JP7205691B2 - Anti-canine procalcitonin antibody and test kit using the same - Google Patents

Description

The present invention relates to monoclonal antibodies or functional fragments thereof that bind to specific epitopes of canine procalcitonin (cPCT). The present invention also provides a polynucleotide encoding the variable region of the antibody or a functional fragment thereof, use of the antibody or a functional fragment thereof for measuring canine procalcitonin, a kit for measuring canine procalcitonin, and a canine The present invention relates to a test kit for sepsis or systemic inflammatory response syndrome.

Sepsis is an acute disease leading to death from shock, disseminated intravascular coagulation (DIC), multiple organ failure, etc., if left untreated. In addition to systemic inflammatory reactions caused by infectious diseases such as sepsis, similar systemic inflammatory reactions are induced by trauma, burns, pancreatitis, and the like. The systemic inflammatory response resulting from both infectious and non-infectious insults is called systemic inflammatory response syndrome (SIRS). Since these diseases progress rapidly, rapid diagnosis is required for their treatment.

In humans, it has been shown that the procalcitonin concentration in blood can evaluate the degree of severity and prognosis (or mortality risk) of sepsis and SIRS (Non-Patent Document 1). In fact, the US FDA has approved human blood procalcitonin as a marker capable of predicting the severity of sepsis.

Sepsis and SIRS are serious diseases requiring urgent treatment even in pets and livestock. In dogs and the like, sepsis and SIRS progress earlier than in humans, and there is a high demand for rapid diagnosis. In addition, the current diagnostic criteria for these diseases in dogs require direct diagnosis by a doctor of heart rate, body temperature, etc., and there are problems in clarifying the criteria and improving diagnostic throughput. Therefore, a method for determining the severity of sepsis in dogs by measuring procalcitonin as a marker for sepsis and SIRS has been proposed (Non-Patent Document 2).

On the other hand, these animals are often examined and treated at veterinary hospitals, and since the scale of these facilities is generally smaller than that for humans, it is required that the method of obtaining diagnostic information be simple. be done.

Harbarth S. et al. American Journal of Respiratory and Critical Care Medicine (2001), vol.164, p.396-402. Troia R et al. BMC Veterinary Research (2018) 14:111 (URL: https://doi.org/10.1186/s12917-018-1427-y)

Non-Patent Document 2 quantifies low concentrations of canine blood procalcitonin by ELISA. However, the antibody used is a polyclonal antibody, and deterioration in measurement sensitivity and variation may occur due to low antigen-binding ability and variation in quality between antibody lots.

The concentration of procalcitonin in the blood of dogs is as low as a fraction of that in humans, both when healthy (about 40 pg/mL) and when suffering from sepsis (about 100 pg/mL). The amount of change in density over time is also small. Therefore, the diagnosis of sepsis or SIRS in dogs requires the ability to quantify blood procalcitonin with higher sensitivity than in humans.

Therefore, the present invention provides a monoclonal antibody having high binding to canine procalcitonin contained in the blood of dogs at a low concentration, and furthermore, canine procalcitonin can be detected easily, rapidly and at a high level using the antibody. The aim is to provide a means to measure with sensitivity.

As a result of extensive studies, the present inventors found that administration of an N-terminal procalcitonin region or katacalcin region having an amino acid sequence unique to canine procalcitonin to an animal as an antigen results in a high binding property to canine procalcitonin. The present inventors have found that a monoclonal antibody having the

Specifically, the present invention provides the following anti-canine procalcitonin antibodies or functional fragments thereof.
A monoclonal antibody against canine procalcitonin or a functional fragment thereof, at least one of which is contained in the amino acid sequence of positions 26-47 (SEQ ID NO: 1) or the amino acid sequence of positions 121-130 (SEQ ID NO: 2) of canine procalcitonin An antibody or functional fragment thereof that binds to an epitope.

The anti-canine procalcitonin antibody or functional fragment thereof of the present invention preferably has a dissociation constant (K _D ) for canine procalcitonin of 2×10 ⁻⁹ M or less.

The anti-canine procalcitonin antibody or functional fragment thereof of the present invention may be labeled and/or tagged.

The present invention also provides a polynucleotide comprising a nucleotide sequence encoding the heavy chain and/or light chain variable region of an anti-canine procalcitonin antibody or a functional fragment thereof.

Furthermore, the present invention provides use of an anti-canine procalcitonin antibody or a functional fragment thereof for detecting or measuring canine procalcitonin. Among them, it is preferably used for measurement by immunochromatography or ELISA from the viewpoint of realizing rapid and highly sensitive measurement by utilizing the high binding property of the antibody.

Furthermore, the present invention provides a detection or measurement kit for canine procalcitonin containing an anti-canine procalcitonin antibody or a functional fragment thereof, and a test kit for canine sepsis or SIRS.

Since the antibody or functional fragment thereof of the present invention has a high binding property to canine procalcitonin, it is possible to detect or measure trace amounts of canine procalcitonin with high sensitivity.
Furthermore, the antibody or functional fragment thereof of the present invention can be applied to immunochromatographic methods. By such application, canine procalcitonin contained in blood or the like can be detected or measured, and can be used for examination of canine sepsis or SIRS.

4 is a graph showing the results of measuring 0 to 0.5 ng/mL canine procalcitonin by immunochromatography using an anti-canine procalcitonin monoclonal antibody. The vertical axis represents absorbance at a wavelength of 525 nm, and the horizontal axis represents canine procalcitonin concentration (ng/mL). 1 is a graph showing the results of measuring 0 to 100 pg/mL canine procalcitonin by immunochromatography using an anti-canine procalcitonin monoclonal antibody. The vertical axis represents absorbance at a wavelength of 525 nm, and the horizontal axis represents canine procalcitonin concentration (pg/mL). 1 is a graph showing the results of measurement of canine procalcitonin by ELISA using an anti-canine procalcitonin monoclonal antibody. The vertical axis represents absorbance at a wavelength of 450 nm, and the horizontal axis represents canine procalcitonin concentration (ng/mL).

[Anti-canine procalcitonin antibody and functional fragment thereof]
The anti-canine procalcitonin (cPCT) antibody of the present invention has an amino acid sequence at positions 26-47 of the cPCT N-terminal procalcitonin region (SEQ ID NO: 1) or an amino acid sequence at positions 121-130 of the katacalcin region (SEQ ID NO: 2). A monoclonal antibody that binds to at least one epitope included and has binding to cPCT.

(antigen)
Canine procalcitonin (cPCT), which is the antigen of the antibody of the present invention, is produced by cleaving the secretory signal peptide at positions 1-25 of precalcitonin (NCBI accession number: NP_001003266), a peptide consisting of 130 amino acid residues. , is a peptide consisting of 105 amino acid residues. In this specification, the residue number of cPCT is represented by the residue number of its precursor, precalcitonin. That is, the full cPCT length starts at position 26 and ends at position 130.

Positions 26-82 of cPCT are referred to herein as the "N-terminal procalcitonin region", positions 83-120 as the "calcitonin region", and positions 121-130 as the "Katacalcin region". Peptides containing the N-terminal procalcitonin domain and katacalcin domain are low in healthy individuals, but increase in inflammatory conditions such as sepsis and SIRS due to the secretion of procalcitonin into the blood. Further, canine calcitonin is a peptide consisting of positions 85 to 116 obtained by further processing after excision of the calcitonin region.

(monoclonal antibody)
Although the class of the monoclonal antibody of the present invention is not particularly limited, it is preferably an IgG class antibody.

The origin of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include rabbits, mice, rats, sheep, goats, cows, horses, and guinea pigs. be.

The monoclonal antibodies of the invention bind to full-length cPCT. On the other hand, the monoclonal antibody of the present invention is not particularly limited, but preferably does not bind to canine calcitonin from the viewpoint of detecting blood cPCT separately from canine calcitonin with high sensitivity. Although not particularly limited, the antibody of the present invention preferably does not bind to full-length human calcitonin.

A specific embodiment of the monoclonal antibody of the present invention has a heavy chain CDR1, sequence heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 4, heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 5, light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and SEQ ID NO: It is an antibody containing a light chain CDR3 consisting of an 8 amino acid sequence. Such an antibody (referred to herein as Ab-cPCT(26-47)Kabat) binds to at least one epitope contained in the amino acid sequence of positions 26-47 of the N-terminal procalcitonin region of cPCT.

A specific embodiment of the monoclonal antibody of the present invention has a heavy chain CDR1 in which the heavy and light chain variable regions consist of the amino acid sequence of SEQ ID NO: 9 as the CDRs defined by Kabat et al. heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 12, light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and the amino acid sequence of SEQ ID NO: 14 Antibodies containing light chain CDR3. Such an antibody (referred to herein as Ab-cPCT(121-130)Kabat) binds to at least one epitope contained in the amino acid sequence of positions 121-130 of the katacalcin region of cPCT.

In a specific embodiment of the monoclonal antibody of the present invention, the heavy and light chain variable regions consist of the heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16 as CDRs defined by IMGT. Heavy chain CDR2, heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 19, and light chain consisting of the amino acid sequence of SEQ ID NO: 20 Antibodies containing chain CDR3. Such an antibody (referred to herein as Ab-cPCT(26-47)IMGT) binds to at least one epitope contained in the amino acid sequence of positions 26-47 of the N-terminal procalcitonin region of cPCT.

In a specific embodiment of the monoclonal antibody of the present invention, the heavy and light chain variable regions consist of the heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22 as CDRs defined by IMGT. Heavy chain CDR2, heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:23, light chain CDR1 consisting of the amino acid sequence of SEQ ID NO:24, light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:25, and light chain consisting of the amino acid sequence of SEQ ID NO:26. Antibodies containing chain CDR3. Such an antibody (referred to herein as Ab-cPCT(121-130)IMGT) binds to at least one epitope contained in the amino acid sequence of positions 121-130 of the katacalcin region of cPCT.

As used herein, "CDRs defined by Kabat et al." refer to Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). , means the CDRs defined under NIH Publication 91-3242.
In addition, as used herein, "CDRs defined by IMGT" refer to Lefranc M.-P., Immunology Today 18, 509 (1997); Lefranc M.-P., The Immunologist, 7, 132-136 ( 1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003).

A specific embodiment of the monoclonal antibody of the invention is that the heavy and light chain variable regions are Ab-cPCT(26-47)Kabat, Ab-cPCT(121-130)Kabat, Ab-cPCT(26- 47) 1 or 2 amino acid residues among all amino acid residues in the set of heavy chain CDR1-3 and light chain CDR1-3 amino acid sequences of either IMGT or Ab-cPCT(121-130)IMGT is an antibody comprising heavy chain CDRs 1-3 and light chain CDRs 1-3 with substitutions, deletions or insertions and binding to cPCT . At this time, the amino acid residue to be substituted, deleted or inserted may be present in a single CDR or may be present in multiple CDRs.

A specific embodiment of the monoclonal antibody of the present invention is an antibody having the amino acid sequence of SEQ ID NO:27 for the heavy chain variable region and SEQ ID NO:28 for the light chain variable region.

A specific embodiment of the monoclonal antibody of the present invention is an antibody having the amino acid sequence of SEQ ID NO:29 for the variable region of the heavy chain and SEQ ID NO:30 for the variable region of the light chain.

The sequences of the framework region and the constant region in the variable region of the monoclonal antibody of the present invention are not particularly limited as long as they do not lose binding to cPCT, and examples thereof include humanized antibodies, mouse chimeric antibodies, human chimeric antibodies, and the like. may

(Functional fragment of monoclonal antibody)
The functional fragment of the monoclonal antibody of the present invention refers to an antibody fragment that has a combination of heavy chain CDRs 1-3 and light chain CDRs 1-3 of the above monoclonal antibody and has cPCT-binding ability.

The types of antibody fragments are not particularly limited, but examples include Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, ScFv fragments and the like. For reviews of these antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134 and Plueckthun, A., In: The Pharmacology of Monoclonal Antibodies, Vol. .), Springer-Verlag, New York (1994), pp. 269-315.

(Modification of antibody or functional fragment thereof)
From the viewpoint of detection of cPCT, the antibodies or functional fragments thereof of the present invention are preferably labeled with microparticles, fluorescent substances, enzymes, biotin, radiolabels, magnetic particles, and the like.

The microparticle is not particularly limited as long as it does not impair the antigen-binding property of the antibody or functional fragment thereof, and examples thereof include metal colloid particles such as gold colloid, colored particles such as colored latex particles, and the like.

The fluorescent substance is not particularly limited as long as it does not impair the antigen-binding property of the antibody or functional fragment thereof. , anthracene-based, phenanthrene-based, pyrene-based, carbazole-based, Cy-based, EvoBlue-based, fluorescein-based or derivatives thereof.

The enzyme is not particularly limited as long as it does not impair the antigen-binding property of the antibody or functional fragment thereof, and includes horseradish peroxidase (HRP), alkaline phosphatase, peroxidase, β-D-galactosidase, microperoxidase and the like.

Examples of radioactive labels include, but are not particularly limited to, ^3H , ^14C , ^15N , ^35S , ^90Y , ^99Tc , ^111In , ^125I , ^131I and the like.

The antibody or functional fragment thereof of the present invention can be tagged. Examples of such tags include FLAG, HA (hemagglutinin), GST (glutathione-S-transferase), Myc, polyhistidine (6×His tag, etc.) and the like. These tags can be used for antibody immobilization, purification, detection, and the like. Tagged antibodies or functional fragments are produced, for example, by adding a polynucleotide encoding a tag to a polynucleotide encoding the antibody of the present invention or a functional fragment thereof, and in vivo such as animal cells or It can be produced by introducing into an in vitro protein expression system such as a cell-free translation system.

(dissociation constant)
The dissociation constant (K _D ) of the antibody or functional fragment thereof for full-length cPCT of the present invention is preferably 2×10 ⁻⁹ M or less, more preferably 1.1×10, from the viewpoint of significantly exhibiting the effects of the present invention. ⁻⁹ M or less, more preferably 1×10 ⁻⁹ M or less, particularly preferably 5×10 ⁻¹⁰ M or less, most preferably 1×10 ⁻¹⁰ M or less. Such dissociation constants are measured by surface plasmon resonance (SPR). Specifically, for example, Biacore 3000 (registered trademark, manufactured by GE Healthcare) is used as an apparatus, and full-length canine procalcitonin with a 6×His tag attached to the N-terminus is used as an antigen and immobilized on a sensor chip using an amine coupling method. Measurements are performed using the phased material, using PBST (PBS with 0.005% Tween 20 added) as the running buffer, at a temperature of 25° C., and at a flow rate of 20 μL/min. The obtained data (sensorgram) was subjected to fitting based on a 1:1 Langmuir binding reaction model to determine the binding rate constant (k _a ) and the dissociation rate constant (k _d ), and from k _d / k _a A dissociation constant (K _D ) is obtained.

(Application)
The antibody or functional fragment thereof of the present invention is used for detection or measurement of cPCT, although not particularly limited.

As used herein, the term "detection" refers to qualitative discrimination to detect the presence or absence of an antigen. As used herein, the term "measurement" refers to determining the amount of antigen (relative quantification) or quantifying the absolute amount of antigen.

The cPCT to be detected or measured is not particularly limited, but is, for example, cPCT contained in canine cell tissue or biopsy material. The biopsy material is not particularly limited. serum, etc.), preferably blood or a isolate or extract thereof, and more preferably serum.

The method of detection or measurement is not particularly limited, and examples thereof include immunochromatography (lateral flow immunoassay, strip test) method, ELISA method, surface plasmon resonance method, latex agglutination method, immunodiffusion method, Western blot method, dot blot method. , immunoprecipitation method (Ouchterlony method, etc.), immunoelectrophoresis method (Mancini method), immunoelectrodiffusion method (Rocket method), etc., but from the viewpoint of significantly exhibiting the effects of the present invention, immunochromatographic method, An ELISA method or a surface plasmon resonance method, more preferably an immunochromatographic method or an ELISA method, and still more preferably an immunochromatographic method.

When the detection or measurement method is immunochromatography or ELISA, the anti-cPCT antibody or functional fragment thereof preferably contains two or more antibodies or functional fragments thereof that recognize different epitopes. Among them, one or more antibodies or functional fragments selected from the group consisting of Ab-cPCT(26-47) Kabat, Ab-cPCT(26-47) IMGT and functional fragments thereof, and Ab-cPCT(121 -130) Kabat, Ab-cPCT(121-130)IMGT, and one or more antibodies or functional fragments selected from the group consisting of functional fragments thereof, as labeled antibodies and antigen-capturing antibodies. preferable. Among them, from the viewpoint of increasing detection sensitivity, one or more antibodies selected from the group consisting of Ab-cPCT(26-47)Kabat, Ab-cPCT(26-47)IMGT and functional fragments thereof, or functionalities thereof The fragment is labeled antibody, Ab-cPCT (121-130) Kabat, Ab-cPCT (121-130) IMGT and one or more antibodies selected from the group consisting of functional fragments thereof or functional fragments thereof as antigens More preferred is the use of capture antibodies.

As used herein, a labeled antibody refers to a labeled antibody or functional fragment thereof. An antigen-capturing antibody refers to an antibody or functional fragment thereof immobilized on a solid phase and capable of binding to an antigen.

The antibody or functional fragment thereof of the present invention is not particularly limited, but can be used, for example, to test for canine sepsis or systemic inflammatory response syndrome, preferably for canine sepsis.

In the measurement of cPCT using the antibody of the present invention or a functional fragment thereof, the lower limit of the measurable concentration of cPCT is not particularly limited. From the viewpoint of use in testing for reaction syndrome, it is preferably 0.05 ng/mL or less, more preferably 0.03 ng/mL or less, still more preferably 0.02 ng/mL or less, and particularly preferably 0.01 ng/mL or less. is.

(Production method)
Antibodies of the present invention are produced from hybridomas derived from B lymphocyte cells obtained by immunizing an animal with a peptide containing the amino acid sequence of positions 26-47 or a peptide containing the amino acid sequence of positions 121-130 in cPCT, for example. can do.

Animals used for immunization are not particularly limited, and examples thereof include rabbits, mice, rats, sheep, goats, cows, horses, guinea pigs, etc. Rabbits are preferred.

In addition, the antibody or functional fragment thereof of the present invention is not particularly limited to a polynucleotide containing a nucleotide sequence encoding the variable region of the antibody described below. cells (COS), HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, human hepatocellular carcinoma cells (e.g., Hep G2), non-animal eukaryotic cells (insect cells, etc.), bacteria ( Escherichia coli, etc.), yeast, insects, plants, transgenic animals (cattle, chicken, etc.), or in vitro translation systems (eukaryote-derived, E. coli-derived cell-free translation systems, etc.) to express antibodies. can do. In producing rabbit-derived antibodies, mouse chimeric antibodies, human chimeric antibodies, humanized antibodies, or functional fragments thereof, it is preferable to produce antibodies by such polynucleotide introduction.

[Polynucleotide]
The polynucleotide of the present invention comprises a base sequence encoding the heavy chain and/or light chain variable region of the above monoclonal antibody. Polynucleotides of the present invention are preferably DNA or mRNA.

As a particular embodiment of the polynucleotide of the invention, the heavy chain variable region comprising the heavy chain CDRs 1-3 of Ab-cPCT(26-47)Kabat and/or the light chain CDRs 1-3 of Ab-cPCT(26-47)Kabat and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region containing 3.
As a particular embodiment of the polynucleotide of the invention, the heavy chain variable region comprising the heavy chain CDRs 1-3 of Ab-cPCT(121-130)Kabat and/or the light chain CDRs 1-3 of Ab-cPCT(121-130)Kabat and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region containing 3.
As a particular embodiment of the polynucleotide of the invention, the heavy chain variable region comprising heavy chain CDRs 1-3 of Ab-cPCT(26-47)IMGT and/or the light chain CDRs 1-3 of Ab-cPCT(26-47)IMGT and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region containing 3.
As a particular embodiment of the polynucleotide of the invention, the heavy chain variable region comprising heavy chain CDRs 1-3 of Ab-cPCT(121-130)IMGT and/or the light chain CDRs 1-3 of Ab-cPCT(121-130)IMGT and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region containing 3.

The polynucleotide of the present invention is reversed from B lymphocyte cell-derived mRNA obtained by immunizing an animal with a peptide containing the amino acid sequence of positions 26-47 or a peptide containing the amino acid sequence of positions 121-130 in cPCT, for example. You can get a copy. Alternatively, it can be obtained by known gene recombination techniques such as chemical synthesis, cleavage, ligation, and PCR.

The polynucleotide of the present invention can be used for producing the above antibody or functional fragment thereof. In such uses, the polynucleotides of the present invention are selected from regulatory sequences (transcriptional or translational regulatory sequences, promoters, enhancers, ribosome binding sites, etc.), replication origins, and selectable genes (antibiotic resistance genes, thymidine kinase genes, etc.). It preferably contains one or more elements that

[Kit containing anti-canine procalcitonin antibody or functional fragment thereof]
The kit of the present invention contains an anti-canine procalcitonin antibody or a functional fragment thereof, and is used for detecting or measuring canine procalcitonin.

Targets for detection or measurement of cPCT, features of detection or measurement, etc., and aspects of preferred antibodies or functional fragments are as described in the section [Anti-canine procalcitonin antibodies and functional fragments thereof].

The kit of the present invention is not particularly limited, and can be appropriately selected depending on the detection or measurement method used. It preferably includes instruments and/or reagents.

When the detection or measurement method is an immunochromatographic method or an ELISA method, the instrument for measurement preferably contains a solid-phase substrate carrying the capture antibody.

When the detection or measurement method is an immunochromatography method or an ELISA method, the labeled antibody may be contained in the measurement instrument, or may be contained in, for example, a sample tube or the like separately from the measurement instrument.

In the embodiment of the immunochromatography kit, the instrument for measurement is not particularly limited, but preferably contains a water-absorbent solid-phase substrate on which one or more anti-canine procalcitonin antibodies or functional fragments thereof are immobilized. . Examples of materials for the solid-phase substrate include nitrocellulose. The solid-phase substrate is not particularly limited, but is preferably accommodated in a cartridge having an opening for sample injection, such as the case disclosed in Japanese Patent Application Laid-Open No. 2014-190926.

In the embodiment of the ELISA kit, the instrument for measurement preferably includes, for example, a plate (eg, 96-well plate) coated with an anti-cPCT antibody or functional fragment thereof. In addition, although not particularly limited, it preferably contains a washing buffer, a plate seal, and the like.

In addition to the above anti-cPCT antibodies and functional fragments thereof, the kit of the present invention contains, as other reagents, water, cPCT (including recombinant proteins), pH buffer components, surfactants, salts (sodium chloride, potassium chloride, etc.), sugars (sucrose, etc.), blocking agents (bovine serum albumin (BSA), casein, serum, etc.), substrates for color development or fluorescence detection, and the like. These components are preferably provided in liquid (aqueous) form.

Examples of pH buffer components include, but are not limited to, acids such as phosphoric acid, boric acid, acetic acid, citric acid, formic acid, and cacodylic acid, or salts thereof; amino acids such as glycine; trishydroxyaminomethane (Tris); , and good buffers such as MES.

Although the surfactant is not particularly limited, nonionic surfactants are preferred, and any of ester ether type, ester type and ether type can be used. More specifically Tween 20, Tween 40, Tween 80, Triton-X100, polyoxyethylene (60) sorbitan monostearate, polyoxyethylene (65) sorbitan tristearate, polyoxyethylene (80) sorbitan monooleate, polyoxyethylene Ethylene alkylphenyl ether, Nonidet P-40, CHAPS and the like. Any of these surfactants may be used alone, or two or more thereof may be used in combination.

The kit of the present invention can contain, for example, a dropper, a syringe, a tube with a cap, etc., as instruments other than the instrument for measurement.

(use, other)
The kit of the present invention can be used for detecting or measuring cPCT, and testing for canine sepsis or systemic inflammatory response syndrome.

Other specific embodiments of the kit of the present invention are as described in the use section of the above section [Anti-canine procalcitonin antibody and functional fragment thereof].

The present invention will be described in more detail below using examples. However, these examples do not limit the invention.

[Example 1. Preparation of anti-canine procalcitonin antibody]
(Preparation of antigen)
As shown in Table 1, as immunostimulatory antigens, a peptide (cPCT(26-47)) having the amino acid sequence at positions 26-47 of cPCT (SEQ ID NO: 1) and an amino acid sequence at positions 121-130 (sequence Peptides with number 2) (cPCT(121-130)) were prepared by peptide synthesis with purities of 81.9% and 86.5%, respectively. Peptide synthesis was outsourced to Cell Engineering Laboratory Co., Ltd. In addition, antigens for evaluating binding properties were prepared by adding 6×His tags to the N-terminus or C-terminus of each of them, expressing them as recombinant proteins in animal cells, and purifying them with Ni columns. These antigens are called N-His-cPCT and C-His-cPCT.

For cPCT(26-47) and cPCT(121-130), keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) as a carrier protein and MBS (m-maleimidobenzoyl-N-hydroxy sulfosuccinimide ester) were reacted and coupled.

(Immunization of animals)
A mixture of KLH-conjugated cPCT (26-47) or KLH-conjugated cPCT (121-130) and an adjuvant (manufactured by Difco, product code 263810) at a volume ratio of 1:1 was administered to rabbits (Slc:JW/CSK, 13 weeks old). ) Each animal received 2.5 mg at the start (after day 0). Furthermore, as a booster antigen, the same antigen peptide as at the start was mixed with an adjuvant (Funakoshi: CYT, G-1) at a volume ratio of 1:1. immunized. After 46 days, blood was collected from the rabbit, and serum and lymphocytes were collected in order to confirm that the antibody titer had increased. Lymphocytes were cultured and the culture supernatant was separated. Antibody titers of the obtained sera and culture supernatants were measured by ELISA using plates on which BSA-bound cPCT(26-47) or BSA-bound cPCT(121-130) were immobilized.

(Isolation of positive cells and isolation of antibody genes)
Lymphocytes derived from the spleen of rabbits in which an increase in antibody titer was confirmed were obtained. Lymphocytes diluted with RPMI medium (10% FBS) were added to the cPCT(26-47) or cPCT(121-130)-immobilized Single-cell ELISA plate so that each well contained only one cell. , incubated for 45 minutes. Positive clones were identified by detecting anti-cPCT antibody bound to the antigen on the plate with a labeled anti-rabbit IgG antibody. 2×10 ⁵ lymphocytes obtained by immunization with cPCT(26-47) and cPCT(121-130) as antigens were screened to obtain 15 and 13 positive clones, respectively. From the positive clones obtained, the IgG heavy chain and light chain genes of each cell were amplified using the PCR method.

(Confirmation of antibody production and antigen specificity in HEK293 cells)
A CAG promoter was added to the obtained heavy chain and light chain genes of each cell using PCR. The resulting polynucleotides containing heavy and light chain genes were introduced into HEK293 cells and cultured in RPMI medium for 3 days. Then, the culture supernatant containing the monoclonal antibody was collected.

The anti-canine procalcitonin antibody titer in the medium supernatant of each clone obtained was measured by ELISA. ELISA plates coated with cPCT, N-His-cPCT, or C-His-cPCT were used as plates, and an anti-rabbit IgG antibody was used as a secondary antibody.

As a result, 4 clones of monoclonal antibodies using cPCT(26-47) as antigens and 9 clones of monoclonal antibodies using cPCT(120-130) as antigens were obtained. All had binding properties to cPCT, N-His-cPCT and C-His-cPCT.

[Example 2. Verification of the applicability of antibodies to immunochromatography (IC)]
Phosphate buffers were prepared by adding N-His-cPCT or C-His-cPCT to 1, 0.1, or 0.01 mg/mL. The resulting solution was linearly applied to each immunochromatography (IC) strip (25 mm × 5 mm, manufactured by Millipore) at a position 10 mm from the edge using an antibody coating machine (manufactured by Musashi Engineering). , and dried under reduced pressure for 90 minutes to form a solid phase.

The culture supernatant of HEK293 cells corresponding to the positive clones finally obtained in Example 1 (4 clones of N-terminal procalcitonin region-binding antibodies and 9 clones of katacalcin region-binding antibodies, total 13 clones) was labeled with colloidal gold. A sample was prepared by adding a mouse-derived anti-rabbit IgG antibody (manufactured by Cell Engineering Laboratories) to a concentration of 2 μg/mL. This results in a colloidal gold-labeled form of the cPCT-binding monoclonal antibody.

The end of the strip coated with each concentration of antigen was immersed in a sample containing one of the 13 types of antibodies described above, and allowed to absorb the sample for about 30 minutes. Strips coated with C-His-cPCT were used for N-terminal procalcitonin region-binding antibodies, and strips coated with N-His-cPCT were used for katacalcin region-binding antibodies. As a control, the strips were soaked with medium without antibody. After drying the strip, the purple coloration derived from colloidal gold aggregating at the antigen-applied portion was visually evaluated. Tables 2 and 3 show the results. ELISA measurements obtained in Example 1 using N-His-cPCT or C-His-PCT as an antigen are also shown.

All four monoclonal antibodies that bind to the N-terminal procalcitonin domain and five out of nine monoclonal antibodies that bind to the katacalcin domain were detectable at an antigen concentration of 0.01 mg/mL. In addition, it was found that the magnitude of the measured value by ELISA and the evaluation by immunochromatography do not necessarily correlate. That is, for antibodies suitable for immunochromatography, in addition to antigen binding strength, other factors such as interaction with strips and antibody stability are presumed to affect analytical sensitivity.

[Example 3. Application of monoclonal antibody to immunochromatography]
cPCT(26-47)-binding clone No.4 antibody obtained above (hereinafter referred to as RAb-cPCT(26-47)-4) and cPCT(121-130)-binding clone No.6 antibody (hereinafter referred to as RAb-cPCT (120-131)-6) was labeled with colloidal gold by physical adsorption.

A phosphate buffer containing 0.5 mg/mL RAb-cPCT(120-131)-6 as an antigen-capturing antibody was prepared. Like the band-shaped layer 15 in FIG. 3 of Japanese Patent Application Laid-Open No. 2014-190926, at a position 10 mm from the first end of a nitrocellulose membrane having a width of 25 mm and a length of 5 mm, using an antibody coating machine A buffer solution containing this antibody was linearly applied at a coating amount of 26 μL/cm and dried at 55° C. for 30 minutes. Next, on the first end of the membrane, as a conjugate pad, a 5 mm wide by 5 mm long strip pre-soaked with 0.16 μg of colloidal gold-labeled RAb-cPCT(26-47)-4 and dried. A glass fiber pad (manufactured by Millipore) was installed. Further, a glass fiber sample pad (width 5 mm x length 20 mm, manufactured by Millipore) was overlaid on the conjugate pad for impregnating the sample. These were sandwiched in a case provided with two openings (a sample injection port and an observation window) as shown in FIG. 4 of Japanese Patent Application Laid-Open No. 2014-190926 to produce a test device. At this time, the antibody-applied portion was exposed in one opening, and the sample pad was exposed in the other opening. In addition, a test device was similarly prepared in which the combination of the labeled antibody and the antigen-capturing antibody was reversed.

Normal beagle pooled serum containing 0.5, 0.25, 0.125, 0.0625, 0.0312, 0 ng/mL of N-His-cPCT was prepared and 100 μL was dropped into the sample inlet of the test device. After 15 minutes had passed, the coloration of the zonal layer portion was quantified by measuring the absorbance at a wavelength of 525 nm using a point reader (registered trademark, manufactured by Ushio Inc.).

In addition, normal beagle dog pooled sera containing 100, 50, 25, 12.5 and 0 pg/mL of N-His-cPCT were prepared, subjected to immunochromatography in the same manner as above, and quantified by measuring absorbance at 525 nm.

Figures 1 and 2 show the measurement results when RAb-cPCT(120-131)-6 was used as the antigen-capturing antibody and gold colloid-labeled RAb-cPCT(26-47)-4 was used as the labeled antibody. Even at an antigen concentration of 12.5 pg/mL, the measured value was sufficiently higher than the background. Although quantification was observed over the entire antigen concentration measured, it was shown to be highly linear with absorbance, especially between antigen concentrations of 0 to 0.125 ng/mL. From the above, it was shown that low-concentration cPCT can be measured by immunochromatography.

When the combination of antigen-capturing antibody and labeling antibody was reversed, the sensitivity was lower than in FIGS. That is, it was shown that the detection sensitivity of cPCT is increased by using an antibody that binds to the katacalcin region as the antigen-labeled antibody and an antibody that binds to the N-terminal procalcitonin region as the labeled antibody.

[Example 4. Application of monoclonal antibody to ELISA]
An ELISA plate was prepared using the antibodies obtained in the above test. 50 μL of phosphate buffer (3 μg/mL) containing RAb-cPCT(120-131)-6 was used to coat the bottom of a 96-well plate. After that, blocking was performed.

As a secondary antibody, RAb-cPCT(26-47)-4 was labeled with HRP using Peroxidase Labeling Kit-SH (manufactured by Dojindo Laboratories).

50 μL of phosphate buffer containing 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0 ng/mL of N-His-cPCT was added to each well of the obtained plate and incubated for 60 minutes to bind the antigen. After washing according to the manual, 50 μL of secondary antibody solution (0.03 μg/mL) was added and incubated for 60 minutes. After washing, a detection reagent was added according to the manual, and absorbance at a wavelength of 450 nm was measured.

The results are shown in FIG. Absorbance sufficiently higher than the background was measured even at an antigen concentration of 0.0312 ng/mL. Furthermore, quantification was observed down to 0.25 ng/mL.

[Example 5. Sequencing of antibody genes]
Of the anti-cPCT monoclonal antibodies obtained, the nucleotide sequences of the heavy and light chain genes of RAb-cPCT(26-47)-4 and RAb-cPCT(120-131)-6 were determined. The amino acid sequences of the heavy and light chain variable regions were obtained from the obtained base sequences of the coding regions. The heavy chain variable region of RAb-cPCT(26-47)-4 was SEQ ID NO:27, and the amino acid sequence of the light chain variable region was SEQ ID NO:28. RAb-cPCT(120-131)-6 had the amino acid sequence of SEQ ID NO: 29 for the heavy chain variable region and SEQ ID NO: 30 for the light chain variable region. Table 4 shows CDRs defined by Kabat et al. and CDRs defined by IMGT obtained from the amino acid sequences of the respective variable regions.

[Example 6. Determination of dissociation constant of antibody for canine procalcitonin]
The dissociation constants of RAb-cPCT(26-47)-4 and RAb-cPCT(120-131)-6 were determined by surface plasmon resonance (SPR) method. Biacore3000 (manufactured by GE Healthcare) was used as an apparatus. 1190 RU of N-His-c-PC was immobilized on a sensor chip (Sensor Chip CM5 (manufactured by GE Healthcare)) by the amine coupling method and blocked with 1.0 M ethanolamine-HCl (pH 8.5). PBST (0.005% Tween 20 in PBS) was used as the running buffer, and the antibody was serially diluted from 50 nM to 1/2 in the running buffer (50, 25, 12.5, 6.13, 3.13, 1.56, 0.781). A solution obtained by diluting RAb-cPCT(26-47)-4 or RAb-cPCT(120-131)-6 with nM) was used as a sample. A sample was flowed at a flow rate of 20 μL/min and subjected to SPR to acquire a sensorgram. From the obtained sensorgrams, the binding rate constant (k _a ) and the dissociation rate constant (k _d ) were obtained using fitting with a 1:1 Langmuir Binding model, and k _d /k _a was taken as the dissociation constant (K _D ). bottom.

As a result, the dissociation constant of RAb-cPCT(26-47)-4 is 2.82 × 10^-11 M (k_a=2.74×10^FiveM.^-1・s^-1, k_d=7.74×10^-6 s^-1), The dissociation constant of RAb-cPCT(120-131)-6 is 1.07×10^-9 M (k_a=1.20×10^Five M.^-1・s^-1, k_d=1.29×10^-Four s^-1)Met.

(SEQ ID NO: 27: RAb-cPCT(26-47)-4 heavy chain variable region; amino acid sequence)
QSVEESGGRLVTPGTPLTLTCTVSGFSLNRYAINWVRQAPGKGLEWIAYISIRGGSSYASWAKGRFTISKTSTTVDLRITSPTTEDTATYFCVRGPTYQIEDIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

(SEQ ID NO: 28: RAb-cPCT(26-47)-4 light chain variable region; amino acid sequence)
DPVLTQTPSSVSAALGGTVTINCQASQSLYNNNFLSWYQQKPGQPPKLLIYQASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGGIDIDGWYFGFGGGTEVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRG

(SEQ ID NO: 29: RAb-cPCT(120-131)-6 heavy chain variable region; amino acid sequence)
QSLEESGGDLVKPGASLTLTCTASGFSFSSSYWAYWVRQAPGKGLEWIGCIVTDTGTPYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRGLLIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

(SEQ ID NO: 30: RAb-cPCT(120-131)-6 light chain variable region; amino acid sequence)
AQVLTQTPSPVSAAVGGTVTINCQSSQSVFGGNYLAWYQQKPGQPPKQLIYDASTLASGVPSRFEGSGSGTQFTLTISGVQCDDAATYYCLGGFSCNSADCNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRG

Claims (12)

A monoclonal antibody against canine procalcitonin or an antigen-binding fragment thereof, which binds to at least one epitope contained in the amino acid sequence of positions 26-47 of canine procalcitonin (SEQ ID NO: 1);
The variable regions of the heavy and light chains are
(A) having the following CDRs as defined by Kabat et al.
a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:3;
a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:4;
a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:5;
a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO:6;
a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and
a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:8; or
(B) having the following CDRs as CDRs defined by IMGT:
a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 15;
a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 16;
a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 17;
a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 18;
a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 19, and
a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:20;
Antibodies or antigen-binding fragments thereof. 2. The antibody or antigen-binding fragment thereof according to claim 1 , which is labeled or tagged. A monoclonal antibody against canine procalcitonin or an antigen-binding fragment thereof, which binds to at least one epitope contained in the amino acid sequence of positions 121-130 of canine procalcitonin (SEQ ID NO: 2);
The variable regions of the heavy and light chains are
(C) having the following CDRs as defined by Kabat et al.
a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:9;
a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 10;
a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 11;
a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 12;
a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 14; or
(D) having the following CDRs as CDRs defined by IMGT:
a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 21;
a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 22;
a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 23;
a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO:24;
a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 26;
Antibodies or antigen-binding fragments thereof. 4. The antibody or antigen-binding fragment thereof according to claim 3 , which is labeled or tagged. A polynucleotide comprising a base sequence encoding the variable region of the heavy chain and/or light chain of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 . Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for detecting or measuring canine procalcitonin in canine cell tissue or biopsy material. 7. Use according to claim 6 , characterized in that said detection or measurement is performed by immunochromatography. A kit for detecting or measuring canine procalcitonin, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 . A test kit for canine sepsis or systemic inflammatory response syndrome, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 . 10. A kit according to claim 8 or 9 , comprising two or more antibodies recognizing different epitopes. The kit according to any one of claims 8 to 10 , which is an immunochromatography kit. The kit according to any one of claims 8 to 11 , wherein the lower limit of measurable canine procalcitonin concentration is 0.05 ng/mL or less.

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