JPWO2015016036A1 - Biomarker for fertilization test of egg and determination using it - Google Patents

Abstract

Providing new biomarkers to test fertilization potential of eggs. In the follicular fluid collected from the subject, the amount of one or more peptides selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4 is measured. A test method of fertilization possibility of an egg.

Description

(Cross-reference of related fields)
This application claims the benefit of priority of Japanese Patent Application No. 2013-156672 filed on Jul. 29, 2013, which is incorporated herein by reference in its entirety.

(Technical field)
The present invention relates to a novel biomarker for examining the fertilization potential of an egg, and a method for examining or determining the fertilization potential of an egg using the same.

  In Japan, where there is a declining birthrate and an aging population, 1 in 50 live births, about 20,000 a year, are born by in vitro fertilization, and in vitro fertilization has recently become a popular medical technology that will stop the aging population with a declining birthrate. Have been doing. On the other hand, the success rate of in vitro fertilization depends on the quality or state of an egg to be collected, such as fertility, but there is currently no method for discriminating an egg having a quality or state suitable for fertilization.

  Conventionally, there has been a method of selecting an ovum by microscopic observation by a doctor after in vitro fertilization processing, but in this case, depending on the experience of the doctor, subjective judgment is entered and it takes time for judgment.

  Recent studies show that the quality of ova changes with aging, which is a significant risk factor for pregnancy and childbirth in older people. Attempts have been made to determine the quality of the ovum, such as fertilization potential, from the components of the follicular fluid obtained simultaneously with the collection of the ovum during in vitro fertilization (Patent Document 1 and Non-Patent Document 1). To date, no suitable biomarker that can be tested with high accuracy has been discovered.

  The rate of in vitro fertilization in elderly births is expected to increase further in the future, so increasing the success rate of fertilization is an urgent issue. If a method for selecting or discriminating eggs with high fertilization potential is developed, it will improve the success rate of in vitro fertilization, which will be good news for couples who want children, and is expected to be further developed in Japan. It is expected that it can be a means of stopping the process.

Special table 2007-530030

Wu et al. Reproductive Biology and Endocrinology 2012, 10: 116

  An object of the present invention is to provide a novel biomarker for examining the fertilization potential of an egg, and a method for examining or determining the fertilization potential of an egg using the biomarker.

  As a result of mass spectrometry of follicular fluid collected from a patient in order to achieve the above object, the present inventors have found a specific peptide whose amount in the follicular fluid varies depending on the degree of maturation of the follicle. These peptides were newly identified as markers for the test or determination of fertilization potential of eggs, and the present invention was completed.

  According to the first aspect of the present invention, the amount of one or more peptides selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4 in the follicular fluid collected from the subject is measured. A test method for fertilization possibility of an egg in the subject is provided.

  In one embodiment, subjecting the follicular fluid to mass spectrometry.

  In another embodiment, the amount of peptide obtained by the measurement is compared with a threshold set based on the amount of peptide in the control sample, and the amount of peptide obtained by the measurement is compared to the peptide in the control sample. The method further includes determining that the fertilization probability of the egg is high when the variation is statistically significantly compared to a threshold set based on the amount of the egg.

  According to the 2nd aspect of this invention, the biomarker for the test | inspection of the fertilization ability of the egg containing the peptide selected from the peptide group which consists of an amino acid sequence represented by sequence number 1-4 is provided.

  According to the 3rd aspect of this invention, the biomarker for the test | inspection of the fertilization possibility of the egg which consists of a peptide selected from the peptide group which consists of an amino acid sequence represented by sequence number 1-4 is provided.

  According to a fourth aspect of the present invention, there is provided a method for examining fertilization potential of an egg, which specifically recognizes a peptide selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1-4. Methods using antibodies are provided.

  According to the fifth aspect of the present invention, there is provided a determination kit for examining fertilization potential of an egg containing an antibody, wherein the antibody comprises a peptide group consisting of amino acid sequences represented by SEQ ID NOs: 1-4. A determination kit is provided that is an antibody that specifically recognizes the selected peptide.

  According to the sixth aspect of the present invention, there is provided an immunodiagnosis or determination device in which an antibody against a peptide selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4 is fixed.

  According to the 7th aspect of this invention, the peptide which consists of an amino acid sequence represented by sequence number 1, sequence number 2, sequence number 3, or sequence number 4 is provided.

  According to the present invention, the fertility of an egg can be determined quickly and with high accuracy without damaging the egg, so that an egg suitable for fertilization, particularly in vitro fertilization can be distinguished, and as a result, the success rate of in vitro fertilization is greatly improved. Can be made.

It is a table | surface which shows the molecular weight in mass spectrometry of the biomarker peptide 4 types obtained in Example 1, the protein name derived, and an amino acid sequence. (A) Plot diagram of peak intensity between unfertilized group (group 1) and fertilized group (group 2) and (B) ROC curve diagram of biomarker (molecular weight 2743) obtained in Example 1. The AUC value, sensitivity (SN; prevalence rate of prevalence) and specificity (SP; prevalence rate of non-morbidity) are shown in FIG. 2A. (A) Plot diagram of peak intensity between unfertilized group (Group 1) and fertilized group (Group 2) and (B) ROC curve diagram of biomarker (molecular weight 2757) obtained in Example 1. The AUC value, sensitivity (SN; prevalence rate of prevalence) and specificity (SP; prevalence rate of non-morbidity) are shown in FIG. 3A. (A) Plot diagram of peak intensity between unfertilized group (group 1) and fertilized group (group 2) of biomarker (molecular weight 2862) obtained in Example 1, and (B) ROC curve diagram. The AUC value, sensitivity (SN; prevalence rate of prevalence) and specificity (SP; prevalence rate of non-morbidity) are shown in FIG. 4A. (A) Plot diagram of peak intensity between unfertilized group (group 1) and fertilized group (group 2) of biomarker (molecular weight 2941) obtained in Example 1, and (B) ROC curve diagram. The AUC value, sensitivity (SN; prevalence rate of disease prevalence) and specificity (SP; prevalence rate of disease prevalence) are shown in the upper part of FIG. 5A. (A), (b) Examples of immunochromat chips.

  In this specification, the singular forms (a, an, the) include the singular and the plural unless specifically stated otherwise or otherwise clearly contradicted by context.

  The present invention provides a novel and useful marker peptide for testing or determining the fertilization potential of an egg.

  Ovulation is the process by which mature oocytes, or ova, are excreted from the ovarian follicle. The present inventors have found that the expression level of each of the following four peptides of the present invention contained in the follicular fluid of the follicle is related to the fertilization potential of the ovum.

  Peptide 1: consisting of the amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence corresponds to the 341st to 367th fragments of human α-2-HS-glycoprotein.

  Peptide 2: consisting of the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence corresponds to the 25th to 48th fragments of human serum albumin.

  Peptide 3: consisting of the amino acid sequence represented by SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 1 is the same as that of 27 amino acids, but in peptide 3, cysteine is further bonded to the 18th cysteine by a disulfide bond.

Peptide 4: consisting of the amino acid sequence represented by SEQ ID NO: 4. It corresponds to the 25th to 50th fragments of human serum albumin.
TVVQPSVGAAAGPVVPPCPGRIRHFKV (SEQ ID NO: 1)
DAHKSEVAHRFKDLGEENFKALVL (SEQ ID NO: 2)
TVVQPSVGAAAGPVVPPCPGRIRHFKV Cysteinylation at position 18 (SEQ ID NO: 3)
DAHKSEVAHRFKDLGEENFKALVLIA (SEQ ID NO: 4)

  Specifically, when the ovum is fertilized or in a state of high fertility, the expression levels of peptides 1, 2, and 4 are increased, and the expression level of peptide 3 is low. Therefore, by confirming that the expression levels of peptides 1, 2, and 4 are high and / or that peptide 3 is low, it is possible to determine whether the egg is fertilized or has high fertility.

Peptides 1 to 4 have average molecular weights of 2743, 2757, 2862, and 2941, respectively, but precise molecular weights of protonated molecules ([M + H ⁺ ]) are 2738.52, 2752.43, 2875.52, and 2936.44, respectively (FIG. 1). ). As described above, the actual measurement value of the molecular weight may slightly vary depending on the measurement method / measurement apparatus used. For example, in the case of a method using a mass spectrometer, it is preferable to measure the peak intensity that appears at the indicated numerical value ± 0.5% (preferably ± 0.3%, more preferably ± 0.1%).

  Since the above peptides 1 to 4 are useful as a test or determination marker for fertilization of an egg, peptides containing amino acid residues of these four peptides are similarly used as a test or determination marker for fertilization of an egg. It is possible.

  The peptide of the present invention includes a peptide having an amino acid sequence substantially the same as the amino acid sequence of the four peptides in addition to the peptide having the entire amino acid sequence of the amino acid sequences of the four peptides. . “Substantially identical” means that one or a few amino acids are modified as compared with the amino acid sequences of the above four peptides, but can still be used as a test or determination marker for fertilization of an egg. To do. The minority refers to 1 to 3, preferably 1 to 2, more preferably 1. Here, the modification means that the amino acid is deleted, added, or substituted with another amino acid, or the amino sequence is changed by a combination thereof. Furthermore, the modification includes addition and phosphorylation of sugar chains and / or fatty acids.

  For example, when the subject patient has a polymorphism or allelic variation that includes one or more amino acid substitutions, deletions, insertions or additions or combinations thereof within the amino acid sequence of the peptide of the present invention, the detection is performed. It is obvious to those skilled in the art that the amino acid sequence of the peptide should be interpreted as “the amino acid sequence having the polymorphism or allelic variation in each amino acid sequence represented by SEQ ID NOs: 1 to 4.”

  The present invention also provides for fertilization of an ovum in the subject by measuring the amount of one or more peptides of the present invention in a follicular fluid from the subject in need of testing for fertilization of the ovum. Provide a test method of possibility. Since the peptide of the present invention fluctuates depending on whether or not fertilization is possible after maturation of the ovum, by adopting a test or diagnosis using the peptide of the present invention as an index, fertilized ova that can be fertilized can be selected. The success rate of in vitro fertilization can be greatly improved. Since fertilized eggs can be differentiated by this method, pregnancy with a probability close to 100% in a patient is possible even with one external fertilization.

  The peptide to be measured is not particularly limited as long as it is selected from the above four peptides of the present invention. Moreover, in order to improve the measurement accuracy, as long as the peptide of the present invention is included, the amount of one or more biological substances that can serve as a test or determination marker for eggs other than the peptide of the present invention may be further measured. .

  The body fluid derived from the subject as the test sample is follicular fluid. The follicular fluid is advantageous in that it can be collected from the follicle without damaging the ovum in the case of artificial insemination. The peptide of the present invention to be detected may be directly used for inspection or determination without pretreatment such as protein removal of the sample, that is, without purification, and if necessary, using a spin column or the like, It is also possible to lightly separate and remove a high molecular weight protein fraction from the sample in advance.

  The detection of the peptide of the present invention in the follicular fluid can be carried out, for example, by measuring various molecular weights of the follicular fluid, such as gel electrophoresis or various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, affinity). Chromatography, reverse phase chromatography, etc.), surface plasmon resonance, ionization (eg, electron impact ionization, field desorption, secondary ionization, fast atom bombardment, matrix-assisted laser desorption / ionization (MALDI)) Methods, electrospray ionization methods, etc., and mass spectrometers (eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, immunology) Combination of mass spectrometers, mass spectrometers with stable isotope peptides as internal standards, etc. Subjected, it can be carried out by detecting a band or spot or peak, consistent with the molecular weight of the peptide, but not limited to.

  A method of purifying the peptides of the present invention to produce antibodies that recognize them and detecting the peptides by ELISA, RIA, immunochromatography, Western blotting, immunomass spectrometry or various immunoassays can also be preferably used. Furthermore, a hybrid detection method with the above detection method is also effective.

One of the particularly preferable measurement methods in the inspection method of the present invention is that a test sample is brought into contact with the surface of a plate used for time-of-flight mass spectrometry, and the mass of a component captured on the plate surface is measured using a time-of-flight mass spectrometer. The method of measuring by is mentioned. A plate compatible with a time-of-flight mass spectrometer has a surface structure (for example, functional group-added glass, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ ) capable of efficiently adsorbing the peptide of the present invention to be detected. , Modified silicon, various gels or polymer coatings).

  In a preferred embodiment, the support used as a plate for mass spectrometry is a substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF (WO 2004/031759 See). Such a substrate is not particularly limited as long as it is used in a plate for mass spectrometry, and examples thereof include insulators, metals, conductive polymers, and composites thereof. Such a plate for mass spectrometry coated with a thin layer with PVDF is preferably a blot chip (BLOTCHIP, registered trademark) manufactured by Protocera. Mass spectrometry using this BLOTCHIP (registered trademark) is the world's first peptome analysis method that enables analysis without pretreatment of specimens (body fluids such as blood and tissue extracts). For details, see Japanese Patent No. 4447458. No., Tanaka, K; BBRC, 379, 110-114. (2009), Y. Araki; Proteomics, 11 (13), 2727-2737. (2011).

  After pretreatment of the test sample-derived follicular fluid from the subject or by simply removing the high molecular weight protein using an antibody column or other method, the test sample is subjected to SDS-polyacrylamide gel electrophoresis or isoelectric focusing. After electrophoresis, the gel is transferred to a plate for mass spectrometry and transferred (blotted). The transfer method itself is known and preferably electrotransfer is used. As a buffer used at the time of electrotransfer, it is preferable to use a known buffer having a pH of 7 to 9 and a low salt concentration (for example, Tris buffer, phosphate buffer, borate buffer, acetate buffer, etc.).

  In a preferred embodiment, the peptide adsorbed to the protein is dissociated by electrophoresis in the presence of a surfactant (SDS), transferred to BLOTCHIP®, and then the chip is analyzed at high speed by MALDI-MS. As a result, the loss of protein complex peptide due to conventional pretreatment (protein removal) does not occur, and the peptidome profile of any sample can be quantitatively reproduced, and many biomarker peptides that have been missed so far can be accurately detected. And can be found with probability.

  The presence and amount of the peptide of the present invention, which is a target molecule, can be identified from the information on the molecular weight by mass spectrometry of molecules in the test sample captured on the support surface by the above method. It is also possible to output information from the mass spectrometer as differential information by using an arbitrary program and comparing it with mass spectrometry data in follicular fluid when fertilization is impossible. It will be appreciated that such programs are well known and those skilled in the art can easily construct or modify such programs using known information processing techniques.

  In order to obtain highly accurate mass spectrometry results, a stable isotope-labeled peptide of the target molecule is synthesized and mixed with a test sample as a known amount of internal standard, and Protocera's BLOTCHIP® system is used. Measurement data with a CV value of 5% or less can be acquired. Of course, mass spectrometry can be performed by a mass spectrometry system other than the BLOTCHIP (registered trademark) system, and this method can be clinically used as a diagnostic apparatus that does not use an antibody.

  In the above-described detection by mass spectrometry, peptides can be identified using a tandem mass spectrometry (MS / MS) method. As such an identification method, an amino acid sequence is determined by analyzing an MS / MS spectrum. And a method of identifying a peptide by performing a database search using partial sequence information (mass tag) contained in the MS / MS spectrum. Further, by using the MS / MS method, the amino acid sequence of the peptide of the present invention is directly identified, and all or a part of the peptide is synthesized based on the sequence information. It can also be used as

  The peptide of the present invention can be measured using an antibody against it. Therefore, the present invention includes a method for examining fertilization ability of an egg using an antibody that specifically recognizes a peptide, and a determination kit for examining fertilization ability of an egg containing an antibody containing such an antibody. Such a test method can detect the peptide with high sensitivity and high accuracy without using a special apparatus such as the mass spectrometer, if an optimized immunoassay system is constructed and a kit is prepared. It is useful in that it can.

  The present invention also includes an apparatus for immunodiagnosis or determination using an antibody against a peptide. Examples of such an apparatus include, but are not limited to, an immunochromatography diagnosis or determination chip in which an antibody against a peptide is fixed in a determination region of the chip. . A preferred embodiment of such a chip is an immunochromatographic diagnostic or determination chip in which an antibody against the peptide of the present invention is immobilized.

  An antibody against the peptide of the present invention can be prepared, for example, by isolating and purifying the peptide of the present invention from a follicular fluid derived from a patient expressing the peptide and immunizing an animal using the peptide as an antigen. Alternatively, when the amount of the peptide obtained is small, the peptide can be prepared in a large amount by a well-known genetic engineering technique such as amplification of a cDNA fragment encoding the peptide by RT-PCR. The peptide of the present invention can also be obtained using a cell-free transcription / translation system as a template. Further, it can be prepared in a large amount by an organic synthesis method.

  The antibody against the peptide of the present invention (hereinafter sometimes referred to as “the antibody of the present invention”) may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. The antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2, ScFv, and minibody.

  For example, a polyclonal antibody is administered about 2 to 4 times every 2 to 3 weeks subcutaneously or intraperitoneally in an animal together with a commercially available adjuvant (for example, complete or incomplete Freund's adjuvant) using the peptide of the present invention as an antigen. It can be obtained by collecting whole blood after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals capable of obtaining the target antibody, such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

A monoclonal antibody can be prepared by a cell fusion method. Description of techniques for the preparation of a monoclonal antibody, Stites et al, Basic and Clinical Immunology. (Lang Medical Publications Los Altos. CA. 4 th Edition) and references therein,, in particular Koehler, G. & Milstein, C. Nature 256, 495-497 (1975). For example, the peptide of the present invention is administered to a mouse 2 to 4 times subcutaneously or intraperitoneally with a commercially available adjuvant, and after the final administration, the spleen or lymph node is collected, and white blood cells are collected.

  The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like. Culture of hybridomas producing monoclonal antibodies can be performed in vitro or in vivo, such as mouse or rat, preferably mouse ascites, and antibodies can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively. .

The test method of the present invention using an antibody is not particularly limited, and detects the amount of an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in a test sample by chemical or physical means, Any measurement method may be used as long as it is a measurement method calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In the measurement, the antibody or antigen can be bound with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, or a luminescent substance. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.
These individual immunoassays can be applied to the quantification method of the present invention by ordinary skill of those skilled in the art.

  Since the peptides of the present invention are composed of proteolytic products, various molecules such as undegraded proteins and similar peptides having a common cleavage site may affect the measurement value. Thus, in the first step, the follicular fluid is immunoaffinity purified with an antibody, and the fraction bound to the antibody is subjected to mass spectrometry in the second step, and so-called immunomass spectrometry, in which identification and quantification are performed based on the precise molecular weight. (See, for example, Rapid Commun. Mass Spectrom. 2007, 21: 352-358). According to immunomass spectrometry, undegraded proteins and similar peptides are completely separated by a mass spectrometer, and can be quantified with high specificity and sensitivity based on the exact molecular weight of a biomarker.

  Alternatively, as another test method of the present invention using the antibody of the present invention, the antibody is immobilized on the surface of a chip that can be adapted to a mass spectrometer as described above, and a test sample is applied to the antibody on the chip. Examples include a method in which a component in a sample captured by the antibody is contacted and subjected to mass spectrometry, and a peak corresponding to the molecular weight of the marker peptide recognized by the antibody is detected.

  The level of the peptide of the present invention in the subject-derived sample measured by any of the above methods varies statistically significantly compared to a threshold set based on the amount of peptide in the control sample. If so, it can be diagnosed or determined that the egg of the subject is highly fertilized. The threshold value set based on the amount of peptide in the control sample may be the amount of peptide in the control sample itself, or in the control sample as long as the reliability of the test or determination method of the present invention is ensured. Another reference value set based on the amount of the peptide may be used.

  In the present specification, the control sample refers to a sample serving as a reference for comparison, and includes a sample in the case of unfertilized. The level of the peptide of the present invention in the control sample may be detected in parallel with the level of the peptide of the present invention in the biological sample of the subject, or the level of the peptide of the present invention in a number of control samples in advance. The level may be measured and the standard expression level in the control sample may be determined statistically. Specifically, for example, the average value ± 2 × standard deviation (S.D.), or the average value ± × standard deviation (S.D.) or the average value ± 3 × standard deviation (S.D.) can be used as the standard value.

  Or you may set the expression level of the peptide of this invention in a control sample using a ROC curve. An ROC curve (receiver operating characteristic curve) is a graph showing the detection sensitivity on the vertical axis and the false positive rate (that is, “1-specificity”) on the horizontal axis. An ROC curve can be obtained by plotting changes in sensitivity and false positive rate when the reference value for determining the expression level of the peptide in the biological sample is continuously changed.

  Based on the obtained ROC curve, it is possible to select a standard value at which desired detection sensitivity and accuracy can be expected. A standard value set statistically by an ROC curve or the like is also called a cut-off value. When detecting fertility of an egg based on a cutoff value, the level of the peptide of the present invention in the subject-derived sample is compared with the cutoff value. Then, when the level of the peptide of the present invention in the sample derived from the subject varies with respect to the cut-off value, the fertility of the egg is detected or determined.

  In addition, in the field of disease diagnosis, the cut-off value is a criterion value that is predicted to have a high risk of suffering from the disease if it is greater than or equal to that value. Determined as negative. If the cut-off value is set low, the sensitivity is high and the specificity is low, and if the cut-off value is set high, the sensitivity is low and the specificity is high. In one embodiment, the cut-off value is a reference range (95% confidence interval, normal distribution) obtained by measuring the number of individuals of a reference value that is a measurement value in a control sample and obtaining a central value including 95% of the reference value. In this case, the upper limit of the measurement average value ± 2 standard deviations in the control sample). However, another value may be set as the cut-off value as long as the sensitivity and specificity required by the determination are satisfied.

  More specifically, the peptide of the invention is either peptide 1, 2, or 4, and the level of the peptide in the sample is statistically significantly increased compared to the peptide level in the control sample And / or if the level of Peptide 3 in the sample is statistically significantly lower than the level of the peptide in the control sample, it is diagnosed or determined that the egg is likely to be fertilized can do.

  The peptide of the present invention, more specifically, the four peptides of the present invention can be used alone or as a diagnostic marker for fertilization potential of an egg. (Correct diagnosis rate) and specificity (no disease correct diagnosis rate) can be further increased.

  When 2 or 4 peptides of the present invention are used as markers, they are determined by a population learning method, or the measured values of each peptide in a sample of a known patient and a control-derived sample in a corresponding number of samples are two-dimensional or three-dimensional. By plotting the coordinates and creating a scatter plot, it is visualized in which region each of the two groups is distributed, and then the measured values of each peptide in the sample derived from the subject are plotted on the scatter plot. Thus, the possibility of fertilization of the subject's egg can be easily determined. The determination can also be made by determining the cutoff value of each peptide based on the scatter diagram and comparing the measured value of each peptide in the subject-derived sample with this.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

  The disclosures of all patent applications and documents cited herein are hereby incorporated by reference in their entirety.

Example 1 Profiling Analysis Using BLOTCHIP (Registered Trademark) Eight female patients who visited Fukui University Obstetrics and Gynecology Department and obtained informed consent were used as subjects. From the follicle collected by in vitro fertilization treatment of the subject, the follicular fluid is sucked by pointing to the injection needle, and the follicular fluid containing the ovum that was not fertilized by in vitro fertilization (unfertilized group, 4 samples of 1-F1 to 1-F4) ) And follicular fluid (fertilization group, 4 samples of 2-F1 to 2-F4) containing eggs fertilized by in vitro fertilization. Confirmation of fertilization was confirmed by a conventional method of microscopic observation of the start of cell division in the fertilized egg. The collected follicular fluid was associated with each ovum on a one-to-one basis, immediately frozen at -80 ° C., and used for subsequent profiling analysis.

Mix 1.5 μL of the follicular fluid from the above 4 fertilized groups and 4 fertilized groups with 4.5 μL of sample processing solution for electrophoresis (NuPAGE (registered trademark) LDS Sample Buffer 4x; Invitrogen) and heat-treat at 70 ° C. for 10 minutes. Then, it was applied to a 4-12% gradient polyacrylamide gel (Invitorigen) and electrophoresed. After the completion of electrophoresis, the gel was cut out, laminated on BLOTCHIP (registered trademark) (Protosera, Inc.), and transferred in an electric transfer buffer (BLOTBuffer ^™ ; Protosera, Inc.) at 90 mA for 120 minutes. After transfer, rinse the surface of the chip with ultrapure water, apply matrix (α-Cyano-4-hydroxy cinamic acid) to the entire chip, and then apply matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) Mass spectrometry was performed with a mass spectrometer (Ultra-FlexII manufactured by Bruker Daltnics). The measurement parameters were Detector voltage 1685V, Supression 1000, Laser Intensity 28-35 Fuzzy mode, 415 points per chip, 500 times laser irradiation per chip, total 207,500 times laser irradiation. Each peak intensity in the obtained spectrum was integrated for each M / z and converted into one integrated spectrum. Peptides giving statistically significant difference (P value less than 0.05) by performing differential profiling analysis between follicular fluid of unfertilized group and fertilized group using ClinProTools 2.2 (Bruker Daltonik GmbH) Groups were selected and four peptides with molecular weights of 2743, 2757, 2862, and 2941 were identified as egg test or decision markers (FIG. 1).

  2A, FIG. 3A, FIG. 4A and FIG. 5A show the peak intensities at molecular weights 2743, 2757, 2862, and 2941, respectively, between the two groups of the four obtained biomarkers, and FIG. 2B, FIG. 3B, FIG. FIG. 5B shows a plot of peak intensity and an ROC curve of the four biomarkers between 1: fertilized group and 2: fertilized group. From these results, it is understood that the peak intensity at the molecular weights 2743, 2757, and 2941 is significantly higher in the fertilized group and the peak intensity at the molecular weight 2862 is significantly lower than that in the unfertilized group, and the four peptides are unique. It has been shown to be useful as a test or determination marker for fertility of typical eggs.

Example 2 Identification of peptides by de novo MS / MS analysis on BLOTCHIP Reflector of MALDI-TOF mass spectrometer Ultraflex II (manufactured by Bruker Daltnics) for the fraction spots with peptides shown in FIGS. Monoisotopic mass [M + H] + was measured in mode. The obtained monoisotopic mass [M + H] + was designated as Parent Mass, and MS / MS measurement was performed in Lift mode (PSD). The measurement result was searched with MASCOT Server, the amino acid sequence was determined by MS / MS analysis, and the peptide was identified (FIG. 1).

  As a result, peptide 1 consisting of the amino acid sequence represented by SEQ ID NO: 1 corresponding to the 341st to 367th fragments of human α-2-HS-glycoprotein, and fragments 25 to 48 of human serum albumin Corresponding peptide 2 consisting of the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence is the same as SEQ ID NO: 1, but peptide 18 in which cysteine is further linked to the 18th cysteine by a disulfide bond, from human serum albumin 25 Peptides 4 corresponding to the 50th fragment and consisting of the amino acid sequence represented by SEQ ID NO: 4 were identified.

Example 3 Example of Apparatus for Immunodiagnosis or Determination FIGS. 6A and 6B are schematic diagrams showing an example of an immunochromat chip that is an apparatus for immunodiagnosis or determination of the present invention. The immunochromatography chip 10 is an immunoassay device for analyzing a specimen using an antigen-antibody reaction, also called an immunostrip or an immunoassay strip, and includes a base material and a specimen provided on the base material as a basic structure. An absorption pad for absorbing and developing a biological sample (in this application follicular fluid) is provided. The base material and the absorbent pad may be made of materials known or known in the art.

  6A and 6B, a region 20 is provided along the longitudinal direction of the immunochromatography chip 10 to which an antibody against the peptide of the present invention is bound.

  When a biological sample containing follicular fluid is absorbed and developed from the lower end of the immunochromatographic chip 10, the biological sample flows from bottom to top (in the direction of the arrow in FIG. 6), as in FIGS. 6 (a) and 6 (b), The fertilization possibility of the ovum in the follicular fluid can be determined. FIG. 6 (a) shows that there is no line in region 20 and that the egg is unfertilized or not suitable for fertilization. FIG. 6 (b) shows that there is a line in the region 20 and that the egg is in a state suitable for fertilization or fertilization.

  Thus, according to the immunochip using the antibody against the peptide of the present invention, the quality or state of the ovum can be determined easily, rapidly and accurately.

  The clinical examination method using the novel ovum test or determination marker of the present invention is useful in that the success rate of fertilization, particularly in vitro fertilization, can be greatly improved because it can quickly and accurately determine the fertility of the ovum. is there.

Claims (9)

  In the follicular fluid collected from the subject, the amount of one or more peptides selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4 is measured. A test method of fertilization possibility of an egg.   The method of claim 1, comprising subjecting the follicular fluid to mass spectrometry.   The amount of peptide obtained by the measurement is compared with a threshold value set based on the amount of peptide in the control sample, and the amount of peptide obtained by the measurement is set based on the amount of peptide in the control sample. The method of claim 1, further comprising determining that an egg is highly fertile if it has a statistically significant variation relative to a determined threshold.   The biomarker for the test | inspection of the fertilization possibility of the egg containing the peptide selected from the peptide group which consists of an amino acid sequence represented by sequence number 1-4.   The biomarker for the test | inspection of the fertilization possibility of the egg which consists of a peptide selected from the peptide group which consists of an amino acid sequence represented by sequence number 1-4. A method for testing fertility of an egg,
A method using an antibody that specifically recognizes a peptide selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4.   A determination kit for examining fertility of an egg containing an antibody, wherein the antibody specifically recognizes a peptide selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4 The determination kit.   An apparatus for immunodiagnosis or determination in which an antibody against a peptide selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4 is fixed.   A peptide comprising the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

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