JP7051096B2 - Antibodies that specifically recognize bovine procalcitonin, their antigen-binding fragments, and their use. - Google Patents

Description

NPMD NITE P-02715 NPMD NITE P-02711 NPMD NITE P-02712 NPMD NITE P-02713 NPMD NITE P-02714

The present invention relates to an antibody that specifically recognizes bovine procalcitonin, an antigen-binding fragment thereof, and its use.

Sepsis is a condition that causes severe systemic symptoms and requires prompt diagnosis. However, it is difficult to distinguish sepsis test findings because there are many test findings that overlap with non-infectious inflammatory diseases (autoimmune diseases, cancer, etc.). Therefore, the development of a specific marker for sepsis has been desired. In recent years, procalcitonin (PCT), which is a precursor protein of calcitonin (CT), has attracted attention as a specific marker for sepsis.
Normally, PCT is produced in C cells of the thyroid gland, but it is rapidly cleaved inside the cells and decomposed into N-terminal aminoprocalcitonin, central calcitonin, and C-terminal catacalcin, mainly. Central calcitonin is released into the blood. On the other hand, PCT is produced in cells throughout the body by the action of cytokines such as lysophosphatidylcholine (LPS), which is a bacterium during sepsis, and IL-1β and TNF-α, which increase during inflammation, and is the full-length form of PCT without being cleaved. It is secreted into the blood while keeping the.
Thus, during sepsis, the blood concentration of PCT rises sharply, and the blood concentration of PCT is maintained for a long period of time. In addition, since the blood concentration of PCT does not change during non-communicable inflammatory disease or virus infection, it has the advantage of being highly specific for sepsis (Non-Patent Document 1).

In animal experiments using hamsters and pigs, administration of anti-PCT serum at the time of sepsis reduced the mortality rate (Non-Patent Documents 2 to 4). Therefore, it is considered that PCT produced during sepsis exacerbates sepsis. Be done. Therefore, the measurement of PCT concentration may be useful as an index for diagnosing the onset of sepsis and determining the prognosis.

In order to specifically detect PCT in blood, it is considered that it should be detected by using an antibody specific to the N-terminal region and C-terminal region of PCT, which is not normally secreted into blood. However, it is insufficient to detect using an antibody corresponding to either the N-terminal region or the C-terminal region. This is because, in this case, not only the full-length PCT but also the N-terminal aminoprocalcitonin or the C-terminal catacalcin will be recognized, and the correct PCT concentration cannot be obtained.

So far, several antibodies for detecting PCT have been reported. For example, Patent Document 1 describes a monoclonal antibody that binds to the 25-37th amino acid region on the N-terminal side of a human PCT (116 residues excluding the signal sequence) and the 53-116th amino acid region on the C-terminal side. A method for detecting a human PCT by a sandwich method using an antibody is disclosed. Further, for example, in Non-Patent Document 5, human PCT is prepared by a sandwich method using a monoclonal antibody that binds to the 3rd to 21st amino acid regions on the N-terminal side and the 99th to 109th amino acid regions on the C-terminal side. Disclose the method of detection.

JP-A-2015-163877

Misa Nakamura et al., “Procalcitonin: Mysterious Protein in Sepsis” Journal of Basis & Clinical Medicine (2013) 2 (1): 7-11 Nylen ES et al, “Mortality is increased by procalcitonin and decreased by an antiserum reactive to procalcitonin in experimental sepsis” Crit Care Med. (1998) 26: 1001-6. Wagner KE et al., “Early immunoneutralization of calcitonin precursors precursors the adverse physiologic response to sepsis in pigs” Crit Care Med (2002) 30: 2313-21. Martinez JM et al., “Late immunoneutralization of procalcitonin arrests the progession of lethal porcine sepsis” Surg Infect (2001) 2 (3): 193-202 Kramer P. M. et al., “Development and characterization of new rat monoclonal antibodies for procalcitonin” Anal Bioanal Chem. (2008) Oct, 392 (4): 727-36. Doi: 10.1007 / s00216-008-2321-4

A method for diagnosing the onset of sepsis and prognosis with high accuracy and simplicity is also an important issue in the livestock industry that handles livestock such as cattle. An object of the present invention is to provide an antibody capable of measuring bovine procalcitonin (boPCT) concentration and a method for detecting bovine procalcitonin using the antibody for clinical application of procalcitonin, which has been attracting attention as a specific marker for sepsis in recent years. do.

First, the present inventors examined the application of an antibody against human procalcitonin in order to solve the above-mentioned problems. Therefore, the amino acid sequences of human procalcitonin and bovine procalcitonin were compared, and the identity between the amino acid sequences was confirmed. As a result, the identity between the amino acid sequences was as low as about 60% (Fig. 1), and it was presumed that it would be difficult to use an antibody that recognizes human procalcitonin as it is for the detection of bovine procalcitonin. Therefore, the present inventors attempted to newly prepare an antibody that recognizes the N-terminal region and the C-terminal region of bovine procalcitonin.
Specifically, bovine procalcitonin (hereinafter referred to as Trx-His-boPCT) to which a thioredoxin-histidine tag (Trx-His tag) has been added is prepared using Escherichia coli, and this purified Trx-His-boPCT is used for immunization of mice. By using this, a hybridoma that produces a monoclonal antibody specific for Trx-His-boPCT was obtained. As a result of screening by the ELISA method, 5 clones (1C11, 1A8, 1A3, 1E12, 2E4) of hybridomas producing anti-boPCT monoclonal antibody could be obtained. As a result of epitope analysis of these antibodies using Western blotting and ELISA, 2E4 recognized the N-terminal region (amino acids 26-96), while the remaining four antibodies recognized the C-terminal region (87-96). 143) was recognized. Furthermore, we succeeded in detecting and quantifying bovine procalcitonin by performing a sandwich ELISA method using the obtained antibody that recognizes the N-terminal region and the antibody that recognizes the C-terminal region.
The present invention has been completed based on the above findings, and includes the following aspects.

The present invention is in one aspect.
[1] An antibody or an antigen-binding fragment thereof that specifically recognizes bovine procalcitonin.
(I) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 26th to 96th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1 or an antigen-binding fragment thereof.
(II) The present invention relates to an antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the amino acid sequence 87 to 143 in the amino acid sequence shown in SEQ ID NO: 1.
Here, the antibody according to the present invention or an antigen-binding fragment thereof is used in one embodiment.
[2] The antibody or antigen-binding fragment thereof according to the above [1].
The antibody of (I) or its antigen-binding fragment is an antibody or its antigen produced by a hybridoma deposited as Accession No. NITE P-02715 to the National Institute of Technology and Evaluation on May 16, 2018. It is a combined fragment and
The antibody of (II) or an antigen-binding fragment thereof is one of the following (i) to (iv):
(I) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02711 on May 16, 2018.
(Ii) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02712 on May 16, 2018.
(Iii) An antibody or an antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02713 on May 16, 2018, or
(Iv) An antibody or antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02714 on May 16, 2018.

Further, the present invention is in another aspect.
[3] A combination of an antibody that specifically recognizes bovine procalcitonin or an antigen-binding fragment thereof.
The present invention relates to a combination of an antibody or an antigen-binding fragment thereof, which comprises the antibody (I) or an antigen-binding fragment thereof according to the above [1] or [2] and the antibody (II) or an antigen-binding fragment thereof.

Further, the present invention is in another aspect.
[4] The present invention relates to a kit for detecting bovine procalcitonin, which comprises a combination of the antibody or an antigen-binding fragment thereof according to the above [3].
Here, the kit according to the present invention is, in one embodiment,
[5] The kit according to the above [4] is used to assist in diagnosing the possibility of suffering from sepsis or the state of prognosis.

Further, the present invention is in another aspect.
[6] NITE P-02715 , NITE P-02711 , NITE P-02712 , NITE P-02713 , or NITE P- 02714 , the Incorporated Administrative Agency, was assigned to the National Institute of Technology and Evaluation. Regarding hybridomas deposited on May 16, 2018.

Further, the present invention is in another aspect.
[7] A method for detecting bovine procalcitonin contained in a sample.
(A) A step of contacting bovine procalcitonin contained in the sample with the following antibodies (I) and (II) or antigen-binding fragments thereof:
(I) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 26th to 96th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1.
(II) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 87th to 143rd amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1.
(A) The present invention relates to a detection method including a step of detecting the binding of an antibody or an antigen-binding fragment thereof to bovine procalcitonin contained in the sample.
Here, the method for detecting bovine procalcitonin contained in the sample according to the present invention is, in one embodiment,.
[8] The detection method according to the above [7].
The antibody of (I) or its antigen-binding fragment is an antibody or its antigen produced by a hybridoma deposited as Accession No. NITE P-02715 to the National Institute of Technology and Evaluation on May 16, 2018. It is a combined fragment and
The antibody of (II) or an antigen-binding fragment thereof is one of the following (i) to (iv):
(I) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02711 on May 16, 2018.
(Ii) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02712 on May 16, 2018.
(Iii) An antibody or an antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02713 on May 16, 2018, or
(Iv) An antibody or antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02714 on May 16, 2018.
Further, the method for detecting bovine procalcitonin contained in the sample according to the present invention is, in one embodiment,.
[9] The detection method according to the above [7] or [8].
It is characterized in that the bovine procalcitonin is detected by the sandwich ELISA method.

Further, the present invention is in another aspect.
[10] A method for assisting in diagnosing the possibility or prognostic condition of sepsis in a subject.
Diseases of sepsis based on the step of quantitatively measuring the procalcitonin concentration contained in the sample derived from the body fluid of the subject and the measured procalcitonin concentration value by using any of the above-mentioned detection methods [7] to [9]. With respect to a process that assists in diagnosing the potential or prognostic condition of, and methods including.

According to the antibody that specifically recognizes bovine procalcitonin or its antigen-binding fragment of the present invention, bovine procalcitonin in a sample can be used together with an antibody that recognizes the N-terminal side or an antibody that recognizes the C-terminal side. It can be detected and quantified separately from the decomposition products aminoprocalcitonin and catacalcin.

FIG. 1 is a diagram showing a comparison of amino acid sequences between human procalcitonin and bovine procalcitonin. FIG. 2 shows the base sequence and amino acid sequence of bovine procalcitonin used in the following Example “1. Preparation of antibody” and a plasmid map for expressing Escherichia coli incorporating the bovine procalcitonin. FIG. 3 is an image showing the results of purification of Trx-His-boPCT expressed in Escherichia coli in the following Example “1. Preparation of antibody” using His GraviTrapTM (GE healthcare) and confirmed by Western blotting. Is. FIG. 4 is a conceptual diagram showing each schedule of immune protocols # 1 and # 2 of BALB / c mice in the following Example “1. Preparation of antibody”. FIG. 5 shows the recognition of the 1A3, 1A8, 1C11, 1E12, and 2E4 antibodies and the boPCT full length, N-terminal fragment, or C-terminal fragment in the following Example “2-1. Direct ELISA method”. It is a graph which shows the result measured by the ELISA method. FIG. 6 shows recognition of each antibody of 1A3, 1A8, 1C11, 1E12, and 2E4 and boPCT full length, N-terminal fragment, or C-terminal fragment in the following Example “2-2. Western blotting”. It is an image which shows the result detected by the Western blotting. FIG. 7 shows an image of the anti-boPCT monoclonal antibody purified in the following Example “3. Detection of bovine procalcitonin by sandwich ELISA” stained with SDS-PAGE and Coomassie Brilliant Blue. FIG. 8 is a conceptual diagram showing the principle of the following Example “3-1. Sandwich ELISA method”. In FIG. 9, purified Trx-His-boPCT was detected using 2E4 (N-terminal recognition) and 1A8 (C-terminal recognition) as anti-boPCT monoclonal antibodies in the following Example “3-1. Sandwich ELISA”. It is a graph which shows the absorbance at 450 nm. FIG. 10 shows 450 nm when purified boPCT was detected using 2E4 (N-terminal recognition) and 1A8 (C-terminal recognition) as anti-boPCT monoclonal antibodies in the following Example “3-1. Sandwich ELISA”. It is a graph which shows the absorbance.

The present invention, in one aspect, provides an antibody or antigen-binding fragment thereof that specifically recognizes bovine procalcitonin. The antibody or antigen-binding fragment thereof can be specified as (I) or (II) below:
(I) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 26th to 96th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1 or an antigen-binding fragment thereof.
(II) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 87th to 143rd amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1.

As used herein, "bovine procalcitonin" is a precursor protein of calcitonin, which is an important parathyroid hormone for calcium metabolism, and is a protein produced in bovine organisms. Synthesis of bovine procalcitonin is initiated by translation of preprocalcitonin (prePCT) (amino acid sequence set forth in SEQ ID NO: 1; FIG. 2), a precursor peptide consisting of 143 residues of amino acids. Bovine procalcitonin refers to a protein consisting of 118 residues of amino acids formed after elimination of a signal peptide (region consisting of amino acids 1 to 25 of prePCT). As mentioned above, bovine procalcitonin is normally produced in the C cells of the thyroid gland and undergoes rapid intracellular cleavage. That is, N-terminal aminoprocalcitonin (region consisting of amino acids 26 to 84 in SEQ ID NO: 1), carcitonin in the center (region consisting of amino acids 87 to 118 in SEQ ID NO: 1), and catacalcin on the C-terminal side (region consisting of amino acids 87 to 118 in SEQ ID NO: 1). It is decomposed into the region consisting of the 123rd to 143rd amino acids in SEQ ID NO: 1. And mainly central calcitonin is released into the blood. On the other hand, when suffering from sepsis, bovine procalcitonin is produced in cells throughout the body and secreted into the blood without undergoing the above-mentioned cleavage while retaining the form of aminoprocalcitonin, calcitonin, and bovine procalcitonin having a region of catacalcin. To.

Further, in the present specification, "bovine procalcitonin" includes a protein consisting of the 26th to 143rd amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1 and a variant thereof. Here, the "variant" is a substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid residues in the amino acid sequence of bovine procalcitonin. , Deletion, and / or addition, meaning a peptide having substantially the same antigenicity as bovine procalcitonin. As used herein, "substantially" means the degree of specificity as specifically recognized by an anti-bovine procalcitonin antibody to the extent that it can be used in the diagnosis of sepsis characterized by the expression of bovine procalcitonin. Typical examples of mutants include genotypic polymorphisms and sequence changes due to splicing and the like.

Antibodies used in the present invention are immunoglobulins and include, for example, IgA, IgD, IgE, IgG ₁ , IgG _2a , IgG _2b , IgG ₃ , IgG ₄ , and immunoglobulins of the class or subclass of IgM. The constant region of the light chain of the antibody may be either λ or κ, preferably κ. The antibody used in the present invention can be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
Further, the antibody according to the present invention may have a partially modified structure such as a chimeric antibody or a humanized antibody, or may be modified with a fluorescent label or the like for the detection of bovine procalcitonin. Chimerization and humanization of antibodies and labeling with fluorescent dyes and chemiluminescent dyes can be appropriately carried out according to known methods. The fluorescent dye or chemiluminescent dye used for the label is not particularly limited, and a known dye used for labeling the antibody can be used.
The antibody or antigen-binding fragment thereof according to the present invention is not limited to the following, but is preferably capable of measuring bovine procalcitonin by using an antibody concentration of 2.0 ng / ml or more, more preferably 0.5 ng / ml or more. be.

Here, the antibody according to the present invention recognizes, in one embodiment, an epitope contained in (I) the 26th to 96th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1. The region consisting of the 26th to 96th amino acids in the amino acid sequence shown in SEQ ID NO: 1 corresponds to the N-terminal region of bovine procalcitonin after the signal peptide is desorbed. Further, the antibody of (I) does not recognize the region consisting of the 87th to 143rd amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1. Therefore, the antibody of (I) specifically recognizes the N-terminal region of bovine procalcitonin.
Further, one embodiment of the antibody according to the present invention recognizes an epitope contained in the amino acid sequence 87 to 143 in the amino acid sequence shown in (II) SEQ ID NO: 1. The region consisting of the 87th to 143rd amino acids in the amino acid sequence shown in SEQ ID NO: 1 corresponds to the C-terminal region of bovine procalcitonin after the signal peptide is desorbed. Further, the antibody of (II) does not recognize the region consisting of the 26th to 96th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1. Therefore, the antibody of (II) specifically recognizes the C-terminal region of bovine procalcitonin.

The antibody that recognizes the epitope contained in the 26th to 96th amino acid sequences in the amino acid sequence shown in (I) SEQ ID NO: 1 is not limited to the following, but is a product of Incorporated Administrative Agency as Accession No. NITE P-02715. An antibody produced by a hybridoma deposited on May 16, 2018 by the National Institute of Technology and Evaluation (referred to as 2E4 in the present specification for convenience) can be used.
Further, the antibody that recognizes the epitope contained in the 87th to 143rd amino acid sequences in the amino acid sequence shown in (II) SEQ ID NO: 1 is not limited to the following, but is independent as (i) Accession No. NITE P-02711. Antibodies produced by hybrid domas deposited with the National Institute of Technology and Evaluation on May 16, 2018 (referred to as 1A3 for convenience in this specification), (ii) Independent as accession number NITE P-02712. An antibody produced by a hybrid doma deposited with the National Institute of Technology and Evaluation on May 16, 2018 (referred to as 1A8 for convenience in this specification), (iii) independent as accession number NITE P-02713. An antibody produced by a hybrid dome deposited with the National Institute of Technology and Evaluation on May 16, 2018 (referred to as 1C11 in this specification for convenience), or (iv) Accession No. NITE P-02714. As a result, an antibody produced by a hybridoma deposited with the National Institute of Technology and Evaluation on May 16, 2018 (referred to as 1E12 in this specification for convenience) can be used.

Antibodies used in the present invention immunize animals (eg, mammals such as mice, rats, guinea pigs, rabbits, horses, sheep, goats, chickens) and collect serum or spleen cells, as detailed below. It can be made by conventional methods, including A method for producing an antibody by immunosensitization of such an animal is known and is not limited to the following, but can be performed as follows, for example. First, a vector for synthesizing a recombinant peptide of bovine procalcitonin (preferably bovine procalcitonin to which a solubilized and / or purified tag such as Thioredoxin-His) is added is constructed using Escherichia coli or the like. The vector is then introduced into host cells and cultured to promote the synthesis of bovine procalcitonin. Then, bovine procalcitonin recovered as a culture is purified, turbidized with an appropriate adjuvant such as Freund's complete adjuvant, and used for immunization of mammals such as mice. Immunization can be performed by repeatedly inoculating the abdominal cavity, subcutaneous or vein of the immunized mammal several times every few weeks.
The polyclonal antibody can be collected as an antiserum from the blood, ascites, etc. of a mammal immunized by the above method. The polyclonal antibody titer in antiserum can be measured according to a known method. For example, the antibody titer in the antiserum can be measured by reacting the labeled bovine procalcitonin with the antiserum and then measuring the activity of the labeling agent bound to the antibody.

Further, for the monoclonal antibody, the spleen is taken out from the mammal 3 to 5 days after the final immunization in the above-mentioned method of immunosensitization, and the antibody-producing cells are collected. At the same time, as a parent cell for fusing with the antibody-producing cell to obtain a hybridoma, a myelogram cell line having an appropriate marker was prepared, and this was fused with the antibody-producing cell to hybridize. -Make a cell. As the medium for preparing the vibrido-ma, commonly used media such as Eagle MEM, Dulbecco's modified medium, and RPMI-1640 can be used. For fusion, first, the parent cells myeloma and splenocytes are prepared at a ratio of about 1: 5. It is said that the fusion rate is high when polyethylene glycol (PEG) is used at a concentration of 50% as the fusion agent. The fusion strain can be selected by the HAT selection method. Screening of the resulting hybridoma is performed by a known method such as an ELISA method directly using the culture supernatant, and the clone of the hybridoma secreting the target immunoglobulin is selected. To ensure the unity of the hybridoma, sow the hybridoma in a cell culture plate such as 96 wells so that there is no more than one hybridoma in one hole, and screen the growing clone again. Perform collateral. By repeating this subcloning, a unity hybridoma can be obtained.

Antibodies are separated and purified by known immunoglobulin separation and purification methods (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchanger (eg, DEAE) adsorption and desorption, and ultracentrifugation. It can be carried out according to a method, a gel filtration method, a specific purification method in which only an antibody is collected by an antigen-binding solid phase or an active adsorbent such as protein A or protein G and the binding is dissociated to obtain an antibody).

The epitope of the obtained monoclonal antibody can be confirmed by a known method. For example, a plurality of peptides that are candidates for epitopes can be prepared, and the epitopes of monoclonal antibodies can be confirmed by a direct ELISA method or Western blotting method using the peptides.

The present invention also provides antigen-binding fragments derived from the antibodies described above. The antigen-binding fragment is a fragment of an antibody that maintains a specific recognition ability for an epitope of the derived antibody. Not limited to such antigen-binding fragments, but specifically, for example, Fab, Fab', F (ab') 2, single chain antibody (scFv), disulfide stabilized V region fragment (dsFv), or CDR. Functional fragments of antibodies such as peptides containing, Diabodies, and Minibodies and the like. The disulfide-stabilized V-region fragment refers to a polypeptide in which one amino acid residue in _VF and _VL is replaced with a cysteine residue, which is bound via a disulfide bond between the cysteine residues. Methods of making such antigen-binding fragments are known to those of skill in the art.

Further, the antibody according to the present invention is an antibody that specifically recognizes the (I) N-terminal region of bovine procalcitonin in order to avoid false positives such as calcitonin after degradation and accurately measure the concentration of bovine procalcitonin. , (II) Used in combination with an antibody that specifically recognizes the C-terminal region.
As described above, in one embodiment, (I) 2E4 can be used as an antibody that specifically recognizes the N-terminal region, and (II) 1A3, 1A8 as an antibody that specifically recognizes the C-terminal region. , 1C11, and 1E12 can be used. The 2E4 antibody may be used in combination with any of the 1A3, 1A8, 1C11, and 1E12 antibodies. A combination of 2E4 as an antibody that specifically recognizes the (I) N-terminal region and 1A8 as an antibody that specifically recognizes the (II) C-terminal region can be preferably mentioned without being limited to the following.

The present invention provides, in one embodiment, a method for detecting bovine procalcitonin contained in a sample.
The method for detecting bovine procalcitonin according to the present invention comprises (a) contacting bovine procalcitonin contained in a sample with the above-mentioned antibodies (I) and (II) or antigen-binding fragments thereof, and (b) the above-mentioned. It comprises a step of detecting the binding of an antibody or an antigen-binding fragment thereof to bovine procalcitonin contained in a sample.

Here, the bovine procalcitonin contained in the sample may be synthesized or derived from a living body. Preferably, the bovine procalcitonin in the sample is of biological origin. As a sample containing bovine procalcitonin derived from a living body, for example, bovine-derived body fluid can be used, and examples thereof include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion. Alternatively, it may be a purified product or an extract derived from these body fluids.
In addition, the detection method of the present invention may contain aminoprocultocytonin, cultoshitonin, and / or catacalcin in the sample. Even in such cases, only bovine procultocitonin can be detected or quantified separately.

The method for detecting bovine procalcitonin of the present invention includes the above steps (a) and (b). In step (a), the antibodies of (I) and (II) or their antigen-binding fragments are brought into contact with bovine procultosine in a sample and bound to the N-terminal region and C-terminal region of the bovine procultosine, respectively. Let me. Then, the antibody (I) and (II) bound to bovine procultocytonin or the antigen-binding fragment thereof is detected by the step (a).
The steps (a) and (b) in the detection method of the present invention can be carried out according to the detection method using a known antibody. Detection methods that can be used in the method for detecting bovine procalcitonin of the present invention are not limited to the following, and include, for example, radioimmunoassay (RIA), chemical luminescence and fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), and Luminex. Includes various types of immunoassays, such as Luminex-based bead arrays, protein microarray assays, and high-speed test formats such as, for example, immunoimmunoassay assay strips. Those skilled in the art can appropriately set the conditions of the step (a) and the step (b) according to the known detection methods to be adopted.
The detection method employed in one preferred embodiment is a sandwich immunoassay, more specifically a sandwich ELISA. For example, when bovine procalcitonin is detected by the sandwich ELISA method, (I) an antibody that specifically recognizes the N-terminal region and (II) an antibody that specifically recognizes the C-terminal region are bound to bovine procalcitonin. Let me. Either (I) an antibody that specifically recognizes the N-terminal region or (II) an antibody that specifically recognizes the C-terminal region may be used as the first antibody or the second antibody, but the first antibody is solid. It can be used in combination with a phase, eg, the surface of a bead, well or other container, chip or strip. Bovine procalcitonin is contacted and bound to the immobilized first antibody, and the second antibody is further bound to the bovine procalcitonin. The second antibody can be, for example, a dye, a radioisotope, or an antibody labeled with a reactive or catalytically active moiety. The amount of labeled secondary antibody bound to the sample is then measured by a suitable method. For example, as described in the following Examples, by modifying the second antibody with biotin, the absorbance of the color-developing reaction by the enzymatic reaction can be determined using avidin-HRP, a commercially available TMB-peroxidase substrate kit, a microplate reader, or the like. It can be measured and quantified bovine procalcitonin. General detection and quantification methods related to sandwich immunoassays have been established and can be carried out by those skilled in the art with appropriate conditions.

As another detection method, one antibody is labeled with a part of a labeling system based on quenching or amplification of fluorescence or chemical emission, and the other antibody is labeled with a labeling system that affects the quenching or amplification. However, a known method for measuring the quenching or amplification of fluorescence or luminescence that changes due to the binding of both antibodies to bovine procalcitonin can also be adopted.

In another aspect, the invention provides a method of assisting in diagnosing a potential or prognostic condition of sepsis in a subject. Methods to assist in diagnosing the possibility or prognostic condition of the sepsis include the step of quantitatively measuring the procalcitonin concentration contained in the sample derived from the body fluid of the subject by the above-mentioned method for detecting bovine procalcitonin. Includes steps to assist in diagnosing the potential or prognostic condition of sepsis based on measured procalcitonin levels.
"Assisting in diagnosing the potential or prognostic condition of sepsis" means whether or not sepsis is present or after treatment for sepsis by measuring the amount of bovine procalcitonin present in the body fluids. It is meant to provide assistive data in the diagnosis of prognostic conditions in. For example, the amount of bovine procalcitonin present in bovine body fluids can be measured and the determination of whether or not the patient has sepsis can be based on known information (eg, Nazli Ercan et al., “Diagnostic value”. of serum procalcitonin, neopterin, and gamma interferon in neonatal calves with septicemic colibacillosis ”(2016) Journal of Veterinary Diagnostic Investigation, Vol. 28 (2) 180–183).

The present invention provides, in one aspect, a kit for detecting bovine procalcitonin. The kit according to the present invention is a combination of (I) an antibody that specifically recognizes the N-terminal region or an antigen-binding fragment thereof and (II) an antibody that specifically recognizes the C-terminal region or an antigen-binding fragment thereof. including. In addition, the kit according to the present invention can include other reagents and instruments necessary for detecting bovine procalcitonin. For example, but not limited to the following, a labeling substance, an antibody, or a solid phase reagent on which the labeling substance is immobilized can be included. In addition, various buffers, various reaction vessels (Eppendorf tube, etc.), blocking agents, experimental operation manuals (instructions), etc. for carrying out the detection method of the present invention may be included.

In one aspect, the present invention is a hybrid doma deposited as (I) National Institute of Technology and Evaluation as accession number NITE P-02715 on May 16, 2018, (II) (i) accession number NITE P. The hybrid doma deposited with the National Institute of Technology and Evaluation as 02711 on May 16, 2018, (ii) as the accession number NITE P-02712 to the National Institute of Technology and Evaluation of Product Evaluation, May 16, 2013. The hybrid doma deposited at the same time, (iii) NITE P-02413 , the hybrid doma deposited at the National Institute of Technology and Evaluation of Products on May 16, 2018, or (iv) NITE P-02714 . We will provide the hybrid doma deposited with the National Institute of Technology and Evaluation of Products on May 16, 2018.
The hybridoma provided by the present invention can be cultured in vivo or in vitro, and an antibody that specifically recognizes the (I) N-terminal region from the recovered product or culture, or (II) C-terminal region. An antibody that is specifically recognized can be obtained. Techniques for culturing hybridomas in vivo or in vitro are known, and those skilled in the art can appropriately carry out the techniques according to known techniques. For example, when culturing in vitro, the medium may be a medium to which serum is added, and after culturing in this medium for several days, a monoclonal antibody can be obtained from the culture supernatant. In addition, when culturing in vivo, the hybridoma can be inoculated into the abdominal cavity of a mammal such as a mouse, and ascites can be collected 1 to 3 weeks later to obtain a monoclonal antibody.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

(1. Preparation of antibody)
To purify the recombinant protein (Trx-His-boPCT) of bovine procalcitonin (boPCT) tagged with Thioredoxin-His (Trx-His), boPCT was added to the EcoRV / XhoI restriction enzyme site of the pET32a plasmid (Novagen). A plasmid for expressing Escherichia coli incorporating the above was prepared (FIG. 2; the base sequence and amino acid sequence of boPCT are also shown in FIG. 2). This plasmid was introduced into BL-21 E. coli (Novagen) and the expression of boPCT was induced by 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After induction of expression and culture by IPTG, Escherichia coli was suspended in a binding buffer (300 mM NaCl, 50 mM sodium phosphate, pH 7.4) and sonicated for 3 minutes 5 times. Then, it was centrifuged at 12,000 × g at 4 ° C. for 10 minutes, and Trx-His-boPCT in the supernatant was purified using His GraviTrapTM (GE healthcare) (Fig. 3). The concentration of the purified protein was measured using Protein assay dye reagent concentrate (Bio-Rad).

Four BALB / c mice (male, 7 weeks old) were purchased from Kyowa Kirin Plus Co., Ltd. and used for immunization one week after the purchase (Fig. 4). On the first day of immunization (Day 0), each mouse was mixed with 50 μg of purified Trx-His-boPCT protein (0.5-1.0 mg / ml) and an equal amount of complete Freund's adjuvant (CFA) (Difco). Was injected intraperitoneally. Of the four mice, one mouse was incomplete Freund's adjuvant in an amount equal to 50 μg of purified Trx-His-boPCT protein on Day 14, as shown in Protocol # 1 in FIG. A mixture of (IFA)) and (Difco) was intraperitoneally administered, and on Day 28, 50 μg of purified Trx-His-boPCT protein mixed with an equivalent amount of PBS was intraperitoneally administered. Of the four mice, the remaining three were mixed with 50 μg of purified Trx-His-boPCT protein and an equivalent amount of incomplete Freund's adjuvant on Day 14 and Day 28, as shown in Protocol # 2 of FIG. Was administered intraperitoneally, and on Day 42, 50 μg of purified Trx-His-boPCT protein mixed with an equivalent amount of PBS was intraperitoneally administered. All mice were collected under anesthesia 3 days after final immunization and the spleen was removed after euthanasia. The collected blood was allowed to stand at 37 ° C for 1 hour and then at 4 ° C overnight. Then, it was centrifuged at 8,500 × g at 4 ° C for 15 minutes, and the supernatant was used as an antiserum in an experiment for confirming antibody production. The removed spleen was hemolyzed by treating with Tris buffer (pH 7.6) containing 0.8% NH ₄ Cl, and the remaining spleen was placed in NS-1 medium (RPMI-1640 medium (Wako)) with 10% fetal bovine serum. Resuspended in 2 mM L-glutamine, 1 mM sodium pyruvate, 50 IU / ml penicillin, 50 μg / ml streptomycin). After resuspension, the obtained splenocytes were mixed with mouse myeloma P3U1 cells at a ratio of 5: 1 (cell number) and centrifuged at 800 × g at room temperature for 5 minutes. After centrifugation, the cells were washed once with NS-1 medium containing no fetal bovine serum. To these cells, 1 ml of 50% (w / v) polyethylene glycol 4000 (Merck) was added with vigorous mixing, then 10 ml of RPMI-1640 medium was added, and the cells were centrifuged at 170 × g at room temperature for 5 minutes. Then, the cells were re-cultured in HAT selective medium (adding HAT supplement (Gibco; containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine) to NS-1 medium) at a density of 5 × 10 ⁶ cells / ml. The cells were turbid, added to a 96-well flat bottom plate (Iwaki) at 100 μl / well (5 × 10 ⁵ cells / well), and cultured in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On days 4 and 6 of culture, 50 μl of HAT selective medium was added to each well. The presence or absence of anti-boPCT antibody in the culture supernatant was examined by direct ELISA, and for wells in which anti-boPCT antibody production was confirmed, HT medium (RPMI-1640 medium with HT supplement (Gibco: 100 μM hypoxanthine, 16 μM) The culture was continued while adding (including thymidin) as appropriate, and the hybridoma was cloned by performing limiting dilution twice in HT medium containing Briclone (Dainippon Sumitomo Pharma). The obtained clones were cryopreserved at -80 ° C using a bun bunker (Nippon Genetics).

(2. Determination of epitope)
The cloned hybridoma was cultured in NS-1 medium, and the culture supernatant was used for epitope analysis. Epitope analysis was performed by direct ELISA (FIG. 5) and Western blotting (FIG. 6) using recombinant Trx-His-boPCT (full-length, N-terminal fragment, C-terminal fragment) protein as an antigen. The N-terminal fragment is composed of the 26th to 96th amino acids of the amino acid sequence in SEQ ID NO: 1, and the C-terminal fragment is composed of the 87th to 143rd amino acids of the amino acid sequence of SEQ ID NO: 1.

(2-1. Direct ELISA method)
Purified Trx-His-boPCT protein used as an antigen is diluted in carbonate buffer (100 mM sodium carbonate, 100 mM sodium hydrogencarbonate, pH 8.7), and then 200 ng / well Trx-His-boPCT protein is added to a 96-well flat-bottomed plate (100 mM sodium carbonate, pH 8.7). In addition to NUNC), it was allowed to stand overnight at 4 ° C. After washing the plate once with PBS (-) (PBS-T) containing 0.1% Tween-20, add PBS (-) containing 2% gelatin (Bio-Rad) and let stand at 37 ° C for 30 minutes. Blocking was performed by. After washing the plate with PBS-T three times, 50 μl of the hybridoma culture supernatant was added, and the plate was allowed to stand at 37 ° C. for 30 minutes. The plate was washed 3 times with PBS-T, HRP-added Goat anti-mouse IgG (Jackson ImmunoResearch) diluted 1: 5000 with PBS (-) was added, and the plate was allowed to stand at 37 ° C. for 30 minutes. After washing the plate with PBS-T three times, color was developed with the TMB peroxidase EIA substrate kit (Bio-Rad), and the absorbance at 450 nm was measured using the iMark microplate reader (Bio-Rad).
The results are shown in FIG. Among the antibodies produced from each hybridoma, the monoclonal antibodies that recognize boPCT are referred to as 2E4, 1A3, 1A8, 1C11, and 1E12, respectively. As shown in FIG. 5, 1A3, 1A8, 1C11, 1E12 recognized the full-length boPCT and the C-terminal fragment. 2E4 also recognized the full-length boPCT and the N-terminal fragment.

(2-2. Western blotting)
Protein samples containing Trx-His-boPCT were mixed with an equal volume of 2 × SDS sample buffer and the proteins were separated by SDS-PAGE using 12% acrylamide gel. Then, it was transferred to a polyvinylidene difluoride (PVDF) membrane (Merck-Millipore) and blocked by shaking in PBS-T containing 2% skim milk. The membrane was immersed in the primary antibody (diluted with PBS-T containing 2% skim milk) and allowed to stand at 4 ° C. for 16 hours. The membrane was washed 3 times with PBS-T, immersed in a secondary antibody (HRP-added Goat anti-mouse IgG (Jackson ImmunoResearch)) diluted 1: 5000 with PBS-T, and shaken at room temperature for 1 hour. After washing the membrane with PBS-T three times, a luminescence reaction was carried out using a luminescent substrate (EzWestLumi plus (Atto)), and luminescence was detected using LAS-3000 mini (Fuji film).
The results are shown in FIG. As shown in FIG. 6, in Western blotting, 1A3, 1A8, 1C11, and 1E12 recognized the full-length boPCT and the C-terminal fragment, and did not recognize the N-terminal fragment, as in the result of the ELISA method. In addition, 2E4 recognized the full-length boPCT and the N-terminal fragment, and did not recognize the C-terminal fragment.
The results of the ELISA method and Western blotting method are summarized in Table 1 below.

また、得られたハイブリドーマにより産生される抗体のアイソタイプ（重鎖および軽鎖）を調べた結果を下記表２に示す。これらの抗体のアイソタイプを確認したところ、免疫グロブリン重鎖は1C11がIgG₁、残る4種がIgG_2bであり、軽鎖は全ての抗体でκであった。

The results of examining the isotypes (heavy chain and light chain) of the antibody produced by the obtained hybridoma are shown in Table 2 below. When the isotypes of these antibodies were confirmed, the immunoglobulin heavy chain was IgG ₁ for 1C11, IgG _2b for the remaining 4 types, and κ for the light chain for all antibodies.

(3. Detection of bovine procalcitonin by sandwich ELISA method)
Anti-boPCT monoclonal antibody is obtained by passing the hybridoma culture supernatant through a HiTrap Protein G HP column (1 ml column volume, GE healthcare) and recovering the antibody molecule bound to Protein G according to the method described in the product description. Purified. The concentration of the obtained monoclonal antibody was measured using Protein assay dye reagent concentrate (Bio-Rad), and the molecular weight and degree of purification were confirmed by SDS-PAGE and Coomassie Brilliant Blue staining (Fig. 7).
Of the purified monoclonal antibodies, 2E4, which recognizes the N-terminal side of boPCT, was used as the detection antibody, so biotin labeling was performed using biotin labeling kit-NH2 (Dojin Kagaku) according to the instruction manual of the product. 100 μg of purified monoclonal antibody (2E4) was mixed with NH ₂ reactive biotin, allowed to stand at 37 ° C. for 10 minutes, and then recovered using 200 μl of WS buffer. The reactivity of the biotinylated monoclonal antibody was confirmed by detecting Trx-His-boPCT on the membrane with the biotinylated anti-boPCT antibody and avidin-HRP in Western blotting (Fig. 8).

(3-1. Sandwich ELISA method)
The purified anti-boPCT monoclonal antibody (1A8, C-terminal recognition) used as a supplemental antibody was diluted to a concentration of 6 μg / ml using a carbonate buffer (100 mM sodium carbonate, 100 mM sodium hydrogen carbonate, pH 8.7). The supplemental antibody was immobilized by adding 300 ng / well (50 μl / well) of the diluted antibody to a 96-well flat-bottomed plate and allowing it to stand at 4 ° C. overnight. After washing the plate once with PBS-T, PBS (-) containing 2% gelatin was added and the plate was allowed to stand at 37 ° C. for 30 minutes for blocking. Plates were washed 3 times with PBS-T, 50 μl / well purified Trx-His-boPCT or purified boPCT (Cusabio) of various concentrations were added and allowed to stand at 37 ° C. for 90 minutes. The plate was washed 3 times with PBS-T, 50 μl / well of biotinylated anti-boPCT monoclonal antibody (2E4, N-terminal recognition) diluted 1:50 with carbonate buffer was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. The plates were washed 3 times with PBS-T, 50 μl / well of avidin-HRP diluted 1: 2000 with carbonate buffer was added, and the plate was allowed to stand at 37 ° C. for 1 hour. After washing the plate with PBS-T three times, a color reaction was carried out using a TMB peroxidase EIA substrate kit (Bio-Rad), and the absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad).

The detection result of purified Trx-His-boPCT is shown in FIG. 9, and the detection result of purified boPCT is shown in FIG. As shown in FIGS. 9 and 10, the absorbance changed in correlation with the concentrations of purified Trx-His-boPCT and purified boPCT, and a calibration curve could be prepared. This result shows that bovine procalcitonin can be detected or quantified by the sandwich ELISA method using the antibody that recognizes the C-terminal side and the antibody that recognizes the N-terminal side produced by the hybridoma obtained in the above example. Is shown.

Claims (7)

An antibody or a combination thereof that is a combination of an antibody that specifically recognizes bovine procalcitonin or an antigen-binding fragment thereof , and comprises the following antibody (I) or an antigen-binding fragment thereof and an antibody (II) or an antigen-binding fragment thereof. Combination of antigen-binding fragments:
(I) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 26th to 96th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1, and is designated as Accession No. NITE P-02715, an independent administrative agency product evaluation technology. Antibodies produced by hybridomas deposited with the National Institute of Technology on May 16, 2018, or antigen-binding fragments thereof, or
(II) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 87th to 143rd amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1, and is any of the following (i) to (iv). Antigen binding fragment:
(I) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02711 on May 16, 2018.
(Ii) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02712 on May 16, 2018.
(Iii) An antibody or an antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02713 on May 16, 2018, or
(Iv) An antibody or antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02714 on May 16, 2018 . A kit for detecting bovine procalcitonin, which comprises a combination of the antibody according to claim 1 or an antigen-binding fragment thereof. The kit according to claim 2, for assisting in diagnosing the potential or prognostic condition of sepsis. NITE P-02715, NITE A-02711, NITE P-02712, NITE P-02713, or NITE P-02714, the Independent Administrative Institution Product Evaluation Technology Infrastructure Organization May 2018 Hybridoma deposited on the 16th of March. A method for detecting bovine procalcitonin contained in a sample.
(A) A step of contacting bovine procalcitonin contained in the sample with the following antibodies (I) and (II) or antigen-binding fragments thereof:
(I) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 26th to 96th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1, and is designated as Accession No. NITE P-02715, an independent administrative agency product evaluation technology. Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology on May 16, 2018,
(II) An antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the 87th to 143rd amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1, and is any of the following (i) to (iv). Antibodies or antigen-binding fragments thereof:
(I) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02711 on May 16, 2018.
(Ii) Antibodies or antigen-binding fragments thereof produced by hybridomas deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02712 on May 16, 2018.
(Iii) An antibody or an antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02713 on May 16, 2018, or
(Iv) An antibody or antigen-binding fragment thereof produced by a hybridoma deposited with the National Institute of Technology and Evaluation as Accession No. NITE P-02714 on May 16, 2018.
(A) A detection method comprising a step of detecting the binding of an antibody or an antigen-binding fragment thereof to bovine procalcitonin contained in the sample. The detection method according to claim 5 .
A detection method for detecting bovine procalcitonin by a sandwich ELISA method. A method of assisting in diagnosing the potential or prognostic condition of sepsis in a subject.
Using the detection method of claim 5 or 6 , the step of quantitatively measuring the procalcitonin concentration contained in the sample derived from the body fluid of the subject and the possibility or prognosis of sepsis based on the measured procalcitonin concentration value. A method that includes steps to assist in the diagnosis of the condition.

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