JP2015072196A - DETECTION METHOD OF DISEASE AND THE LIKE BY MEASURING AUTOTAXIN ISOFORMS δ AND ε - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detection method and a detection agent by measuring autotaxin isoforms δ and ε for diseases such as a chronic liver disease and pregnancy capable of being detected by measuring autotaxin isoforms α, β, γ, δ and ε.SOLUTION: In a detection method of a disease and the like by measuring autotaxin isoforms δ AND ε, autotaxin isoforms δ AND ε in a specimen such as serum is measured by use of an immunochemical measurement method using antibody to detect diseases such as a chronic liver disease and pregnancy capable of being detected by measuring the autotaxin isoforms α, β, γ, δ and ε. Concentration of the autotaxin isoforms δ AND ε is measured and converted, so that concentration of the autotaxin isoforms α, β, γ, δ and ε is estimated.

Description

  The present invention relates to a detection method, a detection drug, and an estimation method for chronic liver diseases, diseases that can be detected by measuring the total concentration of autotaxin including pregnancy, and the like by measuring autotaxin isoforms δ and ε.

  Autotaxin is known to have several isoforms (isoforms, proteins with different structures but the same function). As an example, autotaxin isoforms α, β, γ, δ, ε in which splicing variants α, β, γ, δ, ε have been identified as genes (hereinafter abbreviated as ATXα, β, γ, δ, ε) (Non-Patent Documents 1 and 2).

  As a result of confirming the homology of the amino acid sequence of the isoform, ATXα, β and γ (hereinafter defined as ATXα, β, γ) are Val-Glu-Pro-Lys (SEQ ID NO: 1) in the L2 linker region. And ATXδ and ε (hereinafter defined as ATXδ and ε) have been found to lack the amino acid sequence of SEQ ID NO: 1 in the L2 linker region (Non-patent Document 2). ATXδ is the main isoform after ATXβ and has biochemical properties similar to ATXβ (Non-patent Document 1).

  Regarding the measurement of autotaxin, it is possible to quantitate ATXα, β, γ, δ and ε (hereinafter defined as the total amount of ATX) in human specimens such as human serum easily, in a short time, and with high reliability. Novel methods have been disclosed, and malignant lymphoma, fibrosis of the liver, pancreatic disease, steroid administration effect monitor, pregnancy and pregnancy hypertension syndrome can be detected (Patent Documents 1, 2, 3, 4, 5) However, the concentrations of ATXδ and ε in various diseases have not been known so far.

International Publication No. 2008/016186 Pamphlet International Publication No. 2011/081214 Pamphlet JP 2012-52875 A JP 2012-2588 A JP 2009-162616 A

J. et al. Biol. Chem. , 283, 7776, 2008 J. et al. Biochem. 151, 89-97, 2012

  Accordingly, an object of the present invention is to provide a detection method, a detection drug, and an estimation method for chronic liver diseases, diseases that can be detected by measuring the total amount of ATX such as pregnancy, and the like by measuring ATXδ and ε.

The present invention made in view of the above problems includes the following aspects:
(1) A method for detecting a disease or the like that can be detected by measuring the total amount of ATX, comprising measuring ATXδ and ε.
(2) The detection method according to (1), wherein the disease or the like is chronic liver disease.
(3) The detection method according to (1), wherein the disease or the like is malignant lymphoma.
(4) The detection method according to (1), wherein the disease or the like is pancreatic disease.
(5) The detection method according to (1), wherein the disease or the like is a steroid administration effect monitor.
(6) The detection method according to (1), wherein the disease or the like is pregnancy or pregnancy-induced hypertension syndrome.
(7) Any of (1) to (6), wherein ATXδ and ε are full-length ATXδ and ε, partially cut ATXδ and ε, or ATXδ and ε that have undergone partial gene mutation The detection method of crab.
(8) The detection method according to any one of (1) to (7), wherein the specimen is a blood component or a cell or tissue extract.
(9) The detection method according to any one of (1) to (8), wherein the method is an immunochemical method using an antibody.
(10) A drug for detecting a disease or the like that can be detected by measuring the total amount of ATX, which uses the detection method according to any one of (1) to (9).
(11) A method for estimating ATX total amount concentration, comprising measuring ATXδ and ε concentrations and converting the values.
(12) A method for estimating ATXδ and ε concentrations, wherein the ATX total amount concentration is measured and the value is converted.

  Hereinafter, the present invention will be described in detail.

  The present inventors focused on the ATX isoform and attempted to detect ATXδ and ε in human blood. As a result, in the patients with chronic liver disease, the value showed a significant difference with respect to the normal value consisting of healthy persons. In pregnant women, the value was significantly higher than the normal value consisting of measured values of healthy non-pregnant persons. In malignant lymphoma, the value shows a high value with a significant difference from the normal value of a healthy person. In non-cancer pancreatic disease patients, the value is lower than the normal value of healthy individuals, and in non-cancer pancreatic disease patients, the value is lower than in pancreatic cancer patients. The value is low due to steroid use. In gestational hypertension syndrome, the value is significantly lower than the measured value in the normal pregnancy group.

  ATXδ, ε to be measured includes not only full-length ATXδ, ε, but also partially cut ATXδ, ε, or ATXδ, ε subjected to partial gene mutation. Samples include blood components or cell or tissue extracts.

  In the present invention, the method for measuring ATXδ and ε is not limited as long as ATXδ and ε can be detected. Preferably, it is an immunochemical method in terms of specificity, sensitivity, versatility and the like.

  The anti-ATXδ, ε antibody used at this time is, for example, the amino acid sequence Cys-Asp-Asp-Lys-Asn-Lys-Leu-Asp-Glu-Leu-Asn present on human ATXδ, ε among the amino acid sequences of ATX. -Amino acid sequence Cys-Asp-Asp-Lys-Val-Glu- that specifically recognizes autotaxin including an amino acid sequence consisting of Lys-Arg-Leu (SEQ ID NO: 2) and exists on human ATX α, β, γ It does not recognize autotaxin containing an amino acid sequence consisting of Pro-Lys-Asn-Lys-Leu-Asp-Glu-Leu-Asn-Lys-Arg-Leu (SEQ ID NO: 3).

  Here, the amino acid sequence of SEQ ID NO: 2 is from an oligopeptide (SEQ ID NO: 3) consisting of amino acids 569 to 586 of human ATXβ (GenBank: AAB00855.1, SEQ ID NO: 4) to the fifth valine to 8 This corresponds to an oligopeptide in which amino acids up to the first lysine (that is, the amino acid sequence consisting of SEQ ID NO: 1) are deleted.

  Anti-ATXδ and ε antibodies can be obtained by immunizing animals such as mice, rats, rabbits, etc. with a peptide comprising the amino acid sequence of SEQ ID NO: 2.

  An autotaxin comprising an amino acid sequence consisting of SEQ ID NO: 2 by using an autotaxin measuring reagent comprising an anti-ATXδ, ε antibody and an antibody recognizing a region other than the amino acid sequence consisting of SEQ ID NO: 2 among the amino acid sequences of ATX That is, ATXδ, ε can be specifically detected by sandwich immunoassay.

  The detection method of the present invention can be used to detect a disease or the like that can be detected by measuring the total amount of ATX. In particular, the sandwich immunoassay method for ATXδ and ε as described above can quantitate ATXδ and ε in a short time without being affected by endogenous measurement interference factors or competing enzymes contained in the sample. It is possible to provide the detection agent of the present invention that can be easily and inexpensively diagnosed in a medical facility.

  In addition, ATX δ and ε concentrations had a significant positive correlation with the ATX total amount concentration regardless of the disease, and ATX δ and ε were present at a ratio of about 32% with respect to the ATX total amount. Therefore, the ATX total amount concentration can be estimated by dividing the ATX δ and ε concentrations by 0.32, and the ATX δ and ε concentrations can be estimated by multiplying the ATX total amount concentration by 0.32.

  By using the above method, ATXδ and ε can be quantified in a short time without being affected by endogenous measurement interfering factors and competing enzymes contained in the sample. It is possible to provide the detection agent of the present invention that can be diagnosed at cost. By measuring ATX δ and ε, chronic liver disease and pregnancy can be diagnosed in the same manner as ATX total amount measurement. In addition, since the ATX δ and ε concentrations show a good positive correlation with the ATX total amount concentration, and the ratio of the ATX δ and ε concentrations in the ATX total amount concentration is about 32%, which is almost constant regardless of the disease, By measuring ε, the total amount of ATX can be estimated and vice versa. Therefore, the measurement of ATXδ, ε can also be used for diagnosis of diseases associated with an increase or decrease in the blood concentration of the total amount of ATX, such as malignant lymphoma, pancreatic disease, steroid administration effect monitor, and pregnancy hypertension syndrome). I can expect.

It is a figure which shows ATX (delta) and (epsilon) Index of a healthy person (circle) and a chronic liver disease patient (square). It is a figure which shows ATX total amount Index of a healthy person (circle) and a chronic liver disease patient (square). It is a figure which shows ATX (delta) and (epsilon) density | concentration of a healthy female (circle) and a pregnant woman (square). It is a figure which shows the ATX total amount density | concentration of a healthy female (circle) and a pregnant woman (square). It is a figure which shows the correlation of ATX total amount density | concentration of a healthy person, and ATX (delta) and (epsilon) density | concentration. It is a figure which shows the correlation of ATX total amount density | concentration of a chronic liver disease patient, and ATX (delta) and (epsilon) density | concentration. It is a figure which shows the correlation of ATX total amount density | concentration of pregnant women, ATX (delta), and (epsilon) density | concentration. It is a figure which shows the ratio of ATX (delta) and (epsilon) density | concentration to ATX total amount density | concentration of a healthy person, a chronic liver disease patient, and a pregnant woman.

  Examples are shown below, but the present invention is not limited to the examples described in the Examples. In addition, the human sample used in the following experimental examples was carried out using a sample collected under informed consent. Moreover, the measurement of the total amount of ATX was carried out using an automatic immunoassay device AIA series (manufactured by Tosoh Corporation) based on the method of Patent Document 1 and the measurement of ATXδ and ε concentrations based on the following method.

Reference example:
The immunizing antigen was prepared by conjugating the synthesized peptide (SEQ ID NO: 2) with Image Keyhole Limetic Hemocyanin (PIERCE; Part No. 77153) according to the protocol. This immunizing antigen was used to immunize the hind paw using ether with Freund's complete adjuvant (DIFCO) under ether anesthesia. One month later, cervical lymph nodes and iliac lymph nodes were collected from rats, and B cells were collected and fused with myeloma cells to prepare hybridoma cells.

  For selection of clones, a screening peptide-BSA conjugate antigen was used, and a clone showing reactivity with the oligopeptide-BSA of SEQ ID NO: 2 and showing no reactivity with the oligopeptide-BSA of SEQ ID NO: 3 was selected. As a peptide-BSA conjugate antigen for screening, peptide-BSA (Bovine serum albumin) was prepared by conjugation according to the protocol using Image Bovine serum albumin (PIERCE; product number 77171). The cells in the screening positive wells were monoclonalized by the limiting dilution method and established as hybridomas.

  The anti-ATXδ and ε monoclonal antibodies used in the present invention are clone name ATXR4-127, and the anti-ATX monoclonal antibody (recognizing a region other than the amino acid sequence consisting of SEQ ID NO: 2 in the amino acid sequence of ATX) is disclosed in Patent Document 1. It is the clone name ATXR10.23 obtained by the described method.

  ATXδ and ε measurement reagents were prepared according to the method of Patent Document 1. The prepared reagent can be used with an automatic immunoassay device AIA series (manufactured by Tosoh Corporation). Specifically, the following method was used.

  Physically overnight at 30 ° C. so that the water-insoluble carrier (ethylene vinyl alcohol carrier having a particle diameter of about 1.5 mm kneaded with ferrite inside) anti-ATXδ, ε monoclonal antibody (ATXR4-127) is 100 ng / carrier. Then, the antibody-immobilized carrier was prepared by blocking with a 100 mM Tris buffer (pH 8.0) containing 1% BSA at 40 ° C. for 4 hours. A labeled antibody was prepared by binding an anti-ATX monoclonal antibody (ATXR10.23) to alkaline phosphatase using an alkaline phosphatase labeling kit-NH2 (Dojindo Laboratories, product number: LK12).

  After 12 antibody-immobilized carriers are placed in a magnetically permeable container (capacity 1.2 mL), a buffer solution containing 0.7 μg / mL labeled antibody (Tris buffer solution containing 3% BSA, pH 8.0) 50 μL was added to the container and freeze-dried to obtain a reagent for measuring ATXδ and ε. The ATX measuring reagent was hermetically sealed under nitrogen filling and stored at 4 ° C. until measurement.

  A standard product of known concentration ATXδ was prepared according to the method described in Patent Document 1. Specifically, the following method was used. ATX-free serum was prepared by adsorbing autotaxin in bovine serum using an ATX monoclonal antibody (ATXR10.23) binding column. By using recombinant ATXδ prepared using baculovirus and adding an appropriate concentration to serum not containing ATX, standard products of 5 concentrations (6 concentrations when serum containing no ATX was added) were prepared. The concentration of the standard product was determined using the ATX total amount determination reagent described in Patent Document 1.

  Recombinant ATXδ was prepared using baculovirus according to the method of Patent Document 1. A specific method is shown below. Among the autotaxin-t (Genbank accession number L46720), cDNA (SEQ ID NO: 5) consisting of nucleotides from the 60th to 2648th nucleotides of base number was introduced into the baculovirus transfer vector pFASTBac-HT (Invitrogen), A baculovirus plasmid for autotaxin (ATXβ) expression containing an amino acid sequence consisting of SEQ ID NO: 1 to which histidine was added was prepared. A polyhistidine-added autotaxin (ATXδ) expression plasmid containing no amino acid consisting of SEQ ID NO: 1, using an ATXβ expression baculovirus plasmid as a template, and using KOD-Plus-Mutageness Kit (Toyobo) A plasmid containing a polynucleotide (SEQ ID NO: 6) lacking the 1717th to 1728th nucleotide (corresponding to the 1776th to 1787th nucleotides of GenBank No. L46729) of the polynucleotide consisting of No. 5 was prepared. Recombinant baculovirus was prepared with the Bac-to-Bac system (Invitrogen) using the prepared plasmid. A culture supernatant containing ATXδ was prepared by infecting sf9 insect cells with the prepared baculovirus according to a conventional method. Purification of the expressed protein was performed according to JP 2011-037802. Specifically, the following method was used.

  Anti-autotaxin monoclonal antibody R10.7 (Patent Document 1) was bound to a HiTrap NHS-activated HP column (GE Healthcare Bioscience), and the ATXδ expression culture supernatant was loaded onto the column. Thorough washing was performed with TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). Bound ATXδ was eluted with 100 mM citrate buffer (pH 5.0).

Example 1: Quantification of ATXδ and ε concentrations in healthy human specimens Prior to measurement of patient specimens, ATXδ and ε concentrations in healthy human serum were measured as a control group. ATX δ and ε concentrations in the serum of 81 males (19-61 years old) and 52 females (19-50 years old) were measured using a fully automatic immunoassay apparatus.

  The results are shown in Table 1. The ATXδ and ε concentrations in the serum of women were clearly higher than those of men, and the Mann-Whitney U test showed a significant difference of P <0.001. Since it is necessary to consider the sex difference between men and women when measuring patient samples, the values obtained by dividing patient sample measured values by the 95th percentile upper limit of 0.369 mg / L and 0.489 mg / L for each gender “autotaxin δ, ε The following evaluation was carried out using values corrected for male-female differences by “index” (ATXδ, ε index = ATXδ, ε measured value / 95th percentile upper limit value).

実施例２：健常者検体のＡＴＸ全量濃度の定量
実施例１と同じ健常者検体中のＡＴＸ全量濃度を全自動免疫測定装置にて実施した。

Example 2: Quantification of ATX total amount concentration of healthy subject specimen The ATX total amount concentration in the same healthy subject specimen as in Example 1 was carried out by a fully automatic immunoassay device.

  The results are shown in Table 2. The total amount of ATX in female specimens was clearly higher than that of males, and Mann-Whitney U test showed a significant difference of P <0.001. Because it is necessary to consider gender differences when measuring patient samples, the value obtained by dividing the measured values of patient samples by the 95th percentile upper limit of 1.013 mg / L and 1.354 mg / L for each gender “Autotaxin Total Index” The following evaluation was performed using a value corrected for male and female differences in (ATX total amount index = ATX total amount measured value / 95th percentile upper limit value).

実施例３：慢性肝疾患患者検体のＡＴＸδ、ε濃度とＡＴＸ全量濃度の定量
慢性肝疾患患者血清のＡＴＸδ、ε濃度を健常人と比較した。比較のために、ＡＴＸ全量濃度の測定を行なった。結果を表３（男性及び女性のＡＴＸδ、ε濃度）、表４（性差を考慮したＡＴＸδ、ε Ｉｎｄｅｘ）、表５（男性及び女性のＡＴＸ全量濃度）、表６（性差を考慮したＡＴＸ全量 Ｉｎｄｅｘ）、図１（性差を考慮したＡＴＸδ、ε Ｉｎｄｅｘ）および図２（性差を考慮したＡＴＸ全量 Ｉｎｄｅｘ）に示す。

Example 3: Quantification of ATX δ, ε concentration and ATX total concentration of chronic liver disease patient specimens ATX δ, ε concentrations of serum of chronic liver disease patients were compared with healthy individuals. For comparison, the total ATX concentration was measured. The results are shown in Table 3 (Male and female ATXδ, ε concentrations), Table 4 (ATXδ, ε Index considering gender differences), Table 5 (Male and female ATX total concentrations), and Table 6 (ATX total amounts Index considering gender differences). ), FIG. 1 (ATXδ, ε Index considering gender difference) and FIG. 2 (ATX total amount Index considering gender difference).

  ATX δ, ε concentration and ATX δ, ε Index were confirmed to be significantly higher in patients with chronic liver disease as well as ATX total concentration and ATX total index.

実施例４：妊婦検体におけるＡＴＸ全量濃度とＡＴＸδ、ε濃度
妊婦血清のＡＴＸδ、ε濃度を、健常人女性と比較した。また、比較のために、ＡＴＸ全量濃度の測定を行なった。結果を表７（ＡＴＸδ、ε濃度）、表８（ＡＴＸ全量濃度）、図３（ＡＴＸδ、ε濃度）および図４（ＡＴＸ全量濃度）に示す。

Example 4: ATX total amount concentration and ATX δ, ε concentration in pregnant woman specimens ATX δ, ε concentration of pregnant woman serum was compared with healthy women. For comparison, the total ATX concentration was measured. The results are shown in Table 7 (ATX δ, ε concentration), Table 8 (ATX total amount concentration), FIG. 3 (ATX δ, ε concentration) and FIG. 4 (ATX total amount concentration).

  It was confirmed that the ATXδ and ε concentrations were significantly higher in pregnant women, as was the ATX total concentration.

実施例５：ＡＴＸδ、ε濃度とＡＴＸ全量濃度の相関
健常人検体、慢性肝疾患患者検体、妊婦検体におけるＡＴＸ全量濃度を横軸に、ＡＴＸδ、ε濃度を縦軸にしてプロットし、相関関係を確認した。結果を図５（健常人）、図６（慢性肝疾患患者）、図７（妊婦）に示す。

Example 5: Correlation between ATX δ, ε concentration and ATX total amount concentration Plotting ATX total amount concentration in healthy human specimen, chronic liver disease patient specimen, pregnant woman specimen on the horizontal axis and ATX δ, ε concentration on the vertical axis confirmed. The results are shown in FIG. 5 (healthy person), FIG. 6 (chronic liver disease patient), and FIG. 7 (pregnant woman).

  As shown in FIGS. 5, 6, and 7, ATXδ, ε concentrations and ATX total amount concentrations showed good positive correlation among healthy subjects, patients with chronic liver diseases, and pregnant women.

  Table 9 shows the results of the ratio of ATXδ, ε concentration (ATXδ, ε concentration (mg / L) / ATX total amount concentration (mg / L)) in the total amount of ATX in healthy subjects, patients with chronic liver disease, and pregnant women. As shown in FIG. The average ratios of ATXδ and ε concentrations in the total ATX concentration were 0.317 for healthy subjects, 0.318 for samples of patients with chronic liver disease, and 0.349 for pregnant women. From these facts, the ratio of ATX δ and ε concentration to the total amount of ATX is 0.32, which is almost constant regardless of the disease. Therefore, the concentration of ATX δ is estimated by dividing ATX δ and ε concentration by 0.32. The ATX δ and ε concentrations can be estimated by multiplying the total ATX concentration by 0.32.

Claims (12)

A method for detecting diseases and the like that can be detected by measuring autotaxin isoforms α, β, γ, δ, and ε, characterized by measuring autotaxin isoforms δ and ε. The detection method according to claim 1, wherein the disease or the like is chronic liver disease. The detection method according to claim 1, wherein the disease or the like is malignant lymphoma. The detection method according to claim 1, wherein the disease or the like is pancreatic disease. The detection method according to claim 1, wherein the disease or the like is a steroid administration effect monitor. The detection method according to claim 1, wherein the disease or the like is pregnancy or pregnancy hypertension syndrome. Autotaxin isoforms δ and ε are full-length autotaxin isoforms δ and ε, partially cleaved autotaxin isoforms δ and ε, or autotaxin isoform δ that has undergone partial gene mutation And ε, according to any one of claims 1 to 6. The detection method according to claim 1, wherein the specimen is a blood component or a cell or tissue extract. The detection method according to claim 1, wherein the method is an immunochemical method using an antibody. A detection agent for diseases and the like that can be detected by measuring autotaxin isoforms α, β, γ, δ, and ε, using the detection method according to claim 1. A method for estimating autotaxin isoform α, β, γ, δ and ε concentration, characterized by measuring autotaxin isoform δ and ε concentrations and converting the values. A method for estimating autotaxin isoform δ and ε concentrations, comprising measuring autotaxin isoforms α, β, γ, δ, and ε concentrations and converting the values.

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