WO2010038829A1

WO2010038829A1 - Method, composition and kit for detecting diabetic peripheral vascular disorder

Info

Publication number: WO2010038829A1
Application number: PCT/JP2009/067167
Authority: WO
Inventors: 岩田岳; 松野聖; 棚橋一裕
Original assignee: 独立行政法人国立病院機構; 参天製薬株式会社
Priority date: 2008-10-02
Filing date: 2009-10-01
Publication date: 2010-04-08
Also published as: JP2010085363A

Abstract

Disclosed is a method for detecting a diabetic peripheral vascular disorder such as diabetic macular edema, which comprises measuring at least one component selected from a polypeptide comprising an amino acid sequence depicted in any one of SEQ ID NO:1 to SEQ ID NO:9 shown in the Sequence Listing and a mutant or a fragment of the polypeptide in a biological sample collected from a subject. Also disclosed is a composition or a kit for diagnosing and/or detecting a diabetic peripheral vascular disorder such as diabetic macular edema.

Description

Methods, compositions and kits for the examination of diabetic peripheral vascular disorders

The present invention relates to a test method for diabetic peripheral vascular disorder. The inspection method includes a method for determining or identifying the disorder.

The present invention also relates to a composition and kit useful for diagnosis of diabetic peripheral vascular disorder.

The retina is a thin membrane at the back of the eyeball, consisting of many cells, and numerous fine blood vessels. If the state of high blood sugar continues for a long time due to diabetes or the like, the thin blood vessels of the retina are gradually damaged, various components in the blood leak, and swelling occurs in the macular in the center of the object. Such a condition is called macular edema. Since the macula is a very important place for visual acuity, if macular edema persists for a long time, the visual acuity is greatly reduced and often difficult to recover. In Japan (Japan), with the rapid increase in the elderly population and the westernization of food in recent years, the number of diabetic patients is steadily increasing, and the complications of diabetic macular edema tend to increase.

】 Since this disease is a disease that leads to decreased visual acuity, it is very important to make an early diagnosis. Conventionally, diagnosis of diabetic macular edema has been performed mainly by the fundus examination, and the patient applies eye drops to open the pupil before the examination, or a fluorescent dye that stains the blood vessels of the retina. After injection into a vein, a doctor observes the retina directly with a fundus mirror or fundus camera. However, since the pupil is left open by eye drops, the dull and invisible state may continue for about 3 hours, which is a temporary burden for the patient. Moreover, it cannot be said that there is no possibility of causing side effects such as nausea, vomiting and shock after intravenous injection of fluorescent dyes. Furthermore, direct observation of the retina by a doctor is not necessarily an objective diagnostic method, and the doctor must deal with each patient, so that it is difficult to develop into a mass screening that handles a large number of subjects. Thus, there is a demand for a high-throughput diagnostic method with less patient burden that can more objectively and quantitatively indicate the degree of progression of a patient's disease state and the change over time during treatment.

Diagnostic method using diagnostic markers is one of objective and high-throughput methods. A diagnostic method using VEGF or bFGF in plasma has been disclosed so far (Non-patent Document 1). These techniques cannot determine diabetic macular edema with relatively high specificity using these markers, and are currently at the research level.

With recent advances in genome analysis or proteome analysis, various new marker candidates have been reported. In diabetic macular edema, various novel marker candidates have been reported by proteomic analysis using ocular tissue (Non-patent Document 2). However, a method for diagnosing diabetic macular edema using a highly reliable protein marker for diabetic macular edema or a protein marker in the blood of diabetic macular edema is still unknown. If a marker capable of diagnosing diabetic macular edema and a method capable of diagnosing using the marker can be created, it can be expected to expand to the diagnosis of diabetic peripheral vascular disorders.

The above existing markers and marker candidates are generally not clinically used because of their poor specificity and / or sensitivity and their efficient detection methods from biological samples have not been established. Markers of diabetic macular edema with high specificity and sensitivity are eagerly desired. In addition, a high-throughput inspection method with a low patient burden that can more objectively and quantitatively indicate the degree of progression of the patient's pathological condition and the temporal change after surgery is desired.

An object of the present invention is to provide a composition or kit useful for diagnosis of diabetic peripheral vascular disorder, particularly diabetic macular edema, and a method for examining diabetic macular edema using the composition or kit.

The present inventors have conducted proteomic analysis of vitreous specimens of patients with diabetic peripheral vascular disorders such as diabetic macular edema and patients with other eye diseases, so that they can be detected in vivo specifically for patients with the disorders. The present inventors have found a protein marker and invented a method for examining diabetic peripheral vascular disorder using the protein marker.

<Summary of invention>
The present invention has the following features.

(1) Quantitative or qualitative analysis of any one or more of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 to 9 in the sequence listing contained in a biological sample derived from a subject, its variant or fragment thereof A method for examining diabetic peripheral vascular disorder, comprising measuring and / or detecting the disease.

(2) The assay method according to (1) above, wherein the diabetic peripheral vascular disorder is diabetic macular edema.

(3) The method according to (1) or (2) above, wherein measurement and / or detection of the polypeptide, variant thereof or fragment thereof is performed using mass spectrometry.

(4) The method according to (3) above, wherein the measurement and / or detection is performed using a substance capable of binding to the polypeptide, a variant thereof or a fragment thereof.

(5) The method according to (4) above, wherein the bindable substance is an antibody or a fragment thereof.

(6) The method according to (5) above, wherein the antibody or fragment thereof is labeled with any of an enzyme, a fluorescent substance, a dye, or a radioisotope.

(7) The method according to (5) or (6) above, wherein the antibody or fragment thereof is a monoclonal or polyclonal antibody, or a fragment thereof.

(8) The method according to any one of (1) to (7) above, wherein the biological sample is blood, plasma, serum, urine, tears, vitreous or vitreous humor.

(9) an antibody or a fragment thereof, or a chemically modified derivative thereof, which specifically binds to at least one of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 to 9 in the sequence listing, a variant thereof or a fragment thereof A composition for diagnosis or / and detection of diabetic peripheral vascular disorder, comprising one or more substances.

(10) Fragments that bind to an antibody or an antigen thereof, which specifically bind to at least one of the polypeptides comprising the amino acid sequences represented by SEQ ID NOS: 1 to 9 in the sequence listing, variants or fragments thereof, or their chemistry A kit for the diagnosis and / or detection of diabetic peripheral vascular disorder comprising one or more of the modified derivatives.

(11) A fragment that binds to an antibody or an antigen thereof, which specifically binds to at least one of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 to 9 in the sequence listing, a variant thereof or a fragment thereof, or a chemistry thereof Use of one or more of the modified derivatives in the manufacture of a kit for the diagnosis and / or detection of diabetic peripheral vascular disorders.

<Definition>
The terms used herein have the following definitions:

The variant of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 to 9 in the sequence listing in this specification is one or more of the amino acid sequence represented by SEQ ID NO: 1 to 9 in the sequence listing or a partial sequence thereof. About 80% or more, about 85% or more, preferably about 90% or more, preferably a variant comprising deletion, substitution, addition or insertion of one or several amino acids, or the amino acid sequence or a partial sequence thereof More preferably, it means a variant comprising an amino acid sequence having about 95% or more, about 97% or more, about 98% or more, or about 99% or more identity.

As used herein, the term “% identity” generally refers to an amino acid residue or position when aligned (aligned) with or without introducing a gap between two amino acid sequences. The percentage of the number of identical amino acid residues or positions shared by the sequence relative to the total number. The identity of two amino acid sequences can be determined using a mathematical algorithm, examples of such algorithms are described in Karlin and Altshul, Proc. Natl. Acad. Sci. USA 1990, 87: 2264 and its improved versions, Karlin and Altshul, Proc. Natl. Acad. Sci. USA 1993, 90: 5873-5877. This type of algorithm is incorporated in BLASTN, BLASTX, etc. (Altshul et al., J. Mol. Biol. 1990, 215: 403). In order to obtain an amino acid sequence that is homologous to each amino acid sequence of the polypeptides represented by SEQ ID NOs: 1 to 9 in the sequence listing of the present invention, BLAST protein search is performed using, for example, BLAST protein search with score = 50 and word length = 3. Execute using a program. Moreover, in order to obtain the alignment which introduce | transduced the gap, gap introduction BLAST (gapped BLAST) (Altshul et al., Nucleic Acid Res. 1997, 25: 3389) can be used.

As used herein, the term “several” refers to any integer less than or equal to 10, ie, 10, 9, 8, 7, 6, 5, 4, 3 or 2.

The term “chemically modified derivative” as used herein is not limited to the following, for example, a labeled derivative such as an enzyme, a fluorescent substance, a dye, a radioisotope, or the like, or biotinylation, acetylation , Means derivatives containing chemical modifications such as glycosylation, phosphorylation, ubiquitination, sulfation.

As used herein, the term “composition for diagnosis and / or detection” or “kit for diagnosis and / or detection” refers to the presence or absence of a diabetic peripheral vascular disorder such as diabetic macular edema, Directly for diagnosing and / or detecting the degree of morbidity or the presence or improvement of the disease and / or the degree of improvement, or for screening candidate substances useful for the prevention, amelioration or treatment of diabetic peripheral vascular disorders such as diabetic macular edema Or it can be used indirectly.

The “biological sample” to be detected and / or diagnosed in the present specification includes a target polypeptide that appears or is suspected to be contained in the development of diabetic peripheral vascular disorders such as diabetic macular edema. It refers to a sample collected, for example, a sample of cells, tissues (eg, vitreous), or body fluids (eg, blood, lymph, urine, tears, etc.).

As used herein, “specifically binds” means that an antibody or a fragment thereof forms an antigen-antibody complex only with a target polypeptide that is a marker for diabetic macular edema in the present invention, a variant or a fragment thereof, The other peptidic or polypeptide substance means that it does not substantially form an antigen-antibody complex. Here, “substantially does not form” means that non-specific complex formation can occur to a small extent.

This specification includes the contents described in the specification of Japanese Patent Application No. 2008-257430 which is the basis of the priority of the present application.

The marker for diabetic peripheral vascular disorder such as diabetic macular edema in the present invention is found in a biological sample such as vitreous body, vitreous humor or blood of a diabetic macular edema patient, but there are other factors such as proliferative diabetic retinopathy and macular hole. Since most or no eye diseases are found, it is possible to easily detect diabetic macular edema by simply using the presence or amount of the marker as an index, for example, vitreous or blood. It has the following effects.

Hereinafter, the present invention will be described more specifically.

<Diabetic peripheral vascular disorder marker>
A diabetic macular edema marker for diagnosing and / or detecting diabetic macular edema using a composition or kit for the diagnosis or detection of diabetic peripheral angiopathy such as diabetic macular edema of the present invention is A polypeptide comprising the amino acid sequence represented by SEQ ID NOs: 1 to 9, a variant thereof or a fragment thereof.

Polypeptides comprising the amino acid sequences represented by SEQ ID NOs: 1 to 9 in the sequence listing of the present invention are shown in the following Table 1 together with their protein numbers (UniProtKB / Swiss-Prot Release 52.2 registered name and registration number) and their characteristics. Shown in These polypeptides are specifically detected in the vitreous of patients with diabetic macular edema and are not detected in the vitreous of patients with proliferative diabetic retinopathy and macular hole, or compared to the vitreous of diabetic macular edema Detection was significantly reduced. The amino acid sequences of these polypeptides can be obtained by accessing a data bank such as UniProtKB / Swiss-Prot.

In the present invention, any of the above target polypeptides for detection of diabetic peripheral vascular disorder is detected only in a biological sample such as a vitreous body of a diabetic macular edema patient, or a patient with proliferative diabetic retinopathy and macular hole Is characterized by significantly or exceptionally high in diabetic macular edema patients. Here, “significantly” indicates a statistically significant value, and is a term used when the risk factor (p) is less than 0.05.

Therefore, when any one, preferably two or more of the above-mentioned diabetic macular edema marker polypeptides are detected at a significant level in the biological sample of the subject (that is, the test subject) compared to the above-mentioned comparative control It can be determined that this is a diabetic peripheral vascular disorder.

The polypeptide in the present invention can be produced, for example, by chemical synthesis (for example, peptide synthesis, automatic DNA / RNA synthesis, etc.) or DNA recombination techniques that are conventional techniques in the art. The use of DNA recombination technology is preferred from the viewpoint of procedure and ease of purification.

Briefly, first, a polynucleotide sequence encoding a partial sequence of the polypeptide of the present invention is chemically synthesized using an automatic DNA synthesizer. In general, the phosphoramidite method is used for this synthesis, and single-stranded DNA of up to about 100 bases can be automatically synthesized by this method. Automatic DNA synthesizers are commercially available from, for example, Polygen and ABI.

Using the obtained polynucleotide as a probe or primer, total RNA extracted from a living tissue such as an eye tissue in which the target gene is expressed by a known cDNA cloning, specifically, using an oligo dT cellulose column. A cDNA library is prepared from the poly A (+) RNA obtained by the treatment by RT-PCR, and the target cDNA clone is obtained from this library by screening such as hybridization screening, expression screening, and antibody screening. If necessary, the cDNA clone can be further amplified by PCR. As a result, cDNA corresponding to the target gene can be obtained.

Probes or primers can be selected from continuous sequences in the range of, for example, 15 to 100 bases based on the polypeptide sequences shown in SEQ ID NOs: 1 to 9 in the sequence listing and synthesized as described above. The cDNA cloning technique is described in, for example, Sambrook, J. et al. And Russel, D .; Written by Molecular Cloning, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Vol. 1, published in January 15, 2001, Volumes 7.42-7.45, Volumes 8.9-8.17, Ausubel et al., Current Proto. in Molecular Biology, 1994, John Wiley & Sons.

Next, the cDNA clone obtained as described above is incorporated into an expression vector, and a prokaryotic or eukaryotic host cell transformed or transfected with the vector is cultured, so that the target polymorph is obtained from the cell or the culture supernatant. Peptides can be obtained. At this time, the mature polypeptide can be secreted out of the cell by flanking the nucleotide sequence encoding the secretory signal sequence at the 5 'end of the DNA encoding the target mature polypeptide.

Vectors and expression systems are available from Novagen, Takara Shuzo, Daiichi Chemicals, Qiagen, Stratagene, Promega, Roche Diagnostics, Invitrogen, Genetics Institute, Amersham Bioscience, and the like. Examples of host cells include prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis), yeast (eg, Saccharomyces cerevisiae), insect cells (eg, Sf cells), mammalian cells (eg, COS, CHO, BHK, NIH3T3, etc.) Can be used. Vectors, in addition to the DNA encoding the polypeptide, regulatory elements such as promoters (e.g. lac promoter, trp promoter, _{P L} promoter, _{P R} promoter, SV40 viral promoter, 3-phosphoglycerate kinase promoter, glycolytic System enzyme promoters), enhancers, polyadenylation signals and ribosome binding sites, replication origins, terminators, selectable markers (for example, drug resistance genes such as ampicillin resistance gene and tetracycline resistance gene; auxotrophic complementary markers such as LEU2 and URA3) ) And the like.

In order to facilitate the purification of the polypeptide, an expression product can be generated in the form of a fusion polypeptide in which a labeled peptide is bound to the C-terminus or N-terminus of the polypeptide. Representative labeled peptides include 6-10 residue histidine repeats (His tag), FLAG, myc peptide, GST (glutathione S-transferase) polypeptide, etc., but the labeled peptides are not limited to these. Absent.

When the polypeptide according to the present invention is produced without attaching a labeled peptide, examples of the purification method include a method by ion exchange chromatography. In addition to this, a method combining gel filtration, hydrophobic chromatography, isoelectric point chromatography, high performance liquid chromatography (HPLC), electrophoresis, ammonium sulfate fractionation, salting out, ultrafiltration, dialysis and the like may be used. Furthermore, when a labeled (poly) peptide such as histidine repeat, FLAG, myc, or GST is attached to the polypeptide, a method by affinity chromatography suitable for each generally used labeled peptide can be mentioned. In this case, an expression vector that facilitates isolation and purification may be constructed. In particular, if an expression vector is constructed so that it is expressed in the form of a fusion polypeptide of a polypeptide and a labeled peptide, and the polypeptide is genetically engineered, isolation and purification are easy.

Nucleic acid can be purified by a purification method using agarose gel electrophoresis, a DNA-binding resin column, or the like. Moreover, since an automatic nucleic acid purification apparatus, a nucleic acid purification kit, etc. are marketed, nucleic acid purification can also be performed using these.

As defined above, the variant of the polypeptide of the present invention is a deletion of one or more, preferably one or several amino acids in the amino acid sequence represented by SEQ ID NOs: 1 to 9 or a partial sequence thereof. About 80% or more, about 85% or more, preferably about 90% or more, more preferably about 95% or more, about 97% or more with a mutant comprising deletion, substitution, addition or insertion, or the amino acid sequence or a partial sequence thereof , A variant consisting of an amino acid sequence having% identity of about 98% or more and about 99% or more. Such mutants include, for example, homologues of mammalian species different from humans, natural variants such as mutants based on polymorphic variation between the same mammalian species (for example, race), splice variants, mutants, etc. Variants are included.

In the present invention, the term “subject” is intended to include not only humans but also other mammals.

In the present invention, the polypeptide fragment comprises at least 7, at least 8, at least 10, at least 15, preferably at least 20, at least 25, more preferably at least 30, of the amino acid sequence of the polypeptide. Consists of at least 40, at least 50, at least 100, at least 150, or at least 200 to a total number of consecutive amino acid residues and retains one or more epitopes. Such a fragment is capable of immunospecifically binding to the antibody of the present invention or a fragment thereof. For example, when the polypeptide is present in blood, it is assumed that the polypeptide is present by being cleaved and fragmented by an enzyme such as protease or peptidase.

<Composition or kit for diagnosis or detection of diabetic peripheral vascular disorder>
The present invention relates to an antibody or fragment thereof, or a chemically modified derivative thereof, which specifically binds to a polypeptide comprising the amino acid sequence represented by SEQ ID NOS: 1 to 9 in the sequence listing, a variant thereof or a fragment thereof. For the diagnosis and / or detection of diabetic peripheral vascular disorders, comprising one or more, preferably 3 or more, more preferably 5 or more, even more preferably 7 or more, most preferably 9 different substances A composition is provided.

In the present invention, the term composition refers not only to a mixture of a plurality of antibodies, fragments thereof, and / or chemically modified derivatives thereof, but also to a plurality of antibodies, fragments thereof, and / or chemically modified derivatives thereof. It is intended to include combinations.

An antibody that recognizes a polypeptide that is a marker for diabetic macular edema, a variant thereof, or a fragment thereof can specifically bind to the polypeptide, a variant thereof, or a fragment thereof via the antigen-binding site of the antibody It is. The antibody that can be used in the present invention is conventionally used by using a polypeptide comprising the amino acid sequences of SEQ ID NOs: 1 to 9, a variant thereof, or a fragment thereof, or a fusion polypeptide thereof as one or a plurality of immunogens. It can produce by the technique of. These polypeptides, fragments, variants or fusion polypeptides contain epitopes that elicit antibody formation, but these epitopes may be linear or higher order (intermittent). Epitopes capable of binding to an antibody are generally considered to be present on the hydrophilic surface of the polypeptide structure.

The antibody that can be used in the present invention includes any type, class, and subclass. Such antibodies include, for example, IgG, IgE, IgM, IgD, IgA, IgY, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2.

Furthermore, antibodies of all aspects are induced by the polypeptide according to the present invention. If all or part of the polypeptide or epitope is isolated, both polyclonal and monoclonal antibodies can be prepared using conventional techniques. Methods include, for example, Kennet et al. (Supervised), Monoclonal Antibodies, Hybridomas: Ａ A NewＡ Dimension in Biological Analyzes, Pleumnum Press, New York, 1980.

Polyclonal antibodies can be produced by immunizing animals such as birds (for example, chickens) and mammals (for example, rabbits, goats, horses, sheep, mice, etc.) with the polypeptide of the present invention. The target antibody can be purified from the blood of an immunized animal by appropriately combining techniques such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and the like.

Monoclonal antibodies can be obtained by techniques including producing hybridoma cell lines that produce monoclonal antibodies specific for each polypeptide in mice by conventional techniques. One method for producing such hybridoma cell lines is to immunize an animal with a polypeptide according to the invention, collect spleen cells from the immunized animal, fuse the spleen cells to a myeloma cell line, Generating hybridoma cells and identifying a hybridoma cell line that produces a monoclonal antibody that binds to the polypeptide. Monoclonal antibodies can be recovered by conventional techniques.

Preparation of monoclonal and polyclonal antibodies is described in detail below.

A. Preparation of monoclonal antibody (1) Immunization and collection of antibody-producing cells The immunogen obtained as described above is administered to mammals such as rats, mice (for example, Balb / c of inbred mice), rabbits, and the like. . A single dose of the immunogen is appropriately determined according to the type of animal to be immunized, the route of administration, etc., and is about 50 to 200 μg per animal. Immunization is performed mainly by injecting an immunogen subcutaneously or intraperitoneally. Further, the immunization interval is not particularly limited, and after the initial immunization, booster immunization is performed 2 to 10 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 1 to 4 weeks. After the initial immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA (Enzyme-Linked Immuno Sorbent Assay) method, etc. When the antibody titer reaches a plateau, the immunogen is intravenously or intraperitoneally Inject and give final immunization. Then, antibody-producing cells are collected 2 to 5 days, preferably 3 days after the last immunization. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

(2) Cell fusion Hybridoma cell lines that produce monoclonal antibodies specific for each protein can be produced and identified by conventional techniques. One method for producing such a hybridoma cell line is to immunize an animal with a polypeptide of the invention, collect spleen cells from the immunized animal, and fuse the spleen cells to a myeloma cell line, thereby Generating hybridoma cells and identifying a hybridoma cell line producing a monoclonal antibody that binds to the enzyme. As a myeloma cell line to be fused with an antibody-producing cell, a generally available cell line of an animal such as a mouse can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. The cell line is preferably derived from an animal of the same species as the immunized animal. Specific examples of myeloma cell lines include P3X63-Ag., Which is a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient cell line derived from Balb / c mice. 8 strains (ATCC TIB9).

Next, cell fusion is performed between the myeloma cell line and antibody-producing cells. In cell fusion, antibody-producing cells and myeloma cell lines are mixed at a ratio of about 1: 1 to 20: 1 in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and cell fusion is performed. The fusion reaction is performed in the presence of an accelerator. As a cell fusion promoter, polyethylene glycol having an average molecular weight of 1500 to 4000 daltons can be used at a concentration of about 10 to 80%. In some cases, an auxiliary agent such as dimethyl sulfoxide may be used in combination in order to increase the fusion efficiency. Furthermore, antibody-producing cells and myeloma cell lines can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(3) Selection and cloning of hybridoma The target hybridoma is selected from the cells after cell fusion treatment. As a method for this, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, and then plated on a microtiter plate at about 2 million cells / well, and a selective medium is added to each well. Cultivate by changing the selective medium. The culture temperature is 20 to 40 ° C, preferably about 37 ° C. When the myeloma cells are of HGPRT-deficient strain or thymidine kinase-deficient strain, cells having the ability to produce antibodies and myeloma cell lines can be obtained by using a selective medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine. Only those hybridomas can be selectively cultured and propagated. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

Next, it is screened whether the target antibody is present in the culture supernatant of the hybridoma that has proliferated. Hybridoma screening is not particularly limited, and may be carried out according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma is collected and subjected to enzyme immunoassay (EIA: Enzyme Immuno Assay and ELISA), radioimmunoassay (RIA: Radio Immuno Assay), etc. Can do. Cloning of the fused cells is performed by limiting dilution or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established. The hybridoma is stable in culture in a basic medium such as RPMI-1640 or DMEM, and produces and secretes a monoclonal antibody that specifically reacts with the polypeptide diabetic macular edema marker of the present invention.

(4) Antibody recovery Monoclonal antibodies can be recovered by conventional techniques. That is, as a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (eg, 37 ° C., 5% CO ₂ concentration). Cultivate for 2-10 days and obtain antibody from the culture supernatant. In the case of the ascites formation method, about 10 million hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and the same type of animal, and the hybridomas are proliferated in large quantities. Ascites fluid or serum is collected after 1-2 weeks.

In the above antibody collection method, when purification of the antibody is required, a known method such as ammonium sulfate salting out method, ion exchange chromatography, affinity chromatography, gel filtration chromatography or the like is appropriately selected, or these In combination, a purified monoclonal antibody of the present invention can be obtained.

B. Preparation of polyclonal antibody When preparing a polyclonal antibody, an animal is immunized in the same manner as described above, and 6 to 60 days after the last immunization, enzyme immunoassay (EIA and ELISA), radioimmunoassay (RIA), etc. The antibody titer is measured and blood is collected on the day when the maximum antibody titer is shown to obtain antiserum. Thereafter, the reactivity of the polyclonal antibody in the antiserum is measured by an ELISA method or the like.

In the present invention, an antigen-binding fragment of the above antibody can also be used. Examples of antigen-binding fragments that can be produced by conventional techniques include, but are not limited to, fragments such as Fab and F (ab ′) ₂ , Fv, scFv, dsFv. Also included are antibody fragments and derivatives that can be produced by genetic engineering techniques. Such antibodies include, for example, synthetic antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), single chain antibodies, and the like.

The antibody of the present invention can be used in an assay for detecting the presence of a polypeptide or a (poly) peptide fragment thereof in the present invention, both in vitro and in vivo. The use of monoclonal antibodies is preferred to allow specific detection in the assay, but even polyclonal antibodies can be identified by the so-called absorption method, which involves binding the antibody to an affinity column to which the purified polypeptide is bound. Antibodies can be obtained.

Therefore, the composition of the present invention preferably comprises at least one antibody or fragment thereof that can specifically bind to a polypeptide comprising the amino acid sequence of SEQ ID NOs: 1 to 9 in the sequence listing, a variant thereof, or a fragment thereof. Can include multiple types (two or more, three or more, etc.), more preferably all types.

Preferably, the composition of the present invention is in the form of a kit. Such a kit includes a container for accommodating antibodies or fragments thereof that can specifically bind to each of the above polypeptides, either separately or as a mixture. The antibody or fragment thereof may be attached or bound on a solid phase carrier such as a multi-well plate made of polystyrene, a spherical carrier such as latex beads or magnetic beads.

The antibody or fragment thereof used in the present invention may be bound with a label, for example, a fluorophore, an enzyme, a radioisotope, or the like, if necessary, or such a label may be bound to a secondary antibody. Also good.

Fluorophores include, for example, fluorescein and its derivatives (such as FITC), rhodamine and its derivatives (such as tetramethylrhodamine), dansyl chloride and its derivatives, umbelliferone, and the like.

Enzymes include, for example, horseradish peroxidase, alkaline phosphatase and the like.

Radioisotopes include, for example, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), phosphorus ( ³² P), sulfur ( ³⁵ S), metals (eg, ⁶⁸ Ga, ⁶⁷ Ga, ⁶⁸ Ge, ⁵⁴ Mn, ⁹⁹ Mo, ⁹⁹ Tc, ¹³³ Xe, etc.).

Other labels include, for example, luminescent substances such as luminol, and bioluminescent substances such as luciferase and luciferin.

If necessary, an avidin-biotin system or a streptavidin-biotin system can be used. In this case, for example, biotin can be bound to the antibody of the present invention or a fragment thereof.

The composition of the present invention further comprises an antibody or an antigen-binding fragment thereof that specifically binds to at least one of a polypeptide comprising the amino acid sequence represented by SEQ ID NOs: 1 to 9 in the sequence listing, a variant thereof, or a fragment thereof. Kits for the diagnosis or / and detection of diabetic peripheral vascular disorders comprising one or more of those chemically modified derivatives are provided.

The kit comprises, for example, different containers (for example, vials) containing the above-mentioned antibodies or nucleic acids for detecting a marker of diabetic peripheral vascular disorder individually or appropriately mixed. The antibodies can preferably be contained in a container in lyophilized form.

Alternatively, the kit of the present invention comprises an antibody or antigen-binding fragment thereof that can specifically bind to each of the above polypeptides, such as a multi-well plate, an array, a microtiter plate, a test strip (or test strip), a latex bead. Or a solid support such as a spherical support such as a magnetic bead or a covalently bonded (covalent or non-covalent) bond.

The kit may further contain a buffer, a secondary antibody, an instruction manual, etc. for use in the assay method of the present invention.

Therefore, the present invention further provides a fragment that binds to an antibody or an antigen thereof that specifically binds to at least one of a polypeptide comprising the amino acid sequence represented by SEQ ID NOs: 1 to 9 in the sequence listing, a variant thereof or a fragment thereof. Also provided is the use of one or more of these chemically modified derivatives, in the manufacture of a kit for the diagnosis and / or detection of diabetic peripheral vascular disorders.

<Detection of diabetic peripheral vascular disorder>
According to the present invention, using a substance that can bind to the marker, the polypeptide represented by SEQ ID NOs: 1 to 9 in the sequence listing in a biological sample derived from a subject, a variant or fragment thereof, or the polypeptide , By measuring the amount or presence of one or more of the nucleic acids encoding the variant or fragment thereof in vitro, by a method comprising diabetic peripheral angiopathy (eg diabetic macular edema, simple diabetic retina) , Background diabetic retinopathy, preproliferative diabetic retinopathy, diabetic macular disease, etc.) can be detected. When the diabetic macular edema marker is detected by the method of the present invention, or when it is determined that the gene expression level is significantly higher than the control, the subject has advanced diabetic peripheral vascular disorder and suffers from diabetic macular edema. Can be diagnosed.

In the method of the present invention, the detection of the diabetic macular edema marker may be a single marker, but is preferably performed on a plurality of markers, for example, 2 or more, 3 or more, 4 or more, or 5 or more and 9 or less. . This is to improve the accuracy of disease detection by using a plurality of markers.

The composition or kit of the present invention is useful for diagnosis, determination or detection of diabetic peripheral vascular disorder, that is, diagnosis of the presence or absence of disease and the degree of disease. Diagnosis of diabetic macular edema is performed by comparing with a negative control such as normal cells, tissues, or body fluids, and detecting the presence or amount of the above-mentioned diabetic macular edema marker in a biological sample of a subject. Is significant, the subject is suspected of having diabetic macular edema.

The specimen sample used in the method of the present invention is a biological sample represented by a body fluid such as blood, serum, plasma, urine or tears, or a tissue such as vitreous.

The substance capable of binding to the above-mentioned diabetic macular edema marker is, for example, aptamer, Affibody (trademark) (Affibody), each diabetic macular edema marker receptor, each diabetic macular edema marker, Specific action inhibitors, specific action activators of the respective diabetic macular edema markers, preferably antibodies or fragments thereof, or chemically modified derivatives thereof.

In an embodiment of the present invention, the measurement is performed by contacting an antibody or fragment optionally labeled with a conventional enzyme or fluorophore with a tissue section or homogenized tissue or body fluid, qualitatively or with an antigen-antibody complex. A step of quantitatively measuring can be included. Detection is, for example, a method for measuring the presence and level of a target polypeptide by immunoelectron microscopy, an enzyme antibody method (for example, ELISA), a fluorescent antibody method, a radioimmunoassay method, a homogeneous method, a heterogeneous method, a solid phase method, a sandwich method Or the like by a method of measuring the presence or level of the target polypeptide by a conventional method. Diabetic macular edema is diagnosed when the target polypeptide is present in a body fluid or tissue or cell, preferably blood, or when the level of the target polypeptide is significantly increased or high compared to a negative control.

As a measurement method replacing the immunological method, a method using mass spectrometry is included. Specifically, this method can be performed by the technique described in the examples. That is, a biological sample such as serum or plasma is filtered to remove impurities, diluted with a buffer solution (for example, pH of about 8) and adjusted to a concentration of about 10 mg / ml to about 15 mg / ml, and then a molecular weight of 5 Molecular weight fractionation is performed through a hollow fiber filter (Reference Example (1) below) or a centrifugal flat membrane filter capable of removing more than 10,000 proteins, and the fraction is treated with protease (for example, trypsin) to be peptideized, and mass spectrometry is performed. Based on the mass / charge number and intensity of a specific peak derived from the polypeptide of interest, it can be applied to a diabetic macular edema patient and healthy by applying a meter (a type using matrix-assisted laser desorption ionization or electrospray ionization) The difference in polypeptide abundance in the sample between humans or other eye diseases can be measured.

The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention shall not be limited by this example.

<Reference example>
(1) Production of hollow fiber filter 100 polysulfone hollow fibers having a pore size of about 50,000 on the membrane surface are bundled, and both ends are fixed to a glass tube with an epoxy potting agent so as not to block the hollow part of the hollow fiber. And created a mini-module. The minimodule (module A) is used to remove high molecular weight proteins in serum or plasma, and has a diameter of about 7 mm and a length of about 17 cm. Similarly, a minimodule (module B) used for concentration of low molecular weight proteins was prepared using a membrane having a pore size of about 3,000 for the molecular weight cut off. The mini module has an inlet connected to the hollow fiber lumen at one end, and an outlet at the opposite end. The hollow fiber inlet and outlet are closed circulation system flow paths using silicon tubes, and the liquid is driven and circulated through the flow path by a peristaltic pump. Further, the glass tube of the hollow fiber mantle is provided with a port for discharging the liquid leaking from the hollow fiber, and one module set is configured. Modules were connected by T-shaped connectors in the middle of the flow path, and three modules A and one module B were connected in tandem to form one hollow fiber filter. The hollow fiber filter was washed with distilled water and filled with an aqueous solution of PBS (phosphate buffer containing 0.15 mM NaCl, pH 7.4). The fraction raw material serum or plasma is injected from the flow channel inlet of the hollow fiber filter, and is discharged from the flow channel outlet after fractionation and concentration. Serum or plasma injected into the hollow fiber filter is subjected to molecular sieving with a molecular weight of about 50,000 for each module A, and components having a molecular weight lower than 50,000 are concentrated and prepared in module B. Yes.

<Example 1>
(1) Protein identification of diabetic macular edema, macular hole, and proliferative diabetic retinopathy 16 diabetic macular edema patients with an average age of 61, 16 macular hole patients of the same age, 16 proliferative diabetic retinopathy patients Vitreous bodies were obtained from each and measured for each. The vitreous was centrifuged to remove contaminants. This vitreous was combined into 16 samples for each disease. The vitreous sample was further diluted in a 300 mM ammonium bicarbonate solution, and fractionated by molecular weight using the hollow fiber filter shown in Reference Example (1). 300 μg of protein was used per fractionation, and this was repeated 8 times. Eight fraction collection solutions were combined again. The vitreous sample after fractionation (8 fraction collection solutions: total volume 13.6 mL, containing up to 600 μg of protein) is separated into 3 fractions by reverse phase chromatography with AKTA explorer 10s (GE Healthcare Biosciences) Each fraction was lyophilized and redissolved in 100 mM ammonium bicarbonate buffer. This sample was treated with DTT / iodoacetamide and digested with trypsin in an amount of 1/50 of the protein overnight at 37 ° C. for peptideization. The peptides of each fraction were further fractionated into 8 fractions by an ion exchange column. Each fraction was further fractionated on a reverse phase column, and the eluted peptide was measured using a mass spectrometer LCQ Deca XP plus (Thermo Fisher Scientific Co., Ltd.) linked online. .

(2) Vitreous protein expression comparison of macular hole, diabetic macular edema, and proliferative diabetic retinopathy The data measured in (1) above was obtained from Bioworks (Thermo Fisher Scientific Co., Ltd.), a protein identification software, and By using Phenyx (GENE BIO), protein identification was performed comprehensively. Among the identified proteins, the proteins identified together with two kinds of software were listed, and the proteins were detected from patient vitreous samples of each disease. This was done to eliminate false positive proteins contained in the analysis results of one software by combining two types of software with different algorithms. However, unlike Bioworks, Phenix conducts searches taking into account isoform-specific amino acid sequences due to alternative splicing of the same protein, changes in mass due to post-translational modifications, etc., and therefore identifies peptides that cannot be identified by Bioworks There is a case. Under these conditions, proteins identified only in Phenix were also listed.

Among the proteins listed in each disease, a protein detected in a patient with diabetic macular edema but not detected in a macular hole or proliferative diabetic retinopathy patient was found as a vitreous marker protein. These proteins are polypeptides comprising the amino acid sequences represented by SEQ ID NOs: 1 to 9 shown in Table 1 (above) and the sequence listing, and therefore diabetic peripheral vascular disorders such as diabetic macular edema as diabetic macular edema markers. Has proved useful in the detection and diagnosis during treatment.

The present invention is particularly useful in the pharmaceutical and pharmaceutical industries because it can provide a composition for diagnosing diabetic peripheral vascular disorders such as diabetic macular edema having excellent specificity and sensitivity.

All publications cited in this specification shall be incorporated into this specification as reference.

Claims

Quantitatively or qualitatively measure one or more of the polypeptide comprising the amino acid sequence represented by SEQ ID Nos. 1 to 9 in the sequence listing contained in the biological sample derived from the subject, its variant or fragment thereof, and A method for examining diabetic peripheral vascular disorders, comprising detecting.
The method according to claim 1, wherein the diabetic peripheral vascular disorder is diabetic macular edema.
The method according to claim 2, wherein measurement and / or detection of the polypeptide, a variant thereof or a fragment thereof is performed using mass spectrometry.
The method according to claim 3, wherein the measurement and / or detection is performed using a substance capable of binding to the polypeptide, a variant thereof or a fragment thereof.
The method according to claim 4, wherein the substance capable of binding is an antibody or a fragment thereof.
The method according to claim 5, wherein the antibody or a fragment thereof is labeled with any of an enzyme, a fluorescent substance, a dye, or a radioisotope.
The method according to claim 5 or 6, wherein the antibody or a fragment thereof is a monoclonal or polyclonal antibody, or a fragment thereof.
The method according to any one of claims 1 to 7, wherein the biological sample is blood, plasma, serum, urine, tears, vitreous or vitreous humor.
One of an antibody or a fragment thereof, or a chemically modified derivative thereof, which specifically binds to at least one of a polypeptide comprising the amino acid sequence represented by SEQ ID NOs: 1 to 9 in the sequence listing, a variant thereof or a fragment thereof A composition for diagnosis or / and detection of diabetic peripheral vascular disorder, comprising one or more substances.
A fragment comprising the amino acid sequence represented by SEQ ID NOs: 1 to 9 in the sequence listing, a fragment thereof binding specifically to at least one of its variants or fragments thereof, or a chemically modified derivative thereof; A kit for the diagnosis and / or detection of diabetic peripheral vascular disorders, comprising one or more of the substances.
A fragment comprising the amino acid sequence represented by SEQ ID NOs: 1 to 9 in the sequence listing, a fragment thereof binding specifically to at least one of its variants or fragments thereof, or a chemically modified derivative thereof; Of one or more of the above in the manufacture of a kit for the diagnosis and / or detection of diabetic peripheral vascular disorders.