JP7185895B2 - Diagnostic techniques for neonatal or perinatal infections - Google Patents

Description

The present invention relates to techniques for diagnosing neonatal or perinatal infections. More specifically, leucine rich alpha 2 glycoprotein (LRG) (hereinafter also referred to as LRG) is used as an index (biomarker) for neonatal infections or perinatal infections for examination or diagnosis. Regarding technology.

Neonatal or perinatal infections are broadly defined as diseases of the neonatal or perinatal fetus or of the newborn and/or its mother (maternal mother), which, without early treatment, can affect both mother and child. However, it is difficult to evaluate with existing markers such as CRP due to physiological changes, and it may be difficult to delay treatment and determine efficacy. There are no techniques for diagnosis or detection (Non-Patent Documents 1-2).

Nishikawa T et al., The Journal of Immunology, 2008, 180: 3492-3501. Wasunna, A., et al., 1990. C-reactive protein and bacterial infection in pretern infants. Eur. J. Pediatr. 149; 424-427

INDUSTRIAL APPLICABILITY The present invention enables early detection and early treatment of "neonatal infections or perinatal infections" including neonatal pneumonia, neonatal sepsis, and chorioamnionitis (subjects include neonates, fetuses, and mothers). Methods, kits, test agents, diagnostic agents, and test or diagnostic systems for testing or diagnosing neonatal or perinatal infections using the novel biomarkers that are effective are provided. Pregnant women and neonatal infections are difficult to evaluate with existing markers such as CRP due to physiological changes, and there have been difficulties in delaying treatment and determining efficacy, but the present invention has solved this problem.

More specifically, it is as follows. In order to achieve the above objects, the present inventors measured LRG concentrations in cord blood of newborns with and without neonatal infections or perinatal infections, and compared the values. As a result, LRG concentrations were higher in neonatal or perinatal infections compared to those without neonatal or perinatal infections. This result implicates LRG in neonatal or perinatal infections. Specifically, serum LRG was measured by the ELISA method in blood collected from pregnant women who underwent perinatal management and umbilical cord blood from newborns, and physiological fluctuations and changes at the time of infectious disease complications were investigated. , This study was conducted with the approval of the ethics committee of each institution). As shown in the Examples, serum LRG in uninfected pregnant women showed a higher level (15.18±5.0 μg/ml) than that in non-pregnant women (about 10.0 μg/ml) throughout the entire gestational period. No effects of obstetric complications such as hypertension and intrauterine growth restriction were observed. In addition, LRG in maternal serum is significantly elevated in intrauterine infection complications, and LRG in body fluids (e.g., serum) in pregnant women during the perinatal period serves as a marker for perinatal infections in pregnant women such as intrauterine infections. It has been proven. Cord blood serum LRG in uninfected neonates was 3.47±0.72 μg/ml. In neonatal cases with infection, cord blood serum CRP was not significantly elevated (p=0.06), but LRG was significantly elevated (p<0.01). Serum LRG has been shown to serve as a valid marker in maternal and early neonatal infections.

The results disclosed in the present invention demonstrate the efficacy of the use of LRG as a biomarker for neonatal or perinatal infections, such that its use can be used to test or Diagnostic methods, kits, test agents, diagnostic agents and test or diagnostic systems can be provided.

Thus, the present invention relates to novel uses of LRG. Specifically, the invention relates to a novel use of LRG as a biomarker for perinatal and neonatal neonatal infectious diseases. LRG is a conventionally known gene and protein, but its use as a marker for perinatal, neonatal neonatal infections, and perinatal infections is unknown. As inflammatory markers, CRP and procalcitonin, which are used for clinical examination in adults, are known, but these genes are not expressed or expressed insufficiently in the perinatal period and the neonatal period, and are not used as markers. It was not available (Non-Patent Documents 1 and 2). In general, the metabolic mechanisms in the liver are different between non-pregnant adults and children, and those of pregnant women and newborns. and cannot be used as a marker as it is. Therefore, even today, there are no effective markers of neonatal and perinatal infections available, especially in perinatal to early neonates.

Under such circumstances, the present invention unexpectedly found that LRG is capable of early detection of perinatal and neonatal infections (e.g., detectable in cord blood). It has a remarkable effect as compared with .

The present invention also found that LRG is significantly elevated in cord blood of babies with known funisitis. We also found that other obstetric complications did not necessarily increase LRG. Therefore, LRG of the present invention is more useful than CRP as a marker for meningitis.

Accordingly, the present invention provides the following.
(1) A testing agent, a diagnostic agent, or a testing or diagnostic kit for testing or diagnosing neonatal infections or perinatal infections, containing reagents or means for detecting or measuring LRG gene products or degradation products thereof Or system.
(2) Reagents or means for detecting or measuring LRG gene products or degradation products thereof are nucleic acids, proteins, or antibodies or binding fragments thereof that specifically bind to said LRG gene products or degradation products thereof, as well as NMR and mass spectrometry. , autoradiography, chromatography, Southern blot, Northern blot, Western blot, LTIA, ELISA, RA, ECLIA and electrophoresis. Kits or systems for testing or diagnosis.
(3) the reagent or means for detecting or measuring the LRG gene product or its degradation product,
(i) a nucleic acid probe or nucleic acid primer that specifically binds to a transcript of an LRG gene or a precursor or breakdown product thereof, or (ii) an antibody that specifically binds to an LRG protein or a precursor or breakdown product thereof or binding thereof 3. A test agent, diagnostic agent or kit or system for testing or diagnosis according to item 1 or 2, comprising a sex fragment.
(4) According to any one of items 1 to 3, wherein the neonatal infection or perinatal infection is an infection of a fetus or newborn between 22 weeks of gestation and 4 weeks of age, or the mother's body, or both. test agent, diagnostic agent or kit or system for testing or diagnosis.
(5) The test agent according to any one of items 1 to 4, wherein the neonatal infection or perinatal infection is an infection of a fetus or newborn or its maternal body from 22 weeks of gestation to 7 days after birth; Diagnostic agents or kits or systems for testing or diagnosis.
(6) The test agent according to any one of items 1 to 5, wherein the neonatal infection or perinatal infection is an infection of a fetus or newborn or its maternal body from 22 weeks of gestation to 2 days after birth; Diagnostic agents or kits or systems for testing or diagnosis.
(7) The test agent according to any one of items 1 to 6, wherein the neonatal infection or perinatal infection is an infection of a fetus or newborn or its maternal body from 22 weeks of gestation to 1 day after birth; Diagnostic agents or kits or systems for testing or diagnosis.
(8) The test agent, diagnostic agent or kit for test or diagnosis according to any one of items 1 to 7, wherein the LRG gene product or degradation product thereof is present in the body fluid of the newborn or its mother. Or system.
(9) The bodily fluids include maternal blood, lymphatic fluid, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, aqueous humor, digestive fluid, sweat, tears, nasal mucus, urine, feces and vaginal fluid, and the neonatal fluid. According to item 8, selected from the group consisting of cord blood, blood, lymphatic fluid, tissue fluid, body cavity fluid, cerebrospinal fluid, joint fluid, aqueous humor, digestive fluid, sweat, tears, nasal discharge, urine, feces and vaginal fluid. test agent, diagnostic agent or kit or system for testing or diagnosis.
(10) The test agent, diagnostic agent, test or diagnosis kit or system according to item 8 or 9, wherein the bodily fluid comprises the maternal blood or the umbilical cord blood, or both.
(11) said neonatal or perinatal infection is intrauterine infection, chorioamnionitis, funsiitis, neonatal conjunctivitis, neonatal bacterial meningitis, congenital and perinatal cytomegalovirus infection; Neonatal hepatitis B virus infection, neonatal hepatitis C virus infection, human T-cell leukemia virus infection, chlamydia infection, group B streptococcal infection, neonatal pneumonia, congenital rubella, neonatal herpes simplex virus infection, neonatal sepsis , congenital syphilis, neonatal nosocomial infections, perinatal tuberculosis, congenital toxoplasmosis, chickenpox, HIV, and neonatal listeriosis. 3. The test agent, diagnostic agent, or kit or system for test or diagnosis according to 1.
(12) The test agent or diagnostic agent according to any one of items 1 to 11, wherein the neonatal infection or perinatal infection includes intrauterine infection, chorioamnionitis, neonatal sepsis, or neonatal pneumonia. or a kit or system for testing or diagnosis.
(13) The test or diagnosis includes neonatal infection or perinatal infection, at least one selected from the group consisting of preterm birth, fetal growth restriction (FGR), hypertension of pregnancy, and fetal dysfunction (NRFS) 13. The test agent, diagnostic agent or kit or system for test or diagnosis according to any one of items 1 to 12, which distinguishes between two diseases.
(14) Use of the LRG gene product or degradation product thereof as a diagnostic agent or indicator for diagnosing neonatal infections or perinatal infections.
(14A) Use according to item 14, further comprising one or more features according to any one of items 1-13.
(15) A method for testing or diagnosing neonatal or perinatal infections in a subject, comprising measuring LRG gene products or degradation products thereof in the subject.
(15A) The method of item 15, further comprising one or more features of any one of items 1-13.
(16) Use of reagents or means for detecting or measuring LRG gene products or degradation products thereof for testing or diagnosing neonatal or perinatal infections.
(16A) Use according to item 16, further comprising one or more features according to any one of items 1-13.

The present invention also provides a method of testing for or diagnosing neonatal or perinatal infections, comprising the steps of:
a) detecting or measuring LRG gene products or degradation products thereof in the sample;
b) comparing the level of said LRG gene product or degradation product thereof to a predetermined value, wherein if said level is greater than said predetermined value, determining a neonatal or perinatal infection. , to provide a method. The method may further comprise one or more features according to any one of items 1-13.

In the present invention, it is intended that one or more of the above features may be provided in further combinations in addition to the explicit combinations. Still further embodiments and advantages of the present invention will be appreciated by those skilled in the art upon reading and understanding the following detailed description, if necessary.

According to the present invention, the effectiveness of LRG as a biomarker for neonatal infections or perinatal infections (including neonates, fetuses and maternal (pregnant women)) has been clarified. Detecting or measuring can test for or diagnose neonatal or perinatal infections, including neonates, fetuses and maternal (pregnant women). In addition, the most effective method can be selected for determining the end of drug administration in a subject, changing the administered drug or treatment policy, and the like.

FIG. 1 is a diagram comparing the concentrations of CRP protein and LRG protein between an infected group and a non-infected group. FIG. 2 shows ROC curves of CRP protein and LRG protein in cord blood. FIG. 3 is a comparison of serum LRG protein concentrations between pregnant women with CAM complications (CAM(+)) and those without (CAM(−)). FIG. 4 is an explanatory diagram relating to an example in which the presence or absence of funisitis and cord blood LRG were investigated. When there is maternal infection, if there is no funisitis, the neonate is not infected, and if there is funisitis, the neonate is determined to be infected. FIG. 5 is a diagram showing that the cord blood LRG of a baby with funisitis rises more significantly than CRP and is useful as a marker for funisitis. LRG and CRP in umbilical cord blood were measured for neonates born from pregnant women. Newborns born from mothers with chorioamnionitis were divided into two groups based on the presence or absence of fetal infection based on the histopathological presence or absence of funisitis. The control group consisted of cases in which neither mother nor child was infected. Among neonates born to mothers with chorioamnionitis, LRG was significantly elevated only in the group with funisitis. As for CRP, a significant increase was also seen in the group with funisitis, but a clear increase was seen in only a few cases. Figure 6 shows that LRG is more useful as a marker for meningitis than CRP. 1 ROC curves for specificity and sensitivity were constructed and LRG had much better ROC values than CRP. FIG. 7 shows the results of investigating the relationship between obstetric complications and LRG. It was found that other obstetric complications did not necessarily raise LRG.

The present invention will be described below. It should be understood that throughout this specification, expressions in the singular also include the concept of the plural unless specifically stated otherwise. Thus, articles in the singular (eg, “a,” “an,” “the,” etc. in the English language) should be understood to include their plural forms as well, unless otherwise stated. Also, it should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will control. As used herein, "about" before a numerical value means ±10% of the numerical value that follows.

The present invention is based on the discovery that LRG proteins are associated, at least in part, with neonatal or perinatal infections, including neonatal, fetal and maternal (maternal) infections, including neonatal sepsis. . The findings indicate that LRG can be used not only as a marker for neonatal infections or perinatal infections, but also for selecting the most effective method for decisions at the end of drug administration and changes in administration drugs and treatment strategies. can also Thus, it is useful in early detection and provision of early treatment for neonatal or perinatal infections.

(definition)
The following provides definitions of terms used herein.

As used herein, "leucine rich alpha 2 glycoprotein (LRG)" is a glycoprotein of about 50 kDa, and is known to be contained in healthy human serum at a concentration of about 3.0 μg/mL. It is It has also been reported that LRG is secreted from neutrophils (J Leukoc Biol. 2002 72(3):478-85.). It has also been reported that LRG is expressed in granulocytes, but LRG expression is not induced by IL-6 stimulation. The present inventors have reported that such LRG can be a biomarker for autoimmune diseases such as Behcet's disease, Castleman's disease or rheumatoid arthritis, and ulcerative colitis (Ann Rheum Dis Published Online First: 22 Ｏｃｔｏｂｅｒ ２００９． ｄｏｉ：１０．１１３６／ａｒｄ．２００９．１１８９１９；Ｓｅｒａｄａ Ｓ，Ｎａｋａ Ｔ ｅｔ ａｌ“Ｓｅｒｕｍ ｌｅｕｃｉｎｅ－ｒｉｃｈ ａｌｐｈａ－２ ｇｌｙｃｏｐｒｏｔｅｉｎ ｉｓ ａ ｄｉｓｅａｓｅ ａｃｔｉｖｉｔｙ ｂｉｏｍａｒｋｅｒ ｉｎ ｕｌｃｅｒａｔｉｖｅ ｃｏｌｉｔｉｓ”Ｉｎｆｌａｍｍ Ｂｏｗｅｌ Ｄｉｓ ２０１２ Ｎｏｖ；１８（１１） : 2169-79.doi: 10.1002/ibd.22936.Epub 2012 Feb 28).

The base sequence of LRG is GenBank Accession No. NM_052972.2, etc., and the amino acid sequence is GenBank Accession No. NP_443204.1 and the like. In LRG, the amino acid sequence constituting the protein may be the amino acid sequence specified above, or one to more amino acids may be substituted, deleted, added, or introduced into the amino acid sequence. . Furthermore, it may be the entire LRG protein or a partial protein. Hereinafter, the term "LRG protein" as used herein refers to GenBank Accession No. 2, as described above. A protein composed of an amino acid sequence specified by NP_443204.1, or an amino acid sequence in which one to multiple amino acids are substituted, deleted, added, or introduced from the specified amino acid sequence, and a partial protein of LRG Used in the sense of including.

In addition, transcripts of the LRG gene to be detected or measured in the test method of the present invention include DNA containing the same or substantially the same nucleotide sequence as the above LRG-encoding nucleotide sequence. Examples of DNA substantially identical to the base sequence encoding LRG include, for example, about 50% or more, preferably about 60% or more, more preferably about 70% or more, and particularly preferably about 80% or more, DNA containing a base sequence having a homology of about 90% or more is most preferred. The homology of the nucleotide sequences in this specification is calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; filtering = ON; match Score = 1; mismatch score = -3).

As used herein, "neonatal period" and "early neonatal period" mean less than 28 days of age and less than 7 days of age, respectively, as defined by the World Health Organization. "Perinatal" means from 22 weeks of gestation to less than 7 days of age, as defined by the World Health Organization (ICD-10).

As used herein, "neonatal infection" or "perinatal infection", when referred to collectively, are infections of the fetus or newborn and mother (maternal) during the neonatal or perinatal period. refers to disease. Neonatal or perinatal infections include, for example, diseases transmitted through the cervix or transplacenta, including maternal infections. When fetal and maternal infections are referred to separately, they are referred to herein as "fetal perinatal infections" and "maternal perinatal infections" respectively. Transplacental infections include chorioamnionitis (CAM), a bacterial infection of the fetal appendages of the chorion and amniotic sac, which can lead to premature birth and very low birth weight. It is known to be the causative infectious disease. These infections include, for example, intrauterine infections, chorioamnionitis, funnicitis, neonatal conjunctivitis, neonatal bacterial meningitis, congenital and perinatal cytomegalovirus infection, neonatal hepatitis B virus infection. neonatal hepatitis C virus infection, human T-cell leukemia virus infection, chlamydia infection, group B streptococcal infection, neonatal pneumonia, congenital rubella, neonatal herpes simplex virus infection, neonatal sepsis, congenital syphilis, neonatal Nosocomial infections, perinatal tuberculosis, congenital toxoplasmosis, chickenpox, HIV and neonatal listeriosis. Preferably, the neonatal or perinatal infection comprises intrauterine infection, chorioamnionitis, neonatal sepsis or neonatal pneumonia.

In one embodiment, the tests or diagnoses performed in the present invention include neonatal or perinatal infections, premature birth, fetal growth restriction (FGR), hypertension of pregnancy, fetal dysfunction (NRFS), and others. can be distinguished from at least one, at least two, at least three, or all four of the diseases of It is an unexpected effect that can be distinguished from other obstetric complications such as preterm birth, fetal growth restriction (FGR), hypertension of pregnancy and fetal dysfunction (NRFS).

In one aspect, a neonatal or perinatal infection is an infection of the fetus or neonate or its mother between 22 weeks gestation and 4 weeks (28 days) of age (28 days) in humans. In another aspect, the neonatal or perinatal infection is infection of the fetus or neonate or its mother from 22 weeks of gestation to 7 days of age, preferably up to 6 days, up to 5 days, up to 4 days, up to 3 days. disease. In a preferred aspect, the neonatal or perinatal infection is an infection of the fetus or newborn or its mother between 22 weeks gestation and 2 days of age. In a more preferred aspect, said neonatal or perinatal infection is an infection of the fetus or neonate between 22 weeks of gestation and 1 day of age or of its mother. More preferably, the neonatal infection or perinatal infection is an infection of the fetus or newborn at 22 weeks of gestation to soon after birth (approximately 1 to 6 hours) or the mother's body. These time periods can vary in animal species other than humans. The conversion is easy for those skilled in the art, and can be converted as appropriate.

As used herein, examples of "therapeutic agents for infectious diseases" include ceftriaxone, erythromycin, azithromycin, polymyxin, bacitracin, acyclovir, vidarabine, idoxuridine, povidone-iodine, ganciclovir, valganciclovir, penicillin, and spiramycin. , pyrimethamine, sulfadiazine, acyclovir, trifluridine, idoxuridine, ampicillin, rifampicin, meropenem, trimethoprim, sulfamethoxazole, gentamicin, cefotaxime, amphotericin B, ethambutol, ethionamide, pyrazinamide and amikacin.

The living body to which the method of the present invention can be applied is not particularly limited. Living organisms suffering from infectious diseases or perinatal infections, such as humans, monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats, etc. be done. Humans are preferred. In the case of non-humans, those skilled in the art can appropriately convert the period of neonatal infection or perinatal infection based on that of humans.

The biological sample used in the testing or diagnostic method of the present invention is one isolated from the above-described living body (maternal, fetal or neonatal) to be tested or diagnosed, and is the LRG gene product ( RNA, protein, degradation products thereof, etc.), tissue containing cells, blood (serum, plasma, etc.), urine, cerebrospinal fluid, coelomic aspirate (pleural fluid, ascites), lymph fluid, etc. There are no particular restrictions. For example, less invasive and ethically acceptable: brain, meninges, spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (e.g., large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicles, ovaries, placenta, uterus, bones, joints, adipose tissue, skeletal muscle, lymph nodes, blood (serum, Plasma, etc.), urine, cerebrospinal fluid, coelomic puncture fluid (pleural effusion, ascites), or body fluids such as lymph and body parts can also be used, but lungs, meninges, lymph nodes, and blood (serum, plasma, etc.) can also be used. ), urine, cerebrospinal fluid, body cavity aspirate (pleural fluid, ascites) or lymph, more preferably blood (serum, plasma, etc.) or lymph, particularly preferably serum. When the biological specimen is serum, in order to facilitate detection of LRG, highly expressed serum proteins such as albumin, immunoglobulin G (IgG), transferrin, immunoglobulin A (IgA), haptoglobin, α1-antitrypsin, Pretreatment may be performed to remove fibrinogen, α2 macroglobulin, immunoglobulin M (IgM), α1-acid glycoprotein, complement C3, apolipoprotein AI, apolipoprotein AII, transthyretin, and the like.

As used herein, "bodily fluid" refers to fluid that a living body has in some way. Body fluids include, for example, maternal blood, lymph, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, aqueous humor, digestive fluid, sweat, tears, nasal mucus, urine, feces and vaginal fluid, or the umbilical cord of a newborn baby. blood, lymph, tissue fluid, body cavity fluid, cerebrospinal fluid, joint fluid, aqueous humor, digestive fluid, sweat, tears, nasal discharge, urine, feces and vaginal fluid.

As used herein, the term "sample" refers to any substance obtained from a subject or the like, and includes, for example, nucleic acids (eg, RNA), cells, tissues, organs, and the like. A person skilled in the art can appropriately select a preferable sample based on the description of this specification.

As used herein, the term "means" refers to anything that can be any tool for achieving a certain purpose.

As used herein, "diagnosis" refers to identifying various parameters associated with a disease, disorder, condition, etc. in a subject to determine the current or future status of such disease, disorder, condition. By using the methods, devices, and systems of the present invention, conditions within the body can be investigated, and such information can be used to determine the disease, disorder, condition, treatment or prophylactic formulation to be administered in a subject. Alternatively, various parameters such as method can be selected. In the present specification, "diagnosis" in a narrow sense means diagnosing the current situation, but in a broader sense it includes "early diagnosis", "predictive diagnosis", "pre-diagnosis" and the like. INDUSTRIAL APPLICABILITY The diagnostic method of the present invention is industrially useful because, in principle, it is possible to use what comes out of the body, and it can be performed without the hands of medical professionals such as doctors. In this specification, in order to clarify that it can be performed without the hands of medical personnel such as doctors, "predictive diagnosis, preliminary diagnosis or diagnosis" is sometimes referred to as "assisting".

Procedures for prescribing the diagnostic agent or the like of the present invention as a pharmaceutical or the like are known in the art, and are described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopoeia, and the Pharmacopoeias of other countries. Accordingly, those skilled in the art, given the description herein, will be able to determine the amount to use without undue experimentation.

Detection or quantification of LRG in a biological specimen isolated from a living body is performed by preparing an RNA (e.g., total RNA, mRNA) fraction from the biological specimen, and obtaining the LRG gene transcription product or its reverse transcription product contained in the fraction. can be examined by detecting or quantifying the

(Detection and inspection of LRG)
Therefore, in one embodiment, the testing method of the present invention is characterized by measuring using a nucleic acid probe or nucleic acid primer capable of specifically detecting the transcription product of the LRG gene or its reverse transcription product.

The RNA fraction can be prepared using known techniques such as the guanidine-CsCl ultracentrifugation method and the AGPC method. High-purity total RNA can be prepared quickly and simply from a trace amount of biological sample. Means for detecting the transcript of the LRG gene in the RNA fraction include, for example, a method using hybridization (Northern blot, dot blot, DNA chip analysis, etc.), or PCR (RT-PCR, competitive PCR, real-time PCR, etc.). ) and the like. Quantitative PCR methods such as competitive PCR and real-time PCR are preferable in that expression fluctuations of the LRG gene can be detected quickly, easily, and quantitatively from a small amount of biological sample.

Detection of the LRG gene by Northern blot or dot blot hybridization can be performed using, for example, a nucleic acid probe capable of specifically detecting LRG transcripts. Such nucleic acid probes are about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, even more preferably about 18 to about 50 bases, contained in the aforementioned known LRG nucleotide sequences. A polynucleotide comprising a sequence of contiguous nucleotides of bases or a complementary sequence thereof. The nucleic acid may be DNA, RNA, or a DNA/RNA chimera. DNA is preferred. Also, the nucleic acids used as probes may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA or a DNA:RNA hybrid. If single stranded, the antisense strand can be used.

Also, the nucleic acid probe used in the test method of the present invention is a polynucleotide containing a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence shown for the known LRGs described above. Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, Second Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). can be done. Stringent conditions include, for example, a hybridization reaction in 6×SSC (sodium chloride/sodium citrate) at 45° C., followed by one or more hybridization reactions in 0.2×SSC/0.1% SDS at 65° C. washing, etc. Those skilled in the art can appropriately change the salt concentration of the hybridization solution, hybridization reaction temperature, probe concentration, probe length, number of mismatches, hybridization reaction time, washing solution salt concentration, washing temperature, etc. can be easily adjusted to the desired stringency.

A nucleic acid that functions as a probe capable of detecting the expression of the LRG gene is obtained by using a primer set that can amplify a part or all of the transcription product of the gene, and using a primer set described later, in vivo (e.g., humans, monkeys, mice, rats, dogs, Bovine, horse, pig, sheep, goat, cat, rabbit, hamster, guinea pig, etc.) cells [for example, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, Epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, blood cells, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, obesity) neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary or stromal cells, or ethical progenitor cells of these cells, stem cells or cancer cells, etc.] or any tissue in which those cells are present [e.g., brain, each part of the brain (e.g., olfactory bulb, amygdaloid nucleus, cerebral thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, adipose tissue, skeletal muscle, etc.], and the desired cDNA or genomic DNA is used as a template by PCR. The LRG gene or cDNA is cloned from the above cell/tissue-derived cDNA or genomic DNA library by colony or plaque hybridization or the like, and if necessary, using restriction enzymes or the like. It can be obtained by making a fragment of an appropriate length. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd edition (mentioned above). Alternatively, the nucleic acid may be obtained by chemically converting part or all of the base sequence and/or its complementary strand sequence using a commercially available DNA/RNA automatic synthesizer or the like, based on known base sequence information for LRG. It can also be obtained by synthesis.

The nucleic acid is preferably labeled with a labeling agent to enable detection and quantification of the target nucleic acid. Examples of labeling agents that can be used include radioactive isotopes, enzymes, fluorescent substances, and luminescent substances. Examples of radioactive isotopes include [ ³² P], [ ³ H], and [ ¹⁴ C]. Enzymes that are stable and have a high specific activity are preferred, and examples include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, or the like is used. Luminol, luminol derivatives, luciferin, lucigenin, and the like are used as the light-emitting substance, for example. In addition, biotin-(strept)avidin can be used to bind probes and labeling agents.

In the case of Northern hybridization, the RNA fraction prepared as described above is separated by gel electrophoresis, transferred to a membrane such as nitrocellulose, nylon, polyvinylidene difluoride, etc., and prepared as described above. After specific hybridization in a hybridization buffer containing the labeled probe, the expression level of the LRG gene can be measured by measuring the amount of label bound to the membrane for each band by an appropriate method. can. In the case of dot blotting, the expression level of the LRG gene can be measured by similarly subjecting the membrane on which the RNA fraction is spotted to the hybridization reaction and measuring the labeling amount of the spots.

According to another preferred embodiment, a quantitative PCR method is used as a method for measuring the expression of the LRG gene. Quantitative PCR includes, for example, competitive PCR and real-time PCR.

A set of oligonucleotides used as primers in PCR includes, for example, nucleic acid primers capable of specifically detecting transcripts of the LRG gene. In one preferred embodiment, the nucleic acid primer used in the testing method of the present invention is, for example, about 15 bases or more, preferably about 15 to about 50 bases, more preferably about 15 to about 50 bases, contained in the nucleotide sequence shown for known LRG. has a continuous nucleotide sequence length of about 15 to about 30 bases, more preferably about 15 to about 25 bases, and is designed to amplify a DNA fragment of about 100 bp to several kbp (sense strand ), and a set of oligonucleotides that are capable of hybridizing to a polynucleotide having a nucleotide sequence complementary to said polynucleotide sequence (the antisense strand).

Alternatively, the detection or quantification of LRG protein in a biological sample separated from a living body is performed by preparing a protein fraction from the biological sample and detecting or quantifying the translation product of the gene contained in the fraction (i.e., LRG). can be checked by LRG protein can also be detected or quantified by an immunoassay method (eg, ELISA, FIA, RIA, Western blot, etc.) using an antibody that specifically recognizes LRG protein.

Accordingly, in one embodiment, the testing method of the present invention is characterized by measuring using an antibody capable of specifically detecting the translation product of the LRG gene.

An antibody that specifically recognizes LRG is an LRG polypeptide or a partial peptide thereof having antigenicity, specifically, the entire peptide sequence shown in the known LRG or a partial peptide having a portion corresponding to an epitope as an immunogen. It can be manufactured by an existing general manufacturing method. As used herein, antibodies include polyclonal antibodies, natural antibodies such as monoclonal antibodies (mAb), chimeric antibodies that can be produced using genetic recombination technology, humanized antibodies and single-chain antibodies, and their binding properties. Fragments include, but are not limited to. Preferably, the antibody is a polyclonal antibody, monoclonal antibody or binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specific examples thereof include F(ab') ₂ , Fab', Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996). The antibody class is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD and IgE. IgG or IgM is preferred, and IgG is more preferred in consideration of ease of purification and the like.

Specifically, as a method for measuring the amount of LRG protein, for example, (i) the detection antibody of the present invention is competitively reacted with a sample solution and labeled LRG protein, and bound to the antibody. A method for quantifying LRG protein in a sample solution by detecting a labeled protein, and (ii) a sample solution, a detection antibody of the present invention insolubilized on a carrier, and another labeled protein of the present invention. A method of quantifying the LRG protein in the sample liquid by measuring the amount (activity) of the labeling agent on the insolubilized carrier after simultaneously or sequentially reacting with the detection antibody.

Specifically, as a method for measuring the amount of LRG protein, for example, (i) the detection antibody of the present invention is competitively reacted with a sample solution and labeled LRG protein, and bound to the antibody. A method for quantifying LRG protein in a sample solution by detecting a labeled protein, and (ii) a sample solution, a detection antibody of the present invention insolubilized on a carrier, and another labeled protein of the present invention. A method of quantifying the LRG protein in the sample liquid by measuring the amount (activity) of the labeling agent on the insolubilized carrier after simultaneously or sequentially reacting with the detection antibody.

The LRG protein expression level can be detected and quantified according to known methods such as Western blotting using an antibody that recognizes the LRG protein. In Western blotting, after using an antibody that recognizes the LRG protein as a primary antibody, the primary antibody labeled with a radioactive isotope such as ¹²⁵ I as a secondary antibody, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), etc. It can be carried out by labeling with a binding antibody and measuring signals derived from these labeling substances with a radiometer (BAI-1800II: manufactured by Fuji Film Co., etc.), a fluorescence detector, or the like. In addition, after using an antibody that recognizes the LRG protein as a primary antibody, it is detected according to the protocol using ECL Plus Western Blotting Detection System (manufactured by GE Lifesciences), and measured with a multi-biomegaer STORM860 (manufactured by GE Lifesciences). You can also

The above antibody is not particularly limited in its form, and may be a polyclonal antibody that uses the LRG protein as an immunogen, or a monoclonal antibody. , usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.

Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.12-11.13).

In the quantification method (ii) above, it is desirable that the two antibodies recognize different portions of the LRG protein. For example, if one antibody recognizes the N-terminus of an LRG protein, the other antibody that reacts with the C-terminus of the protein can be used.

Examples of labeling agents used in measurement methods using labeling substances include radioactive isotopes, enzymes, fluorescent substances, and luminescent substances. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], and [ ¹⁴ C]. As the above-mentioned enzyme, those that are stable and have a high specific activity are preferred, and examples thereof include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, or the like is used. Luminol, luminol derivatives, luciferin, lucigenin, and the like are used as the light-emitting substance, for example. Furthermore, a biotin-(strept)avidin system can also be used for binding the antibody or antigen to the labeling agent.

The LRG quantification method using the detection antibody of the present invention is not particularly limited. Any measurement method may be used as long as it is detected by means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used. From the viewpoint of sensitivity and specificity, it is preferable to use, for example, the sandwich method described later.

For insolubilization of antigens or antibodies, physical adsorption may be used, or chemical bonds that are usually used for insolubilizing and immobilizing proteins, enzymes, etc. may be used. Examples of carriers include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicon, and glass.

In the sandwich method, the sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and further reacted with another labeled detection antibody of the present invention (secondary reaction). LRG protein in the sample solution can be quantified by measuring the amount or activity of the labeling agent. The primary and secondary reactions may be carried out in reverse order, may be carried out simultaneously, or may be carried out at different times. The labeling agent and the method of insolubilization can be according to those described above. In immunoassays using the sandwich method, it is not always necessary to use a single type of antibody for immobilized or labeled antibody. may

The detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as competitive methods, immunometric methods, and nephrometry.

In the competitive method, after the LRG protein in the sample solution and the labeled LRG protein are reacted competitively with the antibody, the reacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B/F separation), and by measuring the amount of either B or F labeled, LRG protein in the sample solution is quantified. In this reaction method, a soluble antibody is used as the antibody, and polyethylene glycol or a secondary antibody against the antibody (primary antibody) is used to perform B/F separation, and a solid-phase method is used as the primary antibody. An antibody is used (direct method), or a soluble primary antibody is used and an immobilized antibody is used as a secondary antibody (indirect method).

In the immunometric method, the LRG protein in the sample solution and the immobilized LRG protein are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or The LRG protein is allowed to react with an excess amount of the labeled antibody, then the solid-phased LRG protein is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to quantify the amount of antigen in the sample solution. In addition, in nephrometry, the amount of insoluble sediment produced as a result of antigen-antibody reaction in gel or solution is measured. Even when the amount of LRG protein in the sample solution is very small and only a small amount of sediment can be obtained, laser nephrometry or the like using laser scattering is preferably used.

When applying individual immunoassay methods to the test method of the present invention, no special conditions, operations, etc. are required. A measurement system for LRG may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures in each method. For details of these general technical means, reference can be made to review articles, books, and the like. For example, Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Continued Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. 1953), Eiji Ishikawa et al., "Enzyme Immunoassay" (2nd ed.) (Igakushoin, 1982), Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd ed.) (Igakushoin, Showa published in 1962), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. 73 (Immunochemical Techniques (Part B)), ibid. Vol. 74 (Immunochemical Techniques (Part C)), ibid. Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (published by Academic Press).

In another aspect, the LRG protein is detected by proteome analysis using a combination of an iTRAQ ^TM reagent (ABI) capable of high-throughput protein expression and quantitative analysis and a mass spectrometer. do.

(preferred embodiment)
Preferred embodiments of the present invention are described below. The embodiments provided below are provided for a better understanding of the invention, and it is understood that the scope of the invention should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present invention in light of the description in this specification. It is also understood that the following embodiments of the invention can be used singly or in combination.

It should be noted that the embodiments described below are all comprehensive or specific examples. Numerical values, shapes, materials, components, arrangement positions and connection forms of components, steps, order of steps, and the like shown in the following embodiments are examples and are not intended to limit the scope of the claims. In addition, among the constituent elements in the following embodiments, constituent elements that are not described in independent claims representing the highest concept will be described as optional constituent elements.

In one aspect, the present invention provides a method of testing or diagnosing neonatal or perinatal infections comprising the steps of: a) detecting or measuring LRG gene products or degradation products thereof in a sample; b) comparing the level of said LRG gene product or degradation product thereof to a predetermined value, wherein if said level is greater than said predetermined value, determining a neonatal or perinatal infection. , to provide a method. In another aspect, the method of the invention is a method of diagnosing whether a subject has a neonatal infection or a perinatal infection, comprising: a) LRG gene product or its LRG gene product in a sample obtained from the subject; detecting or measuring a degradation product, b) comparing the level of the LRG gene product or degradation product thereof to a predetermined value, wherein if the level is greater than the predetermined value, the subject has neonatal infection or and determining that it is a perinatal infection. In one aspect of the present invention, the level of the LRG gene product or its degradation product is reduced to a predetermined value by using a means of detection or measurement that does not react to the absence of the LRG gene product or its degradation product above a certain concentration. Neonatal or perinatal infections can be determined without comparison. In other aspects, the methods of the present invention use catalysts that bind to LRG gene products or degradation products thereof, catalysts that bind to LRG gene products or degradation products thereof, as reagents or means for detecting or measuring LRG gene products or degradation products thereof. At least one of a nucleic acid, a protein that binds the LRG gene product or a breakdown product thereof, or a particle that binds the LRG gene product or a breakdown product thereof may be used.

The present invention is a method for determining the therapeutic effect of administering a drug for treating an infectious disease to a subject having an infectious disease, comprising: Detecting or measuring, b) comparing the current level of the LRG gene product or its breakdown product to a level previously measured in the same subject and to a predetermined value, wherein the current level is If the level is equal to or higher than the measured level, administration of the infection-treating agent is discontinued; if lower than the previously measured level and higher than the predetermined value, the administration period is extended; if lower than the predetermined value, administration and determining to terminate the. In one aspect of the invention, if the current level of the LRG gene product or breakdown product thereof is about the same as the previously measured level, administration may not be discontinued, but may be extended. In another aspect, administration can be extended if the current level of the LRG gene product or degradation product thereof is below a predetermined value.

When the LRG gene product or degradation product thereof is an LRG protein, 5.0 μg/ml may be used as the predetermined value for comparison (below about 5.0 μg/ml or about 5.0 μg/ml). /ml.) can be changed as appropriate. For example, values such as about 15 μg/mL and about 20 μg/mL can be used for maternal. Even uninfected pregnant women tend to have a somewhat higher LRG than normal adults, but it has been demonstrated that infected pregnant women show significantly higher LRG values than uninfected pregnant women. It is known that in pregnant women, especially in the later period (including the perinatal period), metabolism fluctuates significantly. It is said that the marker does not exist. Therefore, it would be extremely useful not only for newborns and fetuses, but also for expectant and nursing mothers to be provided with their own infectious disease markers. It is a strong evidence that pregnant women have infectious diseases, and as shown in the examples, existing markers such as CRP do not show significant differences in pregnant women, so it is also a powerful test for pregnant women. and diagnostic agents.

In one aspect of the present invention, test agents, diagnostic agents, test kits for testing or diagnosing neonatal infections or perinatal infections, containing reagents or means for detecting or measuring LRG gene products or degradation products thereof, A diagnostic kit, test system or diagnostic system is provided. In some aspects, the test agent or diagnostic agent, test kit or diagnostic kit or test system or diagnostic system of the present invention uses LRG gene product or its degradation product as a reagent or means for detecting or measuring LRG gene product or its degradation product. Perform nucleic acids, proteins or antibodies or binding fragments thereof that specifically bind to degradation products and NMR, mass spectroscopy, PCR, autoradiography, Southern blotting, Northern blotting, chromatography, electrophoresis, microscopy and next-generation sequencing at least one of the devices for Specific examples include ELISA, LTIA, FIA, RIA, CIA, two-dimensional electrophoresis, Western blotting, and the like.

In another aspect, the present invention provides kits for testing or diagnosing neonatal or perinatal infections comprising reagents or means for detecting or measuring LRG gene products or degradation products thereof.

In one preferred embodiment, reagents or means for detecting or measuring LRG gene products or degradation products thereof include (i) nucleic acid probes or nucleic acid primers that specifically bind to transcripts of LRG genes or precursors or degradation products thereof or (ii) an antibody or binding fragment thereof (eg, an antigen-binding fragment) that specifically binds to the LRG protein or a precursor or degradation product thereof.

In one embodiment, the neonatal or perinatal infection is an infection of the fetus or neonate and/or its mother between 22 weeks gestation and 4 weeks of age, preferably between 22 weeks gestation and 7 weeks of age. Fetal or neonatal infection or maternal infection, more preferably 22 weeks gestation to 2 days after birth or maternal infection, still more preferably 22 weeks gestation to 1 day after birth Fetal or neonatal or maternal infection.

The LRG gene product or degradation product thereof is present in the bodily fluids of said neonate or its mother. Such entities in bodily fluids may be preferred due to the ease of subsequent processing. Body fluids include said maternal blood, lymphatic fluid, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, aqueous humor, digestive juices, sweat, tears, nasal discharge, urine, feces and vaginal fluids, and said newborn's umbilical cord blood, blood , lymphatic fluid, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, aqueous humor, digestive fluid, sweat, tears, nasal discharge, urine, feces and vaginal fluid. Blood, urine, sweat and the like are particularly preferred. Blood is more preferred. Blood may include maternal blood or said cord blood, or both.

Diseases or disorders that can be targeted by the present invention include intrauterine infections, chorioamnionitis, meningitis, neonatal conjunctivitis, neonatal bacterial meningitis, congenital and perinatal cytomegalovirus infection, and neonatal type B. Hepatitis virus infection, neonatal hepatitis C virus infection, human T-cell leukemia virus infection, chlamydia infection, group B streptococcal infection, neonatal pneumonia, congenital rubella, neonatal herpes simplex virus infection, neonatal sepsis, congenital syphilis , neonatal nosocomial infections, perinatal tuberculosis, congenital toxoplasmosis, chickenpox, neonatal or perinatal infections such as HIV or neonatal listeriosis. Preferably, these diseases or disorders may include intrauterine infection, chorioamnionitis, neonatal sepsis or neonatal pneumonia.

In a preferred embodiment, the tests or diagnoses of the present invention detect neonatal or perinatal infections in at least one of the following diseases: preterm birth, fetal growth restriction (FGR), hypertension of pregnancy, fetal dysfunction (NRFS). To distinguish between 1, at least 2, at least 3, or all 4 diseases.

As used herein, the term "kit" refers to a unit provided with parts to be provided (eg, reagents, primers, drugs, labels, instructions, etc.), usually divided into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed form for reasons such as stability, and is preferably used in a mixed form immediately before use. Such kits preferably include the parts provided (e.g., instructions or instructions describing how to use the agent or how to handle the reagents). In the present specification, when the kit is used as a reagent kit, the kit usually includes instructions describing how to use drugs, antibodies, and the like.

As used herein, "instructions" are written instructions to the user how to use the present invention. The instructions contain language that instructs how to use the reverse transcription template-switching PCR and reagents of the invention. In addition, the instructions may include words that instruct the method of use (screening method). The instructions are made in accordance with the form prescribed by the regulatory authority of the country in which the invention is implemented and it is expressly stated that it has been approved by that authority. The instruction is a so-called package insert, and may be provided in a form of paper medium or electronic medium (for example, homepage provided on the Internet, e-mail).

In other aspects, reagents or means for detecting or measuring LRG gene products or degradation products thereof include catalysts that bind LRG gene products or degradation products thereof, nucleic acids that bind LRG gene products or degradation products thereof, LRG gene products or degradation products thereof, It may comprise at least one of a protein that binds its breakdown product or a particle that binds the LRG gene product or its breakdown product. Specific examples include enzymes, hormones, colloids, antibodies or binding fragments thereof, nucleic acid primers, nucleic acid probes, receptors and the like. In yet another aspect, the kit of the present invention comprises (i) a nucleic acid probe or nucleic acid primer capable of specifically detecting an LRG gene transcript or its precursor or degradation product, or (ii) an LRG protein or its precursor. or an antibody that can specifically detect the degradation product, which can detect or measure the LRG gene product or its degradation product, whether the subject has a neonatal infection or a perinatal infection It may be a diagnostic agent, kit or system, or an agent, kit or system for evaluating the efficacy of a treatment against an infectious disease. In one aspect, the agent, kit or system of the present invention can be used to determine the efficacy of treatment for neonatal or perinatal infectious disease, thereby continuing or terminating treatment for infectious disease or ending treatment for infectious disease. You can also select the type.

In other aspects, the kits of the invention may be capable of measuring levels of LRG gene products or degradation products thereof. In a further aspect, the kit of the present invention may be a kit that does not require level measurement for determining whether a neonatal infection or perinatal infection is present or for evaluating therapeutic effects. In some aspects, kits of the invention may include a concentration-adjustable LRG gene product or degradation product thereof.

In another aspect, the kit of the present invention may further comprise an antibody, primer or probe that can specifically detect an LRG gene product or a degradation product thereof. Such antibodies, primers or probes may be labeled. Kits of the present invention include, for example, an enzyme-linked immunosorbent assay (ELISA) kit, a latex immunoturbidimetric kit, a kit for real-time PCR detection, a rapid multiplex count PCR detection kit, an electrochemiluminescence immunoassay (ECLIA) kit. , particle agglutination immunoassay (LTIA) kits or kits for in situ hybridization.

In yet another aspect, the kit of the present invention comprises a nucleic acid probe capable of differentially detecting an LRG gene transcript or its precursor or degradation product as a reagent or means for detecting or measuring an LRG gene product or a degradation product thereof. Alternatively, nucleic acid primers or antibodies capable of specifically detecting LRG proteins or precursors or degradation products thereof may be included. Examples of test or diagnostic agents may include substances bound with reagents or means for detecting or measuring LRG gene products or degradation products thereof, such as antibodies, primers or probes. Examples of such substances include latex, metal colloids, gelatin, liposomes, microcapsules, silica, alumina, carbon black, metal compounds, metals, ceramics, magnetic substances, and the like.

In ELISA, for example, a sandwich method can be used. An anti-LRG antibody is immobilized on a solid phase carrier, an appropriately treated biological sample is added and reacted, and then an anti-LRG antibody that recognizes another epitope labeled with an enzyme is added and reacted. After washing, the LRG concentration can be determined by reacting with an enzyme substrate, developing color, and measuring absorbance. In addition, after reacting the anti-LRG antibody immobilized on the solid-phase carrier with LRG in the biological sample, an unlabeled LRG antibody (primary antibody) is added, and the antibody (secondary antibody) against this unlabeled antibody is enzyme-labeled. may be further added.

When the enzyme is peroxidase, 3,3'-diaminobenzidine (DAB), 3,3'5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used as the enzyme substrate. In the case of, p-nitropheny phosphate (NPP) or the like can be used.

Among the above immunoassays, the ECLIA method is also preferred as a method for measuring protein with high sensitivity. In the ECLIA method, an anti-LRG antibody is immobilized on microbeads, an appropriately treated biological sample is added and reacted, and then an anti-LRG antibody that recognizes another epitope labeled with an electrochemiluminescent substance is added and reacted. After washing the microbeads, the microbeads are captured on the electrode, applied with electrical energy to emit light, and the amount of light emitted is measured to determine the LRG concentration.

Among the above immunoassays, an agglutination method is also preferable as a method for easily detecting a minute amount of protein. The agglutination method includes, for example, a latex agglutination method in which latex particles are bound to an antibody.

When anti-LRG antibodies are bound to latex particles and mixed with a biological sample, the antibody-bound latex particles aggregate if LRG is present. Therefore, the concentration of LRG can be determined by irradiating a sample with near-infrared light and quantifying aggregates by measuring absorbance (turbidimetric method) or scattered light (nephelometric method).

Particle agglutination immunoassay (LTIA) is also preferred. An outline of the LTIA method is as follows.
Four aspects A to D of the measurement reagent (kit) for detecting LRG present in a sample each include at least the following elements:
A. (a) latex particles having a first monoclonal antibody immobilized thereon and (b) latex particles having a second monoclonal antibody immobilized thereonB. (a) latex particles having a first monoclonal antibody immobilized thereon and (b) a second monoclonal antibody C.I. Latex particles to which (a) a first monoclonal antibody and (b) a second monoclonal antibody are immobilizedD. (a) latex particles on which both a first monoclonal antibody and a second monoclonal antibody are immobilized.

These measurement reagents (kits) can be suitably used for the LTIA method. The latex particles used in A to D can be appropriately selected in particle size and type in order to obtain desired performance such as sensitivity improvement. Any latex particles may be used as long as they are suitable for carrying antigens or antibodies. For example, polystyrene, styrene-sulfonic acid (salt) copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic acid ester copolymer A polymer etc. are mentioned. The shape of the latex particles is not particularly limited, but the average particle size is large enough to allow the aggregates resulting from the agglutination reaction between the antibody or antigen on the surface of the latex particles and the object to be measured to be detected with the naked eye or optically. It is preferred to have A preferred average particle size is about 0.02 to about 1.6 μm, more preferably about 0.03 to about 0.5 μm. Particles made of materials such as metal colloids, gelatin, liposomes, microcapsules, silica, alumina, carbon black, metal compounds, metals, ceramics or magnetic substances can be used instead of latex particles.

For example, reagents for the LTIA method used in clinical tests are usually provided in the form of a first test solution and a second test solution, which are sequentially mixed with a test sample and used. Both or one of (a) and (b) in each of the above aspects A to D can be contained in the first reagent solution or the second reagent solution. The method of containing these substances can be appropriately selected in consideration of the specifications of measuring instruments in clinical tests and the design of measuring reagents (performance, ease of use, etc.). In general, it is preferable to contain both (a) and (b) of embodiment A in the second test solution, but (a) of embodiment A is contained in the first test solution and (b) is contained in the second test solution. It can also be suitably used to allow

Among the above immunoassays, an immunochromatographic method can be mentioned as a method that can be easily measured at a bedside or the like.

Immunochromatography, which is a labeled immunoassay method, can also be used.

In a general immunochromatography method, on a sheet-like solid phase support such as a membrane, "1. Test sample supply site" and "2. A labeling reagent site containing a labeling reagent (the first monoclonal antibody is labeled with a labeling substance such as colloidal gold particles) that is developably held on the membrane”, “3. The test sample solution is configured to continuously move through the "capture reagent site where the second monoclonal antibody is immobilized for capturing the immune complex of the monoclonal antibody and LRG" by capillary action.

Specifically, first, when a predetermined amount of a test sample containing LRG is added to the test sample supplying site, the sample penetrates into the labeling reagent site in the process of developing and moving the solid phase support, and LRG becomes the labeling reagent (first 1 monoclonal antibody) to form an immune complex of the LRG-labeled reagent. The LRG-labeling reagent complex moves on the membrane as it is, and when it enters the capture reagent site containing the second monoclonal antibody on the membrane, it is captured by the capture reagent immobilized on the solid phase support. An immune complex of (second monoclonal antibody)-LRG-labeled reagent (first monoclonal antibody) is formed at the capture reagent location. Then, the presence of the analyte is determined by detecting the labeling reagent by an arbitrary method (in the case of visible colloidal gold particles, its aggregate image, in the case of enzymes, a color reaction by adding a substrate). can be done.

For ease of understanding, "1. Test sample supply site" and "2. Labeling reagent containing the first monoclonal antibody (the first monoclonal antibody is labeled with a labeling substance such as colloidal gold particles ), and the labeling reagent site that is developably held on the membrane "is described independently in the order of the movement direction of the test sample. A person skilled in the art can naturally understand that aspects and configurations known to those skilled in the art can be adopted.

In the present invention, LRG can be detected or quantified using a kit that can specifically detect LRG. Examples of the kit include commercially available kits, which can be used for detection or quantification of LRG according to the product instructions of the kit. Examples of such a kit include, but are not limited to, Human LRG Assay Kit (IBL).

(general technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and include those already cited, for example, Sambrook J. et al. al.(1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed.(2001); Ausubel, FM(1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA(1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel,FM (1995).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular. Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999). olecular Biology, Wiley, and annual updates; Sninsky, JJ et al.(1999). PCR Applications: Protocols for Functional Genomics, Academic Press. , which are hereby incorporated by reference in relevant part (which may be in whole).

As used herein, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "or". When "within a range of two values" is stated herein, the range includes the two values themselves.

All references, such as scientific articles, patents, patent applications, etc., cited herein are hereby incorporated by reference in their entireties to the same extent as if each were specifically set forth.

The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described with reference to examples, which are provided for illustrative purposes only and not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.

Examples are described below. When necessary, the standards stipulated by Osaka University were observed, and when clinical research was involved, it was conducted in accordance with the Declaration of Helsinki and ICH-GCP. As the reagents, the products described in the examples were specifically used, but equivalent products from other manufacturers can be substituted.

(Example 1 Relationship between cord blood LRG and funisitis)
Of the neonates born at the Department of Obstetrics and Gynecology, Osaka University Hospital, eight neonates born to pregnant women with chorioamnionitis (CAM) and diagnosed with infectious diseases such as pneumonia and sepsis after birth (infected group) was used as the subject. As a control, 7 neonates (non-infected group) whose mother did not have chorioamnionitis and who did not have postnatal infection were used as subjects. Cord blood specimens and blood specimens on the day of birth (day 0) and the day after birth (day 1) were obtained from infected and non-infected neonates. Serum was separated from the obtained samples, and the CRP protein concentration in the serum was measured by the in-hospital clinical laboratory department and external laboratory SRL. A t-test was used to compare the two groups, and the difference was considered significant when p<0.05. Serum LRG concentrations were measured by sandwich ELISA using two anti-human LRG rabbit monoclonal antibodies (huLRB0091 and rbLRB0048). The two antibodies used were generated using expression vectors into which the variable region genes of LRG-specific antibodies obtained from rabbits immunized with purified recombinant human LRG protein were cloned and inserted.

As a result of measuring and comparing CRP protein in cord blood, 4 out of 7 neonates (57%) in the infected group were CRP negative (CRP < 0.2), and there was a difference between the infected group and the non-infected group. It was found that there was no significant difference in CRP protein concentration between (p=0.06) (Fig. 1). This suggests that using the concentration of CRP protein in cord blood alone is not useful for diagnosing neonatal or perinatal infections, and that other means are needed.

Then, similarly to CRP, LRG protein in cord blood was measured and compared. As a result, the LRG protein concentration in the infected group was found to be significantly higher than that in the non-infected group (p<0.01). (Fig. 1). In addition, the LRG protein concentration in cord blood serum of uninfected neonates was 3.47±0.72 μg/ml. It was found that neonatal or perinatal infections can be successfully diagnosed based solely on the concentration of LRG protein in cord blood without invasive neonatal blood sampling.

(Example 2 ROC curve)
Furthermore, using the data obtained in the above measurements, ROC curve analysis was performed to determine the optimal cutoff values for CRP protein and LRG protein. ROC curves were created and analyzed using Excel Statistics 2012 (Social Information Service Co., Ltd.). The point closest to the upper left corner was used as the cutoff value in the ROC curve analysis. As a result, CRP protein had an optimal cut-off value of 0.12 mg/dl, with sensitivity and specificity of 71.4% and 57.1%, respectively, and an area under the curve (AUC) of 0.6327. It turned out to be LRG protein, on the other hand, has an optimal cut-off value of 5.0 μg/ml (the maximum value for cord blood LRG without infection was 4.9), at which time the sensitivity and specificity are 100% and the AUC was found to be 1.000. This indicates that the diagnosis of postnatal infections is more effective based on the cord blood LRG protein than on the cord blood CRP protein (Fig. 2).

(Example 3 LRG mRNA concentration)
Total RNA is extracted from the cord blood specimens of the infected group and the non-infected group obtained in Example 1 using RNeasy Mini Kit (QIAGEN). The resulting total RNA was reverse transcribed using ReverTraAce (registered trademark) (Toyobo) to prepare cDNA, and qRT-PCR was performed using primers that specifically bind to each LRG and SYBR (registered trademark) Green I. , quantify the amount of LRG mRNA in the sample. Comparing the LRG mRNA levels in the infected group and the non-infected group, it can be seen that the LRG mRNA level in the infected group is significantly higher than in the non-infected group.

(Example 4 Usefulness of markers in pregnant women)
In this example, an example was conducted to confirm the usefulness of the marker in pregnant women.

Blood collected from pregnant women with CAM complications (n = 9) and pregnant women without CAM complications (n = 10) who underwent perinatal management at Osaka University Hospital, blood from healthy volunteers (n = 6) ), the serum LRG protein concentration was measured by ELISA. The same measurement method as in Example 1 was used.

This example was carried out with the approval of the ethics committee of each institution.

(result)
As a result, maternal serum LRG protein levels with CAM complications (26.8±13.1 μg/ml) were significantly higher than those without CAM complications (11.4±2.0 μg/ml). (p=0.017) (Fig. 3).

As a result, it was found that the diagnosis of CAM complications in pregnant women can also be successfully determined based on the LRG protein concentration in the maternal serum.

(Example 5 urine sample LRG protein concentration)
Newborns on the day of birth, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth, etc. Alternatively, the LRG protein concentration in urine specimens obtained from pregnant women who underwent perinatal management is measured by the same method as in Example 1. Comparing the LRG protein concentration in the specimens of the infected group and the non-infected group, it can be seen that the LRG protein concentration of the infected group is significantly higher than that of the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal infections or perinatal infections can be determined based on LRG protein concentrations in urine specimens obtained from neonates, such as those in the United States, or pregnant women undergoing perinatal management.

(Example 6 urine sample LRG mRNA concentration)
Total RNA is extracted from the urine sample obtained in Example 5 using RNeasy Mini Kit (QIAGEN). The resulting total RNA was reverse transcribed using ReverTraAce (registered trademark) (Toyobo) to prepare cDNA, and qRT-PCR was performed using primers that specifically bind to each LRG and SYBR (registered trademark) Green I. , quantify the amount of LRG mRNA in the sample. Comparing the LRG mRNA levels in the infected group and the non-infected group, it can be seen that the LRG mRNA level in the infected group is significantly higher than in the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal infections or perinatal infections can be determined based on LRG mRNA concentrations in urine specimens obtained from neonates such as those in the United States, or from pregnant women who underwent perinatal management.

(Example 7 saliva sample LRG protein concentration)
Newborns on the day of birth, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth, etc. Alternatively, LRG protein concentrations in saliva specimens obtained from pregnant women who underwent perinatal management are measured in the same manner as in Example 1. Comparing the LRG protein concentration in the specimens of the infected group and the non-infected group, it can be seen that the LRG protein concentration of the infected group is significantly higher than that of the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal infections or perinatal infections can be determined based on LRG protein concentrations in saliva specimens obtained from neonates such as those in the United States, or pregnant women undergoing perinatal management.

(Example 8 saliva sample LRG mRNA concentration)
Total RNA is extracted from the saliva sample obtained in Example 7 using RNeasy Mini Kit (QIAGEN). The resulting total RNA was reverse transcribed using ReverTraAce (registered trademark) (Toyobo) to prepare cDNA, and qRT-PCR was performed using primers that specifically bind to each LRG and SYBR (registered trademark) Green I. , quantify the amount of LRG mRNA in the sample. Comparing the LRG mRNA levels in the infected group and the non-infected group, it can be seen that the LRG mRNA level in the infected group is significantly higher than in the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal infections or perinatal infections can be determined based on LRG mRNA concentrations in saliva specimens obtained from neonates such as those in the United States, or pregnant women undergoing perinatal management.

(Example 9 fecal sample LRG protein concentration)
Newborns on the day of birth, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth, etc. Alternatively, the LRG protein concentration in fecal specimens obtained from pregnant women who underwent perinatal management is measured in the same manner as in Example 1. Comparing the LRG protein concentration in the specimens of the infected group and the non-infected group, it can be seen that the LRG protein concentration of the infected group is significantly higher than that of the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal or perinatal infections can be determined based on LRG protein concentrations in stool specimens obtained from neonates such as those with perinatal care, or from pregnant women undergoing perinatal management.

(Example 10 fecal sample LRG mRNA concentration)
Total RNA is extracted from the stool sample obtained in Example 9 using RNeasy Mini Kit (QIAGEN). The resulting total RNA was reverse transcribed using ReverTraAce (registered trademark) (Toyobo) to prepare cDNA, and qRT-PCR was performed using primers that specifically bind to each LRG and SYBR (registered trademark) Green I. , quantify the amount of LRG mRNA in the sample. Comparing the LRG mRNA levels in the infected group and the non-infected group, it can be seen that the LRG mRNA level in the infected group is significantly higher than in the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal or perinatal infections can be determined based on LRG mRNA concentrations in stool specimens obtained from neonates such as E. M. et al., or pregnant women undergoing perinatal management.

(Example 11 sweat sample LRG protein concentration)
Newborns on the day of birth, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth, etc. Alternatively, LRG protein concentrations in sweat samples obtained from pregnant women who underwent perinatal management are measured in the same manner as in Example 1. Comparing the LRG protein concentration in the specimens of the infected group and the non-infected group, it can be seen that the LRG protein concentration of the infected group is significantly higher than that of the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal infections or perinatal infections can be determined based on LRG protein concentrations in sweat specimens obtained from neonates such as those in the United States, or from pregnant women undergoing perinatal management.

(Example 12 sweat sample LRG mRNA concentration)
Total RNA is extracted from the sweat sample obtained in Example 11 using RNeasy Mini Kit (QIAGEN). The resulting total RNA was reverse transcribed using ReverTraAce (registered trademark) (Toyobo) to prepare cDNA, and qRT-PCR was performed using primers that specifically bind to each LRG and SYBR (registered trademark) Green I. , quantify the amount of LRG mRNA in the sample. Comparing the LRG mRNA levels in the infected group and the non-infected group, it can be seen that the LRG mRNA level in the infected group is significantly higher than in the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal or perinatal infections can be determined based on LRG mRNA concentrations in sweat specimens obtained from neonates such as those in the United States, or pregnant women undergoing perinatal management.

(Example 13 Blood LRG protein concentration obtained from newborns)
Newborns on the day of birth, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth, etc. LRG protein concentration in the blood body obtained from is measured by the same method as in Example 1. Comparing the LRG protein concentration in the specimens of the infected group and the non-infected group, it can be seen that the LRG protein concentration of the infected group is significantly higher than that of the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal infections or perinatal infections can be determined based on the LRG protein concentration in blood samples obtained from newborns such as.

(Example 14 Blood LRG mRNA concentration obtained from newborns)
Total RNA is extracted from the blood sample obtained in Example 13 using RNeasy Mini Kit (QIAGEN). The resulting total RNA was reverse transcribed using ReverTraAce (registered trademark) (Toyobo) to prepare cDNA, and qRT-PCR was performed using primers that specifically bind to each LRG and SYBR (registered trademark) Green I. , quantify the amount of LRG mRNA in the sample. Comparing the LRG mRNA levels in the infected group and the non-infected group, it can be seen that the LRG mRNA level in the infected group is significantly higher than in the non-infected group. Birth day, 12 hours after birth, 1 day after birth, 36 hours after birth, 2 days after birth, 3 days after birth, 4 days after birth, 5 days after birth, 6 days after birth, 7 days after birth It can be seen that neonatal infections or perinatal infections can be determined based on LRG mRNA levels in blood specimens obtained from newborns such as.

(Example 15 Umbilical cord blood LRG in babies with funisitis is significantly elevated)
In this example, the relationship between the presence or absence of funisitis and cord blood LRG was investigated. The presence or absence of funisitis and the presence or absence of infection in neonates were investigated by pathologically post-diagnosing the presence or absence of funisitis (=fetal infection). A schematic is shown in FIG.

(Methods and materials)
LRG and CRP in umbilical cord blood were measured in neonates born from pregnant women who had been followed up at Osaka University Hospital and Kagoshima Municipal Hospital. The measurement method was in accordance with Example 1.

Newborns born from mothers with chorioamnionitis were divided into two groups based on the presence or absence of fetal infection based on the histopathological presence or absence of funisitis. The control group consisted of cases in which neither mother nor child was infected.

(result)
The results are shown in FIG. As shown in FIG. 5, among neonates born from mothers with chorioamnionitis, LRG was significantly elevated only in the group in which funisitis was observed. As for CRP, a significant increase was also seen in the group with funisitis, but a clear increase was seen in only a few cases.

(Example 16 ROC curve)
Next, in the present Example, an experiment was conducted to demonstrate that LRG is more useful as a marker for meningitis than CRP.

(material and method)
ROC curves were performed according to the method described in Example 2 (Example 1 if necessary).

(result)
The results are shown in FIG. As shown in Fig. 6, ROC analysis was performed to compare the diagnostic performance of LRG and CRP for meningitis in neonates born to mothers with chorioamnionitis. LRG was clearly more diagnostic than CRP.

(Example 17 Obstetric complications and LRG)
In this example, the relationship between obstetric complications and LRG was investigated.

(Methods and materials)
In this example, details of the control group shown in FIG. 5 are provided. In this group, in addition to normal cases without obstetric complications, preterm birth, fetal growth restriction (FGR), gestational hypertension syndrome, and fetal dysfunction as obstetric complications that cause low birth weight in the baby not due to infection. (NRFS).

This is the same group as the control group in FIG. 50 cases in total). In other words, this measurement result showed the breakdown of the group (control group) in which no infection findings were observed in both mother and child.

(result)
The results are shown in FIG. As shown in Figure 7, no significant increase in LRG was observed in any of the control groups. From these results, we were able to show that LRG can detect cases in which fetuses (newborns) themselves have infections among various obstetric complications that cause low birth weight in newborns. As a result, it can be said that appropriate treatment can be given to newborns with antibiotics and the like.

(annotation)
Although the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments may vary. The present invention contemplates that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.

The contents of all publications, including patents and patent applications mentioned herein, are hereby incorporated by reference to the same extent as if expressly set forth in their entirety. . This application claims priority to Japanese Patent Application No. 2017-126646, and the contents described in the same application are hereby incorporated by reference to the same extent as if all of them were explicitly stated. It is incorporated into the book.

The present invention provides for the alternative use of LRG as a biomarker for neonatal or perinatal infections and to test whether a subject using LRGs has neonatal or perinatal infections. Alternatively, diagnostic methods, kits, test or diagnostic agents and test or diagnostic systems are provided. The use of LRG as a biomarker enables early detection and early treatment of neonatal infections or perinatal infections, which have been difficult to date. In addition, this biomarker can be used to determine the efficacy of treatment for subjects with neonatal or perinatal infections, thereby allowing treatment regimens to be selected.

Claims (16)

A test agent, diagnostic agent or kit or system for testing or diagnosing neonatal infections or perinatal infections, comprising reagents or means for detecting or measuring LRG gene products or degradation products thereof. Reagents or means for detecting or measuring LRG gene products or degradation products thereof are nucleic acids, proteins, or antibodies or binding fragments thereof that specifically bind to said LRG gene products or degradation products thereof, as well as NMR, mass spectrometry, autoradio 2. The test agent, diagnostic agent or test according to claim 1, selected from the group consisting of devices for performing lithography, chromatography, Southern blot, Northern blot, Western blot, LTIA, ELISA, RA, ECLIA and electrophoresis, or A kit or system for diagnosis. a reagent or means for detecting or measuring the LRG gene product or its degradation product,
(i) a nucleic acid probe or nucleic acid primer that specifically binds to a transcript of an LRG gene or a precursor or breakdown product thereof, or (ii) an antibody that specifically binds to an LRG protein or a precursor or breakdown product thereof or binding thereof 2. A test agent, diagnostic agent or kit or system for testing or diagnosis according to claim 1, comprising a sex fragment. The test according to any one of claims 1 to 3, wherein the neonatal infection or perinatal infection is an infection of a fetus or newborn at 22 weeks of gestation to 4 weeks of age, or its mother, or both. diagnostic agents or kits or systems for testing or diagnosis. The test agent, diagnostic agent, or method for testing or diagnosis according to claim 4, wherein the neonatal infectious disease or perinatal infectious disease is an infectious disease of a fetus or a newborn from 22 weeks of gestation to 7 days after birth, or its mother. kit or system. The test agent, diagnostic agent, or method for testing or diagnosis according to claim 4, wherein the neonatal infectious disease or perinatal infectious disease is an infectious disease of a fetus or a newborn or its maternal body from 22 weeks of gestation to 2 days after birth. kit or system. The test agent, diagnostic agent, or method for testing or diagnosis according to claim 4, wherein the neonatal infectious disease or perinatal infectious disease is an infectious disease of a fetus or a newborn or its maternal body from 22 weeks of gestation to 1 day after birth. kit or system. The test agent, diagnostic agent or kit or system for testing or diagnosis according to any one of claims 4 to 7, wherein the LRG gene product or degradation product thereof is present in the body fluids of the newborn or its maternal body. . said bodily fluids include said maternal blood, lymphatic fluid, tissue fluid, coelomic fluid, cerebrospinal fluid, synovial fluid, aqueous humor, digestive juices, sweat, tears, nasal discharge, urine, feces and vaginal fluids, and umbilical cord blood of said newborn; 9. The test of claim 8, wherein the test is selected from the group consisting of blood, lymph, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, aqueous humor, digestive fluid, sweat, tears, nasal discharge, urine, feces and vaginal fluid. agents, diagnostic agents or kits or systems for testing or diagnosis. 10. The test agent, diagnostic agent or kit or system for testing or diagnosis according to claim 9, wherein said bodily fluid comprises said maternal blood or said cord blood, or both. Said neonatal or perinatal infection includes intrauterine infection, chorioamnionitis, meningitis, neonatal conjunctivitis, neonatal bacterial meningitis, congenital and perinatal cytomegalovirus infection, neonatal type B Hepatitis virus infection, neonatal hepatitis C virus infection, human T-cell leukemia virus infection, chlamydia infection, group B streptococcal infection, neonatal pneumonia, congenital rubella, neonatal herpes simplex virus infection, neonatal sepsis, congenital syphilis , neonatal nosocomial infections, perinatal tuberculosis, congenital toxoplasmosis, chickenpox, HIV and neonatal listeriosis according to any one of claims 1 to 10. test agent, diagnostic agent or kit or system for testing or diagnosis. The test agent, diagnostic agent or test according to any one of claims 1 to 10, wherein the neonatal infection or perinatal infection includes intrauterine infection, chorioamnionitis, neonatal sepsis or neonatal pneumonia. Or a kit or system for diagnosis. Said examination or diagnosis includes neonatal infection or perinatal infection with at least one disease selected from the group consisting of preterm birth, fetal growth restriction (FGR), hypertension of pregnancy, and fetal dysfunction (NRFS). A test agent, a diagnostic agent or a kit or system for testing or diagnosis according to any one of claims 1 to 12, which is distinguishable. Use of the LRG gene product or its degradation product as an indicator for testing neonatal infections or perinatal infections , wherein the amount or level of the LRG gene product or its degradation product in a subject is normal. A use wherein a neonatal or perinatal infection is determined when the amount or level of the product or its breakdown products is higher. A method for testing for neonatal or perinatal infections in a subject, comprising measuring an LRG gene product or a degradation product thereof in the subject, the method comprising: A method wherein a neonatal or perinatal infection is determined when the amount or level is higher than the amount or level of the LRG gene product or breakdown product thereof in a normal individual. Use of reagents or means for detecting or measuring LRG gene products or degradation products thereof for testing neonatal or perinatal infections, wherein the amount or level of LRG gene products or degradation products thereof in a subject is A use wherein a neonatal or perinatal infection is determined when the amount or level of the LRG gene product or its degradation product is higher than that of a normal person.

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