize a specific epitope site in the BLV gp51 P4 site, which is a common part of the BLV gp51 P1, P2, P3, and P4 sites.

In addition, western blotting was performed using BLV gp51 P4 recombinant protein to determine the form of amino acid sequence regions recognized by these monoclonal antibodies (conformational type or sequential type). Normal cell lysate was used as a negative control. As a result of the experiment, the BLV gp51 P4 recombinant protein and 7 monoclonal antibodies with blocking ability were all confirmed in Western blot. Therefore, all seven monoclonal antibodies appear to be sequential type antibodies. A photograph of the Western blot result for the monoclonal antibody 9D72 is shown in FIG. 6 (the result photograph excluding the 9D72 clone is omitted).

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As the specific parts of the present invention have been described in detail above, it is clear that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

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Korea Research Institute of Bioscience and Biotechnology KCTC18355BP 20150213 Korea Research Institute of Bioscience and Biotechnology KCTC18356BP 20150213

<110> MEDIAN Diagnostics Inc. <120> Monoclonal Antibodies Specifically Binding to Glycoprotein 51 of Bovine Leukemia Virus and Its Use <130> PN150029 <160> 16 <170> KopatentIn 2.0 <210> 1 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> BLV gp51 P1 A/a <400> 1 Trp Arg Cys Ser Leu Ser Leu Gly Asn Gln Gln Trp Met Thr Ala Tyr 1 5 10 15 Asn Gln Glu Ala Lys Phe Ser Ile Ser Ile Asp Gln Ile Leu Glu Ala 20 25 30 His Asn Gln Ser Pro Phe Cys Ala Lys Ser Pro Arg Tyr Thr Leu Asp 35 40 45 Ser Val Asn Gly Tyr Pro Lys Ile Tyr Trp Pro Pro Pro Gln Gly Arg 50 55 60 Arg Arg Phe Gly Ala Arg Ala Met Val Thr Tyr Asp Cys Glu Pro Arg 65 70 75 80 Cys Pro Tyr Val Gly Ala Asp Arg Phe Asp Cys Pro His Trp Asp Asn 85 90 95 Ala Ser Gln Ala Asp Gln Gly Ser Phe Tyr Val Asn His Gln Ile Leu 100 105 110 Phe Leu His Leu Lys Gln Cys His Gly Ile Phe Thr Leu Thr Trp Glu 115 120 125 Ile Trp Gly Tyr Asp Pro Leu Ile Thr Phe Ser Leu His Lys Ile Pro 130 135 140 Asp Pro Pro Gln Pro Asp Phe Pro Gln Leu Asn Ser Asp Trp Val Pro 145 150 155 160 Ser Val Arg Ser Trp Ala Leu Leu Leu Asn Gln Thr Ala Arg Ala Phe 165 170 175 Pro Asp Cys Ala Ile Cys Trp Glu Pro Ser Pro Pro Trp Ala Pro Glu 180 185 190 Ile Leu Val Tyr Asn Lys Thr Ile Ser Ser Ser Gly Pro Gly Leu Ala 195 200 205 Leu Pro Asp Ala Gln Ile Phe Trp Val Asn Thr Ser Ser Phe Asn Thr 210 215 220 Thr Gln Gly Trp His His Pro Ser Gln Arg Leu Leu Phe Asn Val Ser 225 230 235 240 Gln Gly Asn Ala Leu Leu Leu Pro Pro Ile Ser Leu Val Asn Leu Ser 245 250 255 Thr Ala Ser Ser Ala Pro Pro Thr Arg Val Arg Arg Ser Pro Val Ala 260 265 270 Ala Leu Thr Leu Gly Leu Ala Leu Ser Val Gly Leu Thr Gly Ile Asn 275 280 285 Val Ala Val Ser Ala Leu Ser His Gln Arg Leu Thr Ser Leu Ile His 290 295 300 Val Leu Glu Gln Asp Gln Gln Arg Leu Ile Thr Ala Ile Asn Gln Thr 305 310 315 320 His Tyr Asn Leu Leu Asn Val Ala Ser Val Val Ala Gln Asn Arg Arg 325 330 335 Gly Leu Asp Trp Leu Tyr Ile Arg Leu Gly Phe Gln Ser Leu Cys Pro 340 345 350 Thr Ile Asn Glu Pro Cys Cys Phe Leu Arg Ile Gln Asn Asp Ser Ile 355 360 365 Ile Arg Leu Gly Asp Leu Gln Pro Leu Ser Gln Arg Val Ser Thr Asp 370 375 380 Trp Gln Trp Pro Trp Asn Trp Asp Leu Gly Leu Thr Ala Trp Val Arg 385 390 395 400 Glu Thr Ile His Ser Val Leu Ser Leu Phe Leu Leu Ala Leu Phe Leu 405 410 415 Leu Phe Leu Ala Pro Cys Leu Ile Lys Cys Leu Thr Ser Arg Leu Leu 420 425 430 Lys Leu Leu Arg Gln Ala Pro His Phe Pro Glu Ile Ser Leu Thr Pro 435 440 445 Lys Pro Asp Ser Asp Tyr Gln Ala Leu Leu Pro Ser Ala Pro Glu Ile 450 455 460 Tyr Ser His Leu Ser Pro Val Lys Pro Asp Tyr Ile Asn Leu Arg Pro 465 470 475 480 Cys Pro <210> 2 <211> 363 <212> PRT <213> Artificial Sequence <220> <223> BLV gp51 P2 A/a <400> 2 Trp Arg Cys Ser Leu Ser Leu Gly Asn Gln Gln Trp Met Thr Ala Tyr 1 5 10 15 Asn Gln Glu Ala Lys Phe Ser Ile Ser Ile Asp Gln Ile Leu Glu Ala 20 25 30 His Asn Gln Ser Pro Phe Cys Ala Lys Ser Pro Arg Tyr Thr Leu Asp 35 40 45 Ser Val Asn Gly Tyr Pro Lys Ile Tyr Trp Pro Pro Pro Gln Gly Arg 50 55 60 Arg Arg Phe Gly Ala Arg Ala Met Val Thr Tyr Asp Cys Glu Pro Arg 65 70 75 80 Cys Pro Tyr Val Gly Ala Asp Arg Phe Asp Cys Pro His Trp Asp Asn 85 90 95 Ala Ser Gln Ala Asp Gln Gly Ser Phe Tyr Val Asn His Gln Ile Leu 100 105 110 Phe Leu His Leu Lys Gln Cys His Gly Ile Phe Thr Leu Thr Trp Glu 115 120 125 Ile Trp Gly Tyr Asp Pro Leu Ile Thr Phe Ser Leu His Lys Ile Pro 130 135 140 Asp Pro Pro Gln Pro Asp Phe Pro Gln Leu Asn Ser Asp Trp Val Pro 145 150 155 160 Ser Val Arg Ser Trp Ala Leu Leu Leu Asn Gln Thr Ala Arg Ala Phe 165 170 175 Pro Asp Cys Ala Ile Cys Trp Glu Pro Ser Pro Pro Trp Ala Pro Glu 180 185 190 Ile Leu Val Tyr Asn Lys Thr Ile Ser Ser Ser Gly Pro Gly Leu Ala 195 200 205 Leu Pro Asp Ala Gln Ile Phe Trp Val Asn Thr Ser Ser Phe Asn Thr 210 215 220 Thr Gln Gly Trp His His Pro Ser Gln Arg Leu Leu Phe Asn Val Ser 225 230 235 240 Gln Gly Asn Ala Leu Leu Leu Pro Pro Ile Ser Leu Val Asn Leu Ser 245 250 255 Thr Ala Ser Ser Ala Pro Pro Thr Arg Val Arg Arg Ser Pro Val Ala 260 265 270 Ala Leu Thr Leu Gly Leu Ala Leu Ser Val Gly Leu Thr Gly Ile Asn 275 280 285 Val Ala Val Ser Ala Leu Ser His Gln Arg Leu Thr Ser Leu Ile His 290 295 300 Val Leu Glu Gln Asp Gln Gln Arg Leu Ile Thr Ala Ile Asn Gln Thr 305 310 315 320 His Tyr Asn Leu Leu Asn Val Ala Ser Val Val Ala Gln Asn Arg Arg 325 330 335 Gly Leu Asp Trp Leu Tyr Ile Arg Leu Gly Phe Gln Ser Leu Cys Pro 340 345 350 Thr Ile Asn Glu Pro Cys Cys Phe Leu Arg Ile 355 360 <210> 3 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> BLV gp51 P3 A/a <400> 3 Trp Arg Cys Ser Leu Ser Leu Gly Asn Gln Gln Trp Met Thr Ala Tyr 1 5 10 15 Asn Gln Glu Ala Lys Phe Ser Ile Ser Ile Asp Gln Ile Leu Glu Ala 20 25 30 His Asn Gln Ser Pro Phe Cys Ala Lys Ser Pro Arg Tyr Thr Leu Asp 35 40 45 Ser Val Asn Gly Tyr Pro Lys Ile Tyr Trp Pro Pro Pro Gln Gly Arg 50 55 60 Arg Arg Phe Gly Ala Arg Ala Met Val Thr Tyr Asp Cys Glu Pro Arg 65 70 75 80 Cys Pro Tyr Val Gly Ala Asp Arg Phe Asp Cys Pro His Trp Asp Asn 85 90 95 Ala Ser Gln Ala Asp Gln Gly Ser Phe Tyr Val Asn His Gln Ile Leu 100 105 110 Phe Leu His Leu Lys Gln Cys His Gly Ile Phe Thr Leu Thr Trp Glu 115 120 125 Ile Trp Gly Tyr Asp Pro Leu Ile Thr Phe Ser Leu His Lys Ile Pro 130 135 140 Asp Pro Pro Gln Pro Asp Phe Pro Gln Leu Asn Ser Asp Trp Val Pro 145 150 155 160 Ser Val Arg Ser Trp Ala Leu Leu Leu Asn Gln Thr Ala Arg Ala Phe 165 170 175 Pro Asp Cys Ala Ile Cys Trp Glu Pro Ser Pro Pro Trp Ala Pro Glu 180 185 190 Ile Leu Val Tyr Asn Lys Thr Ile Ser Ser Ser Gly Pro Gly Leu Ala 195 200 205 Leu Pro Asp Ala Gln Ile Phe Trp 210 215 <210> 4 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> BLV gp51 P4 A/a <400> 4 Trp Arg Cys Ser Leu Ser Leu Gly Asn Gln Gln Trp Met Thr Ala Tyr 1 5 10 15 Asn Gln Glu Ala Lys Phe Ser Ile Ser Ile Asp Gln Ile Leu Glu Ala 20 25 30 His Asn Gln Ser Pro Phe Cys Ala Lys Ser Pro Arg Tyr Thr Leu Asp 35 40 45 Ser Val Asn Gly Tyr Pro Lys Ile Tyr Trp Pro Pro Pro Gln Gly Arg 50 55 60 Arg Arg Phe Gly Ala Arg Ala Met Val Thr Tyr Asp Cys Glu Pro Arg 65 70 75 80 Cys Pro Tyr Val Gly Ala Asp Arg Phe Asp Cys Pro His Trp Asp Asn 85 90 95 Ala Ser Gln Ala Asp Gln Gly Ser Phe Tyr Val Asn His Gln Ile Leu 100 105 110 Phe Leu His Leu Lys Gln Cys His Gly Ile Phe Thr Leu Thr Trp 115 120 125 <210> 5 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P1 Nucleotide <400> 5 tggagatgct ccctgtccct aggaaaccaa caatggatga cagcatataa ccaagaggca 60 aaattttcca tctccattga ccaaatacta gaggctcata atcagtcacc tttctgtgcc 120 aagtctccca gatacacctt ggactctgta aatggctatc ctaagatcta ctggcccccc 180 ccacaagggc ggcgccggtt tggagccagg gccatggtca catatgattg cgagccccga 240 tgcccttatg tgggggcaga tcgcttcgac tgcccccact gggacaatgc ctcccaggcc 300 gatcaaggat ccttttatgt caatcatcag attttattcc tgcatcttaa acaatgtcat 360 ggaattttca ctctaacctg ggagatatgg ggatatgatc ccctgatcac cttttcttta 420 cataagatcc ctgatccccc tcaacccgac tttccccagt tgaacagtga ctgggttccc 480 tctgtcagat catgggccct gcttttaaac caaacagcac gggccttccc agactgtgct 540 atatgttggg aaccttcccc tccctgggct cccgaaatat tagtatataa caaaaccatc 600 tccagctctg gacccggcct cgccctcccg gacgcccaaa tcttctgggt caacacgtcc 660 tcgtttaaca ccacccaagg atggcaccac ccttcccaga ggttgttgtt caatgtttct 720 caaggcaacg ccttgttatt acctcctatc tccctggtta atctctctac ggcttcctcc 780 gcccctccta cccgggtcag acgtagtccc gtcgcagccc tgaccttagg cctagccctg 840 tcagtggggc tcactggaat taatgtggcc gtgtctgccc ttagccatca gagactcacc 900 tccctgatcc acgttctgga gcaagatcag caacgcttga tcacagcaat taaccagacc 960 cactataatt tgcttaatgt ggcctctgtg gttgcccaga accgacgggg gcttgattgg 1020 ttgtacatcc ggctgggttt tcaaagccta tgtcccacaa ttaatgagcc ttgctgtttc 1080 ctgcgcattc aaaatgactc cattatccgc ctcggtgatc tccagcctct ctcgcaaaga 1140 gtctctacag actggcagtg gccctggaat tgggatctgg ggctcactgc ctgggtgcga 1200 gaaaccattc attctgttct aagcctgttc ctattagccc tttttttgct cttcctggcc 1260 ccctgcctga taaaatgctt gacctctcgc cttttaaagc tcctccggca ggctccccac 1320 ttccctgaaa tctccttaac ccctaaaccc gattctgatt atcaggcctt gctaccatct 1380 gcaccagaga tctactctca cctctccccc gtcaaacccg attacatcaa cctccgaccc 1440 tgccct 1446 <210> 6 <211> 1089 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P2 Nucleotide <400> 6 tggagatgct ccctgtccct aggaaaccaa caatggatga cagcatataa ccaagaggca 60 aaattttcca tctccattga ccaaatacta gaggctcata atcagtcacc tttctgtgcc 120 aagtctccca gatacacctt ggactctgta aatggctatc ctaagatcta ctggcccccc 180 ccacaagggc ggcgccggtt tggagccagg gccatggtca catatgattg cgagccccga 240 tgcccttatg tgggggcaga tcgcttcgac tgcccccact gggacaatgc ctcccaggcc 300 gatcaaggat ccttttatgt caatcatcag attttattcc tgcatcttaa acaatgtcat 360 ggaattttca ctctaacctg ggagatatgg ggatatgatc ccctgatcac cttttcttta 420 cataagatcc ctgatccccc tcaacccgac tttccccagt tgaacagtga ctgggttccc 480 tctgtcagat catgggccct gcttttaaac caaacagcac gggccttccc agactgtgct 540 atatgttggg aaccttcccc tccctgggct cccgaaatat tagtatataa caaaaccatc 600 tccagctctg gacccggcct cgccctcccg gacgcccaaa tcttctgggt caacacgtcc 660 tcgtttaaca ccacccaagg atggcaccac ccttcccaga ggttgttgtt caatgtttct 720 caaggcaacg ccttgttatt acctcctatc tccctggtta atctctctac ggcttcctcc 780 gcccctccta cccgggtcag acgtagtccc gtcgcagccc tgaccttagg cctagccctg 840 tcagtggggc tcactggaat taatgtggcc gtgtctgccc ttagccatca gagactcacc 900 tccctgatcc acgttctgga gcaagatcag caacgcttga tcacagcaat taaccagacc 960 cactataatt tgcttaatgt ggcctctgtg gttgcccaga accgacgggg gcttgattgg 1020 ttgtacatcc ggctgggttt tcaaagccta tgtcccacaa ttaatgagcc ttgctgtttc 1080 ctgcgcatt 1089 <210> 7 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P3 Nucleotide <400> 7 tggagatgct ccctgtccct aggaaaccaa caatggatga cagcatataa ccaagaggca 60 aaattttcca tctccattga ccaaatacta gaggctcata atcagtcacc tttctgtgcc 120 aagtctccca gatacacctt ggactctgta aatggctatc ctaagatcta ctggcccccc 180 ccacaagggc ggcgccggtt tggagccagg gccatggtca catatgattg cgagccccga 240 tgcccttatg tgggggcaga tcgcttcgac tgcccccact gggacaatgc ctcccaggcc 300 gatcaaggat ccttttatgt caatcatcag attttattcc tgcatcttaa acaatgtcat 360 ggaattttca ctctaacctg ggagatatgg ggatatgatc ccctgatcac cttttcttta 420 cataagatcc ctgatccccc tcaacccgac tttccccagt tgaacagtga ctgggttccc 480 tctgtcagat catgggccct gcttttaaac caaacagcac gggccttccc agactgtgct 540 atatgttggg aaccttcccc tccctgggct cccgaaatat tagtatataa caaaaccatc 600 tccagctctg gacccggcct cgccctcccg gacgcccaaa tcttctgg 648 <210> 8 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P4 Nucleotide <400> 8 tggagatgct ccctgtccct aggaaaccaa caatggatga cagcatataa ccaagaggca 60 aaattttcca tctccattga ccaaatacta gaggctcata atcagtcacc tttctgtgcc 120 aagtctccca gatacacctt ggactctgta aatggctatc ctaagatcta ctggcccccc 180 ccacaagggc ggcgccggtt tggagccagg gccatggtca catatgattg cgagccccga 240 tgcccttatg tgggggcaga tcgcttcgac tgcccccact gggacaatgc ctcccaggcc 300 gatcaaggat ccttttatgt caatcatcag attttattcc tgcatcttaa acaatgtcat 360 ggaattttca ctctaacctg g 381 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P1 Forward Primer <400> 9 cgcgaattct ggagatgctc cctgtcccta ggaaa 35 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P1 Reverse Primer <400> 10 cccgcggccg cccaggttag agtgaaaatt cc 32 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P2 Forward Primer <400> 11 cgcgaattct ggagatgctc cctgtcccta ggaaa 35 <210> 12 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P2 Reverse Primer <400> 12 cgcctcgaga atgcgcagga aacagcaagg ctcatta 37 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P3 Forward Primer <400> 13 cgcgaattct ggagatgctc cctgtcccta ggaaa 35 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P3 Reverse Primer <400> 14 cgcctcgagc cagaagattt gggcgtcc 28 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P4 Forward Primer <400> 15 cgcgaattct ggagatgctc cctgtcccta ggaaa 35 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> BLV gp51 P4 Reverse Primer <400> 16 cgcctcgagc ccagaagatt tgggcgtcc 29

Claims (6)

Monoclonal antibody against glycoprotein 51 of bovine leukemia virus produced by hybridoma cells with accession number KCTC 18355P or KCTC 18356P. Hybridoma cells of the accession number KCTC 18355P or KCTC 18356P producing the monoclonal antibody of claim 1. A composition for diagnosing soleukosis comprising an antibody against the soleukosis virus of claim 1. A kit for diagnosing soleukosis, comprising an antibody against the soleukosis virus of claim 1. In order to provide information on the diagnosis of soleukosis, a method of detecting an antibody to soleukosis virus in a biological sample isolated from cattle by using the monoclonal antibody against the soleukosis virus of claim 1. delete

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