KR102288646B1 - Glycan Biomarker for Diagnosing Osteoarthritis of Pets and Method for Diagnosing Osteoarthritis of Pets Using the Same - Google Patents

Abstract

The present invention relates to a method for diagnosing osteoarthritis, a degenerative disease, and a biomarker thereof by measuring the composition and expression level of sugar chains containing immune information in a trace blood sample of a companion animal. The present invention provides a biomarker for diagnosing osteoarthritis in companion animals and a method for diagnosing osteoarthritis in companion animals for the first time.

Description

Glycan Biomarker for Diagnosing Osteoarthritis of Pets and Method for Diagnosing Osteoarthritis of Pets Using the Same}

The present invention relates to a method for diagnosing osteoarthritis, a degenerative disease, and a biomarker thereof by measuring the composition and expression level of sugar chains containing immune information in a trace blood sample of a companion animal. The present invention provides a biomarker for diagnosing osteoarthritis in companion animals and a method for diagnosing osteoarthritis in companion animals for the first time.

The number of households living with companion animals is increasing, and the perception of companion animals is changing due to changes in the social environment such as high-level industrial socialization, nuclear familyization, late marriage, low fertility, single households, and elderly households. In particular, as the number of elderly companion animals increases, the sales volume of companion animal veterinary treatment/diagnosis is approximately KRW 217.8 billion as of 2010, which is a rapidly growing trend annually. However, the veterinary clinical field depends on the development of feed additives and biologics, or remains at the level of imitating human medical technology.

Osteoarthritis (OA) is a common debilitating and degenerative disease that occurs in 1 in 5 dogs, but there is no early diagnosis method that can prevent the clinical progression of the disease. There is only a method to confirm it and a treatment method by behavioral therapy.

Blood is a widely used sample for various basic diagnostics and non-invasive in vitro diagnostics that do not burden the sample collection. Most of the proteins in the blood are immunoglobulin-type immune glycoproteins. The sugar chains in the blood not only perform the biological functions of various proteins, but also reflect the state of the body by containing a significant amount of immune information. do. In particular, cases have been observed that cause cell mutation and/or loss of function due to abnormal overexpression or overexpression of specific sugar chains or abnormal glycosylation due to catalysts or transfer substances depending on biological change conditions such as diseases. Research on the development of disease biomarkers using glycoproteins and/or sugar chains is becoming a new paradigm.

In the case of rheumatoid arthritis, as an autoimmune disease, there are various diagnostic methods to check the inflammatory marker (blood CRP level) and drug therapy to lower the inflammation level. On the other hand, with regard to osteoarthritis, a degenerative disease caused by decline and aging, pathophysiological studies are currently lacking, so there is no early diagnosis method, and no clear treatment has been proposed other than palliative care through lifestyle therapy. Early diagnosis of arthritis and pain treatment are very important.

Glycosylation is the most widely known post-translational modification (PTM) process and is largely involved in the function of proteins, and about 50% or more of proteins in the body are glycoproteins, which are glycosylated proteins. Glycoproteins are widely present in body fluids such as blood, saliva, and tears, as well as in most living tissues. Sugar chains, the result of glycosylation, are very sensitive to changes in the environment inside and outside the body, and are being used as biomarkers for various types of diseases including cancer. In addition, in biopharmaceuticals, sugar chains are known to play an important role in the duration and efficacy of drugs in the body.

For the analysis of sugar chains, two pre-treatment steps are performed: a sugar chain separation step in which only sugar chains are cut and separated from proteins by enzymatic treatment, and a sugar chain purification step of extracting and concentrating the separated sugar chains. The sugar chains obtained after the pretreatment step can be comprehensively screened through high-sensitivity and high-resolution liquid chromatography mass spectrometry to confirm the composition and distribution.

An object of the present invention is to monitor sugar chains that can represent the immune state of the body using a small amount of companion animal blood, which can be collected relatively easily, and to be specific when a disease state among hundreds of sugar chains contained in blood. It is to accurately diagnose osteoarthritis, a representative disease of companion animals, by comparing the expression level of sugar chains, where changes are observed.

The present inventors used companion animal blood, which is relatively easy to collect, as a sample, and discovered degenerative osteoarthritis-specific sugar chain biomarkers by profiling the sugar chain sensitively reflecting the physical condition.

In the present invention, companion animals refer to companion animals including dogs and cats. In addition, it is revealed that the terms used in the present invention are used in the meaning known to those of ordinary skill in the art to which the present invention pertains unless otherwise specified.

the present invention

(1) (Hex)3(HexNAc)2, theoretical monoisotope mass value 910.3,

(2) (Hex)3(HexNAc)2(Fuc)1, theoretical monoisotope mass value 1056.4,

(3) (Hex)4(HexNAc)4(Fuc)2(NeuAc)1, theoretical monoisotope mass value 2061.8,

(4) (Hex)6(HexNAc)5(NeuAc)1, theoretical monoisotope mass value 2296.8,

(5) (Hex)5(HexNAc)4(Fuc)1(NeuAc)2, theoretical monoisotope mass value 2368.8,

(6) (Hex)6(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2239.8,

(7) (Hex)4(HexNAc)3(NeuAc)1, theoretical monoisotope mass value 1566.6,

(8) (Hex)4(HexNAc)3, theoretical monoisotope mass value 1275.5,

(9) (Hex)3(HexNAc)3(Fuc)1, theoretical monoisotope mass value 1259.5,

(10) (Hex)5(HexNAc)3, theoretical monoisotope mass value 1437.5,

(11) (Hex)6(HexNAc)4(NeuAc)1, theoretical monoisotope mass value 2093.7,

(12) (Hex)5(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2277.7,

(13) (Hex)3(HexNAc)4(Fuc)1, theoretical monoisotope mass value 1462.5,

(14) (Hex)3(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1754.7,

(15) (Hex)4(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1916.7,

(16) (Hex)7(HexNAc)7(Fuc)5(NeuAc)2(NeuGc)2, theoretical monoisotope mass value 4500.6,

(17) (Hex)5(HexNAc)4(Fuc)3, theoretical monoisotope mass value 2078.8,

(18) (Hex)4(HexNAc)5(Fuc)2, theoretical monoisotope mass value 1973.7,

(19) (Hex)7(HexNAc)7(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3901.4,

(20) (Hex)6(HexNAc)7(Fuc)4, theoretical monoisotope mass value 2996.1,

(21) (Hex)7(HexNAc)4, theoretical monoisotope mass value 1964.7,

(22) (Hex)7(HexNAc)6(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3698.3,

(23) (Hex)6(HexNAc)3, theoretical monoisotope mass value 1599.6,

(24) (Hex)7(HexNAc)6(Fuc)1(NeuGc)1, theoretical monoisotopic mass value of 2824.0 and

(25) (Hex)6(HexNAc)3(Fuc)1(NeuAc)1, a sugar chain biomarker for diagnosing osteoarthritis in companion animals selected from theoretical monoisotope mass values of 2036.7 {however, HexNAc, N-acetylhexosamine (N-) acetylhexosamine); Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid (N-glycolylneuraminic Acid)} relates to.

In addition, the present invention relates to a sugar chain biomarker for diagnosing osteoarthritis in companion animals, wherein the biomarker AUC value is 0.9 or more.

In addition, the present invention provides that the biomarkers of (1), (2), (4), (5), (7), (8), (9), (13) and (24) are higher in the patient group than in the normal group. It is characterized in that it is expressed significantly less in

In addition, the present invention provides the above (3), (6), (10), (11), (12), (14), (15), (16), (17), (18), (19), It is characterized in that the biomarkers of (20), (21), (22), (23) and (25) are expressed significantly more in the patient group than in the normal group.

In addition, the present invention relates to a sugar chain biomarker for diagnosing osteoarthritis in companion animals, including a sugar chain having an AUC value of 1 (1).

In addition, the present invention relates to a sugar chain biomarker for diagnosing osteoarthritis in companion animals, which contains one or more sugar chains of (14) or (22) that are not found in the normal group.

Also, the present invention

(A) obtaining serum from a companion animal suspected of osteoarthritis;

(B) denaturing the serum protein;

(C) separating N-saccharide chains by enzymatically treating the denatured serum protein;

(D) mass spectrometry of the separated N-saccharide chain; and

(E) Diagnosing osteoarthritis when, as a result of mass spectrometry, one or more sugar chain biomarkers selected from (1) to (25) above shows a significant difference in expression level compared to the corresponding sugar chain mass spectrometry result of the normal companion animal group It relates to a method for diagnosing osteoarthritis in companion animals, including.

In addition, the present invention confirms the N-saccharide composition with the mass value obtained in step (E) by mass spectrometry, and normalizes the identified N-saccharide chains to the total abundance of each N- sugar chain between the normal companion animal group and the patient. It relates to a method for diagnosing osteoarthritis in companion animals, comprising the step of comparing the sensitivity of sugar chains.

In addition, the present invention is characterized in that the enzyme treatment of step (c) is treated with the enzyme PNGase F enzyme to the denatured serum protein for 16 to 24 hours.

In addition, the present invention relates to a method for diagnosing osteoarthritis in companion animals, wherein in the step (E), when the expression level of the sugar chain biomarker of (1) is significantly lower than that of a normal control group, osteoarthritis is diagnosed.

In addition, the present invention relates to a method for diagnosing osteoarthritis in companion animals, wherein in the step (E), when one or more sugar chain biomarkers of (14) or (22) are expressed, osteoarthritis is diagnosed.

Also, the present invention

a) treating the test material to companion animal-derived chondrocytes; and

b) As a result of the treatment in a), there was a significant difference in the expression level of one or more of the sugar chain biomarkers of claim 1 (1) to (25) compared to the normal control group before treatment, and there was a significant difference with the normal control group after treatment It relates to a screening method for treating companion animal osteoarthritis, including;

Also, the present invention

A) treating a blood sample of companion animal, that is, 25 μl of plasma, with PNGase F enzyme to separate N-saccharide chains in the sample;

b) desalting the N-sugar chains obtained in step a) by extraction of the porous graphitized carbon solid phase and separating, purifying and/or concentrating the N-saccharide chains;

C) Qualitative and quantitative analysis of the separated, purified and/or concentrated N-saccharide chains by porous graphitized carbon-based nano liquid chromatography-high-sensitivity high-resolution mass spectrometry (PGC-nanoLC/Q-TOF-MS);

d) Checking the composition of N-saccharide chains with the mass value obtained by mass spectrometry, standardizing the identified N-saccharide chains with the total abundance, and comparing the sensitivity of each N-saccharide chain between the normal group and the patient group. .

In addition, the present invention provides an enzyme treatment method of step a)

a) taking a 25 μl companion animal plasma sample;

b) denaturing the protein by adding a protein denaturing reagent to the sample and treating it at 90 to 110° C. for 1 to 3 minutes;

c) separating N-saccharide chains by treating the sample with 2 μl of PNGase F enzyme for 16 to 24 hours;

d) precipitating the protein in the sample; and

e) centrifuging the protein-precipitated sample and obtaining a supernatant; characterized in that it comprises sequentially.

In addition, the present invention is the extraction of the porous graphitized carbon solid phase in step b)

a) washing the porous graphitized carbon column twice or more with 2.5 to 3.5 ml of purified water;

b) washing the washed column of a) twice or more with 3 ml of 80% acetonitrile solution containing 0.1% trifluoroacetic acid;

c) washing the column subjected to step b) twice or more with 2.5 to 3.5 ml of purified water;

d) loading a sample into the column after step c);

e) washing the column twice or more with 2.5-3.5 ml of purified water;

f) eluting the sample twice or more with 2.5-3.5 ml of a 10% acetonitrile solution;

g) eluting the sample twice or more with 2.5-3.5 ml of a 20% acetonitrile solution; and

h) eluting the sample twice or more with 2.5 to 3.5 ml of a 40% acetonitrile solution containing 0.05% trifluoroacetic acid;

In addition, the present invention includes the steps of c) porous graphitized carbon-based nano liquid chromatography-high-sensitivity high-resolution mass spectrometry (PGC-nanoLC/Q-TOF-MS) qualitative and quantitative analysis

a) Separation and analysis of N-saccharide chain isomers by liquid chromatography in a graphitized carbon-filled nanocolumn using gradient elution of solvent A (10 mM ammonium formate) and solvent B (100% acetonitrile) ;

b) analyzing ions in the range from m/z 500 to 2500 using a high-sensitivity, high-resolution Q-TOF MS detector: and

c) After mass spectrometry, the LC/MS data are analyzed using a Molecular Feature Extractor algorithm included in Mass Hunter Qualitative Analysis software (version B.07.00 SP2, Agilent Technologies) to confirm the mass value; It is characterized in that it includes.

In addition, the present invention provides a step of comparing the degree of increase or decrease between normal and patient by confirming the composition of N-saccharide chains with the mass value obtained by the above d) mass spectrometry, and standardizing the identified N-saccharide chains with the total abundance.

a) comparing the extracted mass value with the theoretically possible composition of sugar chains within 20ppm (±0.001Da) to give the composition of N-saccharides;

b) normalizing the identified sugar chains based on total abundance; and

c) comparing the normal and patient N-saccharide expression levels; characterized in that it comprises a.

The present invention provides, for the first time, a companion animal degenerative osteoarthritis biomarker and a companion animal osteoarthritis diagnosis method using the same.

The companion animal degenerative osteoarthritis biomarker of the present invention can diagnose osteoarthritis with high accuracy by exhibiting high sensitivity and high selectivity with an AUC of 0.9 or more.

In addition, the companion animal degenerative osteoarthritis diagnosis method using the biomarker of the present invention can double-distinguish the difference between the normal and the patient by calculating the expression rate in addition to the ROC (receiver operating characteristic) curve.

In addition, the companion animal degenerative osteoarthritis-specific N-saccharide biomarker of the present invention can be used for diagnostic kits in the future, and has the potential to be widely applied to disease activity measurement and development of specific therapeutic agents.

1 is a flowchart showing an experimental method for finding a biomarker of the present invention.
2A to 2E show the composition and information of sugar chains of blood sugar chains capable of diagnosing the disease of the present invention, and the expression (box plot) and ROC curves of normal patients.
2a to 2c show the expected structure of major sugar chains as a companion animal osteoarthritis marker of the present invention, distribution differences between normal (black circle on the left), patient (white circle on the right), and ROC curves for use as biomarkers am.
2D and 2E show the expected structure of trace sugar chains as a companion animal osteoarthritis marker of the present invention, differences in distribution between normal (black circle on the left), and patients (white circle on the right), and ROC curves for use as biomarkers am.
In the figures shown in FIGS. 2A to 2E , the green circle indicates mannose (Man), the yellow circle indicates galactose (Gal), and “OAc” indicates O-acetylation, and the blue square indicates N-acetylglucosamine (GlcNAc), The red triangle represents fucose, the purple diamond represents N-acetylneuraminic acid (NeuAc) (also known as sialic acid), and the gray diamond represents N-glycolylneuraminic acid (NeuGc).

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the examples.

(1) sample

Serum samples from a total of 133 dogs, including 41 normal dogs and 92 osteoarthritis dogs, were used in this experiment. 25 μl of the normal sample and the patient sample were the same.

(2) protein denaturation

For smooth separation of N-saccharide chains, serum samples were first removed from the tertiary structure through protein denaturation. _{200mM NH 4} HCO _{3 in 25} μl of serum sample 25 μl of (including 10mM dithiothreitol) was added, stirred, and then treated in hot water at 90°C or higher and cold water at 4°C for 10 seconds each for 2 minutes alternately to induce protein denaturation and to prevent protein accumulation due to excessive heat.

(3) Enzyme treatment for separation of N-saccharide chains

PNGase F (peptide N-glycosidase F; 500,000 unit/ml) purchased from New England BioLabs (Ipswich, MA) was treated in the sample to extract N-saccharide chains from the sample. After the protein denaturation process, 1 μl (500 units) of PNGase F was added to the sample and incubated at 37° C. for 16 hours using a constant temperature water bath to separate N-saccharide chains.

(4) Protein precipitation treatment for protein removal

After adding 200 μl of cold ethanol to the sample on which the enzymatic reaction for separation of N-sugar chains was completed, it was allowed to stand at -45° C. for 1 hour to precipitate the protein remaining after the separation of N-saccharide chains. After protein precipitation, centrifugation was performed at 4° C. at 14,400 rpm per minute for 20 minutes, and then 200 μl of the supernatant was taken from each sample and completely dried.

(5) N-saccharide chain separation, purification and concentration

In order to separate and purify only N-saccharide chains from the sample, a solid phase extraction (SPE) method using a graphitized carbon cartridge was applied. Graphitized carbon cartridges were purchased from Agilent. First, the cartridge was washed with 6 ml of ultrapure water, 6 ml of 80% (v/v) acetonitrile (containing 0.1% trifluoroacetic acid), and 6 ml of ultrapure water in this order. Thereafter, a sugar chain sample dissolved in 1 ml of ultrapure water was injected into the graphitized carbon cartridge, and then 6 ml of ultrapure water was flowed to remove salts. The sugar chains remaining in the cartridge are each 6 ml of 10% (v/v) acetonitrile, 6 ml of 20% (v/v) acetonitrile, and 40% (v/v) acetonitrile (0.05% trifluoro acetic acid) was eluted in the following order. Each fraction was collected, dried by a vacuum centrifugal evaporator, and dissolved in 15 μl of ultrapure water prior to mass spectrometry.

(6) N-sugar chain analysis

The obtained sugar chains were analyzed by nanoLC chip/Q-TOF MS. In more detail, the diluted sugar chains were subjected to an autosampler (maintained at 4°C), a tubular pump, a nanopump, an HPLC-Chip/MS interface and an HPLC Chip/Q-TOF (Chip Quadrupole Time-of-toF) with a 6540 Q-TOF MS detector. Flight) was analyzed using the MS system (Agilent Technologies, Santa Clara, CA). A PGC packed chip was used with a nano-ESI spray tip integrated into the stationary phase of a 4 mm concentration column and a 43×0.075 mm analytical column. Separation by chromatography was performed under optimized separation conditions. Briefly, after loading the sample, 0.3 μl of an elution gradient solution of intact sugar chains was flowed per minute. The gradient solutions are (A) a 3% acetonitrile (v/v) solution with 0.1% formic acid, and (B) a 90% acetonitrile (v/v) solution with 0.1% formic acid, each of the following Time was treated with the following concentrations: 3% B, 0-2.5 min; 3-16% B, 2.5-20 min; 16-44% B, 20-40 min; 44-100% B, 40-45 min; 100% B, 45-55 min; 100-3% B, 55-57 min. Finally, the analytical column was re-equilibrated with 3% B for 18 min. After mass spectrometry, the original data of LC/MS were analyzed using the Deconvolution algorithm (Maximum Entropy) included in Mass Hunter Qualitative Analysis software (version B.06.00 SP2, Agilent Technologies) & Bioconfirm software (version B.06.00, Agilent Technologies). was processed by

(7) Confirmation of N-sugar chain composition

The N-sugar chain composition is given by comparing the mass value obtained by mass spectrometry and the theoretically possible mass value of the sugar chain composition within 20ppm (±0.005Da), and the total abundance of the revealed sugar chains is calculated for each sugar chain. was standardized. Later, the difference was confirmed by comparing the N-saccharide content of the normal and the patient, and the sugar chains with a difference between the normal and the patient ^{less than p value 10 -10} and the difference in expression amount more than 1.5 times were selected as markers.

5. Results

Specific sugar chains showing differences between normal and patients among hundreds of sugar chains obtained by isolating and analyzing N-sugar chains present in 25 μl of blood from a total of 133 blood samples, including 41 normal dogs and 92 dogs with osteoarthritis. was selected and the expression level was observed.

Table 1 shows the number of sugar chains according to the serum sugar chain extraction process of companion animals.

(A) Using the mass values obtained by mass analysis of serum samples from 41 normal and 92 patients, sugar chains were first confirmed by comparing (±0.001 Da) with the theoretical values of sugar chains that can be combined based on the theory of sugar chain biosynthesis (normal). 316 in the group and 597 in the patient group).

(B) Sugar chains with an appearance frequency of 10% or more (4 or more in the normal group, 9 or more in the patient group) were filtered (163 types in the normal group, 463 types in the patient group, 466 types in total).

(C) The signal intensity (peak abundance) of each sugar chain that went through step (B) above was normalized to the total abundance.

NAPI (%) = (abundance of the corresponding sugar chain/sum of the abundance of all sugar chains)X100

(D) After sorting by distribution of standardized abundance,

1) Major sugar chains corresponding to the top 95% were grouped (36 in normal group, 50 in patient group, total of 51).

2) Minor sugar chains corresponding to 95-99% were grouped (30 types in the normal group, 102 types in the patient group, 101 types in total).

3) Trace sugar chains corresponding to the lower 1% were grouped (97 species in the normal group, 311 species, a total of 314 species).

division normal patient Total Number of samples 41 92 - Number
of N- glycans Extracted 316 597 - Filtered (f>10%) 163 463 466 Major class (Acc. NAPI ≤ 95%) 36 50 51 Minor class (Acc. NAPI 95%~99%) 30 102 101 Trace (Acc. NAPI > 99% 97 311 314

Table 2 shows the composition information and expression levels of 34 types of sugar chains, which are significantly different by comparing 316 types of sugar chains found in normal and 597 types of sugar chains found in patients. For direct comparison of sugar chain expression between normal control and patient groups, the normalized abundance (NAPI) of 466 sugar chains with a frequency of 10% or more was integrated and compared as an average value that can represent the overall trend of the two groups. To find the marker, a t-test was performed, and among the 466 types of sugar chains, ^{34 types showing a significant difference (P<10 -10} ) between the normal control group and the patient group were primarily selected as diagnostic markers.

Only when the AUC of the ROC curve was higher than 0.90 among the 34 primary selected marker candidates, it was selected as the final diagnostic marker. Among the 25 diagnostic markers selected by the present inventors, 13 are major sugar chains belonging to the top 95%, and 12 are trace sugar chains belonging to 95-99%. Among the sugar chain markers we identified, sugar chains belonging to the trace trace group were not included. _{The best N-saccharide marker for diagnosing osteoarthritis in companion animals is (Hex) 3} (HexNAc) ₂ , which belongs to the major sugar chain group, shows a statistically significant difference between the two samples, and represents AUC 1.

We additionally converted the expression level of the marker sugar chains in the patient group compared to the normal control group into the ratio, that is, the sugar chain expression level of the patient group/the sugar chain expression level of the normal group. was confirmed to be visible. 2 markers ((Hex) ₃ (HexNAc) ₄ (Fuc) ₃ and (Hex) ₇ (HexNAc) ₆ (Fuc) ₃ (NeuAc) ₂ (NeuGc) ₁ ) In the case of normal, it is a specific sugar chain that is not expressed.

The abbreviations used in Table 2 have the following meanings. m/z, molecular mass; NOTE, Hex_HexNAc_Fuc_NeuAc_NeuGc; AVE, average; SD, standard deviation; CV, coefficient of variation; Acc, accumulation intensity.

The abbreviations used in Table 3 have the following meanings. m/z, molecular mass; NOTE, Hex_HexNAc_Fuc_NeuAc_NeuGc; CLASS, sugar chain type (C: complex, HB: hybrid, C/HB: complex or hybrid, F: fucosylated, S: sialylated); CATEGORY, Major: NAPI≤95%, Minor: NAPI 95~99%; P/C ratio: patient/control intensity ratio; A/P, absent/present; AUC, area under the curve.

Claims (12)

(A) obtaining serum from a dog suspected of osteoarthritis;
(B) denaturing the serum protein contained in the serum;
(C) separating N-saccharide chains by enzymatically treating the denatured serum protein;
(D) mass spectrometry of the separated N-saccharide chain; and
(E) Diagnosis of osteoarthritis when the mass spectrometry result shows a difference in expression level of one or more sugar chain biomarkers selected from the following (1) to (25) compared with the corresponding sugar chain mass spectrometry result of the normal dog group; including; canine osteoarthritis diagnosis method;
(1) (Hex)3(HexNAc)2, theoretical monoisotope mass value 910.3,
(2) (Hex)3(HexNAc)2(Fuc)1, theoretical monoisotope mass value 1056.4,
(3) (Hex)4(HexNAc)4(Fuc)2(NeuAc)1, theoretical monoisotope mass value 2061.8,
(4) (Hex)6(HexNAc)5(NeuAc)1, theoretical monoisotope mass value 2296.8,
(5) (Hex)5(HexNAc)4(Fuc)1(NeuAc)2, theoretical monoisotope mass value 2368.8,
(6) (Hex)6(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2239.8,
(7) (Hex)4(HexNAc)3(NeuAc)1, theoretical monoisotope mass value 1566.6,
(8) (Hex)4(HexNAc)3, theoretical monoisotope mass value 1275.5,
(9) (Hex)3(HexNAc)3(Fuc)1, theoretical monoisotope mass value 1259.5,
(10) (Hex)5(HexNAc)3, theoretical monoisotope mass value 1437.5,
(11) (Hex)6(HexNAc)4(NeuAc)1, theoretical monoisotope mass value 2093.7,
(12) (Hex)5(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2277.7,
(13) (Hex)3(HexNAc)4(Fuc)1, theoretical monoisotope mass value 1462.5,
(14) (Hex)3(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1754.7,
(15) (Hex)4(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1916.7,
(16) (Hex)7(HexNAc)7(Fuc)5(NeuAc)2(NeuGc)2, theoretical monoisotope mass value 4500.6;
(17) (Hex)5(HexNAc)4(Fuc)3, theoretical monoisotope mass value 2078.8,
(18) (Hex)4(HexNAc)5(Fuc)2, theoretical monoisotope mass value 1973.7,
(19) (Hex)7(HexNAc)7(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3901.4,
(20) (Hex)6(HexNAc)7(Fuc)4, theoretical monoisotope mass value 2996.1,
(21) (Hex)7(HexNAc)4, theoretical monoisotope mass value 1964.7,
(22) (Hex)7(HexNAc)6(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3698.3,
(23) (Hex)6(HexNAc)3, theoretical monoisotope mass value 1599.6,
(24) (Hex)7(HexNAc)6(Fuc)1(NeuGc)1, theoretical monoisotopic mass value of 2824.0 and
(25) (Hex)6(HexNAc)3(Fuc)1(NeuAc)1, theoretical monoisotopic mass value 2036.7. {However, HexNAc, N-acetylhexosamine (N-acetylhexosamine); Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic Acid}.
The method according to claim 1,
The sugar chain biomarker has an AUC value of 0.9 or more, a method for diagnosing osteoarthritis in dogs.
The method according to claim 1,
One or more sugar chain biomarkers of (1), (2), (4), (5), (7), (8), (9), (13) and (24) above showed osteoarthritis compared to the normal dog group. A method for diagnosing canine osteoarthritis, which is less common in suspected dogs.
The method according to claim 1,
(3), (6), (10), (11), (12), (14), (15), (16), (17), (18), (19), (20), ( 21), (22), (23) and (25), one or more sugar chain biomarkers are more expressed in dogs with osteoarthritis than in the normal dog group, a method for diagnosing canine osteoarthritis.
The method according to claim 1,
A method for diagnosing canine osteoarthritis including a sugar chain biomarker of (1) with an AUC value of 1.
The method according to claim 1,
A method for diagnosing canine osteoarthritis comprising one or more sugar chain biomarkers in (14) or (22) that are not found in the normal canine group.
The method according to claim 1,
In step (E), the N-saccharide chain composition was confirmed with the mass value obtained by mass spectrometry, and the N-saccharide chain sensitization was determined between the normal dog group and the osteoarthritis-suspected dog group by normalizing the identified N-saccharide chains to the total abundance. A method for diagnosing canine osteoarthritis comprising the step of comparing.
The method according to claim 1,
The enzyme treatment in step (c) is a dog osteoarthritis diagnosis method, characterized in that the denatured serum protein is treated with PNGase F enzyme.
The method according to claim 1,
In the step (E), (1) of claim 1, when the expression level of the sugar chain biomarker is lower than that of the normal dog group, the dog suspected of osteoarthritis is diagnosed as having osteoarthritis, the dog osteoarthritis diagnosis method.
The method according to claim 1,
In the step (E), when one or more sugar chain biomarkers of claim 1 (14) or (22) are expressed, the canine osteoarthritis diagnosis method for a dog suspected of osteoarthritis is diagnosed as having osteoarthritis.
a) treating the test material to dog-derived chondrocytes; and
b) As a result of treatment with the test substance in a), there was a significant difference in the expression level of one or more of the sugar chain biomarkers of claim 1 (1) to (25) compared to the normal control before the test substance treatment, but after the test substance treatment A method of screening for a treatment for canine osteoarthritis, comprising a; selecting a test material that exhibits a change that is not significantly different from the normal control. delete

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