JP2021085883A - Method for collecting data for determining whether subject has biliary atresia and diagnostic kit for biliary atresia - Google Patents

Abstract

To provide a technique for noninvasively and simply determining whether a subject has biliary atresia.SOLUTION: A method for collecting data for determining whether a subject has biliary atresia includes a detection step for detecting the presence of a marker protein in the feces of the subject. When the marker protein is one kind of more proteins selected from proteins of a first group and a second group and the marker protein is any of the proteins of the first group, the amount of presence of the marker protein being significantly higher than the amount of presence in the feces of comparison or a set threshold indicates that the subject has biliary atresia, and when the marker protein is any of proteins of the second group, the amount of the presence of the marker protein being significantly lower than the amount of the presence of the feces of the comparison or the set threshold indicates that the subject has biliary atresia.SELECTED DRAWING: None

Description

The present invention relates to a method for collecting data for determining whether or not a subject has biliary atresia and a kit for diagnosing biliary atresia.

Biliary atresia is an intractable disease that develops from the neonatal period to the early infancy, and is a disease in which bile cannot be excreted into the intestinal tract. Successful surgery to clear the bile duct obstruction (Kasai surgery) improves jaundice, but if the surgery is unsuccessful, it progresses to biliary cirrhosis and further liver failure. Liver transplantation is the only life-saving measure for children with liver failure (see, for example, Non-Patent Document 1).

Diagnosis of biliary atresia is complicated, and it is difficult to determine it as another disease presenting with jaundice, which ultimately requires intraoperative cholangiography. Therefore, there is a need for a non-invasive and simple technique for determining whether a child has biliary atresia or a child who has jaundice and does not have biliary atresia.

Jane L Hartley, et al., Biliary atresia, Lancet, 374 (9702), 1704-1713, 2009.

An object of the present invention is to provide a technique for easily and non-invasively determining whether or not a subject has biliary atresia.

The present invention includes the following aspects.
[1] A method for collecting data for determining whether or not a test child has biliary tract obstruction, which comprises a detection step of detecting the presence of a marker protein in the feces of the test child. One or more proteins selected from the group consisting of the proteins listed in Table 1-1 below, Table 1-2 below, Table 1-3 below, Table 2-1 below and Table 2-2 below. When the marker protein is any of the proteins listed in Table 1-1 below, Table 1-2 below, and Table 1-3 below, the abundance of the marker protein is the control stool. Significantly higher than the threshold set based on the abundance in the stool or the abundance in the control stool indicates that the subject suffered from biliary atresia, and the marker protein. In the case of any of the proteins listed in Table 2-1 below and Table 2-2 below, the abundance of the marker protein is based on the abundance in the control stool or the abundance in the control stool. A method of indicating that the subject suffers from biliary atresia if it is significantly lower than the threshold set in.

[2] The method according to [1], wherein the control is a healthy child, a child having jaundice symptoms and not suffering from biliary atresia, or a child after surgical treatment of biliary atresia.

[3] The method according to [1] or [2], wherein the detection step is performed by an enzyme immunoassay method or an immunochromatography method.

[4] A kit for diagnosing biliary atresia, which contains a specific binding substance to a marker protein, wherein the marker protein is shown in [1], Table 1-1, Table 1-2, Table 1-3, and so on. One or more proteins selected from the group consisting of the proteins shown in Table 2-1 and Table 2-2, wherein the specific binding substance detects the presence of the marker protein in the stool of a test child. When the marker protein is any of the proteins listed in Table 1-1, Table 1-2, and Table 1-3, the abundance of the marker protein is used. However, significantly higher than the abundance in the control stool or the threshold set based on the abundance in the control stool indicates that the subject suffered from biliary atresia. When the marker protein is any of the proteins listed in Table 2-1 and Table 2-2, the abundance of the marker protein is the abundance in the control stool or the control stool. A kit in which a significantly lower threshold compared to a threshold set based on the abundance of the subject indicates that the subject has biliary atresia.

[5] The kit according to [4], wherein the control is a healthy child, a child having jaundice symptoms and not suffering from biliary atresia, or a child after surgical treatment of biliary atresia.

[6] The kit according to [4] or [5], wherein the specific binding substance is placed on a plate for enzyme immunoassay or on a membrane for immunochromatography.

[7] The kit according to any one of [4] to [6], wherein the specific binding substance is an antibody.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a technique for easily and non-invasively determining whether or not a subject has biliary atresia.

In Experimental Example 2, it is a heat map showing the relative abundance of a typical protein in the feces of a biliary atresia patient and a non-biliary atresia patient.

[How to collect data]
In one embodiment, the invention is a method of collecting data for determining whether a subject has biliary atresia and detects the presence of a marker protein in the stool of the subject. Including the detection step, the marker protein is selected from the group consisting of the proteins listed in Table 1-1, Table 1-2, Table 1-3, Table 2-1 and Table 2-2. If the marker protein is one or more of the proteins listed in Table 1-1, Table 1-2, and Table 1-3, the presence of the marker protein. Significantly higher amounts compared to control stool abundance or thresholds set based on control stool abundance indicate that the subject suffered from biliary atresia. When the marker protein is any of the proteins listed in Table 2-1 and Table 2-2, the abundance of the marker protein is the abundance in the control stool or the control stool. Significantly lower than the threshold set based on the abundance in the subject provides a method of indicating that the subject suffers from biliary atresia.

As will be described later in the Examples, the inventors will provide stools for children with biliary atresia, children with jaundice and not with biliary atresia, and children after surgical treatment for biliary atresia. Comprehensive protein analysis was performed as a sample, and by comparing these, proteins that are markers of biliary atresia were identified.

Table 1-1, Table 1-2, Table 1-3, Table 2-1 and Table 2-2 show the representative biliary atresia marker proteins identified by the inventors this time. In these tables, "Uniprot ID" indicates the accession number in the public database UniProt Knowledgebase (https://www.uniprot.org/), and "gene name" indicates the name of the gene encoding the protein. Shown.

Since the stool can be collected non-invasively, it is possible to easily determine whether or not the subject has biliary atresia by the method of the present embodiment.

In the method of the present embodiment, the control includes a healthy child, a child having jaundice symptoms and not suffering from biliary atresia, or a child after surgical treatment of biliary atresia.

In the method of the present embodiment, the step of detecting the marker protein in stool is preferably performed by an enzyme immunoassay method or an immunochromatography method. The enzyme-linked immunosorbent assay (ELISA method) may be performed using a commercially available kit.

The immunochromatography method is also called a lateral flow immunoassay, a strip test, or the like. The target protein in the sample introduced into the sample inlet of the assay device advances while soaking into the membrane by capillary force, and the conjugate bound to the secondary antibody impregnated in the membrane in advance serves as a judgment line on the membrane. It binds to the immobilized antibody, is captured in-situ, and is visualized and detected as a line. The secondary antibody that is not bound to the target protein passes through the determination line as it is and flows to the next control line, where it is visualized as a control line, indicating that the assay was performed normally.

In the detection step, the absolute amount of the marker protein in the feces of the test child may be quantified, or the relative abundance of the marker protein may be detected without quantification. By the detection step of the method of the present embodiment, the abundance of the marker protein in the stool of the subject is compared with the threshold set based on the abundance of the marker protein in the control stool or the abundance in the control stool. It suffices if it can be determined whether the amount is large or small.

In the case of the immunochromatography method, the abundance of the marker protein can be detected, for example, by visually measuring the coloration concentration of the visualized determination line or by measuring with a measuring instrument such as a densitometer. In the case of the ELISA method, the absolute amount of marker protein can be quantified from the standard curve.

In addition, it is not necessary to simultaneously detect the marker protein in the control-derived stool sample each time the step of detecting the marker protein in the stool sample of the test child is performed, and the presence of the marker protein in the control stool measured in advance. A threshold of the marker protein in the control stool may be set based on the amount and compared with the threshold to determine whether or not the subject has biliary atresia.

[Diagnosis kit for biliary atresia]
In one embodiment, the present invention is a kit for diagnosing biliary atresia, which comprises a specific binding substance to a marker protein, wherein the marker protein is the above Table 1-1, the above Table 1-2, the above Table 1. -3, one or more proteins selected from the group consisting of the proteins listed in Table 2-1 and Table 2-2 above, and the specific binding substance is the marker protein in the stool of a test child. When the marker protein is any of the proteins listed in Table 1-1, Table 1-2, and Table 1-3, the above is used to detect the presence of the above. The above-mentioned subject suffering from biliary atresia that the abundance of the marker protein is significantly higher than the abundance in the control stool or the threshold set based on the abundance in the control stool. When the marker protein is any of the proteins listed in Table 2-1 and Table 2-2, the abundance of the marker protein is the abundance in the control stool. Alternatively, a kit is provided that indicates that the subject suffers from biliary atresia if it is significantly lower than the threshold set based on the abundance in the control stool. The kit of the present embodiment can preferably carry out the above-mentioned method of collecting data for determining whether or not a subject has biliary atresia.

Specific binding substances include, for example, antibodies, antibody fragments, aptamers and the like. The antibody may be prepared by immunizing an animal such as a mouse, or may be prepared by screening an antibody library such as a phage library. Examples of the antibody fragment include F (ab') 2, Fab', Fab, Fv, scFv and the like. The aptamer is not particularly limited as long as it has a specific binding ability to the target substance, and examples thereof include nucleic acid aptamers and peptide aptamers. The specific binding agent may be an antibody.

In the kit of the present embodiment, the control includes a healthy child, a child having jaundice symptoms and not suffering from biliary atresia, or a child after surgical treatment of biliary atresia.

In the kit of this embodiment, the specific binding substance may be placed on a plate for enzyme immunoassay or a membrane for immunochromatography.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Materials and methods]
(patient)
This study was a retrospective observational study analyzing proteins in the stool of biliary atresia patients (hereinafter sometimes referred to as "BA patients") and non-BA patients. This study was approved by the institutional review committee of the University of Tokyo School of Medicine (IRB No. 20109010NI), and informed consent was obtained from all subjects.

Kasai: Four BA patients and three non-BA patients prior to surgery were recruited for stool proteome analysis. Stools spontaneously defecated by the patient were stored at -80 ° C.

All patients with BA were classified as type III (obstruction of the most proximal part of the extrahepatic biliary tract with hilar vein occlusion). Each of the three non-BA patients suffered from neonatal intrahepatic cholestasis caused by citrine deficiency, cholestasis after repair of abdominal wall rupture, and cholestasis after repair of venous obstruction.

Table 3 shows the clinical characteristics of the subjects in this study. In Table 3, "BA" indicates bilirubin obstruction, "NICCD" indicates intrahepatic cholestasis caused by citrine deficiency, "GS" indicates gastric wall rupture, and "VOD" indicates venous obstruction. , "AST" indicates aspartate aminotransferase (U / L), "ALT" indicates alanine aminotransferase (U / L), "T-Bil" indicates total bilirubin (mg / dL), "D-Bil" directly indicates bilirubin (mg / dL), "GGTP" indicates γ-glutamyl transpeptidase (U / L), and "Non-BA" indicates cholestasis other than biliary atresia. "Age" indicates the number of days after birth.

(Proteome analysis)
Soluble proteins in stool prepared in phosphate buffered saline (PBS) containing protease inhibitors were incubated on ice for 30 minutes and then extracted by pipetting and inversion.

After removing insoluble substances by centrifugation at 15,000 xg at 4 ° C. for 15 minutes, the supernatant is transferred to a new tube, trichloroacetic acid precipitates (final concentration 12.5% v / v), and then acetone. After washing with, the lid of the container was opened and dried.

The dried sample is redissolved in 0.5% sodium dodecanoate and 100 mM Tris-HCl (pH 8.5) using a water bath sonicator (product name "Biooptor UCD-200", Sonic Bio Co., Ltd.). did.

Subsequently, the total protein amount was measured by the biciconic acid assay (BCA assay), and the protein concentration was adjusted to 1 mg / mL. To 20 μL of the prepared sample, 2 μL of 110 mM dithiothreitol was added, and the mixture was allowed to stand at 50 ° C. for 30 minutes to reduce the disulfide bond of the protein. Further, 2 μL of 360 mM iodoacetamide was added, and the mixture was allowed to stand at room temperature for 30 minutes to alkylate the cysteine side chain.

Then, 2 μL of 780 mM cysteine was added, and the mixture was allowed to stand at room temperature for 10 minutes to stop the reaction of iodoacetamide. Next, 150 μL of 50 mM ammonium bicarbonate was added, and 2 μL of each of 0.25 mg / mL Lys-C and 0.25 mg / mL trypsin was added, and the mixture was allowed to stand at 37 ° C. for 15 hours to digest the protein. 30 μL of 5% trifluoroacetic acid (TFA) was added to the digested product to precipitate sodium dodecanoate, which inhibits LC-MS analysis, and 15000 × g was centrifuged for 10 minutes, and only the supernatant was recovered. ..

The collected sample was desalted by a C18 spin column, the eluate was completely dried by a centrifugal evaporator, and then redissolved in 20 μL of 3% acetonitrile-0.1% formic acid, and a liquid chromatography mass spectrometer (LC / MS) was used. ) Analyzed 1 μL. For LC / MS LC, UltraMate 3000 RSLCnano System manufactured by Thermo Fisher Scientific Co., Ltd. is used, the flow velocity is 100 nL / min, and the column has an inner diameter of 75 μm ID × 250 mm and C18 2.6 um core shell particles (manufactured by Osaka Soda Co., Ltd.). I used the packed one. For MS, Q Active HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer manufactured by Thermo Fisher Scientific Co., Ltd. was used. For the MS data obtained in the LC / MS analysis time, the amino acid sequence matching the MS data was searched from the protein sequence database using the Scaffold DIA manufactured by Proteome Software, and the protein was identified. The identification threshold was set so that the false positive rate of peptide-level and protein-level identification was 1% or less, respectively. Quantitative values were calculated from the area values of MS / MS chromatography for the identified proteins, and the protein variability of each sample was analyzed.

(Data analysis)
Statistical p-values (Mann-Whitney U test, p <0.05) were used in the data analysis to determine the difference in protein content in the stools of patients with biliary atresia and those of non-biliary atresia. The heatmap was created using the R function "heatmap2" based on the Z-score calculated from protein quantification.

[Experimental Example 1]
Biliary atresia patients (“BA.1” in Table 3), stools before Kasai surgery, stools after biliary atresia treatment by Kasai surgery, and non-biliary atresia patients (Table 3) The proteome analysis of the stool of "non-BA.1") was performed.

The results show a list of proteins that are significantly higher in the stools of patients with biliary atresia before Kasai surgery than in the stools of patients with non-biliary atresia, and Kasai than in the stools after treatment of biliary atresia by Kasai surgery. The proteins shown in Table 4 below were identified as proteins commonly included in the list of proteins that are significantly abundant in the stool of patients with biliary atresia before surgery.

Also, a list of proteins contained significantly less in the stools of patients with biliary atresia before Kasai surgery than in the stools of patients with non-biliary atresia, and Kasai than in the stools after treatment of biliary atresia by Kasai surgery. The proteins shown in Table 5 below were identified as proteins commonly included in the list of proteins that are significantly less contained in the stool of patients with biliary atresia before surgery.

Therefore, the abundance of the proteins listed in Table 4 below in the feces of the test infants is controlled (healthy infants, children with jaundice symptoms and not suffering from biliary atresia, or biliary atresia treated by surgery. If it is significantly higher than the stool abundance of the child) or the threshold set based on the control stool abundance, it is determined that the subject has biliary atresia. be able to.

In addition, the abundance of the proteins listed in Table 5 below in the stool of the test child is significantly lower than the abundance in the control stool or the threshold set based on the abundance in the control stool. If so, it can be determined that the subject has biliary atresia.

[Experimental Example 2]
Mr. Kasai of biliary atresia patients ("BA.1", "BA.2", "BA.3", "BA.4" in Table 3) Stool before surgery and patients with non-biliary atresia (Table 3) "Non-BA.1", "non-BA.2", "non-BA.3") stool proteomics analysis was performed.

As a result, the proteins shown in Table 6-1 below and Table 6-2 below were identified as proteins contained significantly more in the stools of patients with biliary tract obstruction before Kasai surgery than in the stools of patients with non-biliary tract obstruction.

In addition, the proteins shown in Table 7 below were identified as proteins contained in the stools of patients with biliary atresia before Kasai surgery significantly less than the stools of patients with non-biliary atresia.

Therefore, the abundance of the proteins listed in Table 6 below in the feces of the test infants is controlled (healthy infants, children with jaundice symptoms and not suffering from biliary atresia, or biliary atresia treated by surgery. If it is significantly higher than the stool abundance of the child) or the threshold set based on the control stool abundance, it is determined that the subject has biliary atresia. be able to.

In addition, the abundance of the proteins listed in Table 7 below in the stool of the test child is significantly lower than the abundance in the control stool or the threshold set based on the abundance in the control stool. If so, it can be determined that the subject has biliary atresia.

FIG. 1 is a heat map showing the relative abundance of representative proteins in feces of patients with biliary atresia and patients with non-biliary atresia. In FIG. 1, each column shows one patient. In FIG. 1, "BA.1", "BA.2", "BA.3", "BA.4", "non-BA.1", "non-BA.2", "non-BA.3" Corresponds to each patient shown in Table 3 above. In FIG. 1, the rightmost column shows the gene name encoding each protein.

Table 8-1 below and Table 8-2 below show significantly abundant proteins in the feces of patients with biliary atresia. In Table 8-1 below and Table 8-2 below, "BA / non-BA" divides the abundance of each protein in the stool of patients with biliary atresia by the abundance of each protein in the stool of patients with non-biliary atresia. Indicates the value.

Table 9-1 below and Table 9-2 below show significantly less protein in the feces of patients with biliary atresia. In Table 9-1 below and Table 9-2 below, "BA / non-BA" divides the abundance of each protein in the stool of patients with non-biliary atresia by the abundance of each protein in the stool of patients with biliary atresia. Indicates the value.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a technique for easily and non-invasively determining whether or not a subject has biliary atresia.

Claims (7)

A method of collecting data to determine if a subject has biliary atresia.
A detection step of detecting the presence of a marker protein in the feces of the subject is included.
The marker protein is one or more selected from the group consisting of the proteins listed in Table 1-1 below, Table 1-2 below, Table 1-3 below, Table 2-1 below and Table 2-2 below. Is a protein
When the marker protein is any of the proteins listed in Table 1-1 below, Table 1-2 below and Table 1-3 below, the abundance of the marker protein is the presence in the control stool. Significantly higher than the threshold set based on the amount or abundance in the control stool indicates that the subject has biliary atresia.
When the marker protein is any of the proteins listed in Table 2-1 below and Table 2-2 below, the abundance of the marker protein is the abundance in the control stool or the control stool. A method in which a significantly lower threshold compared to a threshold set based on the abundance of the subject indicates that the subject has biliary atresia.

The method according to claim 1, wherein the control is a healthy child, a child having jaundice symptoms and not suffering from biliary atresia, or a child after surgical treatment of biliary atresia. The method according to claim 1 or 2, wherein the detection step is performed by an enzyme immunoassay method or an immunochromatography method. A diagnostic kit for biliary atresia
Contains specific binding agents for marker proteins
One or more of the marker proteins selected from the group consisting of the proteins shown in Table 1-1, Table 1-2, Table 1-3, Table 2-1 and Table 2-2 according to claim 1. Is a protein of
The specific binding substance is used to detect the presence of the marker protein in the feces of a test child.
When the marker protein is any of the proteins listed in Table 1-1, Table 1-2 and Table 1-3, the abundance of the marker protein is the presence in the control stool. Significantly higher than the threshold set based on the amount or abundance in the control stool indicates that the subject has biliary atresia.
When the marker protein is any of the proteins listed in Table 2-1 and Table 2-2, the abundance of the marker protein is the abundance in the control stool or the control stool. A kit that indicates that the subject suffers from biliary atresia when significantly lower than a threshold set based on the abundance of The kit according to claim 4, wherein the control is a healthy child, a child having jaundice symptoms and not suffering from biliary atresia, or a child after surgical treatment of biliary atresia. The kit of claim 4 or 5, wherein the specific binding agent is located on a plate for enzyme immunoassay or on a membrane for immunochromatography. The kit according to any one of claims 4 to 6, wherein the specific binding substance is an antibody.

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