KR20100070776A - Biomarkers for the diagnosis of recurrent pregnancy loss - Google Patents

Abstract

PURPOSE: A biomarker for diagnosing habitual abortion by combining a group of biomarkers is provided. CONSTITUTION: A biomarker for diagnosing habitual abortion is alpha-2 macroglobulin, alpha-1 antichymotrypsin(AACT), insulin-like growth factor-binding protein(IGFBP1), or inter-alpha trypsin inhibitor-heavy chain 4(ITI-H4). The ITI-H4 is uncut type 120 kDa, cut 85 kDa or cut 36 kDa. A habitual abortion composition for diagnosis comprises an agent measuring mRNA or protein level of biomarker for diagnosing habitual abortion. The agent measuring is primer pair or specific probe which is specific to mRNA. The agent measuring is an antibody which is specific to protein of the gene.

Description

Biomarkers for the diagnosis of habitual miscarriage

The present invention relates to a habitual miscarriage diagnostic biomarker, a habitual miscarriage diagnostic composition comprising an agent for measuring the expression of the biomarker at the mRNA or protein level and a method for diagnosing the habitual miscarriage using the biomarker.

Habitual miscarriage occurs in 2-5% of women who are pregnant with reproductive diseases [Coulam, CB et al., Am. J. Reprod. Immunol . 1997, 38 , 57-74. It is defined as habitual miscarriage at least three consecutive abortions within 20 to 28 weeks of gestation [Crosignani, PG et al., Hum. Reprod. 1991, 6 , 609-610; Harlap, S. et al., Lancet 1980, 2 , 173-176; Pandey, MK et al. Arch. Gynecol. Obstet . 2005, 272 , 95-108. Despite many studies, the exact mechanism of habitual miscarriages by the various circumstances of pregnancy is not yet known. To date, studies have shown that abortion is caused by environmental factors such as immunity, anatomy, endocrine, genetics, infections, chromosomal aberrations, alcohol and tobacco [Pandey, MK et al., Arch. Gynecol. Obstet . 2005, 272 , 95-108; Baek, KH et al., Mol. Hum. Reprod. 2004, 10 , 291-297; Caetano, MR et al., Sao Paulo Med. J. 2006, 124 , 181-185. In addition, various causes influence implantation and placental formation by the complex signaling of habitual abortions.

Many researchers have reported many genes that are expressed differently in patients with habitual miscarriage. Previous studies have linked these genes to maternal immune responses, angiogenesis, apoptosis, and thrombus formation [Choi, HK et al., Mol. Reprod. Dev. 2003, 66 , 24-31; Jasper, MJ et al.,. J. Reprod. Immunol . 2007, 73 , 74-84. Maternal natural killer cells secrete many cytokines adjacent to embryos [Quenby, S. et al., Reprod. Biomed. Online 2006, 13 , 24-28. In particular, cytokines such as the IL-1β, IL-6, IL-11, and TGF-β superfamily promote implantation during the immune response [Dimitriadis, E. et al., Hum. Reprod. Update 2005, 11 , 613-630; Girardi, G. et al., J. Exp. Med. 2006, 203 , 2165-2175. In contrast, genes associated with apoptosis, such as p53, affect implantation failure [Kay, C. et al., Reprod. Biomed. Online 2006, 13 , 492-496. After translation, most proteins undergo modification after translation.

Proteomics studies allow for the identification of posttranslational modifications, particularly in diseases. Therefore, recent proteomics studies have been recognized as a useful way to understand how diseases cause modified protein expression and to identify proteins involved in the etiology. Customary abortion has also been applied to this method and recent studies have identified proteins associated with immunity and thrombus formation in association with customary abortion [Kim, YS et al., Proteomics 2006, 6 , 3445-3454; Baek, KH et al., Trends Mol. Med. 2007, 13 , 310-317. In addition, in the previous study, proteomic analysis using follicular fluid of habitual abortion patients, complement component C3c chain E, fibrinogen γ, antithrombin, angiotensinogen , And five protein expressions of hemopexinprecursor were confirmed differently (Kim, YS et al., Proteomics 2006, 6 , 3445-3454). Previous studies have used follicle fluids because they are an important source of control of the follicle formation process [Angelucci, S. et al., Biochim. Biophys. Acta 2006, 1764 , 1775-1785. Recent studies have shown that amniotic fluid is involved in human reproductive fluids such as follicle fluids [Queloz, PA et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007, 850 , 336-342. However, these body fluids are difficult to collect and use from patients. For that reason, proteins that are differentially expressed in blood samples of habitual miscarriage patients experiencing IVF have been identified. Blood is a body fluid that acts as a transport system for the body to transport oxygen, hormones, and immune-related substances to the mother. Plasma is one of the most complex tissues that contains a variety of cellular proteins 10 to ¹² times in the body [Lee, HJ et al., Curr. Opin. Chem. Biol. 2006, 10 , 42-49. Recently, Sabatini et al. Reported that high concentrations of antiprothrombin (aPT) antibodies in plasma are associated with habitual miscarriage [Sabatini, L. et al., Clin. Chem. 2007, 53 , 228-232.

The discovery of biomarkers that can be used to observe or diagnose the prognosis of habitual miscarriage is required.

It is to provide a meaningful biomarker that can easily and effectively diagnose habitual miscarriage from human blood samples using proteomic analysis. Specifically, 2-DE is performed using a blood sample of a normal person and a habitual abortion patient, and the selected point is analyzed by MALDI-TOF after comparative analysis through image analysis. Proteins were screened and assayed for effectiveness as markers for them.

The present invention is a diagnostic biomarker for habitual miscarriage, α-2 macroglobulin (α-2 macroglobulin), α-1 antichymotrypsin (α-1-antichymotrypsin: AACT), insulin-like growth factor-binding protein (insulin-like growth) factor-binding protein: IGFBP1) and inter-α trypsin inhibitor-heavy chain 4: ITI-H4.

In the present invention, a "marker" or "biomarker" is a substance that shows an increased, decreased or modified form of expression in a patient whose habitual abortion can be used for screening, diagnosis, and prognosis of a habitual abortion disease. The habitual abortion diagnostic marker provided by the present invention is α-2 macroglobulin, α-1 antichymotrypsin, IGFBP1, ITI-H4 and fragments thereof. These markers can be used as habitual miscarriage risk indicators or prognostic indicators, and can be used for general diagnosis, including treatment as well as monitoring of disease progression. The number, type, and combination of markers in a subject sample is a potent indication of whether the subject has a habitual miscarriage risk or prognosis. This information can be used as a useful guide to medical treatment.

Specifically, α-2 macroglobulin, α-1 antichymotrypsin and IGFBP1 show increased expression in patients with habitual miscarriage compared to normal. In the case of ITI-H4, 120 kDa normal protein is expressed in normal subjects, but 120 kDa protein expression is relatively reduced in habitual abortion patients, and 36 kDa and 85 kDa truncated proteins are detected that are not seen in normal subjects.

In other words, ITI-H4 provides habitual miscarriage markers of 36 kDa cleavage and 85 kDa cleavage, as well as 120 kDa total protein. Decreased 120 kDa protein, increased 85 kDa protein and / or increased 36 kDa protein can be used to diagnose habitual miscarriage.

ITI-H4 is a very useful marker in that it shows a different protein expression pattern from that of normal people. Since cleaved proteins that are not expressed in normal people are expressed in patients with habitual miscarriage, habitual miscarriage can be diagnosed without quantitative comparison with normal controls by detecting the cleaved proteins. For example, in Western blot analysis, habitual miscarriage can be diagnosed with high reliability and significance without a process compared with a normal control group only by confirming the presence and pattern of cleaved proteins on the gel.

As used herein, the marker ITI-H4 refers to the 85 kDa and 36 kDa proteins in truncated form as well as the uncleaved 120 kDa.

The present invention can be selected and used in various combinations without particularly limiting the number and combination of markers selected for habitual miscarriage diagnosis. Specifically, the α-2 macroglobulin, α-1 antichymotrypsin, IGFBP1 or ITI-H4 may be used alone, or 2, 3 or 4 markers may be selected and used to suit the purpose. That is, the present invention provides a wide range of markers to enable various selections, and those skilled in the art can select and use the types and numbers of markers in various combinations suitable for making medical judgment. Preferably, a highly significant marker, ITI-H4, is used alone or is added as a marker of 1 to 3, essentially comprising ITI-H4 and selected from α-2 macroglobulin, α-1 antichymotrypsin and IGFBP1. It can be used to include.

The habitual miscarriage can be diagnosed by measuring the expression level of the biomarkers provided in the present invention at the mRNA or protein level. Since the nucleic acid and protein sequences of the markers of the present invention are already known, marker detection for diagnosis can be usefully detected using agents capable of measuring mRNA or protein levels, based on known sequences. . The present invention provides a composition capable of detecting and / or quantitatively measuring the habitual miscarriage detection marker of the present invention in a biological sample of a patient.

Specifically, the present invention is a habitual abortion diagnostic composition comprising an agent for measuring the mRNA level of the habitual lactic acid diagnostic biomarker selected from the group consisting of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4. It is about.

Agents for measuring mRNA levels of the markers in the art are preferably, but not limited to, primers or probes.

A primer is a nucleic acid sequence having a short free 3 'hydroxyl group, which refers to a short nucleic acid sequence capable of forming complementary templates and base pairs and serving as a starting point for template strand copying.

Probe refers to a natural or modified nucleic acid fragment, such as RNA or DNA, which can hybridize to mRNA and a specific nucleotide sequence and can be labeled to confirm the presence or absence of a specific mRNA. Probes used for hybridization may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like. Conditions suitable for hybridization can be determined by adjusting the temperature, ionic strength (buffer concentration), the presence of compounds such as organic solvents, and the like. Such stringent conditions can be determined differently depending on the sequence being hybridized.

Those skilled in the art can design primer pairs that specifically amplify a specific region of the gene or probes that specifically recognize a specific region of the gene from known marker gene nucleic acid sequences of the present invention and are known in the art. It can be synthesized chemically. In addition, it may be modified using a label such as radioisotopes, fluorescent molecules, biotin and the like to provide a detectable signal directly or indirectly.

The present invention also relates to a habitual lactic acid diagnostic composition comprising an agent for measuring the protein level of the habitual lactic acid diagnostic biomarker selected from the group consisting of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4. .

Agents measuring marker protein levels in the art preferably include all polyclonal, monoclonal and recombinant antibodies that specifically bind to α-2 macroglobulin, AACT, IGFBP1 and ITI-H4 as antibodies. do. In addition, the antibodies of the present invention are functional fragments of the antibody molecule with the complete form having two full length light chains and two full length heavy chains, for example Fab, F (ab '), F (ab') 2 And forms of Fv.

Each antibody specific for α-2 macroglobulin, AACT, IGFBP1 and ITI-H4 includes both conventional antibodies as well as antibodies that can be prepared by known methods. For example, the ITI-H4 specific antibodies used in the present invention were able to detect uncleaved 120 kDa and truncated 36 kDa proteins, but could not recognize 85 kDa truncated proteins. However, by methods known to those skilled in the art, one can construct antibodies that specifically recognize 85 kDa cleaved protein.

Specifically, the 85 kDa ITI-H4 specific polyclonal antibody is prepared by a method well known in the art of injecting the above 85 kDa ITI-H4 antigen into an animal and collecting blood from the animal to obtain a serum containing the antibody. can do. In addition, 85 kDa ITI-H4 specific monoclonal antibodies can be prepared using hybridoma methods or phage antibody library techniques.

In the case of the ITI-H4 marker, the band pattern detected will differ depending on the nature of the antibody used in the experiment to recognize 120 kDa, 85 kDa and / or 36 kDa.

The composition of the present invention may be used as a quantitative control gene, which is expressed in approximately the same amount in a normal sample and a patient sample, and thus may further include an agent for detecting mRNA or protein levels of the quantitative control.

The composition for detecting a habitual miscarriage marker of the present invention may be prepared as a kit for detecting a variety of habitual miscarriage diagnostic biomarkers further comprising a component, a detection reagent or a device suitable for each type of kit.

As a specific example, a test tube or other suitable container in addition to the specific primer pairs for the habitual miscarriage diagnostic marker gene selected from the group consisting of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4 of the present invention, Provide kits for RT-PCR, including reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerases and reverse transcriptases, DNAse inhibitors, RNAse inhibitors, DEPC-water, sterile water and the like do.

The present invention also provides a kit for a DNA chip comprising a substrate on which a cDNA corresponding to the marker gene or a fragment thereof is attached with a probe. The DNA microarray chip substrate may be coated with active groups such as amino-silane, poly-L-lysine, and aldehyde. Further, the substrate may be a slide glass, plastic, metal, silicon, nylon film, or nitrocellulose film. In addition, the kit may include a buffer used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, labeling reagents such as cNTPs and rNTP, chemical inducers of fluorescent dyes, washing buffers and the like. rNTPs can be premixed and supplied separately.

As another example, specific antibodies to the marker proteins of the invention and additionally include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes and substrates thereof. It provides a kit for ELISA.

As another example, there is provided a kit for a protein chip in which an antibody specific for the marker protein of the present invention is arranged at a predetermined position on a substrate and immobilized at a high density. Kits for protein chips may include buffers, labeling reagents, wash buffers, and the like, used for hybridization.

In addition to the kits exemplified above, it includes several types of diagnostic kits that are made of a rapid diagnostic kit capable of detecting the markers of the present invention.

Kits of the present invention may comprise agents for detecting mRNA levels or protein levels of quantitative controls.

The present invention also relates to a method for diagnosing habitual miscarriage by a method for detecting a biomarker for diagnosing habitual miscarriage.

Specifically, mRNA levels of the biomarkers in biological samples using primer pairs or probes specific for the habitual abortion diagnostic biomarkers selected from the group consisting of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4. It relates to a method for diagnosing a habitual miscarriage, comprising the step of measuring the amount. In addition, by measuring the protein level of the biomarker in a biological sample using an antibody specific for the habitual lactic acid diagnostic biomarker selected from the group consisting of the α-2 macroglobulin, AACT, IGFBP1 and ITI-H4. A method of diagnosing a habitual miscarriage, comprising the step.

Diagnosing addictive abortion by checking whether α-2 macroglobulin, α-1 antichymotrypsin and IGFBP1 increase expression, ITI-H4 decreases expression of 120 kDa protein and increases cleavage expression of 36 kDa and 85 kDa can do.

MRNA detection of the marker is performed by amplification or hybridization reactions known in the art using marker specific primers or probes. Known detection methods include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, northern blotting, DNA chip, primer extension method, oligonucleotide extension assay (OLA), RNase mismatch cleavage ), Single strand conformation lymorphism (SSCP) and the like, but is not limited thereto, and may be performed by any method.

Detection of a marker protein is performed by contacting a sample with an antibody that specifically recognizes the protein and measuring its antigen-antibody complex formation. The amount of antigen-antibody complex formed can be measured quantitatively through the magnitude of the signal of the detection label. Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but is not necessarily limited thereto.

Known assays for determining protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoproliferation, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, complement fixation assays, Competitive or non-competitive assays such as pricipitin response, gel diffusion pricipitin response, aggregation assay, fluorescence immunoassay, Protein A immunoassay, FACS, protein chip, etc. can be used, but are not limited to, any method Can be performed by

The ELISA is a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody attached to a solid support, and reacts with another antibody that recognizes an antigen in a complex of an antibody attached to a solid support and an antigen. And various ELISA methods, such as indirect sandwich ELISA using a labeled secondary antibody which recognizes this antibody.

The biological sample used for the test is not particularly limited and may be tissue, cell or bodily fluid and is prepared by treatment by methods well known in the art. The present invention is useful in that a gene which is easily detected in blood is used as a marker. Blood can be taken by methods known in the art, for example from veins, arteries or capillaries. Typically, blood is obtained by taking venous blood, for example, from the subject's arm via a syringe. Blood includes a sample obtained from blood by further purification and separation methods, such as serum.

In addition, using a sample of a normal person as a control may include comparing the gene expression in the biological sample of a suspected habitual abortion. This can be compared with absolute or relative differences in the amount of expression between the control and the biological sample.

It is preferred to carry out the combination measurements according to the invention so that parallel measurements of the markers are carried out on one or more samples obtained from the subject. One or more tests are sequentially irradiated simultaneously or immediately on one or more samples collected from the patient, such as blood or serum samples.

The habitual miscarriage detection and measurement method provided by the present invention is not limited to the above-mentioned method, and modification and application may be made by a combination of various methods known in the art.

Through various analysis methods for the above-described detection, the habitual miscarriage marker expression can be analyzed qualitatively or quantitatively at the mRNA or protein level, and can be analyzed to diagnose the habitual inheritance of the suspected habitual miscarriage.

According to the present invention, the habitual miscarriage can be efficiently diagnosed by a method of detecting a group of biomarkers specifically overexpressed in a habitual abortion patient alone or in combination.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Materials and Methods

1-1: Test subject and sample

Protein expression differentiation assays using isoelectric focusing (IEF) and various commercial resources are described previously [Kim, YS et al. Proteomics 2006, 6 , 3445-3454; Lee, HJ et al., Curr. Opin. Chem. Biol. 2006, 10 , 42-49. Female patients with RPL were recruited from Gangnam Tea Hospital in Seoul, Korea. Initial three patients were used for 2-DE, and a total of 17 patients, including 14, were used for Western blot analysis. Three other normal participants were enrolled and considered as controls. Written informed consent was received and accepted by all patients. Plasma samples were prepared as previously described [Angelucci, S, et al., Biochim. Biophys. Acta 2006, 1764 , 1775-1785. Venous blood samples were obtained from patients with RPL and collected in sterile plastic test tubes with EDTA surface-treated as anticoagulants and immediately centrifuged. All samples were stored at -80 degrees Celsius before use.

1-2: 2-DE and Video Analysis

As previously described [Kim, YS et al., Proteomics 2006, 6 , 3445-345], the elimination of the six most abundant proteins in the follicular fluid (albumin, transferrin, IgG, IgA, haptoglobin, antitrypsin) was confirmed by MARC ( Multiple Affinity Removal Column) (Agilent, Wilmington, DE, USA). 4.6 mm x 50 mm MARC was used with adhesion to 20 μl of follicular fluid. Chromatographic separation of enriched proteins using MARC was performed with mobile phase reagent tools according to standard LC manuals provided from suppliers. In brief, the untreated follicular follicles were diluted five times with Buffer A with proteolytic inhibitors (Complete ^™ , Roche, Manheim, Germany), 10.2 g through a 0.22-μm centrifugal filter at room temperature. Filtered through centrifugation for 1-2 minutes. Samples were injected and the passages were collected and stored at -20 ° C until use. In order to separate the specific protein-free follicle protein onto the 2-DE gel, the portion passed through the MARC was collected and precipitated in a pre-cooled 10% TCA solution at -20 ° C for 1 hour. After washing with ice-cooled acetone, the precipitate was dissolved again in 2-DE sample buffer.

IEF was performed using the IPGphor system (Amersham Biosciences, Uppsala, Sweden). Copies containing 1 mg of follicle protein were mixed with a sufficient amount of rehydration buffer (7M urea, 2M thiourea, 4.5% CHAPS, 100 mM DTE, 20 mM Tris, pH 8.8) and the final volume was adjusted to 350 uL. . Samples were injected in 18 cm Immobiline Drystrips (pH 3-10 nonlinear) (Amersham Biosciences, Uppsala, Sweden) by gel internal rehydration. IEF was performed at about 80000 Vh. Two-dimensional separation was performed by applying a fixed 40 mA of current for about 5 hours on a 9-16% linear gradient polyacrylamide gel. After 1 hour protein fixation in 40% methanol, 5% phosphoric acid, the gels were stained with Comassie brilliant blue G-250 (CBB G-250) for 12 hours. Stained gels were transferred to image files via a GS-710 imaging densitometer (Bio-Rad, Hercules, Calif., USA) and were then imaged using Image Master ^™ 2D Platinum software (Amersham Bioscience, Uppsala, Sweden). Analyzed.

1-3: In-gel Degradation and Mass Spectrometry

After spotting the spot pieces from the gel, all spots were previously described as described by Kim, YS et al., Proteomics 2006, 6 , 3445-3454; Cho, SY et al., Proteomics 2005, 5 , 3386-3396] trypsin (Promega, Madison, Wis., USA) to digest proteins. MALDI-TOF-MS was used to identify peptide sequences as previously described [Kim, YS et al., Proteomics 2006, 6 , 3445-3454]. The poros R2 and oligo R3 columns (Applied Biosystems, Foster City, CA, USA) were used to decontaminate and concentrate the peptides. In addition, CHCA (a-cyano-4-hydrozycinnamic acid) matrix and 4700 TOF / TOF spectrophotometer (Applied Biosystems) were used. Peptide mass spectrometry was aligned using MASCOT (http://www.matrixscience.com) and MS-Fit (http://prospector.ucsf.edu) monoisotopic peaks.

1-4 Biological Information

After MALDI-TOF analysis, MS spectroscopy was obtained. To identify each protein from the spectroscopic results, bioinformatics analysis was performed using the Spectrum Mill MS Proteomics Workbench as previously described (Kim, YS et al., Proteomics 2006, 6 , 3445-3454). ) (Rev A.03.00.015, Agilent). The analytical method is the same as previously described [Kim, YS et al., Proteomics 2006, 6 , 3445-3454; Cho, SY et al., Proteomics 2005, 5 , 3386-3396].

1-5 Western Blot Analysis

Human serum proteins were diluted 1/2 in PBS buffer and subjected to SDS-PAGE. Protein was loaded into each lane of 10% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were incubated with a blotting solution comprising anti-IGFBP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-AACT (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1: 200. It was also detected using an α-2 macroglobulin antibody (Abcam, Cambridge, UK) diluted 1: 2000. The membrane was also incubated with a blocking solution containing anti-ITI-H4 antibody (sc-21987 and sc-34471, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1: 200. Secondary antibody reactions were treated with a blotting solution comprising a horseradish peroxidase-conjugated secondary antibody diluted 1: 10000. ECL system (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for signal detection. Bands of western blot were scanned and digitized using Fluor-S ^™ MultiImager (Bio-Rad, Hercules, Calif., USA).

Example 2: 2-DE Assay Using Blood of Patients with Habitual Abortion

Considering the difficulty in obtaining tissue or follicles from patients with habitual miscarriage, blood samples were obtained from normal patients and patients with habitual miscarriage, and the proteomic method was performed. Table 1 describes the clinical characteristics of the patients. For this study, the major proteins present in the obtained serum were removed using an immunoaffinity column (MARC). In addition, the IEF technology was introduced through a known proteomic assay to find proteins showing differences in expression between normal patients and habitual abortion patients (FIG. 1).

Example 3: Identification of Proteins with Different Expressions in Patients with Habitual Abortion

To determine the difference in expression between the proteins, we used one normal patient sample set made by mixing three habitual abortion patient samples and three normal patient samples. In this study, 549 spots, 563 spots, and 553 spots, which are expressed differently from normal patient samples, were obtained by 2-DE analysis in the samples of patients with habitual miscarriage. It became. Fifteen spots were more than twice as strong as normal patients and six spots were less than twice as weak (FIGS. 2 and 3). Fifteen spots that are strongly expressed in habitual abortion patients are 305 in section A, 530 in section B, 669, 684, 704, 714 and 956 in section C, and 778 in section D. 590, 616 and 653 in part E, 402, 428, 453 and 475 in part F. The six weakly expressed spots are 215, 238, 320 and 351 in part A of FIG. Are indicated by 702 and 709 in the section. The spots thus obtained were analyzed by MALDI-TOF method, and Table 2 and Table 3 show 21 proteins analyzed. Of these, ITI-H4 at Spots 475 and 669 and AACT at Spot 402 are involved in the immune response, IGFBP-1 at Spot 305 is involved in metabolism, and α-2 macroglobulin at Spot 714 is involved in thrombus. In the case of ITI-H4, unlike other spots, the spots of different sizes were obtained at 475 and 669, so they became more interested for further experiments, and this phenomenon may be related to addictive abortion patients. Estimated.

Example 4: Western Blot Analysis Using Serum

Western blot analysis was performed on four strongly expressed proteins, ITI-H4, AACT, IGFBP-1, and α-2 macroglobulin, to confirm the expression level using the sera of habitual abortion patients. In FIG. 4, the expression of α-2 macroglobulin, AACT and IGFBP1 was different in each patient. Interestingly, ITI-H4 was found to be weaker in patients with habitual miscarriage, unlike the 2-DE results in FIG. 5. Therefore, Western blot analysis was performed again to find the cleaved 85 kDa and 36 kDa, and it was confirmed that the cleaved form of 36 kDa size was strongly expressed in FIG. 5A. This confirmed that Spot 475, which appears to be strongly expressed in 2-DE, would be 85 kDa truncated rather than 120 kDa ITI-H4. Western blot analysis failed after the purchase of commercially available antibodies to reconfirm 85 kDa. In addition, Western blot analysis was performed using the sera of more habitual abortion patients to support the results of FIG. 5A (FIG. 5B). These analyzes revealed that 36 kDa cleavage of ITI-H4 was found in 13 of 17 addictive abortions and 4 patients with different outcomes, but were able to diagnose 76.5% of addictive abortions. We therefore suggest that ITI-H4 can be used as a biomarker in patients with habitual miscarriage.

Pregnancy is a complex process and is regulated through various intracellular processes, such as immunity from the mother and blood vessel formation, which leads to many problems such as habitual miscarriage due to abnormalities in this process during pregnancy. Currently, 50-60% of habitual miscarriages are reported to be caused by chromosomal abnormalities, anatomical abnormalities and infections of the fetus, but 40-50% of them have no known etiological causes. The present inventors recently attempted to detect abnormal expression of a specific protein by performing 2-DE and MALDI-TOF using follicular fluid of a habitual abortion patient, and through this, a proteomic method such as 2-DE was used for a habitual abortion patient. It is suggested that it is a good technique to investigate the etiology of the disease.

In the present invention, the inventors obtained a total of 21 expression proteins in the blood through the proteomics method, the five proteins were confirmed by Western blot analysis the expression difference between normal patients and habitual abortion patients. Among them, ITI-H4 was confirmed to be cleaved to 85 kDa and 36 kDa in patients with habitual miscarriage. Therefore, in the patients with habitual miscarriage, the uncut 120 kDa was decreased and the cut type was increased. Since the cleavage of ITI-H4 has been reported as an important diagnostic phenomenon in uterine or breast cancer patients, it can be seen that the cleavage of ITI-H4 is an important diagnostic phenomenon in patients with habitual miscarriage. Although the function of ITI-H4 has not yet been clearly identified, it has been reported to be involved in the early inflammatory response, and there are reports of pregnancy-related studies in pig studies, suggesting that ITI-H4 is a major factor related to pregnancy maintenance. It is considered to be. In addition, ITI-H4 is reported to be regulated by interleukin-6, which is consistent with the report that cytokines and hormones play an important role in the maternal and fetal liver, so the association between ITI-H4 and interleukin-6 may be important in pregnancy. see.

The results of the above examples show that 76.5% of 36 kDa truncated forms of ITI-H4 are strongly expressed in patients with habitual miscarriage (FIG. 5). This suggests that ITI-H4 may be an important etiological factor given that habitual miscarriage is caused by a variety of causes. Thus, ITI-H4 is an important diagnostic marker for habitual miscarriage.

The use of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4, which is newly identified as a biomarker for the diagnosis of addictive abortions, enables easy and efficient diagnosis of addictive abortions. do.

1 is a 2-DE image of human serum. Normal and RPL patient sera were separated by 2-DE after MARC. 2-DE images of 1 normal (A) and 3 RPL patient (B-D) sera. Molecular weight is shown as kDa on the left side.

2 shows an increase of 15 proteins in RPL patients. (A) Spot 305 (B) Spot 530 (C) Spots 669, 684, 704, 714, and 956 (D) Spot 778 (E) Spots 590, 616, and 653 (F) Spots 402, 428, 453, and Increased from 475

3 shows six spots reduced in RPL patients. In the patient's 2-DE gel, the reduced spot appeared weak. (A) spots 215, 238, 320, and 351 (B) spots 702 and 709.

4 shows α-2 macroglobulin (163 kDa), α-1 antichymotrypsin (AACT) (68 kDa), and IGFBP1 (alpha) in RPL patients and normal control serum. As a result of Western blot analysis of 36 kDa), individual differences in protein expression can be seen.

5 is Western blot results for ITI-H4 in RPL patients and normal controls. ITI-H4 (36 kDa) was detected in 13 of 17 patients and 4 RPL patients expressed ITI-H4 (120 kDa) as in normal subjects. (A) 3 normal and 7 RPL patients (B) 3 normal and 10 RPL patients.

Claims (9)

α-2 macroglobulin, α-1 antichymotrypsin (AACT), insulin-like growth factor-binding protein (IGFBP1) and inter- A biomarker for the diagnosis of habitual miscarriage selected from 1 to 4 selected from the group consisting of α-trypsin inhibitor-heavy chain 4 (inter-α trypsin inhibitor-heavy chain 4: ITI-H4). The biomarker of claim 1, wherein the ITI-H4 is non-cleaved 120 kDa, truncated 85 kDa or truncated 36 kDa. The habitual lactic acid diagnostic composition comprising an agent for measuring the mRNA or protein level of the habitual lactic acid diagnostic biomarker selected from the group consisting of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4 of claim 1. 4. The composition of claim 3, wherein the ITI-H4 is uncleaved 120 kDa, truncated 85 kDa or truncated 36 kDa. 4. The composition of claim 3, wherein the agent for measuring mRNA level is a primer pair or probe specific for mRNA of said gene. The composition of claim 3, wherein the agent for measuring protein level is an antibody specific for a protein of said gene. MRNA levels of the biomarkers in biological samples using primer pairs or probes specific for the habitual abortion diagnostic biomarker selected from the group consisting of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4 of claim 1 Measuring the habitual miscarriage. Measuring the protein level of the biomarker in a biological sample using an antibody specific for the habitual lactic acid diagnostic biomarker selected from the group consisting of α-2 macroglobulin, AACT, IGFBP1 and ITI-H4 of claim 1 Comprising the steps of diagnosing a habitual miscarriage. The method of claim 7 or 8, wherein the biological sample is blood or plasma.

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