WO2023101447A1 - Biomarker for diagnosis of pancreatitis in companion dog - Google Patents

Abstract

The present invention relates to a composition for diagnosis of canine pancreatitis. Leading to the present invention, a discovery was made of a biomarker capable of diagnosing pancreatitis with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins. Specifically, the present inventors performed proteomic analysis using high-resolution mass spectrometry on plasma proteins of dogs suffering from pancreatitis, and selected biomarkers showing significant expression differences compared to normal controls. It was observed that the pancreatitis diagnostic model established using the selected biomarker can diagnose pancreatitis with high specificity, sensitivity, and accuracy. Therefore, as a non-invasive method using a small amount of blood, which enables prediction and early diagnosis of the risk of pancreatitis in dogs, the present invention is expected to preserve the health of dogs, reduce the burden of owner medical expenses, and contribute to the improvement of the quality of veterinary consulting.

Description

Biomarkers for diagnosis of pancreatitis in dogs

The present invention relates to a biomarker for diagnosing canine pancreatitis and a method for diagnosing canine pancreatitis using the same.

The present invention claims priority based on Korean Patent Application No. 10-2021-0170185 filed on December 1, 2021 and Korean Patent Application No. 10-2022-0164050 filed on November 30, 2022, All contents disclosed in the specification and drawings of the applications are incorporated into this application.

As of 2019, among households raising companion animals in Korea, 39.3% of households raising dogs aged 6 years or older and 18% raising dogs aged 10 years or older accounted for 18%. Interest in therapies is growing rapidly. In particular, for incurable diseases, early diagnosis of diseases is important not only for extending the health life span of companion animals, but also for reducing guardians' medical expenses. In addition, in order to extend the healthy lifespan of companion animals, it is necessary to continuously develop technologies to treat or alleviate the progress of intractable diseases.

Pancreatitis is a disease in which pancreatic secretory glands are destroyed by pancreatic enzymes or the like, or inflammation occurs locally or entirely in the pancreas. Pancreatitis is a disease commonly observed in humans as well as other animals including dogs. Pancreatitis is divided into acute and chronic pancreatitis. Acute pancreatitis occurs when the exocrine function of the pancreas is impaired, digestive enzymes are activated, and this attacks the pancreas and surrounding tissues, causing severe inflammation. Dogs suffering from acute pancreatitis cause vomiting, diarrhea, abdominal pain, etc., and severe symptoms such as bloody stool can lead to a poor prognosis, but if detected early and actively treated, the survival rate can be increased. Chronic pancreatitis is characterized by impairment of both exocrine and endocrine functions due to chronic inflammation. It is accompanied by mild vomiting and diarrhea, and the condition may improve or worsen repeatedly. Canine pancreatitis has a mortality rate of about 40%, which is very high, and can cause complications such as diabetes, so timely treatment is very important.

As a method for diagnosing acute pancreatitis in dogs, measurement of serum amylase or lipase concentration is mainly used, but since the level of these enzymes tends to be increased in other diseases, there is a problem of inaccuracy in diagnosis. Therefore, in order to extend the healthy lifespan of dogs and reduce the burden of treatment costs on guardians, there is a need for a method capable of diagnosing or screening for pancreatitis in dogs in a simpler and non-invasive way. However, a non-invasive method capable of diagnosing canine pancreatitis with high accuracy has not yet been developed.

The present invention was made to solve the above problems, and was completed by discovering a biomarker capable of diagnosing pancreatitis in dogs with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins.

Accordingly, an object of the present invention is to provide a composition for diagnosing canine pancreatitis, which contains, as an active ingredient, an agent for measuring protein or mRNA levels of the biomarkers.

Another object of the present invention is to provide a kit for diagnosing canine pancreatitis, including an agent for measuring protein or mRNA levels of the biomarkers.

Another object of the present invention relates to a method for diagnosing canine pancreatitis by measuring protein or mRNA levels of the biomarkers.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

The present invention relates to a dog product comprising, as an active ingredient, an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG (Fibrinogen gamma chain). A composition for diagnosing pancreatitis is provided.

In one embodiment of the present invention, the protein or mRNA level of SERPINA3 may be increased in pancreatitis subjects compared to normal subjects, but is not limited thereto.

In another embodiment of the present invention, the protein or mRNA level of the PDE4DIP or FGG may be reduced in pancreatitis subjects compared to normal subjects, but is not limited thereto.

In another embodiment of the present invention, the composition comprises Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B) , Complement C3 (LOC484960), and may further include an agent for measuring the level of at least one protein or mRNA selected from the group consisting of Complement factor I, but is not limited thereto.

In another embodiment of the present invention, the agent for measuring the protein level may be an antibody or an aptamer specific to the protein, but is not limited thereto.

In another embodiment of the present invention, the agent for measuring the mRNA expression level may be a primer set or a probe that specifically binds to the mRNA, but is not limited thereto.

In another embodiment of the present invention, the protein or mRNA level may be at least one level selected from the group consisting of blood, whole blood, plasma, serum, and lymph, but is not limited thereto.

In addition, the present invention provides a kit for diagnosing canine pancreatitis, comprising the agent or composition according to the present invention.

In addition, the present invention provides a method for diagnosing canine pancreatitis, comprising the following steps:

(S1) measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject; and

(S2) the protein or mRNA level of SERPINA3 measured in step (S1) is increased compared to the control group; Diagnosing pancreatitis or determining that there is a risk of developing pancreatitis when the protein or mRNA level of PDE4DIP or FGG is reduced compared to the control group.

In one embodiment of the present invention, the method comprises Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, A step of measuring the level of at least one protein or mRNA selected from the group consisting of Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960), and Complement factor I may be further included, but is not limited thereto.

In another embodiment of the present invention, the biological sample may be at least one selected from the group consisting of blood, whole blood, plasma, serum, and lymph, but is not limited thereto.

In another embodiment of the present invention, the protein level is measured by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) assay, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoretic assay, liquid chromatography-mass At least one method selected from the group consisting of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS), western blotting, enzyme linked immunosorbentassay (ELISA), and FACS It may be measured as, but is not limited thereto.

In another embodiment of the present invention, the mRNA level is measured from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chip, and RNA chip. It may be measured by one or more selected methods, but is not limited thereto.

In addition, the present invention is an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG, for preparing an agent or kit for diagnosing canine pancreatitis. provides the use of

In addition, the present invention provides the use of an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG for the diagnosis of canine pancreatitis. .

The present invention relates to a composition for diagnosing canine pancreatitis, and was completed by discovering a biomarker capable of diagnosing pancreatitis with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins. Specifically, the present inventors performed proteomic analysis using high-resolution mass spectrometry on plasma proteins of dogs suffering from pancreatitis, and selected biomarkers showing significant expression differences compared to normal controls. It was confirmed that the pancreatitis diagnostic model established using the selected biomarker can diagnose pancreatitis with high specificity, sensitivity, and accuracy. Therefore, the present invention is a non-invasive method using a small amount of blood, which enables prediction and early diagnosis of the risk of pancreatitis in dogs, thereby preserving the health of dogs, reducing the burden of guardians' medical expenses, and contributing to the improvement of the quality of veterinary consulting. It is expected that

1a is a diagram illustrating the principle of removing the top 14 abundant plasma proteins using a MARS14 column.

Figure 1b shows the result of pre-processing the top 14 abundant plasma proteins and trace proteins and confirming the UV spectrum.

Figure 1c is a flow chart showing the process of preparing plasma proteins into peptides by the suspension trap method.

1d is a diagram showing parameters used in SWATCH LC-MS analysis.

2 is a result of performing quantitative analysis using a nano drop on plasma samples separated from plasma samples of companion dogs.

3 is a graph showing the number of proteins quantified in canine plasma samples.

Figure 4 is a Volcano plot showing increased or decreased proteins in pancreatitis subjects compared to normal controls.

5 is a table showing information on 16 proteins increased or decreased in pancreatitis subjects compared to normal controls.

6a and 6b are diagrams showing box plots and ROC curves of SERPINA3 (FIG. 6a) and Angiotensinogen (FIG. 6b), which are proteins increased in pancreatitis subjects compared to normal controls.

7 shows PLS-DA results using 15 proteins showing differential expression levels in subjects with pancreatitis compared to normal controls.

8 is a result of screening 23 proteins showing differential expression levels in a normal control group, a chronic pancreatitis group, and an acute pancreatitis group using the Kruskal Wallis test.

9 is a box plot comparing the expression levels of Angiotensinogen, SERPINA3, and PDE4DIP in a normal control group, a chronic pancreatitis group, and an acute pancreatitis group (from the left, in order, expression in the acute pancreatitis group, chronic pancreatitis group, and normal group). represents).

10 shows the results of AUC, specificity, and sensitivity analysis in a logistic regression model using three proteins of PDE4DIP, FGG, and SERPINA3.

The present invention relates to a composition for diagnosing canine pancreatitis, and was completed by discovering a biomarker capable of diagnosing pancreatitis in dogs with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins. Specifically, the present inventors performed quantitative and qualitative analysis on plasma proteins of normal and diseased dogs through high-resolution mass spectrometry, and through this, biomarkers showing significant differences between individuals were selected. In addition, it was confirmed that the diagnostic model established by combining the above biomarkers can distinguish between diseased individuals and normal individuals with high specificity, sensitivity, and accuracy. Therefore, through the above biomarkers and their combination, it is not only possible to accurately diagnose pancreatitis in dogs, but also to easily screen the risk of pancreatitis even in normal times, thereby extending the life span and reducing the guardian's medical expenses through early diagnosis of pancreatitis in dogs. Expect to be able to do it. In particular, the diagnosis method according to the present invention is an alternative to conventional invasive diagnosis and examination methods, and has the advantage of being able to diagnose diseases through a relatively simple and economical method of blood sampling.

Therefore, the main object of the present invention is to provide an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG as an active ingredient, It is to provide a composition for diagnosing pancreatitis in an individual.

In the present invention, "subject" means a subject requiring risk prediction, diagnosis, prognosis prediction, or treatment of a disease, and more specifically, non-human primates, mice, rats, dogs , It may mean mammals such as cats, horses, and cows, but preferably means dogs (dogs; scientific name, Canis lupus familiaris).

In the present invention, pancreatitis is a disease in which pancreatic secretory glands are destroyed by pancreatic enzymes or the like, or inflammation occurs locally or entirely in the pancreas. Pancreatitis according to the present invention includes both acute pancreatitis and chronic pancreatitis. Acute pancreatitis occurs when the exocrine function of the pancreas is impaired, digestive enzymes are activated, and this attacks the pancreas and surrounding tissues, causing severe inflammation. Dogs suffering from acute pancreatitis cause vomiting, diarrhea, abdominal pain, and severe symptoms such as bloody stools. The prognosis may be poor, but early detection and active treatment can increase the survival rate, so early diagnosis is more important than anything else. do. Chronic pancreatitis is characterized by impairment of both exocrine and endocrine functions due to chronic inflammation. It is accompanied by mild vomiting and diarrhea, and the condition may improve or worsen repeatedly. Since pancreatitis has no clearly identified cause, it is important to screen for the risk of developing it or diagnose it early.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological state, and confirming the presence or absence of pancreatitis, whether pancreatitis has occurred, the possibility of developing pancreatitis (risk of occurrence), the prognosis of pancreatitis, etc. It can mean including.

More specifically, the term "diagnosis" used in the present invention refers to determining the susceptibility of a subject to a specific disease or disorder, and determining whether a subject currently has a specific disease or disorder. , determining the prognosis of a subject suffering from a particular disease or condition (e.g., identifying tumor status, determining the staging of a tumor, or determining the responsiveness of a cancer to treatment), or therametrics (e.g., treatment monitoring the state of the object to provide information about efficacy).

PDE4DIP (phosphodiesterase 4D interacting protein) is a protein that serves to fix phosphodiesterase 4D to the Golgi/centrosome region of cells. In humans, abnormal PDE4DIP induces myeloproliferative disorders associated with eosinophilia and the like, and is known to be associated with adult pineoblastoma and adult pineal parenchymal tumor. However, there have been no reports of canine pancreatitis and PDE4DIP to date. The present inventors confirmed that the level of PDE4DIP was significantly reduced in the pancreatitis group compared to the normal group through the canine plasma proteomic analysis. Specific information on PDE4DIP can be found in public databases such as Uniprot (Uniprot registration number, A0A8C0SUT4).

In the present invention, SERPINA3 (Serpin family A member 3) is a member of the serpin family, a protein group that inhibits serine proteases. Polymorphisms in SERPINA3 appear tissue-specific and may affect protease targeting. In humans, SERPINA3 abnormalities have been reported to cause Alzheimer's disease, liver disease, Parkinson's disease, etc., but the correlation between SERPINA3 expression level and pancreatitis in dogs has not been known to date. The present inventors confirmed that it was increased in the pancreatitis group compared to the normal group through the dog plasma proteomic analysis. Specific information on SERPINA3 can be found at Uniprot, etc. (Uniprot registration number: A0A8I3MJD4).

In the present invention, FGG (Fibrinogen gamma chain or fibrinogen gamma gene) is a protein that is polymerized with Fibrinogen alpha (FGA) and Fibrinogen beta (FGB) to form an insoluble fibrin matrix. FGG is one of the major components of blood clots and plays an important role in hemostasis. It functions in the early stages of wound repair to stabilize the lesion and regulate cell migration during re-epithelialization. The present inventors confirmed that it was reduced in the pancreatitis group compared to the normal group through the dog plasma proteomic analysis. Specific information on FGG can be found in Uniprot, etc. (Uniprot registration number: P12800).

In one embodiment of the present invention, the composition is Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B), It may further include an agent for measuring the level of at least one protein or mRNA selected from the group consisting of Complement C3 (LOC484960) and Complement factor I. The above proteins (genes) are markers confirmed to be statistically significantly increased or decreased in dogs suffering from pancreatitis compared to normal individuals.

Among them, "Pentaxin" is a protein also referred to as "Pentraxin", which includes a Pentraxin protein domain and is known to be involved in an acute immunological response. Pentaxin is produced by tissue cells, macrophages, monocytes, and dendritic cells at the site of infection or inflammation, and plays a role in suppressing infections such as fungi, bacteria, and viruses. However, nothing is known about the relationship between canine pancreatitis and Pentaxin, but the present inventors confirmed that the level of Pentaxin was significantly increased in pancreatitis subjects compared to normal subjects through canine plasma protein analysis. Specific information on Pentaxin can be found in public databases such as Uniprot (Uniprot registration number: A0A8C0T016).

Also, “Angiotensinogen” is a component of the renin-angiotensin system (RAS), a hormone system that regulates blood pressure and body fluid balance. Angiotensinogen is also known as a renin substrate and is a non-inhibitory member of the serpin family. Although nothing is known about the association between canine pancreatitis and angiotensinogen, the present inventors confirmed that the level of angiotensinogen was significantly increased in pancreatitis subjects compared to normal subjects through canine plasma proteomic analysis. Specific information on angiotensinogen can be found in public databases such as Uniprot (Uniprot registration number: A0A8C0PY30).

In the present invention, the Pentaxin, SERPINA3, and Angiotensinogen may be increased in subjects suffering from pancreatitis compared to normal controls. In addition, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960), Complement factor I, or PDE4DIP It may be characterized as being reduced in individuals suffering from pancreatitis compared to normal controls.

In the present specification, "the level is increased" means that the undetected is detected, or the detected amount is relatively higher than the normal level. For example, a level "increased" means that the level in the experimental group is at least 1%, 2%, 3%, 4%, 5%, 10% or more, such as 5%, compared to that in the control group. , 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or higher, and/or 0.5x, 1.1x, 1.2x, 1.4x, 1.6x , 1.8 times higher or higher. Specifically, compared to that of the control group, 1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5 times. 5.5 times, 5.5 to 6 times, 6 to 6.5 times, 6.5 to 7 times, 7 to 7.5 times, 7.5 to 8 times, 8 to 8.5 times, 8.5 to 9 times, 9 to 9.5 times, 9.5 to 10 times, or 10 times It may mean an increase of more than double, but is not limited thereto. The meaning of the opposite term can be understood as having the opposite meaning according to the above definition by those skilled in the art.

As used herein, the term "measurement" refers to measuring and confirming the presence (expression) of a substance of interest (mRNA or protein of a pancreatitis biomarker gene in the case of the present invention), or the presence level (expression of a substance of interest) level) is meant to include both measuring and confirming changes. That is, measuring the expression level of the protein means measuring whether or not it is expressed (that is, measuring whether or not it is expressed) or measuring the level of qualitative or quantitative change of the protein. The measurement can be performed without limitation including both qualitative methods (assays) and quantitative methods. Types of qualitative and quantitative methods for measuring protein levels are well known in the art, and include the experimental methods described herein. Specific protein level comparison methods for each method are well known in the art. Therefore, detecting the target protein means detecting the presence or absence of the collagen protein, or confirming an increase (up-regulation) or decrease (down-regulation) of the protein level.

As used herein, the term "analysis" may preferably mean "measurement", and the qualitative analysis may mean measuring and confirming the presence or absence of a target substance, and the quantitative analysis may mean It may mean measuring and confirming changes in the presence level (expression level) or amount of the desired substance. In the present invention, analysis or measurement may be performed without limitation including both qualitative and quantitative methods, and preferably, quantitative measurement may be performed.

When the composition is for measuring the level of mRNA, the agent for measuring the level of mRNA may be a primer set or a probe that specifically binds to mRNA. The composition of the present invention including the mRNA-specific primer set or probe of the biomarker genes according to the present invention may further include an agent required for a known RNA detection method. The mRNA level of the biomarkers can be measured in a subject by using a known method for detecting RNA using the composition of the present invention without limitation.

In the present invention, the "primer" is a fragment that recognizes a target gene sequence, and includes a forward and reverse primer pair, preferably a primer pair that provides an analysis result having specificity and sensitivity. High specificity can be imparted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that the primer amplifies only the target gene sequence containing a complementary primer binding site and does not cause non-specific amplification. .

In the present invention, the "probe" means a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of a target substance in a sample through the binding. The type of probe is not limited as a material commonly used in the art, but preferably may be peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, RNA or DNA, and most preferably Most likely it is PNA. More specifically, the probe is a biomaterial, including one derived from or similar to a living organism or manufactured in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.

Since the information (amino acid sequence, nucleic acid sequence, etc.) of the biomarker (protein or gene) according to the present invention is known, a primer, probe, or antisense nucleotide that specifically binds to the gene encoding the protein based on this information (amino acid sequence, nucleic acid sequence, etc.) can be easily designed. Primers or probes may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods, and include all functional equivalents. In addition, primers or probes may be variously modified according to methods known in the art to the extent that hybridization with mRNA is not hindered. Examples of such modifications are methylation, capping, substitution of one or more homologs of the natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and binding of fluorescent or enzymatic labeling materials.

When the composition of the present invention is for measuring the level of a protein, the agent for measuring the level of the protein may be an antibody or an aptamer specific to the protein.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. In addition, as long as they have antigen-antibody binding properties, a part of the entire antibody is also included in the antibody of the present invention, and all types of immunoglobulin antibodies that specifically bind to the protein of the present invention are included. For example, complete antibodies with two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, i.e. Fab, F(ab'), F(ab') with antigen-binding function 2 and Fv; Furthermore, antibodies of the present invention include special antibodies such as humanized antibodies and chimeric antibodies and recombinant antibodies as long as they can specifically bind to the protein of the present invention.

As used herein, the term "aptamer" refers to a substance capable of specifically binding to an analyte to be detected in a sample, and a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure in itself. nucleic acid), it is possible to specifically confirm the presence of a target protein in a sample. The aptamer is synthesized by determining the sequence of an oligonucleotide that is selective and highly binding to the target protein to be identified according to the general aptamer preparation method, and then the 5' or 3' end of the oligonucleotide is applied. It may be made by modification with -SH, -COOH, -OH or NH ₂ so that it can bind to the functional group of the timer chip, but is not limited thereto.

The composition of the present invention may additionally include an agent required for the method of detecting the biomarker protein, and the subject (or biological obtained from the subject) may be used without limitation by using a known protein detection method using the present composition. The expression level of the protein in the sample) can be measured.

The composition according to the present invention can be used for analysis of a biological sample isolated from a subject in need of diagnosis. In the present invention, the "biological sample" may be included without limitation as long as it is collected from a subject to diagnose pancreatitis or predict the risk of developing it. For example, the biological sample may be selected from liquid, whole blood, plasma, urine, saliva, tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen. can Preferably, the composition according to the present invention is for non-invasive diagnosis, and the biological sample may be at least one selected from the group consisting of blood, whole blood, plasma, serum, and lymph.

The biological sample may be pretreated before being used for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. The sample may be prepared to increase the detection sensitivity of a protein marker, for example, a sample obtained from a subject may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography , it may be pretreated using methods such as sequential extraction or gel electrophoresis.

In addition, the present invention can provide a kit for diagnosing canine pancreatitis, comprising the composition according to the present invention.

In the present invention, "kit" refers to a device or set for diagnosis that can diagnose pancreatitis by measuring the amount of mRNA or protein of the biomarkers according to the present invention, which are included in a sample. There is no limitation as long as the expression level of the genes can be measured from a biological sample isolated from an individual. For example, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a Rapid kit, a mass spectrometry kit, a microarray kit, or a multiple reaction monitoring (MRM) kit, but is limited thereto. it is not going to be Accordingly, the kit of the present invention may include an antibody recognizing a target protein as a marker, a primer set recognizing mRNA as a marker, or a probe, as well as one or more other component compositions, solutions, or devices suitable for an analysis method. At this time, the substance for detecting the expression level of the gene, mRNA, or protein can be acted on one or more times without limitation, there is no limitation before and after applying each substance, and the application of each substance may be performed simultaneously or microscopically. may proceed to

In addition, the kit according to the present invention may further include instructions describing the diagnostic method and the like according to the present invention.

Another object of the present invention is to provide a method for diagnosing pancreatitis in an individual or a method for providing information for diagnosing pancreatitis in a dog, including the following steps:

(S1) measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject; and

(S2) the protein or mRNA level of SERPINA3 measured in step (S1) is increased compared to the control group; If the protein or mRNA level of PDE4DIP or FGG is reduced compared to the control group, diagnosing pancreatitis or judging that there is a risk (high probability of developing) pancreatitis.

In one embodiment of the present invention, the method comprises Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, A step of measuring the level of at least one protein or mRNA selected from the group consisting of Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960), and Complement factor I may be further included, but is not limited thereto.

In the method, if the protein or mRNA level of Pentaxin, SERPINA3, and / or Angiotensinogen in the subject is increased compared to the control group, pancreatitis is diagnosed or pancreatitis is diagnosed as having a risk (high probability of developing) Further steps may be included.

In addition, the method, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960) of the subject , Complement factor I, and / or the level of PDE4DIP is reduced compared to the control group, the step of diagnosing pancreatitis or determining that there is a risk of developing pancreatitis (high probability of developing) may be further included.

In the diagnostic method according to the present invention, when analyzing receiver operating characteristics, the area under curve may be 0.7 or more, 0.75 or more, 0.8 or more, 0.85 or more, 0.9 or more, 0.93 or more, 0.95 or more, 0.96 or more, 0.97 or more, 0.98 or more, or 0.99 or more. there is. In addition, the diagnostic method according to the present invention has a specificity and / or sensitivity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more or more, 98% or more, or 99% or more.

For example, when the diagnostic method according to the present invention includes the step of measuring the protein or mRNA level of SERPINA3, the diagnostic specificity may be 70% or more or 75% or more, and the diagnostic sensitivity is 70% or more, 75% or more, Or it may be 80% or more, and the ROC AUC may be 0.75 or more, 0.8 or more, or 0.85 or more, but is not limited thereto.

When the diagnostic method according to the present invention includes the step of measuring the protein or mRNA level of the angiotensinogen, the diagnostic specificity may be 75% or more, 80% or more, or 85% or more, and the diagnostic sensitivity may be 70% or more or 75% or more. % or more, and the ROC AUC may be 0.75 or more or 0.8 or more, but is not limited thereto.

In particular, when the level of the biomarker combination according to the present invention is measured, the diagnostic accuracy can be further increased.

For example, when the diagnostic method according to the present invention includes the step of measuring the protein or mRNA levels of PDE4DIP, FGG, and SERPINA3, the diagnostic sensitivity is 80% or more, 85% or more, 90% or more, 93% or more, or 95% or greater, diagnostic specificity may be 0.7 or greater, 0.75 or greater, or 0.8 or greater, and ROC AUC may be 80% or greater, 85% or greater, 90% or greater, 93% or greater, or 95% or greater. it is not going to be

In addition, the diagnostic method according to the present invention can be used to determine the type (chronic or acute) of pancreatitis in a subject diagnosed with pancreatitis.

For example, the diagnostic method according to the present invention includes measuring the mRNA or protein level of SERPINA3, and at this time, if the mRNA or protein level of SERPINA3 in the subject is increased compared to the control group, the subject has acute pancreatitis can be diagnosed as Here, the control group may be a normal individual or an individual suffering from chronic pancreatitis.

In addition, the diagnostic method according to the present invention includes the step of measuring the protein or mRNA level of PDE4DIP, and at this time, if the protein or mRNA level of PDE4DIP in the subject is reduced compared to the control group, the subject is diagnosed with acute pancreatitis. can be diagnosed. Here, the control group may be a normal individual or an individual suffering from chronic pancreatitis.

Since the detailed description related to the biomarker, biological sample, subject, etc. according to the present invention has been described above, it will be omitted.

As used herein, the term "method for providing information" refers to a method for providing information on diagnosis of a disease by analyzing a biological sample of a subject or increasing or decreasing the level of biomarkers according to the present invention. It refers to a method of obtaining information on the onset or onset (risk) of a disease by checking For example, a method for measuring the level of a biomarker according to the present invention and comparing it with a control group to provide information on whether an individual is likely to develop pancreatitis, has a relatively high likelihood of developing pancreatitis, or has already developed pancreatitis As a result, the risk of developing pancreatitis, that is, the high-risk group, can also be predicted through the method, and it can also be used as a method of providing information on the prevention and treatment of pancreatitis.

In the present invention, a "control" may be a biological sample isolated from a normal subject or a subject suffering from pancreatitis.

In the present invention, the method for measuring the protein level is not particularly limited as long as it is by a protein measuring method known in the art, but protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, Complement fixation method, two-dimensional electrophoretic analysis, liquid chromatography-Mass Spectrometry (LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blotting, ELISA ( enzyme linked immunosorbent assay), and FACS.

In the present invention, the method for measuring the mRNA level is not particularly limited as long as it is by a method for measuring mRNA known in the art, but PCR, RNase protection assay, Northern blotting, Southern blotting, It can be measured by methods such as in situ hybridization, DNA chip, and/or RNA chip.

In addition, the present invention may provide a device for diagnosing pancreatitis in an individual (eg, dog).

The measuring unit of the diagnostic device of the present invention measures the expression level of at least one protein selected from the group consisting of a biomarker according to the present invention, for example, PDE4DIP, SERPINA3, and FGG, or a gene encoding the same, with respect to a biological sample obtained from a subject of interest. The expression level of a protein or gene can be measured using a measurement agent. Pancreatitis can be diagnosed or diagnosed as having a high risk of developing pancreatitis by checking the expression level of the protein or gene using the preparation in the measuring unit.

The diagnostic device of the present invention may further include a detection unit that predicts and outputs the presence, stage, or type of pancreatitis of an individual based on the expression level of the protein or gene obtained from the measurement unit.

In the present invention, the detection unit can diagnose pancreatitis by generating and classifying information on pancreatitis according to the category of the expression level of the protein or gene obtained by the measurement unit.

In addition, the present invention provides a method for preventing or treating pancreatitis in dogs, comprising the following steps:

(S1) measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject;

(S2) the protein or mRNA level of SERPINA3 measured in step (S1) is increased compared to the control group; diagnosing the subject as suffering from pancreatitis when the protein or mRNA level of PDE4DIP or FGG is decreased compared to a control group; and

(S3) treating pancreatitis in the subject diagnosed as suffering from pancreatitis in step (S2).

In addition, the present invention is a method for characterizing a respondent (i.e., dog) to a therapeutic agent for canine pancreatitis (i.e., companion diagnostic method), comprising the following steps:

(a) measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject; and

(b) the protein or mRNA level of SERPINA3 measured in step (a) is increased compared to the control group; If the protein or mRNA level of PDE4DIP or FGG is decreased compared to the control group, determining the subject as a responder to a treatment for canine pancreatitis;

The canine pancreatitis treatment agent is at least one selected from the group consisting of maropitant, ondansetron, Panoquell-CA1, and metoclopramide.

In the present invention, the "treatment" refers to all activities that improve or beneficially change the desired disease and metabolic abnormalities, and include methods such as chemotherapy, radiation therapy, surgical operation, biological therapy, or antibiotic administration. can be used. For example, the treatment may be performed through fluid treatment, drugs, plasma transfusion, and dietary control.

In the present invention, the chemotherapy (Chemotherapy) refers to the act of using a chemical substance for the treatment of a specific disease and the entire drug used at that time. Examples of the drug include antiemetics, gastroprotective drugs, antibiotics, and the like, and more specifically, maropitant, ondansetron, Panoquell-CA1, and metoclopramide.

In the present invention, the surgical operation includes any therapeutic or diagnostic procedure involving the methodical action of the hands or instruments in conjunction with the body of a subject to achieve a curative, therapeutic or diagnostic effect. .

In the present invention, the biological therapy refers to a treatment method that directly or indirectly uses the body's immune system using a biological agent containing a material derived from a living organism or a material produced using a living organism, and the biological agent is a physical , vaccines, allergens, antigens, hormones, cytokines, enzymes, blood and plasma, immune sera, monoclonal antibodies, fermented products, antitoxins, and laboratory diagnostics for which potency and safety cannot be assessed by chemical testing alone.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Dog blood collection

A veterinarian collected blood from each individual and after serum separation, the serum was collected in a separate tube and stored at -80 ° C until analysis. Blood samples were collected from a total of 114 dogs.

Example 2. Pretreatment of canine blood samples

14 high concentrations ( High abundant plasma proteins were removed and low abundant proteins were isolated. The isolated low-concentration protein was subjected to a pretreatment process for producing peptides using an automated liquid dispensing system (Bravo Automated Liquid Handling Platform; Agilent) (FIG. 1a).

That is, 40 μL of plasma sample was injected into the MARS14 column and the top 14 abundant proteins (albumin, IgA, IgG, IgM, α1-antitrypsin, α1-acid glycoprotein, apolipoprotein A1, apolipoprotein A2, Complement C3, transferrin, α2-macroglobulin , transthyretin, haptoglobin, and fibrinogen) were removed (Fig. 1b). To this end, the mixture was diluted 4-fold with Agilent's buffer A (#5185-5987) and loaded onto the MARS14 column of the Shimadzu HPLC LC20AT system. After freeze-drying the unbound low-concentration plasma protein fraction, it was reabsorbed and prepared as a peptide using the suspension trap method (FIG. 1c). The mixture was reabsorbed with 5% SDS (50 mM TEAB) buffer, 1,4-dithiothreitol was added to a concentration of 10 mM, and reacted at 95° C. for 10 minutes to reduce disulfide bonds. Thereafter, for alkylation, iodoacetamide was added to a concentration of 20 mM, reacted for 30 minutes at room temperature under dark conditions, and digested with 6 μg of a trypsin/rice seed mixture at 37° C. for 18 hours. The eluted petide was freeze-dried with a cold trap (CentriVap Cold Traps, LABCONCO) and stored at minus 80°C until use.

Example 3. LC-MS analysis

SWATCH LC-MS analysis was performed on the peptides obtained through the above examples. To this end, a spectrum was obtained by performing LC-MS analysis using the parameters shown in FIG. 1d.

Specifically, the iRT standard provided by the iRT-Kit (Biognosys AG, Schlieren, Switzerland) was added to the prepared Peptide sample in 1/10 volume to the sample, following the supplier instructions (Reference: https://pubmed.ncbi.nlm Retention times were calibrated according to .nih.gov/22577012/). The total amount of each sample was 40 μg and was dissolved in 40 μL of solution.

A 4 μL sample injected was analyzed using a SCIEX TripleTOF 5600+ system mass spectrometer with the following LC-MS/MS settings. For LC separation, a nanoLC 425 (Eksigent, Dublin, CA, USA) was used as an analytical column with an Eksigent Micro Trap C18 column (ChromXP C18CL, 5 μm, 120 Å) and an Eksigent column (C18-CL, 0.3 × 150 mm, particle size 3). μm, pore size 120 Å), the column temperature was maintained at 40° C. and the sample was loaded onto the trap column using buffer A at a flow rate of 10 μL/min. After 10 minutes, the peptide mixture was separated through a concentration gradient using buffer A and buffer B (0.1% formic acid in HPLC acetonitrile) at a flow rate of 5 μL/min by 57 minutes. Change Buffer B 3-25% for 38 min, 25-32% change for 5 min, 32-80% change for 2 min, hold at 80% for 3 min, 80-3% for 3 min and held at 3% for 8 minutes.

For individual samples, all mass spectrometry runs were performed using 100 variable windows and operating in sequential window acquisition in all theoretical mass spectra (SWATH) mode. The SWATH parameters were set as follows: Lower m/z limit 400; m/z upper limit 1250; Window overlap (Da) 1.0; The CES was 5 for the small window, 8 for the large window, and 10 for the largest window. MS2 spectra were collected in the range 100–1500 m/z for 2.5 ms in high-sensitivity mode, with a total cycle time of 2.8 seconds. Other MS parameters were set as follows: ion source gas 1 (GS1) 15 psi; ion source gas 2 (GS2) 20 psi; Curtain Gas (CUR) 30 psi; Temperature (TEM) 250 °C; Floating Ion Spray Voltage (ISVF) 5500V.

Example 4. Statistical analysis

Through the above example, SWATH LC-MS analysis was performed on each sample to obtain a spectrum result, and quantitative and quantitative analysis of proteins from the spectra was performed using DIA-NN software and Pan-human proteome library. Sexual information was extracted. As a result of analyzing the amount information of normalized proteins based on the median value for each sample of the normal control group and the pancreatitis group on a log scale, it was confirmed that reproducibility was shown, and statistical analysis was performed using quantitative information of proteins.

[Experimental Example]

Experimental Example 1. Collection of blood samples

After receiving consent from the guardian at the veterinary hospital, blood was collected by the veterinarian, and after serum separation, the serum was collected in a separate tube and stored at -80 ° C until analysis, and blood samples were collected from a total of 116 companion dogs. The group information of the collected companion dogs is shown in Table 1 below.

group	number
skin disease	One
obesity	6
chronic pancreatitis	17
lymphoma	18
diabetes	4
acute pancreatitis	19
overweight	2
health control group	33

Experimental Example 2. Analysis of the total amount of proteins isolated from plasma samples

According to Example 2, based on S-trap, 116 plasma samples were prepared as peptides with trypsin/LysC, and the amount of peptides was quantified using Nanodrop (FIG. 2). As a result, it was confirmed that a minimum of 214 μg of peptide was obtained at a maximum of 1,023 μg. Considering that only about 8 μg of peptide is usually analyzed during protein analysis, it was found that a sufficient amount of peptide was prepared for the study.

Experimental Example 3. Selection of proteins showing differential levels in pancreatitis individuals compared to normal groups

Sciex's Quadupole Time of Flight (Q-TOF) mass spectrometry was performed on the peptide samples isolated from the plasma of dogs, and the spectrum was obtained by Data-independent acquisition (DIA) method. The obtained spectrum was compared and analyzed using a spectrum library obtained by Data Dependent Acquisition (DDA) method from pooled protein samples of previously secured dogs, and qualitative and quantitative information of the protein was extracted with the DIA-NN algorithm.

As a result of SWATH LC-MS analysis, a total of 701 proteins could be quantitatively analyzed in 116 samples, and the number of quantifiable proteins for each sample is shown in FIG. 3 . There was no difference in the number of quantified proteins by group, and if there was a missing value, the analysis was performed by imputation with a random normal distribution based on 80% frequency.

A total of 16 proteins (Differentially expressed proteins, DEPs) were found in the pancreatitis blood compared to the normal control group, and a total of 3 proteins were increased and 13 proteins were decreased in the pancreatitis blood plasma compared to the normal control group (Fig. 4). The p value and fold change for 16 DEPs are shown in FIG. 5 . Among them, Pentaxin is a C-Reactive protein, which the present inventors have identified as a biomarker that is equally increased in lymphoma. CRP and SERPINA3 are also proteins identified in DEP in lymphoma. In addition, it was confirmed that 10 proteins, including PEPD, were commonly reduced in disease groups.

Since these proteins are DEPs that commonly show expression changes in lymphoma and pancreatitis, disease specificity may be low, but they have the advantage of being used as universal biomarkers for high-risk disease groups.

Experimental Example 4. Screening of Protein Marker Combinations for Diagnosis of Canine Pancreatitis

Among the 16 DEPs showing differential levels in the pancreatitis group compared to the normal group, SERPINA3 (Serpin family A member 3) and Angiotensinogen are both proteins that are increased in pancreatitis. SEPINAA3 showed a specificity of 78.1% and a sensitivity of 81.8%, and ROC AUC was 0.857, indicating excellent discrimination power (FIG. 6a). In addition, angiotensinogen had a sensitivity of 78.8% and a specificity of 87.5%, and the ROC AUC was 0.848, confirming that the disease discrimination power was similarly excellent (FIG. 6b).

In addition, as a result of PLS-DA using the 15 top DEPs identified in the above experimental example, it was confirmed that the pancreatitis group (acute and chronic) and the normal control group have a probability that they can be distinguished from each other using quantitative characteristics of proteins (Fig. 7). F1PAL5, which means angiotensinogen, is marked on the right-axis graph of FIG. 7 .

Experimental Example 5. Marker selection for diagnosis of acute or chronic pancreatitis in dogs

A Kruskal Wallis test was performed to derive DEPs capable of distinguishing the normal control group from acute pancreatitis and chronic pancreatitis, and as a result, 23 DEPs could be derived (FIG. 8).

Among the 23 proteins derived, angiotensinogen was found to be increased in both chronic pancreatitis and acute pancreatitis, and SERPINA3 showed quantitative characteristics with a higher level in normal, chronic, and acute stages. In addition, PDE4DIP was high in the normal group and decreased in the chronic and acute pancreatitis groups (FIG. 9). That is, it was confirmed that a panel for distinguishing chronic pancreatitis from acute pancreatitis could be developed using the quantitative characteristics of the proteins.

As a result of analyzing the diagnostic accuracy using a combination of two or more proteins among DEPs using logistic regression, when PDE4DIP (phosphodiesterase 4D interacting protein), FGG and SERPINA3 proteins were combined, the sensitivity was 97.0% and the specificity was 84.4%. and the ROC AUC was 0.951, which was found to have very good discrimination power (FIG. 10). In view of the importance of sensitivity, which is a criterion for classifying diseases, it can be seen that the biomarker combination has very good discrimination compared to 82% and 59%, which are the sensitivity and specificity of conventional pancreatitis diagnosis.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

The present invention relates to a composition for diagnosing canine pancreatitis, and was completed by discovering a biomarker capable of diagnosing pancreatitis with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins. Specifically, the present inventors performed proteomic analysis using high-resolution mass spectrometry on plasma proteins of dogs suffering from pancreatitis, and selected biomarkers showing significant expression differences compared to normal controls. It was confirmed that the pancreatitis diagnostic model established using the selected biomarker can diagnose pancreatitis with high specificity, sensitivity, and accuracy. Therefore, the present invention is a non-invasive method using a small amount of blood, which enables prediction and early diagnosis of the risk of pancreatitis in dogs, thereby preserving the health of dogs, reducing the burden of guardians' medical expenses, and contributing to the improvement of the quality of veterinary consulting. It is expected that

Claims (15)

1. A composition for diagnosing pancreatitis in dogs, comprising, as an active ingredient, an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG (Fibrinogen gamma chain) .
2. According to claim 1,

   The composition for diagnosing canine pancreatitis, characterized in that the protein or mRNA level of SERPINA3 is increased in pancreatitis subjects compared to normal subjects.
3. According to claim 1,

   The composition for diagnosing canine pancreatitis, characterized in that the protein or mRNA level of PDE4DIP or FGG is reduced in pancreatitis subjects compared to normal subjects.
4. According to claim 1,

   The composition includes Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960), and Complement A composition for diagnosing canine pancreatitis, further comprising an agent for measuring the level of at least one protein or mRNA selected from the group consisting of factor I.
5. According to claim 1,

   The composition for diagnosing canine pancreatitis, characterized in that the agent for measuring the protein level is an antibody or an aptamer specific to the protein.
6. According to claim 1,

   The composition for diagnosing canine pancreatitis, characterized in that the agent for measuring the mRNA expression level is a primer set or a probe that specifically binds to the mRNA.
7. According to claim 1,

   The protein or mRNA level is a level in at least one selected from the group consisting of blood, whole blood, plasma, serum, and lymph, composition for diagnosing canine pancreatitis.
8. A kit for diagnosing canine pancreatitis, comprising the composition of any one of claims 1 to 7.
9. A method for diagnosing pancreatitis in dogs, comprising the following steps:

   (S1) measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject; and

   (S2) the protein or mRNA level of SERPINA3 measured in step (S1) is increased compared to the control group; Diagnosing pancreatitis or determining that there is a risk of developing pancreatitis when the protein or mRNA level of PDE4DIP or FGG is reduced compared to the control group.
10. According to claim 9,

    The method includes Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B) in a biological sample isolated from the subject. , Complement C3 (LOC484960), and Complement factor I, further comprising measuring the level of at least one protein or mRNA selected from the group consisting of, the method for diagnosing canine pancreatitis.
11. According to claim 9,

    The method of diagnosing canine pancreatitis, wherein the biological sample is at least one selected from the group consisting of blood, whole blood, plasma, serum, and lymph.
12. According to claim 9,

    The protein level is measured by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) assay, radioimmunoassay, radioimmuno-diffusion method, Oucteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoretic assay, liquid chromatography-Mass Spectrometry (LC) -MS), LC-MS / MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western blotting, ELISA (enzyme linked immunosorbentassay), and FACS, characterized in that measured by one or more methods selected from the group consisting of, How to diagnose pancreatitis.
13. According to claim 9,

    The mRNA level is measured by one or more methods selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chip, and RNA chip. A method for diagnosing canine pancreatitis.
14. Use of an agent for measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and Fibrinogen gamma chain (FGG) for diagnosis of canine pancreatitis.
15. Use of a preparation for measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and Fibrinogen gamma chain (FGG) for the manufacture of a preparation for diagnosis of canine pancreatitis .

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