WO2014010261A1 - 乳腺疾患の検査方法 - Google Patents
乳腺疾患の検査方法 Download PDFInfo
- Publication number
- WO2014010261A1 WO2014010261A1 PCT/JP2013/052955 JP2013052955W WO2014010261A1 WO 2014010261 A1 WO2014010261 A1 WO 2014010261A1 JP 2013052955 W JP2013052955 W JP 2013052955W WO 2014010261 A1 WO2014010261 A1 WO 2014010261A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cypa
- milk
- mammary gland
- antibody
- mastitis
- Prior art date
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/99—Isomerases (5.)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/365—Breast disorders, e.g. mastalgia, mastitits, Paget's disease
Definitions
- the present invention relates to a method for examining a mammary gland disease such as mastitis in a breast or a milk chamber of a subject.
- the bovine breast is divided into left and right sides by a thick central ligament, and front and back by a thin septum, which are independent quarters.
- the bovine breast consists of a number of mammary cysts, and a single layer of mammary epithelial cells is arranged on the mammary gland side of the mammary cyst.
- Mammary epithelial cells of healthy cows are tightly bound by an intercellular adhesion factor such as tight junction.
- Mammary epithelial cells form a layer and perform a physical barrier function that separates the inside and outside of the mammary gland. Due to the mammary epithelial cell layer, the mutual inflow of substances, such as blood to milk or milk to blood, is prevented.
- the mammary epithelial cells mainly (1) synthesize milk protein and lactose and secrete them from the alveolar space, (2) transport lipid droplets to the alveolar space, and project into the alveolar space Thus, it has a function of inducing milk fat secretion and (3) a function of transferring serum albumin and immunoglobulin from blood to milk.
- epithelial cells are involved in defense mechanisms that prevent foreign substances such as pathogenic microorganisms from entering the body.
- Epithelial cells not only physically prevent the invasion of pathogens, but also chemically inhibit the invasion and proliferation of pathogenic microorganisms by lactic acid, mucin, lysozyme, antibacterial peptides, etc. produced by epithelial cells.
- epithelial cells are also involved in the induction of early immune responses by producing various cytokines.
- Non-Patent Document 4 cellular immunity involving ⁇ + T cells and CD8 + T cells mainly functions during lactation. Therefore, in contrast to mucosal immunity such as the intestinal tract where IgA production is immediately performed by B cells, innate immunity has attracted attention for the immune mechanism of mammary epithelium (see Non-Patent Documents 5 and 6 below, description of the document) Is hereby incorporated by reference).
- Mastitis is an inflammatory disease caused by pathogens entering the breast. Invasion of microorganisms such as bacteria, fungi, and yeast from the nipple hole causes inflammation in the mammary gland and mastitis. Compared with a normal milk quarter, the milk quarter which developed mastitis is generally enlarged and the tissue is destroyed. Symptoms of mastitis include (1) an increase in the number of somatic cells in milk due to the mobilization of immune cells, and (2) a decrease in milk yield and quality due to interstitial hypertrophy and atrophy of the mammary gland. In addition to these, individuals with mastitis may develop systemic symptoms such as loss of appetite and diarrhea and even death.
- Mastitis is roughly classified into clinical mastitis and latent mastitis in which the number of somatic cells in milk increases without showing clinical symptoms.
- Latent mastitis accounts for about 80% of the mastitis damage.
- Escherichia coli a gram-negative bacterium, causes severe inflammation and clinical mastitis, whereas gram-positive bacteria such as S. aureus often cause latent mastitis.
- Subclinical mastitis has a problem that it is difficult to find and treat because the frequency of clinical manifestation is relatively small, and it spreads to the population without realizing it. Symptoms may progress and may be clinical.
- the main treatment for mastitis is the administration of antibiotics.
- S. aureus mastitis is generally considered difficult to cure with antibiotics because some S. aureus is resistant to antibiotics and forms microabscesses in the mammary gland. ing.
- Antibiotics are therapeutically effective in preventing infection.
- the administration of antibiotics is a coping therapy and cannot directly prevent damage to breast tissue.
- Other treatment methods for mastitis include treatments using biologically active substances such as cytokines (GM-CSF, CXCL8, hIFN- ⁇ ) and natural substances with antibacterial action (stevia extract fermentation extract, defensin, Vimulon). Has been tried.
- a PL test As a method for diagnosing mastitis, a PL test is employed in which changes in pH and viscosity caused by aggregation and denaturation due to the number of white blood cells in milk are used as indices.
- somatic cell number inspection method using as an index the number of immune cells released into milk against pathogenic bacteria that have entered the mammary gland.
- a method of detecting a weak potential that appears when somatic cells are active as an indicator by applying somatic cell count testing that is, a method for measuring chemiluminescence (CL ability) is known. (See Non-Patent Document 14 below, the description of which is incorporated herein by reference).
- the CL ability measurement method has a measurement principle of measuring the amount of active oxygen released from neutrophils by chemiluminescence.
- mastitis several cases of changes in lymphocyte subsets in milk due to infiltration of immune cells such as lymphocytes in response to bacteria in milk have been reported (see below). (See Non-Patent Documents 8 and 9, the description of which is incorporated herein by reference).
- Leukocyte migration refers to a phenomenon in which leukocytes stimulated by migration factors such as chemokines, cytokines, and bioactive lipids secreted during inflammation infiltrate into the tissue from the blood vessel and migrate and accumulate at the site of inflammation.
- migration factors such as chemokines, cytokines, and bioactive lipids secreted during inflammation infiltrate into the tissue from the blood vessel and migrate and accumulate at the site of inflammation.
- chemokines which are a family of running factor cytokines
- Another factor is cyclophilin, which is one of running factors. Cyclophilin is expressed in all cells of any organism, including prokaryotes and eukaryotes.
- Cyclophilin is generally an intracellular protein, has peptidyl-prolyl cis-trans isomerase activity, which is a factor involved in protein folding, and is known as an FK-506 binding protein. There are two main cyclophilins, 18 kDa cyclophilin A (CyPA) and 21 kDa cyclophilin B (CyPB). Cyclophilin A is a cytoplasmic protein having no signal sequence, and cyclophilin B is linked to the endoplasmic reticulum through an N-terminal signal sequence (see Non-Patent Document 10 below, the description of which is incorporated herein by reference) .
- CyPA is the most abundant cyclophilin and is said to occupy about 0.1 to 0.4% of the total intracellular protein, and is a protein group belonging to the intracellular binding factor of cyclosporin A which is an immunosuppressant. It is known as a member (see Non-Patent Documents 11 and 12 below, the description of which is incorporated herein by reference). Further, during the onset of inflammation, CyPA is released and secreted from dead and living cells and functions extracellularly (see Non-Patent Documents 13 to 16 below, the description of which is incorporated herein by reference). .
- leukocyte subsets such as monocytes, eosinophils, neutrophils and T-lymphocytes
- An increase in CyPA has been reported in diseases such as sepsis, rheumatism, arthritis, pneumonia, and aneurysms (see Non-Patent Documents 19 and 20 below, which are incorporated herein by reference).
- rheumatism there is a report that there is a correlation between the amount of CyPA and the number of neutrophils (see Non-Patent Document 21 below, the description of which is incorporated herein by reference).
- CyPA is expressed and secreted in lung epithelial cells and vascular endothelial cells.
- extracellular CyPA is a powerful chemotactic factor for human monocytes, neutrophils, eosinophils and T cells (see Non-Patent Document 21 below, the description of which is disclosed herein) As a).
- CyPA may induce inflammation because it is released by an acute inflammatory reaction such as neutrophil mobilization when bacteria enter the body (see Non-Patent Document 11 below). Reference, the description of this document is hereby incorporated by reference).
- Livestock mastitis especially dairy cow mastitis
- the decrease in milk yield and the increase in treatment costs such as antibiotics but also the disuse and death of infected cattle as mastitis progresses.
- Mastitis is regarded as one of the most incurable diseases of livestock in the world, and the economic loss caused by mastitis is estimated to be 80 billion yen per year in Japan and more than $ 1.8 billion per year in the United States.
- MRSA Menticillin-resistant Staphylococcus aureus
- the current standard method for detecting mastitis is based on milk in the middle stage of mastitis, where degeneration has already been observed. Sensitivity for discovery is lacking. Therefore, the CL ability measurement method is promising as an early detection method for mastitis.
- the CL capacity measurement method is not suitable as a method that each dairy farmer performs on a daily basis from the viewpoint of economy and difficulty. Thus, there is no known method for early detection of mastitis to some extent that can be carried out daily by dairy farmers.
- Cyclophilin A is said to occupy about 0.1 to 0.4% of the total intracellular protein, and expression in lung epithelial cells and vascular endothelial cells has been reported in inflammation such as pneumonia and aneurysms. However, there are no reports that cyclophilin A has been used as a biomarker to identify inflammatory sites.
- an object of the present invention is to provide a method and a biomarker that contribute to early detection of mastitis more easily and quickly than in the conventional method.
- Another object of the present invention is to provide a kit for using the method, a component of the kit, and a product that can be used for production of the component.
- the present inventors have made extensive studies on the molecular mechanism of immune response in the mammary gland with the aim of early detection and early treatment of mastitis. At that time, attention was paid to various cells and biological materials, and repeated trials and errors were repeated. For example, we focused on several chemokines and cytokines but did not lead to early detection or early treatment of mastitis. The present inventors eventually came to focus on cyclophilin A (CyPA).
- CyPA cyclophilin A
- the expression and possibility of CyPA in mammary epithelial cells that have developed mastitis have not been known.
- the present inventors may be able to play a role of mobilizing immune cells to the inflamed site by releasing CyPA as a leukocyte migratory factor when inflammation occurs in some tissues in vivo. Focusing on the relationship between mastitis and CyPA, I thought it would be a clue to early detection of mastitis. Therefore, the present inventors first examined the expression level of CyPA in the mammary gland tissue that developed mastitis using an immunohistochemical technique. Surprisingly, the present inventors have succeeded in obtaining a result that the expression level of CyPA varies depending on the degree of onset of mastitis.
- the present inventors examined the expression level of CyPA protein in milk derived from a milk tub that developed mastitis using Western blotting. Surprisingly, the present inventors have succeeded in obtaining a result that the expression level of CyPA varies depending on the degree of mastitis in milk. These results indicate that measurement of CyPA in mammary gland tissue and milk can identify a milk compartment that develops mastitis. Furthermore, by conducting parallel experiments with the PL method and the CL ability measurement method, which were methods for identifying the mastitis of the previous mastitis, milk in the early stage of mastitis that cannot be identified by these methods The quarter was successfully identified as a milk compartment that developed mastitis or a milk compartment that could develop mastitis. The present invention is a completed invention based on these findings and successful examples.
- a method for examining a mammary gland disease including the following steps (1) and (2) is provided.
- (1) A step of obtaining cyclophilin A amount in milk by detecting cyclophilin A in milk collected from the breast or milk compartment of the subject (2) Based on the amount of cyclophilin A in the milk, Determining the onset or likelihood of developing mammary gland disease in the breast or breast chamber
- step (2) when the amount of cyclophilin A in the milk is larger than the amount of cyclophilin A in milk collected from a healthy breast or a milk tub, It is a step of determining that a mammary gland disease has developed or is likely to develop in the breast or breast quarter.
- step (2) when the amount of cyclophilin A in the milk is twice or more than the amount of cyclophilin A in milk collected from a healthy breast or milk tub, It is a step of determining that a mammary gland disease has developed or is likely to develop in the subject's breast or breast chamber.
- a method for examining a mammary gland disease including the following steps (1 ′) and (2 ′) is provided.
- (1 ′) a step of obtaining cyclophilin A amount in the mammary gland by detecting cyclophilin A in the mammary gland collected from the breast or milk compartment of the subject (2 ′) based on the amount of cyclophilin A in the mammary gland Determining the onset or likelihood of developing mammary gland disease in the breast or breast chamber of the body
- the step (2 ′) is performed when the amount of cyclophilin A in the mammary gland is larger than the amount of cyclophilin A in mammary gland tissue collected from a healthy breast or a breast chamber. It is a step of determining that a mammary gland disease has developed in the breast or breast chamber of the body, or that a mammary gland disease may develop.
- the breast disease is an infectious breast disease.
- the breast disease is mastitis.
- the subject is a human or non-human animal.
- the non-human animal is selected from the group consisting of cow, goat, buffalo, yak, sheep, horse and camel.
- the cyclophilin A is detected by Western blotting, enzyme-linked immunosorbent assay, enzyme immunoassay (EIA method), fluorescence immunoassay (FIA method), radioimmunoassay (RIA method), fluorescence polarization immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, electrochemiluminescence immunoassay, ELISPOT method, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, agglutination immunoassay, latex agglutination And a method selected from the group consisting of a chromatographic method and a chromatographic method.
- EIA method enzyme immunoassay
- FFA method fluorescence immunoassay
- RIA method radioimmunoassay
- fluorescence polarization immunoassay fluorescence polarization immunoassay
- chemiluminescence immunoassay chemiluminescence enzyme immunoassay
- a reagent for examining a mammary gland disease comprising an anti-cyclophilin A antibody.
- the anti-cyclophilin A antibody is an anti-cyclophilin A antibody immobilized on an insoluble carrier.
- the insoluble carrier is a bead, a plate, or a thin film.
- the breast disease is an infectious breast disease.
- the breast disease is mastitis.
- kits for examining a mammary gland disease containing the reagent of the present invention.
- a hybridoma having the ability to produce an anti-cyclophilin A antibody is provided.
- an anti-cyclophilin A antibody produced by the hybridoma of the present invention.
- a marker for detecting mammary gland disease comprising cyclophilin A is provided.
- the breast disease is an infectious breast disease.
- the breast disease is mastitis.
- the method of the present invention by using the expression level of cyclophilin A in milk or mammary gland tissue as an index, it is possible to easily and quickly identify a diseased site of a mammary gland disease in a subject as compared with the conventional method. .
- the method of the present invention is particularly useful for identifying breasts or milk quarters that are in the early stages of mastitis.
- the method of the present invention since the method of the present invention is a non-invasive method, it may be applicable to the prevention of mastitis.
- the method of the present invention is a method that can be carried out simply and quickly, it is possible to perform on-site measurement on a dairy farm. Mastitis can be diagnosed early, stop milking early, start treatment with a small amount of antibiotics, shorten the duration of the disease, and resume milking early, reducing treatment costs and reducing income from milk production. Economic losses such as declines in dairy farmers can be minimized.
- Dairy cows are most likely to suffer from mastitis one month after delivery, and the prevalence is said to be about 10%.
- Early detection of the onset or likelihood of mastitis in dairy cows can not only initiate early treatment but also consider more expedited disuse and in any event minimize economic losses. Therefore, the method of the present invention is a method that contributes to reducing the economic loss of the entire dairy industry.
- the kit of the present invention is a kit for carrying out the method of the present invention
- the abundance of cyclophilin A in whey obtained by centrifuging milk collected from a subject. Can be measured easily and rapidly by antigen-antibody reaction.
- the hybridoma of the present invention and the antibody of the present invention are for producing an anti-cyclophilin A antibody provided in the method of the present invention and the kit of the present invention, or are themselves. Therefore, the hybridoma of the present invention and the antibody of the present invention can contribute to easy and rapid early detection of mastitis through the method of the present invention and the kit of the present invention.
- the marker of the present invention is a biomarker excellent in early prediction of mastitis.
- FIG. 1 For normal bovine mammary glands in SEC-administered experimental mastitis sections, sections of mammary gland tissue of the mammary parenchyma were prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to localize the expression of CyPA.
- FIG. The figure shows that in normal bovine mammary tissue, the mammary cysts are large and the stroma is small. The bar is 500 ⁇ m.
- the figure shows that mammary cysts are wide, mammary epithelial cells are flat, and all have expression of CyPA.
- the bar is 100 ⁇ m.
- sections of mammary gland tissue of the mammary parenchyma were prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to localize the expression of CyPA.
- FIG. The figure shows that CyPA expression is weakly observed in cells synthesizing milk from mammary epithelial cells.
- the bar is 100 ⁇ m.
- the figure shows that abnormal mammary epithelial cells become associated with atrophy of mammary cysts, and CyPA expression is high.
- the bar is 100 ⁇ m.
- the figure shows that CyPA expression is further enhanced at sites of strong immune cell infiltration, and that large amounts of CyPA are secreted in the intrafollicular milk.
- the bar is 100 ⁇ m. S.
- the figure shows that mammary cysts are wide and mammary epithelial cells are flat.
- the bar is 500 ⁇ m.
- S. For normal bovine mammary glands in the experimental mastitis treated with A, sections of mammary gland tissue of the mammary parenchyma were prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to show the localization of CyPA expression. It is a figure.
- the figure shows that the majority of mammary epithelial cells express CyPA and there is slight CyPA expression in the secreted milk.
- the bar is 100 ⁇ m. S.
- a section of mammary gland tissue of the mammary parenchyma was prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody), and the site of CyPA expression. It is the figure which showed presence. The figure shows that there are sites where CyPA expression is confirmed and sites where CyPA expression is low. The bar is 500 ⁇ m. S.
- a section of mammary gland tissue of the mammary parenchyma was prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody), and the site of CyPA expression. It is the figure which showed presence. The figure shows that at sites where CyPA is confirmed, CyPA is expressed at the sites of mammary epithelial cells, milk and immune cell infiltration. The bar is 100 ⁇ m. S.
- FIG. 1 For normal bovine mammary glands in latent mastitis due to Lf administration, a section of mammary gland tissue of mammary parenchyma was prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to localize the expression of CyPA.
- FIG. The figure shows that there are few stroma and mammary follicles are wide but there are sites of immune cell infiltration. The bar is 500 ⁇ m.
- a section of mammary gland tissue of mammary parenchyma was prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to localize the expression of CyPA.
- FIG. 1 For normal bovine mammary glands in latent mastitis due to Lf administration, a section of mammary gland tissue of mammary parenchyma was prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to localize the expression of CyPA.
- the figure shows that CyPA expression is low in flat mammary epithelial cells, while CyPA expression is high in mammary epithelial cells of the alveoli where immune cell infiltration sites and infiltration cells are seen.
- the bar is 100 ⁇ m.
- slice of the mammary gland tissue of a mammary gland part about the normal bovine mammary gland which administered lactic acid bacteria production peptide, and performing immunostaining using anti-CyPA (2H5-F3 antibody) is there.
- the figure shows that there are sites that are atrophic and non-atrophyed in the mammary follicles.
- the bar is 500 ⁇ m.
- FIG. 3 shows the localization of CyPA expression by preparing a section of mammary gland tissue of the mammary gland parenchyma and performing immunostaining with anti-CyPA (2H5-F3 antibody) for the bovine mammary gland administered with lactic acid bacteria-producing peptide.
- the figure shows that CyPA expression is low at sites where the mammary follicle is atrophied and the stroma is enlarged, and that CyPA is strongly expressed in immune cells in the stroma.
- the bar is 100 ⁇ m.
- FIG. The figure shows that there are many interstitial sites of immune cells infiltration, although there are few stroma and mammary follicles are wide.
- the bar is 500 ⁇ m.
- a section of mammary gland tissue in the mammary parenchyma was prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to show the localization of CyPA expression.
- FIG. The figure shows that there is interstitial hypertrophy and there is a difference in CyPA expression at sites of immune cell infiltration and non-infiltration. The bar is 100 ⁇ m.
- a section of mammary gland tissue in the mammary parenchyma was prepared, and immunostaining was performed using anti-CyPA (2H5-F3 antibody) to show the localization of CyPA expression.
- FIG. 1 The figure shows that CyPA expression is strong in mammary epithelial cells and atypical epithelial cells ( ⁇ ) having immune cell infiltration sites.
- the bar is 100 ⁇ m.
- the left part shows the analysis result of protein in milk by non-reduction treatment, and the right part shows the analysis result of protein in milk subjected to reduction treatment.
- An arrow ( ⁇ ) in the vicinity of 60 kDa indicates a state in which 160 kDa EgG is reduced by the reduction treatment in analysis number 9 which is inflammatory quarter milk.
- FIG. 1 An arrow ( ⁇ ) near 50 kDa indicates that the band is expressed even when the protein is reduced. It is the figure which showed the result analyzed using the western blotting method about the CyPA protein in the whey by the presence or absence of a reduction process using the sample of the bovine body number 81 in Table 1.
- FIG. The figure shows that when no anti-CyPA antibody is used, there is no non-specific reaction by the secondary antibody regardless of the reduction treatment. It is the figure which showed the result analyzed using the western blotting method about the CyPA protein in the whey by the presence or absence of a reduction process using the sample of the bovine body number 81 in Table 1.
- FIG. 7 shows the results of protein analysis of CyPA in milk using Western blotting after reducing milk of analysis numbers 1 to 48 (including blind milk specimen 19). The figure shows that the expression of CyPA was confirmed in healthy cow quarter milk (analysis numbers 33 to 48).
- FIG. 7 shows the results of protein analysis of CyPA in milk using Western blotting after reducing milk of analysis numbers 1 to 48 (including blind milk specimen 19).
- FIG. 7 shows the results of protein analysis of CyPA in milk using Western blotting after reducing milk of analysis numbers 1 to 48 (including blind milk specimen 19).
- the figure shows that the expression level of CyPA in the mastitis-onset quarter milk is larger than the expression level of CyPA in the healthy cow quarter milk (analysis numbers 33 to 48) in FIG. 10A. It is the figure which showed the correlation with the chemiluminescent ability and the expression level of CyPA in milk. Specifically, using the analysis results of FIGS.
- the method of the present invention is a method for examining mammary disease using the amount of cyclophilin A in the mammary gland or milk as an index.
- the test object in the method of the present invention is breast disease.
- Breast disease is a general term for pathological conditions that cause abnormalities in the breasts and mammary glands in the milk compartment.
- the mammary gland diseases include infectious mammary gland diseases caused by foreign substances such as bacteria, and neoplastic mammary gland diseases such as breast cancer and fibroadenoma.
- the mammary gland disease in the method of the present invention is not particularly limited, but since an increase in cyclophilin A level was observed in mammary tissue and milk that developed mastitis, it is preferably an infectious mammary gland disease and is mastitis It is more preferable.
- Mastitis is an inflammatory disease caused by pathogens entering the breast. Mastitis is caused by (1) inflammation in the mammary gland due to invasion of bacteria such as S. aureus and E. coli, molds, and yeasts from the teat foramen, and / or Or (2) It is presumed that the disease develops due to an inflammatory reaction caused by colonization of mammary epithelial cells with bacterial toxins such as LPS and SEs produced by the above-described microorganisms, coagulase and other proteins.
- bacteria such as S. aureus and E. coli, molds, and yeasts from the teat foramen
- / or Or It is presumed that the disease develops due to an inflammatory reaction caused by colonization of mammary epithelial cells with bacterial toxins such as LPS and SEs produced by the above-described microorganisms, coagulase and other proteins.
- Mastitis is roughly divided into clinical mastitis and latent mastitis.
- Clinical mastitis can be determined by observing the breast and milk with the naked eye when symptoms such as swelling and fever are seen in the breast, or degeneration such as looseness and viscosity is seen in the milk.
- Latent mastitis does not show the abnormalities as described above, and can be determined by performing a milk test.
- a milk test a test method for detecting pathogenic bacteria in milk, a PL test based on an increase in somatic cell count (SCC) and changes in the pH of milk (CMT modified method), in milk It has been carried out by a CL capacity measurement method for measuring the chemiluminescence capacity (Chemical Luminescence: CL capacity). Since the method of the present invention is assumed to be a method performed in place of or in parallel with the conventional method performed at the time of the milk test, the mammary gland disease in the method of the present invention is assumed. More preferred is latent mastitis.
- the method of the present invention can be used to determine the possibility of developing mammary gland diseases including prediction of the risk of developing mammary gland diseases and early prediction in addition to the determination of the development of mammary gland diseases including mastitis.
- the mammary gland as a specimen sample is not particularly limited as long as it is meant to be used by those skilled in the art, and usually refers to a secretory gland having a function of secreting milk inside the breast.
- a single layer of mammary epithelial cells is arranged on the cavity side of the mammary cyst and has a barrier function to separate the inside and outside.
- Mammary epithelial cells are known to produce cytokines and antimicrobial peptides.
- the mammary gland contains mammary intraepithelial lymphocytes (mIEL).
- the mammary gland which is a specimen sample may be a part constituting mammary gland tissue such as mammary epithelial cells in addition to the mammary gland tissue itself.
- the milk in the method of the present invention is not particularly limited as long as it is meant by those skilled in the art, and usually refers to a humoral substance secreted from the mammary gland.
- Cyclophilin A is a protein having peptidyl-prolyl cis-trans isomerase activity, which is a factor involved in protein folding.
- the molecular weight of CyPA is about 18 kDa.
- GenBank GenBank
- NBI National Center for Biotechnology Information
- the bovine-derived one is ACCESSION: DAA30468
- the sheep-derived one is ACCESSION: AAP03083
- the mouse-derived one is ACCESSION: AAD50966, the rhesus monkey-derived one is ACCESSESSION: NP_0010279811, the chimpanzee-derived one is ACCESSESSION: ABB77876, the rat-derived one is ACCESSION: NP_058797, and it is derived from orangutan
- the thing is ACCESSION: NP_001126060, And those derived from human ACCESSION: NP_066953 a NP_001008741
- the amount of cyclophilin A (CyPA) in the method of the present invention is not particularly limited as long as it is the amount of CyPA present.
- the amount of CyPA may be expressed as an absolute value and / or a relative value, and may be determined physically, chemically and / or biologically. Therefore, the method of the present invention is not limited to a specific step, and is performed by using the amount of CyPA in the mammary gland or milk as an index.
- the amount of CyPA present in the living body is not used as an index, but the amount of CyPA in the mammary gland or milk collected from the living body is used as an index. Therefore, the marker of the present invention to be described later uses a mammary gland collected from a living body or CyPA separated from milk as a mastitis detection marker.
- One aspect of the method of the present invention is a method for examining a mammary gland disease, which includes the following steps (1) and (2).
- the subject in the method of the present invention is not particularly limited and may be a human or a non-human animal, but is preferably a non-human animal that is difficult to determine the possibility of developing latent mastitis.
- Cattle, goats, buffalos, yaks, sheep, horses, camels and the like, which are daily milked livestock animals, are more preferred, and cattle are more preferred.
- the breast encloses the mammary lobe, which is a mass of the mammary gland, and the milk produced by the mammary cysts in the mammary gland is secreted from the nipple to the outside of the body via the milk duct. While the breast is based on the above structure, the breast has a different structure depending on the animal species. For example, a human has a pair of left and right independent breasts, while a cow has four independent milk compartments separated by left and right by a thick central ligament and further separated by a thin septum.
- the breast and the milk compartment refer to the meanings commonly known by those skilled in the art, but as a specific example, a human breast refers to a pair of left and right breasts that a human has, Milk quarter refers to any of the four independent cows in cows.
- a method for collecting milk from the subject's breast or milk compartment is not particularly limited, and means commonly used by those skilled in the art, such as squeezing the breast at room temperature, can be employed.
- the amount of milk to be collected is not particularly limited as long as it is an amount capable of detecting CyPA, and can be appropriately changed by a means for detecting CyPA.
- the timing (timing) of collecting milk is not particularly limited, but it is obtained from the fact that the amount of CyPA increased with high sensitivity in milk obtained from a milk compartment that developed mastitis or from a milk compartment that is suspected of having mastitis.
- the possibility of milking or milking it is preferred to collect milk continuously or periodically from a breast or milk tub with a lip.
- the onset of mammary gland disease can be predicted at an early stage by using the milk collected before the start of milking or at an early stage from the start of milking.
- the onset of mastitis is observed in any breast or milk chamber. It is preferred to target subjects who have not been tested.
- the method for detecting CyPA is not particularly limited, and can be carried out by applying a method for detecting a specific protein or polypeptide or mRNA known by those skilled in the art.
- detecting cyclophilin A is synonymous with measuring the amount of CyPA.
- the amount of CyPA may be measured semi-quantitatively. That is, it is only necessary to detect the amount of CyPA for an amount comparable to the reference amount to be compared.
- the technique for detecting CyPA may be, for example, a technique in which the stage of separating CyPA and the stage of obtaining the separated CyPA amount are performed separately, or a technique of performing these in one stage.
- the method for reducing the specimen sample containing CyPA is not particularly limited, and reduction methods known to those skilled in the art can be used.
- reducing properties such as ascorbic acid, ⁇ -mercaptoethanol, dithiothreitol, etc.
- Reduction treatment using substances can be mentioned, and among these, reduction treatment using ⁇ -mercaptoethanol is preferred.
- the detection of CyPA is performed by, for example, high-performance liquid chromatography or gel filtration by obtaining a CyPA profile in advance in addition to immunological techniques such as Western blotting, sandwich method, and competitive method. It can be carried out by an analytical chemistry method based on chromatography, such as a chromatography method.
- CyPA adenosine-linked immunosorbent assay
- enzyme immunoassay enzyme immunoassay
- FFA method fluorescence immunoassay
- RIA method radioimmunoassay
- fluorescence Polarization immunoassay chemiluminescence immunoassay
- chemiluminescence immunoassay chemiluminescence enzyme immunoassay
- electrochemiluminescence immunoassay ELISPOT method
- immunoprecipitation assay precipitation reaction, gel diffusion precipitation reaction, agglutination immunoassay, latex agglutination method and chromatography method Etc.
- an immunological technique is preferably used.
- immunological techniques there is a technique that can measure the amount of CyPA quickly, conveniently and with high sensitivity.
- a substance having specific affinity for CyPA such as an anti-CyPA antibody is used.
- an anti-CyPA antibody not only the anti-CyPA antibody but various substances can be used as long as they have specific affinity for CyPA and can measure the amount of binding to CyPA.
- detection of CyPA in addition to a method of directly detecting CyPA in a specimen with a labeled antibody, a method provided with various stages such as antigen capture and detection may be employed.
- An example of a method for detecting CyPA is Western blotting.
- the Western blot method is one of the methods performed separately from the separation and quantification of the target protein.
- the Western blotting method for detecting CyPA is not particularly limited, but the following method can be adopted as an example. First, a whey sample obtained by centrifuging milk is separated from CyPA and other proteins by SDS-PAGE. The band (protein) on the gel is then transferred to the membrane. The membrane is then subjected to an anti-CyPA antibody. Next, an antibody against the anti-CyPA antibody labeled on the membrane is provided. Next, the amount and activity of the labeling substance in the band corresponding to CyPA on the membrane are measured.
- Western blotting for detecting CyPA is not limited to the above-described method.
- an antibody against a labeled anti-CyPA antibody may be enzyme-labeled or fluorescently labeled.
- Various procedures and conditions can be modified as appropriate by methods known to those skilled in the art.
- Examples of immunological techniques that can measure the amount of CyPA rapidly, conveniently and with high sensitivity include FIA and EIA.
- FIA fluorescently labeled antibody
- EIA enzyme-labeled antibody
- an immune complex is detected using color development or luminescence based on an enzyme reaction as a signal.
- the ELISA method has many advantages such as high detection sensitivity, high specificity, excellent quantitativeness, simple operation, and suitability for simultaneous processing of multiple samples.
- the ELISA method includes a competitive method, a sandwich method, a direct adsorption method and the like.
- the competition method is a technique based on the principle of competing for the formation of immune complexes by adding an antigen together with a specimen.
- sandwich method usually, two types of antibodies having different epitopes (a primary antibody as a capture antibody and a secondary antibody as a detection antibody) are used. The outline of the procedure will be described below, but these methods are not limited to the following procedure.
- CyPA protein and labeled CyPA protein in a whey sample are reacted competitively with an anti-CyPA antibody, and then unreacted labeled CyPA (F) and labeled CyPA bound to the antibody ( B) is separated (B / F separation), and then the amount of either B or F labeling is measured to quantify the CyPA protein in the whey sample.
- a soluble antibody is used as an antibody, and a B / F separation is performed using a secondary antibody against polyethylene glycol or an anti-CyPA antibody (primary antibody).
- a solid-phase antibody is used as a primary antibody.
- a direct immobilization method using a primary antibody is soluble and an indirect solid phase immobilization method using a solid phase antibody as a secondary antibody is used, but there is no particular limitation.
- a whey sample is reacted with a first anti-CyPA antibody immobilized on a carrier (primary reaction), and then an epitope different from that of the first anti-CyPA antibody is recognized and labeled.
- the second anti-CyPA antibody is reacted (secondary reaction), and then the amount or activity of the labeling agent on the carrier is measured.
- the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times.
- the labeling agent can be appropriately selected by those skilled in the art, and for the antibody immobilized on the carrier or the labeled antibody, a mixture of two or more kinds of antibodies may be used for the purpose of improving the measurement sensitivity.
- an anti-CyPA antibody is immobilized on an insoluble carrier. Specifically, for example, the surface of the microplate is sensitized (coated) with an anti-CyPA antibody (primary antibody). A specimen sample obtained by centrifuging milk or mammary gland epithelial cells is brought into contact with the antibody thus immobilized. As a result of this operation, if an antigen (CyPA) against the immobilized anti-CyPA antibody is present in the sample, an immune complex is formed. After removing non-specific binding components by washing operation, an immune complex is labeled by adding an antibody to which an enzyme is bound (secondary antibody), and then the enzyme substrate is reacted to cause color development. Then, immune complexes are detected using the color development amount as an index. Details of the ELISA method are described in many books and papers, and can be referred to when setting the experimental procedure and experimental conditions of each method.
- step (2) based on the amount of cyclophilin A in milk obtained by detecting cyclophilin A in milk (measured CyPA amount), breast disease in the subject's breast or milk compartment is examined. Determine the onset or likelihood of developing breast disease. Determination of the onset of mammary gland disease or the possibility of onset of mammary gland disease may be either qualitative or quantitative. Examples of qualitative judgment and quantitative judgment are shown below. However, the determination shown below can be performed programmatically, automatically, or mechanically without depending on the determination of those skilled in the art, as is apparent from the determination criteria.
- the measured CyPA amount when the measured CyPA amount is larger than the reference value, it is determined that “a mammary gland disease has developed or is highly likely to occur”, and the measured CyPA amount is higher than the reference value. Is smaller, it is determined that “the mammary gland disease has not developed or is less likely to develop”.
- the qualitative determination when it is “positive” in which an increase in the amount of measured CyPA is recognized, it is determined that “a mammary gland disease has developed or is highly likely to develop”, and a measured CyPA In the case of “negative” in which no increase in the amount is observed, it is determined that “a mammary gland disease has not developed or is less likely to develop”.
- the possibility of developing mammary gland disease (%) is set in advance for each range of the measured CyPA amount, and the possibility of developing mammary gland disease (%) is determined from the measured CyPA amount.
- an index (PL test, CL ability, etc.) for determining other mammary gland diseases can be used.
- the onset of mammary gland disease or the onset possibility of mammary gland disease based on the amount of CyPA and CL ability first, the onset of mammary gland disease or its onset possibility is determined based on the measured amount of CyPA, and the onset of mammary gland disease is possible.
- the possibility of onset of mammary gland disease by additionally detecting the CL ability when the determination result that the sex is high is obtained, and comprehensively judging the measured CyPA amount and the detection result of the CL ability Judgment finally. That is, the measured CyPA amount is used for primary determination, and the detection result of CL ability is used for secondary determination or final determination.
- the detection result of the index for determining other breast diseases such as CL ability may be used for primary determination.
- step (2) in the method of the present invention when the measured amount of CyPA is larger than the amount of cyclophilin A (reference CyPA amount) in milk collected from a healthy breast or a milk tub, the subject's It is a step of determining that a mammary gland disease has developed or is likely to develop in the breast or breast quarter.
- a healthy breast or breast chamber is not particularly limited as long as it is a breast or milk chamber that does not currently develop breast disease or has no possibility of developing breast disease.
- a breast or a breast chamber that has not previously developed a mammary disease or has not previously been confirmed to have a mammary disease is preferable.
- breasts or breast chambers that have had breast disease or have had the possibility of developing breast disease in the past breasts that have not developed breast disease or have no possibility of developing breast disease with subsequent appropriate treatment
- breasts that have not developed breast disease or have no possibility of developing breast disease with subsequent appropriate treatment
- it if it is recognized as a milk quarter, it can be used as a healthy breast or milk quarter, but it is desirable to carefully determine its identity.
- the method and timing of collecting milk from a healthy breast or milk compartment are not particularly limited. For example, simultaneous collection by the same method as that for collecting milk from a subject's breast or milk compartment. Or it can be implemented at a different time.
- the method and timing of detection of the amount of cyclophilin A in milk collected from a healthy breast or milk quarter are not particularly limited. For example, in the milk collected from the breast or milk compartment of a subject It can be carried out simultaneously or at the same time by the same method for detecting cyclophilin A.
- the milk of analysis number 42 is used as the milk collected from a healthy breast or milk bun. Further, in the method of the present invention, like the milk of analysis number 42, the milk collected from each quarter of the same individual of the subject (quarter milk) is (1) negative for the PL test, respectively. , (2) CL ability is less than 1 ⁇ 10 6 cpm / mL, and (3) each quarter milk is “healthy” if it meets the requirements of no abnormalities in milk quality (pox, viscosity) It may be “milk collected from the breast or milk bun”. That is, in the examples described later, each of the milks of analysis numbers 41 to 44 including the milk of analysis number 42 can be adopted as “milk collected from a healthy breast or milk chamber”.
- FIG. 11 shows that the measured CyPA amount of milk (analysis number 2, 7, 8, 9, 13, 15, 23, 27, 28, 32) positive for the PL test was collected from a healthy breast or milk compartment The fact that it was greater than the reference CyPA amount of the milk (analysis numbers 41-44) was shown.
- step (2) in the method of the present invention it is determined whether or not the measured CyPA amount is larger than the reference CyPA amount. That is, when the measured amount of CyPA is larger than the reference amount of CyPA, it is determined that a mammary gland disease has developed in the subject's breast or milk compartment, or that mammary gland disease may develop.
- the minimum value (4.043; analysis number 8) of the measured CyPA amount of the milk having a positive PL test in FIG. 11 is 2.8 which is the maximum value (1.419; analysis number 44) of the reference CyPA value. It was 3.8 times the average value (1.065) of the standard CyPA value. Therefore, based on these results, in a preferred embodiment of step (2) in the method of the present invention, more preferably, when the measured CyPA amount is twice or more of the reference CyPA amount, the measured CyPA amount is more preferably When the reference CyPA amount is 2.8 times or more, still more preferably when the measured CyPA amount is 3.3 times or more the reference CyPA amount, and particularly preferably, the measured CyPA amount is 3.8 times the reference CyPA amount. If it is greater than or equal to a factor of 2, it is determined that a mammary gland disease has developed or is likely to develop in the subject's breast or breast quarter.
- Another aspect of the method of the present invention is a method for examining a mammary gland disease, which includes the following steps (1 ′) and (2 ′).
- (1 ′) a step of obtaining cyclophilin A amount in the mammary gland by detecting cyclophilin A in the mammary gland collected from the breast or milk compartment of the subject (2 ′) based on the amount of cyclophilin A in the mammary gland Determining the onset or likelihood of developing mammary gland disease in the breast or breast chamber of the body
- the step (1 ') in the method of the present invention can be performed by applying the step (1) in the method of the present invention.
- the method of collecting the mammary gland from the subject's breast or breast chamber is not particularly limited.
- a puncture aspiration method or mammary gland in which a part of the mammary gland is collected by inserting a needle into the breast and sucking mammary epithelial cells can be employed.
- Means commonly used by those skilled in the art, such as a method of excising a part of tissue, can be employed.
- the method for detecting CyPA in the mammary gland is not particularly limited as long as it is a means for detecting a specific protein, polypeptide or mRNA from cells or tissues.
- step (1 ') in the method of the present invention the following procedure may be mentioned. That is, a needle is inserted into the subject's milk compartment and the mammary epithelial cells are aspirated. Next, a labeled anti-CyPA antibody is provided to the mammary epithelial cells obtained by aspiration. Next, using the labeled substance of the labeled CyPA antibody as an index, the absolute amount of CyPA or the amount of CyPA per cell is detected by fluorescence microscopy or flow cytometry.
- the amount of CyPA may be detected by using an immunohistochemical technique on the collected mammary gland tissue, and from the crushed material such as the collected mammary epithelial cells, You may detect the amount of CyPA by the method similar to the detection of the amount of CyPA.
- the collection of the mammary gland and the detection of the amount of CyPA are not particularly limited as long as the amount of CyPA to be detected can be compared with the reference amount to be compared.
- the step (2 ') in the method of the present invention can be performed by applying the step (2) in the method of the present invention.
- step (2 ′) in the method of the present invention when the amount of cyclophilin A in the mammary gland is greater than the amount of cyclophilin A in mammary gland tissue collected from a healthy breast or breast chamber, It is a step of determining that a mammary gland disease has developed or is likely to develop in the breast or breast quarter.
- steps other than those described above can be employed. That is, another step may be provided between the steps (1) and (2) or between the steps (1 ') and (2').
- the method of the present invention can be modified in various ways as long as those skilled in the art can conceive from the state of the art.
- the method of the present invention can be used for various purposes, for example, a method for confirming the prognosis of a mammary gland disease, a method for confirming the course of treatment of a mammary gland disease, a method for confirming eligibility of treatment for a mammary gland disease, It can also be used as a method for confirming the therapeutic effect of breast diseases, a method for screening therapeutic agents for breast diseases, and the like.
- the step of obtaining the amount of cyclophilin A in milk or mammary gland by detecting cyclophilin A in milk collected from the breast or milk quarter of a subject to which a therapeutic agent for mammary gland diseases is administered;
- the amount of cyclophilin A in the mammary gland for example, the amount of cyclophilin A in milk or mammary gland collected from a healthy breast or breast quarter, or in milk or mammary gland observed over time, day, week, month or year And confirming or determining the course, eligibility, or effect of treatment of breast diseases in the breast or breast chamber of the subject based on the amount of cyclophilin A in the subject.
- a step of evaluating the effect of treatment and / or prevention of mammary gland disease based on the amount of cyclophilin A in milk or mammary gland, and a step of determining a test substance evaluated as having high therapeutic effect / prevention as a therapeutic agent for mammary gland disease The method containing these is mentioned.
- the reagent for testing breast disease of the present invention comprises an anti-cyclophilin A antibody (anti-CyPA antibody).
- anti-CyPA antibody The type and origin of the anti-CyPA antibody is not particularly limited as long as it has specific affinity for CyPA.
- the anti-CyPA antibody may be either a monoclonal antibody or a polyclonal antibody.
- a polyclonal antibody in addition to an IgG fraction derived from an antiserum collected from an immunized animal, an antibody obtained from an antiserum by affinity purification using CyPA as an antigen can be used.
- Commercially available antibodies such as abcam (registered trademark) rabbit-anti human CyPA (polyclonal antibody) can also be used.
- the above-mentioned commercially available antibody is an antibody that recognizes human CyPA antibody, it can specifically bind to CyPA in bovine milk. Thus, even if the antigen recognition site is a heterologous anti-CyPA antibody, it can be used as long as it can specifically bind to CyPA in the subject.
- the structure of the anti-CyPA antibody is not particularly limited as long as it has a specific affinity for CyPA.
- the globulin type of the anti-CyPA antibody is not particularly limited, and may be, for example, IgG, IgM, IgA, IgE, IgD and the like. Further, it may be a fragmented antibody such as Fab, Fab ′, F (ab ′) 2 , scFv, or dsFv antibody.
- the anti-CyPA antibody can be prepared using an immunological method, a phage display method, a ribosome display method, or the like.
- Monoclonal antibodies and polyclonal antibodies by immunological techniques can be prepared with reference to the following description, but are not limited thereto.
- an antigen of CyPA or a partial peptide thereof is administered to a site capable of producing an antibody in mammals, together with a carrier or a diluent.
- Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration.
- the method for obtaining the antigen is not particularly limited, but is a recombinant produced by genetic engineering based on separation and purification from a biological sample such as mammary epithelial cells or the information on the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing.
- Can be obtained as In the recombinant for example, a gene encoding an antigen or a part thereof is introduced into an appropriate vector, then the recombinant vector is introduced into an appropriate host, and CyPA or a part thereof is expressed in the obtained transformant. Can be produced.
- the antigen is usually administered to mammals once every 2 to 6 weeks, for a total of about 2 to 10 times.
- mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and the like, and mice and rats are preferably used.
- Monoclonal antibody-producing hybridomas can be prepared by fusing the obtained antibody-producing cells with, for example, homologous or heterologous animal myeloma cells.
- the fusion operation can be performed according to known methods, for example, the method of Kohler and Milstein (Nature, 256, 495 (1975)).
- a fusion promoter is not specifically limited, For example, polyethylene glycol (PEG), Sendai virus, etc. can be mentioned, Preferably PEG is used.
- Myeloma cells are not particularly limited, and examples thereof include mammalian myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and SP2 / 0 is preferably used.
- a preferred ratio between the number of antibody-producing cells (preferably spleen cells) and the number of myeloma cells is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000, more preferably PEG 4000) is 10 to 80%.
- Cell fusion can be carried out efficiently by adding at a moderate concentration and incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 2 minutes.
- Hybridoma culture can be carried out using an animal cell medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
- HAT hyperxanthine, aminopterin, thymidine
- RPMI-1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal calf serum, no hybridoma culture
- a serum medium SFM-101, Nissui Pharmaceutical Co., Ltd.
- the culture temperature is usually 20 to 40 ° C., preferably about 37 ° C.
- the culture time is usually 4 days to 2 weeks, preferably about 1 week. Culturing can usually be performed in the presence of 5% carbon dioxide gas.
- Hybridomas are monoclonalized, and then a clone that produces an anti-CyPA antibody having high specificity for CyPA is selected.
- the target monoclonal anti-CyPA antibody can be obtained by purifying the culture solution of the selected clone.
- the hybridoma After the hybridoma has been grown to a predetermined number or more, it is transplanted into the abdominal cavity of a mammal (such as a mouse) and grown in ascites to obtain the desired monoclonal anti-CyPA antibody. You can also.
- affinity chromatography columns for purifying monoclonal IgM For purification of culture medium or ascites, affinity chromatography columns for purifying monoclonal IgM, affinity chromatography using protein G, protein A, and the like are used. In addition, affinity chromatography in which CyPA as an antigen is immobilized may be used.
- monoclonal antibodies are known per se, for example, salt separation methods, ammonium sulfate fractionation, alcohol precipitation methods, isoelectric precipitation methods, electrophoresis methods known to those skilled in the art as methods for separating and purifying immunoglobulins.
- Absorption and desorption methods using ion exchangers such as DEAE, ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A or protein G, and collecting the antibody alone to obtain the antibody Separation and purification can be performed by employing a specific purification method or the like. These methods are used alone or in any combination.
- a polyclonal antibody against an antigen of CyPA or a partial peptide thereof can be produced according to a method known per se. For example, it is obtained when a immunizing antigen itself or a complex of an antigen and a carrier protein is prepared, and then a mammal is immunized in the same manner as the monoclonal antibody preparation method described above, and then the antibody titer rises moderately.
- a polyclonal antibody against CyPA can be obtained by collecting anti-CyPA antibody-containing material from the immunized animal and then separating and purifying the antibody.
- a complex of an immune antigen and a carrier protein may be used.
- carrier proteins that can be used include KLH (Keyhole Limeto Hemocynin), BSA (Bovine Serum Albumin), and OVA (Ovalbumin).
- KLH Keyhole Limeto Hemocynin
- BSA Bovine Serum Albumin
- OVA Optalbumin
- a carbodiimide method, a glutaraldehyde method, a diazo condensation method, a maleimidobenzoyloxysuccinimide method, or the like can be used for binding the carrier protein.
- an antigen whose structure is partially modified such as a fusion protein with a histidine (His) tag or the like, can also be used. Modified antigens such as fusion proteins can be made and purified by general methods known by those skilled in the art.
- the type of carrier protein and the mixing ratio of the carrier and the hapten are not particularly limited as long as the antibody can be efficiently produced against the hapten that has been immunized by crosslinking with the carrier, but for example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. by weight ratio.
- a method of coupling to hapten 1 at a ratio of about 0.1 to 20, preferably about 1 to 5 is used.
- various condensing agents such as glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and active ester reagent containing a dithiobilidyl group are used.
- Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably blood.
- the polyclonal antibody titer in the antiserum obtained by centrifuging blood obtained from the immunized animal was reacted with a labeled CyPA labeled with a labeling agent such as an enzyme or fluorescence and the antiserum. Then, it can carry out by measuring the quantity and activity of the labeling agent couple
- Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.
- the antiserum can be affinity purified to a polyclonal antibody.
- the anti-CyPA antibody can be prepared by genetic engineering techniques or molecular biological techniques. For example, an antigen recognition site in an antibody recognizing CyPA derived from a subject such as a bovine is analyzed, and a nucleic acid encoding the recombinant antibody having the antigen recognition site and the other part derived from the subject Can be produced. In addition, a gene involved in antibody production of a subject such as a bovine is transferred to another animal species, for example, mouse embryo or mouse-derived antibody-producing cell, and thus obtained mouse or mouse antibody-producing cell and antigen By using bovine-derived CyPA as a complete bovine antibody can be obtained. As the genetic engineering technique and molecular biological technique used in these methods, methods known so far can be used without limitation.
- the anti-CyPA antibody may have been subjected to various modifications under the condition that it retains specific affinity for CyPA.
- modified antibodies can also be used as the reagent of the present invention.
- the amount of bound antibody can be directly detected using the labeling agent as an index. Accordingly, one aspect of the anti-CyPA antibody is a labeled anti-CyPA antibody.
- the labeling agent is not particularly limited, but enzymes such as peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase and glucose-6-phosphate dehydrogenase; fluorescein isothiocyanate (FITC), fluorescent substances such as tetramethylrhodamine isothiocyanate (TRITC), europium, phycoerythrin (PE), Cy2, Cy3 and Cy5; chemiluminescent substances such as luminol, isoluminol and acridinium derivatives; coenzymes such as NAD; biotin and the like Specific proteins; radioactive materials such as 131I and 125I, and the like.
- enzymes such as peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -D-galactosi
- DAB dibenzoidine hydrochloride
- OPD o-Phenylenediamine
- chromogenic substrate when peroxidase is used as a labeling agent, DAB (3,3'-Diaminobenzoidine hydrochloride) or OPD (o-Phenylenediamine) can be used as the chromogenic substrate.
- alkaline phosphatase is used as the labeling agent, Bromochoro indole phosphate / nitro blue tetazolium or NPP (p-Nitrophenyl phosphate disodium salt hydrate) can be used as the chromogenic substrate.
- the reagent of the present invention may be solid-phased on an insoluble carrier in accordance with its use.
- the insoluble carrier used for solid-phase immobilization is not particularly limited.
- An insoluble carrier insoluble in water, such as a functional material can be used.
- the method for supporting the antibody on the insoluble carrier is not particularly limited, and can be carried out by a technique based on physical adsorption or chemical adsorption, which is generally known to those skilled in the art.
- the kit of the present invention contains the reagent of the present invention as a main component.
- reagents buffer solution, blocking reagent, enzyme substrate, coloring reagent, etc.
- CyPA as a standard sample in the kit.
- a PL test reagent, instrument and / or device can be included in the kit to increase the accuracy of determining mammary gland diseases.
- an instruction manual is attached to the kit of the present invention in the same manner as an ordinary commercially available kit.
- the anti-CyPA antibody may be labeled.
- an anti-CyPA antibody that is a primary antibody that binds to an antigen is labeled, there may arise a problem that the detection sensitivity of the antigen is lowered. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling agent is bound, a method using a carrier to which a secondary antibody and a labeling agent are bound, or the like.
- the secondary antibody is not particularly limited as long as the antibody has specific affinity for the anti-CyPA antibody.
- an anti-CyPA antibody is prepared as a rabbit antibody
- an anti-rabbit IgG antibody can be used. Labeled secondary antibodies that can be used for various types of antibodies such as rabbits, goats, and mice are commercially available from, for example, Takara Bio Inc. and Cosmo Bio Inc. Accordingly, an appropriate one can be appropriately selected and included in the kit of the present invention.
- kits of the present invention are kits comprising an anti-CyPA antibody (primary antibody) which is the reagent of the present invention and a labeled antibody (secondary antibody) against the anti-CyPA antibody.
- the secondary antibody preferably has an antigen recognition site that recognizes an epitope different from that of the primary antibody.
- a reagent for detecting an immune complex such as a chromogenic substrate or a chromogenic reagent is included depending on the type of the labeling agent.
- kits of the present invention are kits comprising a porous carrier comprising a site A to which a sample is applied, a site B containing a labeled antibody, and an antigen detection site C.
- the site B containing the labeled antibody contains a labeled antibody against CyPA that can move in a wet state.
- An anti-CyPA antibody against CyPA that recognizes a site different from the labeled antibody is immobilized on the antigen detection site C.
- the anti-CyPA antibody forms a complex (labeled antibody-CyPA-anti-CyPA antibody) in a sandwich form with the labeled antibody bound with CyPA via CyPA.
- the specimen application site A and the labeled antibody site B may be located at the same site.
- CyPA when CyPA is present in the liquid sample, when the liquid sample is applied to the sample application site A, CyPA and the labeled antibody are bound to each other at the labeled antibody-containing site B, and then this conjugate is detected as an antigen. When moved to the site C, it is collected by forming a complex with the immobilized anti-CyPA antibody. Therefore, when a labeling substance that can be visualized with the naked eye, such as colored latex, dye sol, and gold colloid, is used, the presence and / or amount of CyPA in the specimen can be confirmed by the presence or absence of labeling at the antigen detection site C.
- a labeling substance that can be visualized with the naked eye such as colored latex, dye sol, and gold colloid
- the hybridoma of the present invention has the ability to produce an anti-cyclophilin A antibody.
- the antibody of the present invention is an anti-cyclophilin A antibody produced by the hybridoma of the present invention.
- the hybridoma of the present invention is not particularly limited as long as it is a fused cell obtained by fusing an antibody-producing cell capable of producing an anti-CyPA antibody and a tumor cell such as a myeloma cell or an immortalized cell.
- the antibody of the present invention is not particularly limited as long as it has specific affinity for CyPA.
- the confirmation method, the production method, and the use method the above-mentioned description regarding the anti-CyPA antibody or the hybridoma producing the anti-CyPA antibody can be referred to.
- a method for obtaining the hybridoma and the antibody of the present invention in addition to the above-described method, a method specific to M-BIE cells derived from a bovine intestinal epithelial cell line (BIE cell) as described in Examples described later. It can be obtained by screening monoclonal antibodies. That is, M-BIE cells are immunized to mammals (eg, mice), and then antibody-producing cells are excised from the immunized animals. Next, antibody-producing cells are fused with myeloma cells of the same kind of animal (mouse) as the immunized animal, and a hybridoma is obtained by culturing using a HAT medium.
- mammals eg, mice
- antibody-producing cells are excised from the immunized animals.
- myeloma cells of the same kind of animal (mouse) as the immunized animal a hybridoma is obtained by culturing using a HAT medium.
- hybridomas producing antibodies that specifically recognize FAE in bovine intestinal epithelium are selected and cloned by using immunohistochemical staining.
- the cloned cells are then administered into the peritoneal cavity of the mouse, and the monoclonal antibody (2H5-F3) derived from the ascites of the immunized mouse is purified and separated.
- the 2H5-F3 monoclonal antibody and the protein recovered from the cytoplasmic fraction and the cell membrane fraction of M-BIE cells are mixed and subjected to immunoprecipitation.
- the protein lysate obtained by immunoprecipitation is separated by SDS-PAGE, and the band is stained.
- the marker of the present invention is a marker for detecting mammary gland disease consisting of cyclophilin A.
- the marker of the present invention refers to a biomolecule that serves as an index of the onset or possibility of onset of mammary gland diseases, particularly mastitis, which is an infectious mammary gland disease.
- the marker of the present invention is not the cyclophilin A itself present in the living body, but cyclophilin A isolated from the mammary gland and milk collected from the living body is used as the marker of the present invention.
- the marker of the present invention is particularly useful for early detection of mastitis.
- the usefulness of the markers of the present invention for the early detection of mastitis is that bovine mammary epithelial cells in mammary tissue that has developed mastitis compared to normal mammary tissue by carefully observing the development of mastitis and its phenomenon. It can be inferred from the facts found for the first time by the present inventors that an increase in the expression level of CyPA is observed in immune cell infiltrating sites and also in milk. In particular, the present inventors have found that a comparatively high concentration of CyPA is present in the quarter milk that has been determined to develop mastitis using the PL test and CL ability, which are conventional mastitis testing methods. Confirmed that there was an event. In view of this phenomenon, the marker of the present invention is a marker used for determining the onset of latent mastitis in addition to clinical mastitis.
- the present inventors have found that the milk protein composition is not observed in milk having a negative PL test and low CL ability, but an increase in the amount of CyPA was confirmed. Furthermore, the inventors have obtained the fact that some of the milk buns from which these milks have been collected have developed mastitis.
- the marker of the present invention is highly sensitive to the possibility of developing mastitis, which is difficult to determine depending on the PL test and CL ability in addition to early detection of mastitis, using CyPA as an index. It is a marker that can be detected. That is, by using the marker of the present invention, it is possible to identify a site in the early stage of mastitis.
- SA Staphylococcus aureus
- a treatment experimental group was designated.
- Lf latent mastitis in which lactoferrin (Lf) or lactic acid bacteria-producing peptide was dissolved in 10 mL of PBS in a 10 mg / mL or 20 mg / mL Lf solution from the nipple to the milk compartment of Holstein lactating cows (embedded with PLP-fixed paraffin) The mammary gland tissue; Holstein lactating cow; number of days after delivery (about 305 days: 5 days after the introduction of dry milk) was designated as an experimental group for Lf administration.
- BIE cell bovine intestinal epithelial cell line
- M-BIE cell a method for inducing differentiation into in vitro M cell
- M-BIE cells adjusted to 4.0 ⁇ 10 6 cells / ml-PBS was administered from the tail vein of the same mouse and boosted.
- spleen cells were collected from the immunized mice and fused with SP2 / 0-ag14-K13 mouse myeloma cells using 50% (w / v) polyethylene glycol (PEG4000).
- the fused hybridomas were selected using HAT medium (RPMI-1640 containing 2 mM glutamate, 0.2% glucose, 10% FBS, 100 ⁇ M hypoxanthine, 0.4 ⁇ M aminopterine, 16 ⁇ M thymidine).
- Protein lysates obtained by immunoprecipitation were separated by SDS-PAGE and stained with Silver Stain MS Kit (Wako).
- a 2H5-F3 monoclonal antibody-specific bunt was extracted and subjected to LC-MS / MS analysis. Further, the protein obtained by immunoprecipitation was subjected to Western blotting, and the reactivity with the 2H5-F3 monoclonal antibody was analyzed. As a result, it was identified that the antigen recognized by the 2H5-F3 antibody was cyclophilin A.
- A. Day 1 The mammary gland tissue obtained by deparaffinizing the sections was washed with water for 5 minutes, and then subjected to antigen activation treatment at 121 ° C. for 5 minutes using Target Retrieval Solution Low pH (Dako). The treated mammary tissue was washed with PBS for 3 minutes and this was repeated three times. The washed mammary gland tissue was subjected to a blocking treatment for 20 minutes using 3% normal goat serum / PBS. The treated mammary tissue was subjected to a primary antibody reaction at 4 ° C. for 14 hours using an anti-CyPA antibody 2,000-fold diluted / PBS.
- Dako Target Retrieval Solution Low pH
- the washed mammary tissue was subjected to hematoxylin counterstaining for 20 seconds at room temperature.
- the stained mammary tissue was washed with running water for 60 minutes.
- the mammary gland tissue after washing was dehydrated and permeated, and encapsulated with marinol (Muto Chemical).
- the specimen of the mammary gland tissue after the above immunohistochemical staining was observed using an optical microscope (Ax70, Olympus).
- CyPA expression in mastitis-onset mammary tissue in the SEC-administered experimental group was observed in mammary epithelial cells, as in the control normal mammary tissue (FIG. 2A). However, CyPA was more strongly expressed in mammary epithelial cells, milk, and cell infiltrating sites in mastitis-onset mammary tissues compared to control normal mammary tissues (FIGS. 2B and 2C).
- CyPA expression in mammary vesicles and mammary epithelial cells with enlarged stroma and atrophy was decreased compared with mammary cysts and mammary epithelial cells that were not atrophic, as in the mastitis-onset mammary gland in the SA-treated experimental group (Fig. 6C).
- CyPA was highly expressed in mammary epithelial cells, milk, and immune cell infiltrating sites as compared with the normal mammary tissue, as in the experimental mastitis tissue described above. (FIGS. 7A-C).
- CyPA is present as an intracellular protein in mammary epithelial cells and milk in mammary gland tissue collected from a normal breast. Also, SEC, which is a bacterial toxin, and S., which is a bacterium. CyPA was similarly expressed in mammary gland tissues collected from the breasts that developed A and mastitis. In particular, CyPA expression in mammary epithelial cells, milk, and immune cell infiltrating sites in mastitis-induced mammary tissue was stronger than in normal mammary tissue.
- the expression level of CyPA decreased in mammary follicles and mammary epithelial cells that were atrophic due to enlargement of the stroma found in the last stage of milking, compared with mammary follicles and mammary epithelial cells that were not atrophy.
- CyPA was analyzed by Western blotting using a commercially available polyclonal antibody, anti-CyPA antibody (rabit-anti human CyPA; Abcam (registered trademark)).
- CyPA protein was analyzed by the following procedure.
- the washed membrane was subjected to a blocking treatment (3% normal goat serum / TBS-T) for 60 minutes.
- the treated membrane was washed with TBS-Tween by repeating 10 minutes three times.
- the washed membrane was subjected to a primary antibody reaction (anti-CyPA antibody 1,000-fold dilution / TBS-T) at 4 ° C. for 14 hours.
- the membrane after the reaction was washed by repeating 10 minutes three times using TBS-Tween.
- the washed membrane was subjected to a secondary antibody reaction at room temperature for 60 minutes using an alkaline phosphatase-linked goat-anti rabbit IgG (ZYMED).
- the membrane after the reaction was washed by repeating 10 minutes three times using TBS-Tween.
- the washed membrane was further washed with TBS for 5 minutes.
- the washed membrane was developed for 5 minutes at room temperature using an ECF substrate diffusion buffer (BD Healthcare). A band was detected from the membrane after color development by using Molecular Imager FX (BioRad).
- the washed membrane was subjected to a blocking treatment (3% normal goat serum / TBS-T) for 60 minutes. Washing was performed by repeating 10 minutes three times using TBS-Tween. The washed membrane was subjected to a primary antibody reaction (anti-CyPA antibody 1,000-fold diluted / TBS-T) at 4 ° C. for 14 hours.
- a blocking treatment 3% normal goat serum / TBS-T
- Washing was performed by repeating 10 minutes three times using TBS-Tween.
- the washed membrane was subjected to a primary antibody reaction (anti-CyPA antibody 1,000-fold diluted / TBS-T) at 4 ° C. for 14 hours.
- washing was performed by repeating 10 minutes three times using TBS-Tween.
- the washed membrane was subjected to a secondary antibody reaction at room temperature for 60 minutes using alkali phosphorylase-linked goat-anti rabbit IgG (ZYMED).
- the membrane after the reaction was washed by repeating 10 minutes three times using TBS-Tween.
- the washed membrane was washed with TBS for 5 minutes.
- the washed membrane was developed for 5 minutes at room temperature using an ECF substrate diffusion buffer (BD Healthcare). A band was detected from the membrane after color development by using Molecular Imager FX (BioRad).
- C. Commassie Brilliant Blue (CBB) staining Proteins in whey were analyzed by the following procedure using whey samples quantified by protein. Proteins in the whey samples were separated by SDS-PAGE using PAGEL (ATTO; E-T520L) so that the protein concentration was 21 ⁇ g / lane. The separated gel was repeatedly washed with MQ for 5 minutes three times. The washed gel was subjected to a color development treatment at room temperature for 20 minutes using ULTRA-FAST Coomassie Stain (NRV, USA). A band in the gel after the treatment was confirmed.
- PAGEL A band in the gel after the treatment was confirmed.
- CyPA expression analysis in milk samples From the above (1) CyPA protein was detected in milk samples derived from lactating cows suffering from mastitis. However, analysis number 19 was during the treatment of blind milk), and reduction treatment was performed, and the CyPA protein in the obtained whey sample was analyzed. CyPA protein was detected in the analysis using the healthy cow quarter milk sample (Analysis No. 33-48) and the mastitis onset milk sample (Analysis No. 1-32) (FIGS. 10A to 10C). Healthy cow's quarter milk had less CyPA protein than mastitis-induced quarter milk.
- non-infected quarter milk (analysis number 1, 3-6, 10-12, 14, 16, 18-22, 24-26, 29) of mastitis onset milk is mastitis onset milk (analysis number). 2,7-9,13,15,17,23,27,28,30-32), there was less CyPA protein.
- Non-infected quarter milk samples from mastitis-causing cows had lower amounts of CyPA protein compared to mastitis-old quarter milk samples. Further, the amount of CyPA protein in the quarter milk sample (analysis number 2, 8, 9, 13, 15, 18, 27, 28, 32) having a positive PL test and a high CL ability was very large. This is consistent with the trend of intensity of CyPA expression in mammary epithelial cells and mammary alveoli in normal and mastitis-induced mammary tissue.
- the CL value of the milk sample (analysis number 1, 3, 12, 16, 18, 20-22, 26) is based on the quarter milk sample (analysis number 8, 27) diagnosed as mastitis onset by the PL test result. Due to the high price, these quarter milk samples are likely to be early symptoms of mastitis. Actually, in the milk quarter from which the milk sample of analysis number 26 was collected, the onset of mastitis was confirmed.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
(1)被験体の乳房または乳分房から採取された乳汁におけるシクロフィリンAを検出することにより乳汁中のシクロフィリンA量を得る工程
(2)前記乳汁中のシクロフィリンA量に基づいて、被験体の乳房または乳分房における乳腺疾患の発症または乳腺疾患の発症可能性を判定する工程
(1’)被験体の乳房または乳分房から採取された乳腺におけるシクロフィリンAを検出することにより乳腺中のシクロフィリンA量を得る工程
(2’)前記乳腺中のシクロフィリンA量に基づいて、被験体の乳房または乳分房における乳腺疾患の発症または乳腺疾患の発症可能性を判定する工程
ラフィー法からなる群から選択される手法により実施される。
1.乳腺疾患の検査方法
本発明の方法は、乳腺または乳汁におけるシクロフィリンA量を指標とした、乳腺疾患の検査方法である。
(1)被験体の乳房または乳分房から採取された乳汁におけるシクロフィリンAを検出することにより乳汁中のシクロフィリンA量を得る工程
(2)前記乳汁中のシクロフィリンA量に基づいて、被験体の乳房または乳分房における乳腺疾患の発症または乳腺疾患の発症可能性を判定する工程
(1’)被験体の乳房または乳分房から採取された乳腺におけるシクロフィリンAを検出することにより乳腺中のシクロフィリンA量を得る工程
(2’)前記乳腺中のシクロフィリンA量に基づいて、被験体の乳房または乳分房における乳腺疾患の発症または乳腺疾患の発症可能性を判定する工程
本発明の乳腺疾患検査用試薬は、抗シクロフィリンA抗体(抗CyPA抗体)からなる。抗CyPA抗体は、CyPAに対する特異的親和性を有する限り、その種類や由来などは特に限定されない。また、抗CyPA抗体は、モノクローナル抗体およびポリクローナル抗体のいずれであってもよい。ポリクローナル抗体としては、免疫動物から採取した抗血清由来のIgG画分のほか、抗血清からCyPAを抗原とするアフィニティー精製して得た抗体を使用できる。また、市販されている抗体、たとえば、abcam(登録商標) rabbit-anti human CyPA(ポリクローナル抗体)を使用できる。なお、上記市販抗体は、ヒトCyPA抗体を認識する抗体ではあるが、ウシの乳汁中のCyPAに特異的に結合することができる。このように、抗CyPA抗体としては、抗原認識部位が異種のものであっても、被験体におけるCyPAに特異的に結合することができれば、使用することができる。
本発明のキットは、主要構成要素として本発明の試薬を含む。乳腺疾患の検査法を実施する際に使用するその他の試薬(緩衝液、ブロッキング用試薬、酵素の基質、発色試薬など)に加えて、器具や装置(容器、反応装置、蛍光リーダーなど)などをキットに含めてもよい。また、標準試料としてCyPAをキットに含めることが好ましい。さらには、CyPAの検出以外の乳腺疾患の検査法に用いるための試薬、器具、装置などをキットに含めることも可能である。たとえば、乳腺疾患の判定精度を高めるために、PLテスト用の試薬、器具および/または装置をキットに含めることができる。本発明のキットには、通常の市販されているキットと同様に、取り扱い説明書が添付されることが好ましい。
本発明のハイブリドーマは、抗シクロフィリンA抗体を産生する能力を有する。本発明の抗体は、本発明のハイブリドーマによって生産される抗シクロフィリンA抗体である。本発明のハイブリドーマは抗CyPA抗体を産生する能力を有する抗体産生細胞と骨髄腫細胞などの腫瘍細胞または不死化細胞とを融合させて得られる融合細胞であれば特に限定されない。本発明の抗体は、CyPAに対して特異的親和性を有する抗体であれば特に限定されない。本発明のハイブリドーマや抗体の構造および機能、確認方法、製造方法ならびに使用方法については、上記した抗CyPA抗体や抗CyPA抗体を産生するハイブリドーマに関する記載を参照できる。
本発明のマーカーは、シクロフィリンAからなる、乳腺疾患検出用マーカーである。本発明のマーカーは、乳腺疾患、特に感染性の乳腺疾患である乳房炎の発症またはその発症可能性の指標となる生体分子のことをいう。本発明のマーカーは、生体中に存在するシクロフィリンAそのものではなく、生体から採取された乳腺や乳汁から分離されたシクロフィリンAが本発明のマーカーとして利用される。本発明のマーカーは、乳房炎の早期検出に特に有用である。
1.材料および方法
(1)試料
乳房炎発症乳腺組織として、下記A~Cに示す三種の実験区のホルスタイン泌乳牛のうち、実験的乳房炎または潜在性乳房炎の発症が認められた乳房から採取した乳腺組織を用いた。乳腺組織は、ウシを屠殺した後直ちに採取し、すみやかにPLP固定液またはリン酸緩衝ホルマリン固定液を用いて、4℃で一晩固定した。固定後70%エタノール、80%エタノール、90%エタノール、95%エタノールにそれぞれ12時間浸し、さらに100%エタノールに24時間組織を浸し、段階的に脱水を行った。脱水後、トルエン、パラフィンにそれぞれ6時間浸した後、パラフィンに包埋した。また、各実験区のホルスタイン泌乳牛のうち、PBSを投与して処理した乳房から採取した乳腺組織を対照正常乳腺組織として用いた(Toshinobu Kuroishiら、Clin Diagn Lab Immunol.2003;10:1011-1018;Kai Kら、J Vet Med Sci.2002;64:873-8を参照、該文献の記載はここに開示として援用される)。乳酸菌産生ペプチドは、カワイらの文献(Kawai Yら,Biosci Biotechnol Biochem.1997;61:179-82、該文献の記載はここに開示として援用される)に記載の方法にしたがって精製した。
Staphylococcal Endotoxins C(SEC)をPBS 10mLに溶かした0.1μg/μl SEC溶液を乳頭よりホルスタイン泌乳牛の乳分房へ投与して乳房炎を発症させた実験的乳房炎(PLP固定パラフィン包埋乳腺組織;ホルスタイン泌乳牛;分娩後日数 約300日)をSEC投与実験区とした。
Staphylococcas aureus(S.A)をPBS 10mLに溶かした154cfu/ml S.A溶液を乳頭よりホルスタイン泌乳牛の乳分房へ投与して乳房炎を発症させた実験的乳房炎(リン酸緩衝ホルマリン固定パラフィン包埋乳腺組織およびバイオプシーより得た乳腺組織;ホルスタイン泌乳牛;分娩後日数 約60日)をS.A投与実験区とした。
ラクトフェリン(Lf)または乳酸菌産生ペプチドをPBS 10mLに溶かした10mg/mLまたは20mg/mL Lf溶液を乳頭よりホルスタイン泌乳牛の乳分房へ投与した潜在性乳房炎(PLP固定パラフィン包埋乳腺組織;ホルスタイン泌乳牛;分娩後日数 約305日:乾乳導入5日)をLf投与実験区とした。
本発明者らが作製した抗CyPA抗体(2H5-F3抗体)を用いて、免疫組織化学染色により、乳腺組織におけるCyPAの局在を確認した。2H5-F3抗体は以下[i]および[ii]の手順により作製した。
本発明者らは、ウシ腸管上皮細胞株(BIE細胞)の樹立とそのin vitro M細胞(M-BIE細胞)への分化誘導法を確立している。(Characterization of newly established bovine intestinal epithelial cell line,K.Miyazawa,Histochem Cell Biol (2010)vol.133,p125-134、該文献の記載はここに開示として援用される)。M-BIE細胞を2.7×106cells/ml-PBSの濃度に調整し、BALB/cマウスの腹腔内に0.5mlを免疫した。14日後、4.0×106cells/ml-PBSに調整したM-BIE細胞の0.3mlを同一マウスの尾静脈から投与し、追加免疫した。その5日後に、免疫したマウスより脾細胞を採取し、50%(w/v) ポリエチレングリコール(PEG4000)を用いて、SP2/0-ag14-K13マウスミエローマ細胞と融合した。融合したハイブリドーマは、HAT培地 (2mM glutamate,0.2% glucose,10% FBS,100μM hypoxanthine,0.4μM aminopterine,16μM thymidineを含むRPMI-1640)を用いて選抜した。選抜したハイブリドーマから、下記の免疫組織化学染色によってウシ腸管上皮においてFAEを特異的に認識する抗体を産生するウェルを選抜し、限界希釈法によって更なるクローニングを行った。クローニングにより最終的に得られたクローン化細胞をBALB/cマウスの腹腔に投与した。BALB/cマウスの腹水由来のモノクローナル抗体は、HiTrap IgM Purification HP(GE Healthcare Bio-Science AB,Uppsala,Sweden)を用いて精製した。抗体のサブクラスはmouse monoclonal antibody isotyping kit(Dainippon Sumitomo Pharma)で決定し、2H5-F3モノクローナル抗体とした。
Transmembrane Protein Extraction Kit(Novagen)を用いてM-BIE細胞の細胞質画分および細胞膜画分、それぞれのタンパク質を回収した。抽出した細胞膜画分のタンパク質を60μl(2.0μg/μl)と2H5-F3モノクローナル抗体1.0μl(2.45μg/μl)を混合し、4℃で一晩静置した。次にμMACS protein G(Milteny Biotech)50μlと混合し、4℃で1時間静置した後、免疫沈降を行った。免疫沈降で得られたタンパク質溶解液をSDS-PAGEで分離し、Silver Stain MS Kit(Wako)で染色した。2H5-F3モノクローナル抗体特異的なバントを抽出し、LC-MS/MS解析を行った。また、免疫沈降で得られたタンパク質をウェスタンブロッティングして、2H5-F3モノクローナル抗体との反応性を解析した。その結果、2H5-F3抗体の認識する抗原はシクロフィリンAであることを同定した。
各実験区のパラフィン包埋乳腺組織から厚さ4μmの切片を作製し、以下の手順でCyPAについて免疫組織化学染色を行った。
切片を脱パラフィン化して得た乳腺組織を5分間水洗し、次いでTarget Retrieval Solution Low pH(Dako)を用いて121℃で5分間の抗原賦活化処理に供した。処理後の乳腺組織を、PBSで3分間洗浄し、これを3回繰り返した。洗浄後の乳腺組織を、3% 正常ヤギ血清/PBSを用いて20分間のブロッキング処理に供した。処理後の乳腺組織を、抗CyPA抗体2,000倍希釈/PBSを用いて、4℃で14時間の一次抗体反応に供した。
反応後の乳腺組織を、PBSで3分間洗浄し、これを3回繰り返した。洗浄後の乳腺組織を、Histofine Simplestain MAX-PO(M)(Nichirei)を用いて室温で20分間の二次抗体反応に供した。反応後の乳腺組織を、PBSで3分間洗浄し、これを3回繰り返した。洗浄後の乳腺組織を、0.0025% 3.3’-diaminobenzidine(同仁、熊本、日本)+0.006% H2O2/0.05M Tris-HCl(pH 7.5)を用いて、室温で1分間のDAB発色反応に供した。反応後の乳腺組織を蒸留水(DW)で数秒間洗浄した。洗浄後の乳腺組織を、室温にて20秒間のヘマトキシリン対比染色に供した。染色後の乳腺組織を、60分間、流水により洗浄した。洗浄後の乳腺組織を、脱水および透徹し、マリノール(武藤化学)を用いて封入した。
(1)SEC投与実験区の乳腺組織の観察
ウシ乳腺におけるCyPAの発現を免疫組織化学染色により観察した。SEC投与実験区の対照正常乳腺組織において、CyPAは乳腺胞に局在していた(図1A)。乳腺胞の乳腺上皮細胞で均一にCyPAが発現していた(図1B)。微量であるが乳腺胞内にある乳汁でCyPAが弱く発現していた(図1C)。
ウシ乳腺におけるCyPAの発現を免疫組織化学染色により観察した。S.A投与実験区の対照正常乳腺組織において、SEC投与実験区の対照正常乳腺組織と同様に、乳腺胞の乳腺上皮細胞でCyPAが均一に発現していたこと、および乳汁で微量にCyPAが発現していたことが確認された(図3A~3C)。
ウシ乳腺におけるCyPAの発現を免疫組織化学染色により観察した。治療のためにラクトフェリンを投与したLf投与実験区の対照正常乳腺組織において、SECおよびS.A投与実験区のPBS投与乳腺組織(対照正常乳腺組織)と同様に、乳腺上皮細胞および乳汁でCyPAの発現が確認された(図5A~B、図6A~C)。間質が肥大し萎縮した乳腺胞および乳腺上皮細胞のCyPA発現は、SA投与実験区の乳房炎発症乳腺と同様に、萎縮していない乳腺胞および乳腺上皮細胞と比べて減少していた(図6C)。また潜在性乳房炎発症した乳腺組織では、上記の実験的乳房炎組織と同様に、正常乳腺組織と比較すると、乳腺上皮細胞、乳汁および免疫細胞浸潤部位でCyPAが高発現していた。(図7A~C)。
上記の結果から、正常な乳房から採取した乳腺組織において、CyPAが細胞内タンパク質として乳腺上皮細胞および乳汁に存在することが確認された。また、細菌毒素であるSECおよび細菌であるS.Aを投与して乳房炎を発症させた乳房から採取した乳腺組織においても、同様にCyPAが発現した。特に、正常乳腺組織と比べて、乳房炎発症乳腺組織における乳腺上皮細胞、乳汁および免疫細胞浸潤部位のCyPA発現は強かった。搾乳最後期などに見られる間質が肥大化することにより萎縮した乳腺胞および乳腺上皮細胞では、萎縮していない乳腺胞や乳腺上皮細胞と比較すると、それぞれCyPAの発現量が低下した。
1.材料および方法
(1)試料
供試牛として宮城県畜産試験場で飼育されたホルスタイン泌乳牛を用いた。PLテスターおよびCL能の結果より乳房炎を発症していると診断されたホルスタイン泌乳牛8頭および罹患歴がない健常ホルスタイン泌乳牛4頭の合計12頭の供試牛を用いた。乳汁サンプルとして全分房乳、計48サンプル(内1分房は盲乳処置中)を使用した。表1に、試験で使用した乳汁サンプルのデータを示した。
ウェスタンブロット法でのCyPAの解析には、市販のポリクローナル抗体である抗CyPA抗体(rabbit-anti human CyPA;Abcam(登録商標))を用いた。
A.乳汁サンプル中の乳清タンパク質の抽出およびタンパク定量
次の手順で乳汁サンプル中の乳清タンパク質を抽出および定量した。乳汁サンプルを1,100G、4℃、20分間の遠心処理に供した。処理後の乳汁サンプルから乳脂肪および沈殿物を除去し、乳清を採取した。採取した乳清をPierce(登録商標)BCA Protein Assay Kit(Thermo Sientific)を用いて、37℃で30分間インキュベートした。インキュベート後の乳清について、DS PHARMA BIOMEDICALを用いてタンパク濃度を測定したところ、14μg/μlとの定量結果を得た。
タンパク定量した後の乳清サンプルを用いて、以下の手順でCyPAタンパク質の解析を行った。
1日目として、タンパク定量した後の乳清サンプル中のタンパク質を、PAGEL(ATTO;E-T520L)を用いて、タンパク質濃度が21μg/laneになるようにSDS-PAGEによって分離した。分離したタンパク質をImmobion-P Transfer membrames(Millipore)のメンブランとセラミドライシステム(Bio-Rad)を用いて、60分間、1.2mA/cm2で該メンブランへ転写した。転写後のメンブランをTBS-Tweenで、10分間を3回繰り返すことにより洗浄(0.1%Tween20/TBS;TBS-T)した。洗浄後のメンブランを、60分間のブロッキング処理(3% normal goat serum/TBS-T)に供した。処理後のメンブランを、TBS-Tweenにて、10分間を3回繰り返すことにより洗浄した。洗浄後のメンブランを、4℃で14時間の一次抗体反応(抗CyPA抗体1,000倍希釈/TBS-T)に供した。
1日目として、5% 2MEを加えたタンパク定量後の乳清サンプル中のタンパク質を、PAGEL(ATTO;E-T520L)を用いて、タンパク質濃度が21μg/laneになるように、SDS-PAGEによって分離した。分離したタンパク質をImmobion-P Transfer membrames(Millipore)のメンブランとセラミドライシステム(Bio-Rad)を用いて、60分間、1.2mA/cm2でメンブランへ転写した。転写後のメンブランをTBS-Tween(0.1%Tween20/TBS;TBS-T)で10分間を3回繰り返すことにより洗浄した。洗浄後のメンブランを、60分間のブロッキング処理(3% normal goat serum/TBS-T)に供した。TBS-Tweenを用いて10分間を3回繰り返すことにより洗浄した。洗浄後のメンブランを4℃で14時間の一次抗体反応(抗CyPA抗体1,000倍希釈/TBS-T)に供した。
タンパク定量した乳清サンプルを用いて、次の手順で乳清中のタンパク質の解析を行った。乳清サンプル中のタンパク質を、PAGEL(ATTO;E-T520L)を用いてタンパク質濃度が21μg/laneになるようにSDS-PAGEによって分離した。分離後のゲルを、5分間のMQ洗浄を3回繰り返した。洗浄後のゲルを、ULTRA-FAST Coomassie Stain(NRV,USA)を用いて、室温で20分間の発色処理に供した。処理後のゲルにおけるバンドを確認した。
上記(3)B(B)の手順で、乳清サンプル中のタンパク質を分離した。分離後のゲルから、Molecular Imager FXを用いてバンドを検出および撮影した。撮影して得たイメージファイルを、Photshop 5.0 LE.を用いてグレースケールにより白黒反転させた。白黒反転させて得たイメージファイルを、NIHイメージ(NIH imager Ver.1.62,USA)のgel plotting Macrosを用いて解析し、バンド強度を測定した。バンド強度を測定して得たCyPAの濃度は、表1の牛体番号108の右後分房の乳汁サンプル(解析番号42)を基準として数値化した。また、得られたCyPA濃度とCL値との相関関係を解析した。
(1)乳汁中CyPAの検出方法の確立
牛体番号81の分房別乳汁サンプル(解析番号9-12)を用いて、タンパク質解析を行った。Coomassie Brilliant Blue(CBB)染色を行った結果、還元処理した非感染分房乳汁サンプル(解析番号10-12)および乳房炎発症分房乳汁サンプル(解析番号9)の解析結果として、65kDa付近にてバンドが検出された(図8)。これは本来160kDa程度のIgGが還元されて得たものと推測される。また、非還元処理で見られた50kDa付近のタンパク質は、還元処理後には検出されなかった。
上記(1)から、乳房炎に罹患している泌乳牛に由来する乳汁サンプル中にCyPAタンパク質が検出されたことから、全乳汁サンプル(解析番号1-48、ただし解析番号19は盲乳処置中)について還元処理を行い、得られた乳清サンプル中のCyPAタンパク質を解析した。健常牛分房乳汁サンプル(解析番号33-48)および乳房炎発症乳汁サンプル(解析番号1-32)を用いた解析では、CyPAタンパク質が検出された(図10A~C)。健常牛分房乳は乳房炎発症分房乳と比べ、CyPAタンパク質が少なかった。一方、乳房炎発症乳の非感染分房乳(解析番号1,3-6,10-12,14,16,18-22,24-26,29)は、乳房炎発症分房乳(解析番号2,7-9,13,15,17,23,27,28,30-32)より、CyPAタンパク質が少なかった。
健常牛の分房乳汁サンプルで乳性状、PLテストおよびCL能が低かった牛体番号108の解析番号42の乳汁サンプルを標準として、全乳汁サンプルのCyPA発現強度を測定した。解析番号1-48(ただし解析番号19は盲乳処置中)のCyPA発現の相対値を算出した(表2)。CL能とCyPA発現強度には相関関係が見られた(p<0.0001)(図11)。CL能の上昇に伴い、CyPA発現強度も増加することが明らかになった。
非還元処理した乳清サンプルのウェスタンブロットでは、非感染分房乳汁サンプルにおいてCyPAは検出されなかった。しかし、高いCL能を示す乳房炎発症分房乳汁サンプルからは、CyPAを大量に検出した。一方、乳清タンパク質の還元処理を行ったところ、健常牛由来乳汁サンプルおよび乳房炎発症牛由来乳汁サンプルの乳清中にCyPAが検出された。
Claims (14)
- 乳腺または乳汁におけるシクロフィリンA量を指標とした、乳腺疾患の検査方法。
- 下記(1)および(2)の工程を含む、乳腺疾患の検査方法。
(1)被験体の乳房または乳分房から採取された乳汁におけるシクロフィリンAを検出することにより乳汁中のシクロフィリンA量を得る工程
(2)前記乳汁中のシクロフィリンA量に基づいて、被験体の乳房または乳分房における乳腺疾患の発症または乳腺疾患の発症可能性を判定する工程 - 前記工程(2)は、前記乳汁中のシクロフィリンA量が、健常な乳房または乳分房から採取した乳汁中のシクロフィリンA量よりも大きい場合に、被験体の乳房または乳分房において乳腺疾患が発症している、または乳腺疾患が発症する可能性があると判定する工程である、請求項2に記載の方法。
- 前記工程(2)は、前記乳汁中のシクロフィリンA量が、健常な乳房または乳分房から採取した乳汁中のシクロフィリンA量の2倍以上である場合に、被験体の乳房または乳分房において乳腺疾患が発症している、または乳腺疾患が発症する可能性があると判定する工程である、請求項2に記載の方法。
- 前記乳腺疾患が、感染性の乳腺疾患である、請求項2に記載の方法。
- 前記乳腺疾患が、乳房炎である、請求項2に記載の方法。
- 抗シクロフィリンA抗体からなる、乳腺疾患検査用試薬。
- 前記抗シクロフィリンA抗体が、不溶性担体に固相化されている抗シクロフィリンA抗体である、請求項7に記載の試薬。
- 前記乳腺疾患が、感染性の乳腺疾患である、請求項7に記載の試薬。
- 前記乳腺疾患が、乳房炎である、請求項7に記載の試薬。
- 請求項7に記載の試薬を含む、乳腺疾患検査用キット。
- シクロフィリンAからなる、乳腺疾患検出用マーカー。
- 前記乳腺疾患が、感染性の乳腺疾患である、請求項12に記載のマーカー。
- 前記乳腺疾患が、乳房炎である、請求項12に記載のマーカー。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014524659A JP6176741B2 (ja) | 2012-07-12 | 2013-02-08 | 乳腺疾患の検査方法 |
US14/413,855 US10012649B2 (en) | 2012-07-12 | 2013-02-08 | Detection method of mammary gland disease |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2012-156163 | 2012-07-12 | ||
JP2012156163 | 2012-07-12 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2014010261A1 true WO2014010261A1 (ja) | 2014-01-16 |
Family
ID=49915740
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2013/052955 WO2014010261A1 (ja) | 2012-07-12 | 2013-02-08 | 乳腺疾患の検査方法 |
Country Status (3)
Country | Link |
---|---|
US (1) | US10012649B2 (ja) |
JP (1) | JP6176741B2 (ja) |
WO (1) | WO2014010261A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150192585A1 (en) * | 2012-07-12 | 2015-07-09 | Tohoku University | Method of examining mammary gland disease |
JP2016088918A (ja) * | 2014-11-11 | 2016-05-23 | 大山乳業農業協同組合 | 乳房炎治療・予防用組成物、並びに、乳房炎の治療・予防方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7105306B1 (en) * | 2000-08-10 | 2006-09-12 | Trustees Of The University Of Pennsylvania | Methods and compositions for modulating somatolactogenic functions |
WO2010053816A2 (en) * | 2008-10-29 | 2010-05-14 | The Regents Of The University Of Colorado, A Body Corporate | Biomarkers for diagnosis of breast cancer |
US10012649B2 (en) * | 2012-07-12 | 2018-07-03 | Tohoku University | Detection method of mammary gland disease |
-
2013
- 2013-02-08 US US14/413,855 patent/US10012649B2/en active Active
- 2013-02-08 JP JP2014524659A patent/JP6176741B2/ja active Active
- 2013-02-08 WO PCT/JP2013/052955 patent/WO2014010261A1/ja active Application Filing
Non-Patent Citations (2)
Title |
---|
KLASTRUP: "Bovine mastitis", DEFINITION AND GUIDELINES FOR DIAGNOSIS, KIELER MILCHWIRTSHAFTLICHE FORSCHUNGSBERICHTE, vol. 37, no. 3, 1985, pages 254 - 260 * |
THE JAPANESE SOCIETY FOR ANIMAL SCIENCES DAI 115 KAI TAIKAI KOEN YOSHI, 28 March 2012 (2012-03-28), pages 233 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150192585A1 (en) * | 2012-07-12 | 2015-07-09 | Tohoku University | Method of examining mammary gland disease |
US10012649B2 (en) * | 2012-07-12 | 2018-07-03 | Tohoku University | Detection method of mammary gland disease |
JP2016088918A (ja) * | 2014-11-11 | 2016-05-23 | 大山乳業農業協同組合 | 乳房炎治療・予防用組成物、並びに、乳房炎の治療・予防方法 |
Also Published As
Publication number | Publication date |
---|---|
US20150192585A1 (en) | 2015-07-09 |
JPWO2014010261A1 (ja) | 2016-06-20 |
US10012649B2 (en) | 2018-07-03 |
JP6176741B2 (ja) | 2017-08-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5710699B2 (ja) | オートタキシン測定による慢性肝疾患の検査方法および検査薬 | |
US10126312B2 (en) | Diagnostic method for urinary tract infection | |
US11892457B2 (en) | Proteoliposome-based ZnT8 self-antigen for type 1 diabetes diagnosis | |
EP2054443B1 (en) | Antibodies to an epitope of agr2, assays and hybridomas | |
JP6176741B2 (ja) | 乳腺疾患の検査方法 | |
EP2692735B1 (en) | Antibody reacting with native cochlin-tomoprotein (ctp) and method for measuring ctp using same | |
CN106153935B (zh) | 一种定量检测CD79α的酶联免疫试剂盒 | |
US20200386773A1 (en) | Antibody for detecting early damage to blood-brain barrier in ischemic stroke and use thereof | |
WO2020184409A1 (ja) | 生体試料中のβ-D-グルカンの免疫学的分析方法、及びβ-D-グルカン分析用キット | |
JP4567890B2 (ja) | メグシンタンパク質の検出方法およびその用途 | |
JP7306661B2 (ja) | 消化管間質腫瘍を検出する方法および検出試薬 | |
CN115819548B (zh) | 一种检测炎症相关疾病的标志物和方法 | |
WO2022054753A1 (ja) | 敗血症原因細菌の免疫学的分析キット | |
JP2016114360A (ja) | ヒトt細胞白血病ウイルスhbz蛋白質の検出方法 | |
US20230133318A1 (en) | Novel biomarkers of membranous glomerulonephritis | |
JPWO2004009640A1 (ja) | 抗菌性ペプチドに対する抗体及びその利用 | |
JP2013096783A (ja) | 肺腺癌を判定するためのデータ検出方法、診断薬、及び診断用キット | |
WO2022054754A1 (ja) | 敗血症原因細菌の免疫学的分析方法及び該方法に用いるモノクローナル抗体 | |
JP2018084524A (ja) | 疥癬の検査方法、並びにそれに用いる薬剤及びデバイス | |
JP2023138363A (ja) | 妊孕性を判定するためのバイオマーカー及びそれを用いた判定方法 | |
CN117129680A (zh) | 用于预测肿瘤免疫治疗效果的生物标志物及其应用 | |
CN115280146A (zh) | 包含人iv型胶原7s结构域的片段的测定方法以及用于该测定方法的试剂盒 | |
JP2013203733A (ja) | イヌ糸状虫22kDaタンパク質を利用したイヌ糸状虫感染の検出 | |
JP2001289859A (ja) | 多発性硬化症の診断方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13816068 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2014524659 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14413855 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 13816068 Country of ref document: EP Kind code of ref document: A1 |