CN112748249A

CN112748249A - Application of neonatal biliary tract occlusion diagnostic marker

Info

Publication number: CN112748249A
Application number: CN202011510885.1A
Authority: CN
Inventors: 贾伟; 谢国祥; 周科军
Original assignee: Shenzhen Huiyun Biological Technology Co ltd
Current assignee: Shenzhen Huiyun Biological Technology Co ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-05-04

Abstract

The invention discloses an application of a neonatal biliary atresia diagnostic marker in preparing a tool for evaluating and diagnosing neonatal biliary atresia. The diagnostic product can be medical apparatus, kits, test paper, medical devices and the like. The invention also provides a kit for detecting the biomarkers and a use method thereof. The invention provides an effective means for early screening of neonatal biliary atresia risk and diagnosis of biliary stasis caused by other reasons, improves the efficiency and accuracy of neonatal biliary atresia diagnosis, strives for time for taking treatment measures in time, and is beneficial to improving the treatment effect and prognosis of neonatal biliary atresia.

Description

Application of neonatal biliary tract occlusion diagnostic marker

Technical Field

The invention relates to the technical field of biological medical treatment, in particular to a diagnostic marker for evaluating the biliary atresia risk of a newborn and application thereof in the fields of monitoring, screening and diagnosing the biliary atresia of the newborn, and also relates to a method and a device for detecting the diagnostic marker.

Background

The progressive and idiopathic extrahepatic Biliary system disease of Biliary Atresia (BA) in neonatal period is a destructive inflammatory fibrous obstruction of bile ducts of neonates, affecting intrahepatic and extrahepatic bile ducts of different lengths, the leading cause of obstructive jaundice of neonates, and the leading cause of hepatic transplantation of children, and about 60% of children are caused by hepatic transplantation. If the children with congenital biliary atresia is not treated in time, more than 80% of children with congenital biliary atresia die due to liver function failure and exhaustion within 1 year of age. Many children have severe liver fibrosis already when biliary atresia is diagnosed and cirrhosis rapidly develops, eventually leading to liver failure. Therefore, early diagnosis of biliary atresia is more urgent. Gamen jejunostomy (Kasai radical surgery) is the first treatment option for biliary atresia. The duration of the operation has a great influence on the efficacy of the Kasai operation. Research shows that when Kasai operation is performed within 30 or 45 days after birth, the disappearance rate of jaundice after operation and the survival rate of autologous liver in five years are obviously increased, and the requirement on liver transplantation is obviously reduced. Unfortunately, at present, biliary atresia cannot be diagnosed in time, and the time of general operation is up to 2 months later. Early screening and diagnosis of biliary atresia are of great significance to patients, and can save valuable treatment time of patients and improve prognosis. The current clinical common preoperative diagnostic methods comprise liver biopsy, pancreaticocholangiography, hepatobiliary angiography, duodenal examination and the like. These techniques, however, have certain limitations. Abnormal gallbladder (gallbladder shrinkage, poor contraction) and hepatic fibrous mass are characteristic manifestations of liver of infant with biliary tract occlusion under ultrasonic examination [ Ta, etc. The biliary tract occlusion is diagnosed by using the hepatic portal fibroid and the length of the gallbladder. Pediatric imaging, 2000,30(2):69-73], but hepatic portal fibroid masses do not necessarily occur in every infant patient, and ultrasound examinations vary widely depending on the observations made by different doctors, machines, etc. Therefore, the sensitivity and specificity of diagnosing biliary atresia reported in the literature are different. Recently, Meta analysis reported that the specificity of both cholecystokinesis and hepatic fibroid mass was 99%, whereas the corresponding sensitivities were 28% and 80%.

The bile duct of the baby within 3 months of age has small diameter and less liquid in the bile duct, and the high false positive rate of biliary tract occlusion diagnosis by magnetic resonance pancreaticobiliary tract imaging examination is inevitable. In addition, the pathological features of biliary atresia are small bile duct hyperplasia, bile embolism formation, cholestasis of bile capillary duct and liver cells, fibrosis around the region of the sink or lobules, and hepatic lobular structure is visible in early stage. Therefore, when the method is clinically applied to the identification of biliary atresia and other neonatal cholestasis, the diagnosis and treatment of biliary atresia are delayed due to low detection accuracy or complex process. In the early stage of the disease, the clinical manifestations, biochemical indicators, imaging and histological features of BA overlap with those of other cholestatic diseases, and the differential diagnosis process is complicated, complex and costly, easily resulting in delayed diagnosis and increasing the economic burden on the patient's family. Liver biopsy and duodenum examination involve unnecessary surgery and cause additional injury to suspected children. Therefore, the exploration of simple and specific diagnostic methods is never stopped, and the exploration of simple and effective diagnostic biomarkers has important significance for the early screening and diagnosis of biliary atresia.

Current serological indicators for screening and diagnosing biliary atresia include: firstly, total bilirubin in serum is directly bilirubin; which serves as an initial screening indicator. The neonatal serum total bilirubin is more than 2.0mg/dL (42-51 mu mol/L), or the direct bilirubin is more than 1.0mg/dL (17 mu mol/L), the biliary tract occlusion examination is required, but the specificity for diagnosing the biliary tract occlusion is poor; ② GGT: large sample investigation and research show that the GGT content of the biliary tract occlusion group is obviously higher than that of other cholestasis disease groups [ Chen X and the like ] in different age groups. The value of gamma-GT in conjunction with age in diagnosing biliary atresia. Pediatric gastroenterology and nutrition, 2016,63(3): 370-. Liu et al reported GGT >300U/L in the value of gamma-GT for early diagnosis of biliary atresia with an accuracy of 60% -85% diagnosis. GGT is the most common screening index for biliary atresia, but the difference between different children patients in clinical cases is large, and the accuracy is limited; other indexes: the serum cholic acid, the prothrombin concentration and the platelet measurement influence more factors.

Biomarkers for neonatal biliary atresia diagnosis have been explored in the prior art. Chinese patent document CN101221129B discloses a sulfonated bile acid enzyme fluorescence capillary assay and an enzyme fluorescence quantitative kit, which are suitable for rapid screening and diagnosis of liver and gall diseases, and are particularly suitable for early discovery of neonatal jaundice and congenital biliary atresia. CN108267585A discloses MMP-7 in biological samples as diagnostic marker. CN102818866B discloses the ratio of taurochenodeoxycholic acid and chenodeoxycholic acid in serum as a diagnostic marker for neonatal biliary atresia.

Aiming at the defects of the prior art, the invention explores a convenient, reliable method or a way capable of evaluating the neonatal biliary tract occlusion risk, and provides a feasible scheme for the clinical neonatal biliary tract occlusion fast and efficient screening and diagnosis.

Disclosure of Invention

The invention provides a technical scheme which can conveniently, reliably and efficiently evaluate, screen and/or diagnose the neonatal biliary tract occlusion risk. The invention provides a biomarker for evaluating the neonatal biliary tract occlusion risk, and also provides an evaluation method of the neonatal biliary tract occlusion risk. The invention provides a product, a method or a device for preparing a biomarker for evaluating the risk of neonatal biliary atresia, which can be used for clinically screening a patient with high risk of neonatal biliary atresia.

According to all the technical schemes for evaluating the neonatal biliary atresia risk, whether to take further clinical diagnosis and treatment measures can be determined according to the evaluation result. Based on the concepts, methods, and data provided herein, the present invention enables early rapid screening for neonatal biliary atresia.

The invention explores the relevance of bilirubin and its metabolites to the risk of a newborn suffering from biliary atresia. The invention determines that bilirubin and its metabolite are related to neonatal biliary tract occlusion risk by measuring bilirubin and its metabolite in blood samples of healthy neonates and neonates with biliary tract occlusion. In conjunction with bioinformatic methods, the levels of the biomarkers of the invention are correlated with the risk of detecting biliary atresia in a subject. As an example, bilirubin monoglucuronide is used as a biomarker, the content of bilirubin monoglucuronide in a test sample of a healthy newborn and a newborn patient with biliary tract occlusion is detected, and a cutoff value (cutoff value) is determined; when the content of bilirubin monoglucuronide in a test sample of the same test subject is higher than the cut-off value, the test subject is indicated to have a high risk of biliary atresia, indicating the necessity of further clinical diagnosis and treatment.

The invention provides a biomarker related to neonatal biliary atresia, and further provides application of the biomarker in neonatal biliary atresia risk assessment, screening and/or diagnosis, and further provides a tool and a method for assessing neonatal biliary atresia risk, and further more particularly provides application of the biomarker in preparation of a diagnostic product for neonatal biliary atresia risk assessment, and a related diagnostic product and a using method.

The present invention describes the course of research and results of biomarker screening and validation. The invention finds that the quantitative detection value of the bilirubin monoglucuronide can be used as an index for risk assessment to distinguish normal newborns, suspected biliary tract occluded newborns and newborns with biliary stasis diseases caused by other reasons by quantitatively determining the content difference of bilirubin and components thereof in the blood samples of the newborns with biliary stasis diseases caused by the normal newborns, the biliary tract occluded newborns and other reasons. Based on the creative research result, the inventor further explores an application way of using the content of bilirubin monoglucuronic acid ester as a detection index for early screening and diagnosis of clinical neonatal biliary atresia, so that the neonatal can quickly, conveniently and efficiently screen biliary atresia after birth, and further medical measures can be taken quickly for high-risk sick children.

The inventor proposes that bilirubin monoglucuronide can be used as a diagnostic marker for a method for rapid screening of clinical neonatal biliary tract occlusion; and as one example, the inventor proposes bilirubin monoglucuronide as a diagnostic marker, which can be used for preparing a suitable diagnostic product for neonatal biliary atresia risk assessment, the diagnostic product taking bilirubin monoglucuronide as a quantitative detection index, comprising a bilirubin monoglucuronide quantitative detection reagent, and optionally, comprising a reagent for processing a blood sample of a subject and other detection reagents.

Based on the above findings and concepts, the present invention provides in a first aspect the use of bilirubin monoglucuronide as a diagnostic marker in the manufacture of a diagnostic device for assessing the risk of neonatal biliary atresia, characterized in that said diagnostic device carries out a quantitative detection of bilirubin monoglucuronide.

In some specific embodiments, the diagnostic device is selected from the group consisting of a medical device, a kit, a strip, and a test device.

In some specific embodiments, the diagnostic device takes a newborn infant as a test object, and a blood sample of the test object is used for testing. The blood sample is selected from the group consisting of whole blood, plasma, serum, and dried blood spots of a subject.

In some embodiments, a preferred test sample is provided, wherein the test sample of the test method is dispensed into a system comprising a first stabilizing agent and a second stabilizing agent.

The first stabilizer is selected from antioxidants having a solubility in non-polar media. The first stabilizer can be one or more of Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), Propyl Gallate (PG) and tert-butylhydroquinone (TBHQ).

The second stabilizer is selected from antioxidants having a certain solubility in polar media, and as a specific embodiment, the second stabilizer is selected from ascorbic acid. As a preferred example, the system comprising the first antioxidant and the second antioxidant comprises a first stabilizer of dibutylhydroxytoluene (BHT) and a second stabilizer of ascorbic acid.

As a specific embodiment, the content of the first stabilizer and the second stabilizer in the system is in a weight ratio range of 1: 1-1: 50, preferably 1: 10-1: 45, more preferably 1: 20-1: 40, more preferably 1: 30-1: 40.

in some embodiments, the test sample is selected from an organic solvent dispersion of a biological sample. The organic solvent of the organic solvent dispersion system is selected from one or a combination of several of methanol, ethanol, acetone, propylene glycol and acetonitrile. The biological sample may be selected from whole blood, plasma, serum and dried blood spots. After the biological sample is dispersed in an organic solvent dispersion system, centrifuging and taking supernatant fluid to carry out detection according to the detection method provided by the invention.

In some embodiments, the biological sample may be processed as follows: adding bilirubin taurate into a biological sample as an internal standard, adding an organic solvent dispersion system containing a first stabilizer and a second stabilizer, shaking in the dark, centrifuging, and taking supernatant for detection.

In some preferred embodiments, the organic solvent dispersion containing the first stabilizer and the second stabilizer may be a methanol-acetonitrile mixed solution of BHT and ascorbic acid; as a specific embodiment, the content of the first stabilizer and the second stabilizer in the system is in a weight ratio range of 1: 1-1: 50, preferably 1: 10-1: 45, more preferably 1: 20-1: 40, more preferably 1: 30-1: 40.

preferably, the shaking and centrifugation is carried out at 0-10 deg.C, optionally 0 deg.C, 4 deg.C, 10 deg.C; the oscillation condition is 1000-2000rpm, preferably 1400-1500rpm, and the oscillation time is 10-30 minutes, preferably 15-25 minutes; the centrifugal force is 100000-250000g, and the centrifugal time is 10-30 minutes.

In some embodiments, the diagnostic device may optionally be used to assess the risk of neonatal biliary atresia in combination with other measures of the subject, the other measures being selected from one or more of free bilirubin, bilirubin diglucuronate, biliverdin, gamma glutamyltransferase, MMP-7, and other measures that are directly or indirectly diagnostic of neonatal biliary atresia for the purpose of said use.

The invention provides a kit for evaluating the neonatal biliary tract occlusion risk, which is characterized by comprising a bilirubin monoglucuronide quantitative detection reagent. The quantitative detection reagent comprises a bilirubin monoglucuronic acid ester quality control product and an internal standard product.

In some embodiments, the bilirubin monogluconate quality control product is a bilirubin monogluconate standard dispensed in a system comprising a first stabilizer and a second stabilizer. Preferably, the first stabilizer is one or more selected from Butyl Hydroxy Anisole (BHA), dibutyl hydroxy toluene (BHT), Propyl Gallate (PG) and tert-butyl hydroquinone (TBHQ). Preferably, the second stabilizer is selected from ascorbic acid.

In some embodiments, preferably, the system of the first stabilizer and the second stabilizer is an organic solvent dispersion system containing dibutylhydroxytoluene (BHT) and ascorbic acid. Preferably, the organic solvent of the organic solvent dispersion system is selected from one or more of methanol, ethanol, acetone, propylene glycol and acetonitrile.

In other specific embodiments, the system is a solid system comprising a solid support. Preferably, the solid support is a filter paper sheet, preferably selected from analytical filter paper, qualitative analytical filter paper and slow quantitative ashless filter paper. When the filter paper sheet is used as a solid carrier, it is preferably subjected to the following pretreatment: soaking in organic solvent dispersion system containing first stabilizer and second stabilizer, and drying in the shade.

The use method of the kit for evaluating the neonatal biliary atresia risk comprises the following steps,

(1) obtaining a test sample by taking a newborn infant as a detection object;

(2) determining the expression level of the biomarker in the test sample by using a quantitative detection method;

(3) analyzing the expression level of the biomarker for risk assessment, wherein the analysis can be compared with a cut-off value of a quantitative detection method, and the risk assessment conclusion can be used for assessing the high and low biliary atresia of a subject.

Optionally, in the step (3) of risk assessment, the neonatal biliary atresia risk may be assessed in combination with other test indicators of the test subject, the other test indicators being selected from one or more of free bilirubin, bilirubin diglucuronate, biliverdin, gamma-glutamyl transferase, MMP-7, and other indicators having a diagnostic ability directly or indirectly on neonatal biliary atresia.

The quantitative detection method is the liquid chromatography-tandem mass spectrometry quantitative detection method.

The test sample is selected from the group consisting of whole blood, plasma, serum, and dried blood spots of a subject.

In a third aspect, the present invention provides a method of assessing the risk of biliary atresia in a newborn, comprising:

(1) obtaining a test sample by taking a newborn infant as a detection object;

(2) determining the expression level of the diagnostic marker in the test sample using a quantitative detection method; the diagnostic marker comprises bilirubin monoglucuronate, and optionally other detection indexes of the detection object are combined to evaluate the neonatal biliary tract atresia risk, and the other detection indexes are selected from one or more of free bilirubin, bilirubin diglucosuronate, biliverdin, gamma-glutamyltransferase, MMP-7 and other indexes which have the capacity of directly or indirectly diagnosing the neonatal biliary tract atresia;

(3) analyzing the expression level of the diagnostic marker for risk assessment, wherein the analysis can be compared with a cutoff value (cutoff value) of a quantitative detection method, and the conclusion of the risk assessment can be used for assessing whether the biliary atresia risk of the subject is high or low;

(4) the test subjects are classified into a high risk group and a low risk group according to the evaluation conclusion, wherein the high risk group suggests that further clinical diagnosis is necessary to determine whether the test subjects have biliary atresia.

Preferably, the cutoff value of the quantitative determination method in step (3) is a value determined by statistical analysis of the biomarker level in the healthy newborn test sample and the corresponding biomarker level in the biliary atresia newborn test sample in advance by using the quantitative determination method in step (2).

Preferably, the test sample is selected from whole blood, plasma, serum or dried blood spots.

Preferably, the quantitative detection method is selected from any one of the above-mentioned quantitative detection methods of liquid chromatography tandem mass spectrometry. And (3) a detection method.

In a fourth aspect, the present invention provides a device for predicting the risk of neonatal biliary atresia using the expression level of bilirubin monoglucuronate, comprising:

(1) a module for receiving a test sample of a test subject;

(2) a module for detecting data on the expression level of a diagnostic marker; wherein the diagnostic markers comprise at least bilirubin monoglucuronate, optionally in combination with free bilirubin, bilirubin diglucuronate, biliverdin, gamma-glutamyltransferase, MMP-7, and other clinical indicators of diagnostic capacity for biliary atresia in the test sample;

(3) a module for generating a risk score based on the expression levels of the diagnostic markers input to a database comprising control expression profiles associated with the test sample and the test method; the control expression profile is obtained in advance according to the detection sample and the detection method, and can be expressed as a cutoff value of the detected diagnostic marker; the risk assessment is carried out by comparing the expression level of the biomarkers in the test sample with a cut-off value of high performance liquid chromatography tandem mass spectrometry as a detection method, and the detection object is considered to have high risk of biliary atresia if the cut-off value is higher than the cut-off value.

The tool is selected from the group consisting of diagnostic products and analytical test devices.

The 'diagnosis product' of the invention can be medical apparatus, kit, test paper, medical device and the like. As known to those of ordinary skill in the art, the definitions of the medical devices, kits, etc. relate to the regulations of local related laws and regulations, which have different classification methods and meanings in different countries. The terms of the medical apparatus, kit, test paper, etc. of the present invention are only used to describe the application form of the diagnostic marker of the present invention, and are not defined as the meanings below; the diagnostic product may be a medical product that is locally registered in association, or a product or combination of products that are used in a temporary manner and form by the average technician, as long as it is consistent with the objects of the present invention.

The "test sample" as used herein may include body fluid, blood, plasma, serum derived from a subject. Blood, plasma, serum samples are preferred. And as a prominent effect of the present invention, it was found that the biomarkers provided by the present invention can be detected in a small amount of test sample by testing the subject, such as heel blood or fingertip blood, or a dried blood spot prepared from heel blood or fingertip blood.

The "blood sample" of the present invention may be whole blood, plasma, serum, heel blood or fingertip blood taken from a test subject or a clinically commonly used dried blood spot which may be taken from heel blood or fingertip blood of a test subject.

As one example of the invention, the invention provides a kit for evaluating the neonatal biliary atresia risk, which comprises a bilirubin monoglucuronide quantitative detection reagent, such as a quality control product, an internal standard product and an internal standard diluent. As an embodiment, the quality control product comprises a bilirubin monoglucuronide-containing quality control product, the internal standard comprises a taurine bilirubin standard, and the internal standard diluent comprises a methanol acetonitrile solution containing 2, 6-di-tert-butyl-4-methylphenol. And may further comprise an aqueous solution comprising ammonium acetate and acetic acid.

As one example of the present invention, dry blood spots prepared by taking blood from the heel or fingertip of a newborn are used as a test sample, and under the test conditions of the embodiment of the present invention, when the content of bilirubin monoglucuronide is more than 2.5. mu.M, the test subject can be considered to have a high risk of biliary atresia in the newborn. As a specific embodiment, the applicant can rapidly detect and evaluate the neonatal biliary atresia risk by using the bilirubin monoglucuronide content of more than 2.5 μ M as a cutoff value (also called cutoff value) in the blood sample of the subject under the detection conditions described in the examples. The Cutoff values in the examples are standards determined under the conditions of the test methods, test conditions, standards, and the like. According to the teaching of the present invention, a person of ordinary skill in the art can screen and optimize a quantitative detection method of bilirubin monoglucuronide, including but not limited to a high performance liquid chromatography technique, such as a gas chromatography-mass spectrometry detection technique, and under the teaching of the present invention, can also perform evaluation of neonatal biliary atresia risk by optimizing and determining a cutoff value based on the detection method and detection conditions thereof. In the screening of the cutoff values of the different detection methods described above, the detection method of the invention can also be used as a reference and a comparison, in particular to determine the cutoff values of the other detection methods in the absence of clinical samples. Therefore, the bilirubin monoglucuronide content is used as an index for assessing the neonatal biliary tract occlusion risk, and regardless of differences in the detection method and cutoff value, it is understood to be within the scope of the present invention as can be achieved by one of ordinary skill in the art in light of the teachings herein.

The technical scheme provided by the invention can effectively evaluate the neonatal biliary tract occlusion risk and can realize the neonatal biliary tract occlusion early and early screening in clinic. In clinical use, the newborn with high risk of biliary atresia can be screened quickly by quantitatively measuring the content of the biomarker in a blood sample of the newborn. The biomarker of the invention can be used as a marker for screening neonatal biliary atresia, and can also be optionally combined with other detection indexes and detection means to further evaluate the neonatal biliary atresia risk, and can be optionally combined with other detection indexes and detection means in the diagnosis process. Other detection criteria are selected from the group consisting of gamma glutamyl transferase, MMP-7, and clinical criteria.

The invention adopts high performance liquid chromatography-tandem mass spectrometry to quantitatively detect bilirubin and metabolite thereof in a blood sample as a neonatal biliary tract occlusion diagnostic marker. The blood sample may be selected from whole blood, plasma, serum and dried blood spots. Dried blood spots were collected from neonatal heel blood or fingertip blood. As one embodiment, the inventors have explored diagnostic products with dried blood spots as the test sample. The dry blood spot sampling is convenient, is favorable for the newborn to rapidly carry out screening of biliary atresia after birth, and is further favorable for the newborn to rapidly carry out large-area popularization of biliary atresia screening after birth. From another aspect, even if resampling is required, because the preparation technique for dried blood spots is mature, sampling is convenient, and little or no trauma to the newborn is particularly advantageous even when repeated testing is required.

The invention firstly provides the application of the biomarker as a diagnostic marker for screening and risk evaluation of biliary tract occlusion of a newborn, can be used for evaluating biliary tract occlusion risk of the newborn, can be clinically used for screening and further diagnosing biliary tract occlusion risk of the newborn in the early stage, strives for the optimal operation time for infants suffering from biliary tract occlusion of the newborn, and improves operation prognosis.

The invention adopts high performance liquid chromatography-tandem mass spectrometry to quantitatively detect the biomarker in the blood sample as the neonatal biliary tract occlusion diagnostic marker, and has the advantages of convenient sampling, high efficiency and reliable detection result. The detection method can adopt whole blood, serum and plasma as biological samples for detection, and can also adopt dry blood spots prepared by sampling blood from fingertips or heels as detection samples for detection. In addition, compared with the traditional biochemical method, the high performance liquid chromatography-mass spectrometry quantitative detection method provided by the invention has the characteristics of high accuracy and low detection limit.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety and for all purposes. Moreover, the CAS numbers cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each such number were specifically and individually indicated to be incorporated by reference in its entirety and for all purposes.

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. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a component" includes a combination of one or more components, and the like.

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below by way of embodiments with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Drawings

FIG. 1 is a chromatogram of bilirubin and its metabolites, from which it can be seen that each target analyte can be efficiently separated.

FIG. 2 is a liquid chromatogram of an ammonium formate-formic acid system as a mobile phase A, from which it can be seen that each target detection substance can be efficiently separated.

FIG. 3 is a liquid chromatogram of mobile phase A of trifluoroacetic acid system, from which it can be seen that each target detection substance can be effectively separated.

FIG. 4 shows a standard curve prepared from quality control materials in the detection of target components by HPLC-MS.

FIG. 5 is the area under the ROC curve for each metabolite control to distinguish biliary atresia neonates from normal neonates.

FIG. 6 is a set of scatter plots showing the difference in bilirubin monoglucuronide levels in biliary atresia neonate and normal neonate test samples and the area of bilirubin monoglucuronide under the ROC curve that distinguishes biliary atresia neonate from normal neonate controls.

FIG. 7 is a set of scatter plots showing the difference in bilirubin monoglucuronide levels in cholestatic newborn and other infant cholestasis test samples and the area of bilirubin monoglucuronide under the ROC curve that distinguishes cholestatic newborn and other infant cholestasis.

Detailed Description

The major bilirubins in the human blood include 4 bilirubin components, namely alpha, beta, gamma, delta bilirubin (bilirubin IX alpha, B alpha; bilirubin beta-monogluuronide, BMG; bilirubin gamma-diglucuronide, BDG; delta-bilirubin, B delta). Wherein alpha bilirubin is unconjugated bilirubin (UCB) and beta, gamma, and delta bilirubin are Conjugated Bilirubin (CB). Normally, alpha bilirubin in liver cells mainly comes from two sources, namely, alpha bilirubin is absorbed and transported into liver cells from peripheral blood through an organic anion transport polypeptide transporter on a liver cell basement membrane, and alpha bilirubin is generated by oxidative degradation of heme in liver cells. 98-99% of alpha bilirubin in the liver cell is converted into beta or gamma bilirubin through glucuronidation of glucuronic acid transferase 1A1 (UDP-glucuronic transferase 1A1, UGT1A1) and is combined with one or two glucuronic acids, and the beta or gamma bilirubin is discharged to a bile duct through multidrug resistance-associated protein 2 (MRP 2) transporters on the membrane of the capillary bile duct. In case of pathological bile deposition, bile secreted from liver cell can not normally enter bile duct, resulting in accumulation and increase of peripheral blood bile acid and combined bilirubin.

The inventor finds that the correlation between the conjugated bilirubin and the conjugated bilirubin-related component and neonatal biliary atresia is not disclosed in the prior art and is not researched, and the conjugated bilirubin-related component is used as a marker to evaluate neonatal biliary atresia risk; nor does the prior art provide any technical solutions or teachings for bilirubin binding and bilirubin binding components for neonatal biliary atresia diagnosis.

The inventor researches and explores the relevance of the bilirubin and the metabolite related to the bilirubin and the neonatal biliary atresia through a large amount of experiments, and explores the application of the bilirubin and the metabolite related to the bilirubin in the field of clinical neonatal biliary atresia risk assessment.

The inventor firstly explores a technical scheme for quantitatively detecting each component of bilirubin. In the embodiment, the qualitative and quantitative detection of each component of bilirubin is realized by the research works of selecting liquid phase conditions, setting mass spectrum conditions, preparing standard solutions, optimizing analysis methods, drawing standard curves, preprocessing samples and the like. The following examples are given to illustrate the present invention.

The sources of reagents used in the examples herein are as follows:

ditaurine disodium salt: cat No. 201102, purchased from Sigma-Aldrich China

Biliverdin: cat No. 30891, purchased from Sigma-Aldrich China

Free bilirubin: cat No. B4126, purchased from Sigma-Aldrich China

Methanol: cat No. 900688, purchased from Sigma-Aldrich China

Acetonitrile: cat No. 900667, purchased from Sigma-Aldrich China

2, 6-di-tert-butyl-4-methylphenol (BHT): cat No. B1378, purchased from Sigma-Aldrich China

Ammonium acetate: cat No. 73594, purchased from Sigma-Aldrich China

Acetic acid: cat No. 45754, purchased from Sigma-Aldrich China

Ammonium formate: cat No. 70221, purchased from Sigma-Aldrich China

Formic acid: cat No. 5330020050, purchased from Sigma-Aldrich China

Trifluoroacetic acid: cat No. 302031, purchased from Sigma-Aldrich China

Trichloroacetic acid cat # T6399 available from Sigma-Aldrich China

Bilirubin monoglucuronide, self-made in applicant's laboratory, CAS number 27071-67-6, molecular formula C39H44N4O12, average molecular weight 760.786, purity of more than or equal to 90%

Bilirubin diglucuronate, self-made in the laboratory of the applicant, with the CAS number of 17459-92-6, the molecular formula of C45H52N4O18, the average molecular weight of 936.921 and the purity of more than or equal to 90 percent

Example 1 establishment and optimization of liquid chromatography-tandem Mass Spectrometry method

In this embodiment, the establishment process of the qualitative and quantitative detection method for bilirubin in a biological sample based on liquid chromatography-tandem mass spectrometry is described for illustrative purposes, and includes selecting a liquid phase condition, setting a mass spectrometry condition, preparing a standard solution, optimizing an analysis method, and drawing a standard curve. The detection method has high sensitivity, high selectivity and strong anti-interference capability, and can be used as an effective tool for qualitative and quantitative analysis of each component of bilirubin.

Bilirubin samples contain a variety of components. The inventor finds that when the pH value of a mobile phase A of liquid chromatography is 3-4.5, main components of free bilirubin, biliverdin, bilirubin monoglucuronide and bilirubin diglucuronate in a bilirubin sample can be well separated, and the method is suitable for wide chromatographic columns, particularly C8 and C18 silica gel columns, and has good applicability to column temperature and sample injection amount.

The mass spectrum can be selected from a quadrupole mass spectrum, a time-of-flight mass spectrum, an ion hydrazine mass spectrum and a high-resolution orbital hydrazine mass spectrum; the conditions of the mass spectrum and the setting of the qualitative and quantitative detection mode of the mass spectrum comprise the following steps: selecting an electrospray ionization source (ESI) to select an ion scanning mode based on a response to detecting a target compound; and selecting a multi-reaction monitoring method (MRM) and setting a multi-reaction monitoring mode parameter.

First, research of detection conditions

Instruments and reagents: acetonitrile (chromatographically pure grade), methanol (chromatographically pure grade), isopropanol (chromatographically pure grade), ammonium acetate (chromatographically pure grade), acetic acid (chromatographically pure grade), manual or automatic pipettes (10-200. mu.L, 100. mu.L, 1000. mu.L), are all commercially available.

Liquid phase condition 1: mobile phase A: ammonium acetate + acetic acid adjusted ph3.5 ultrapure water solution, mobile phase B: acetonitrile: methanol: isopropanol-8: 1:1, column temperature: 40 ℃, column: acquity BEH C18(2.1 × 50mm,1.7 μm), sample size: 5 μ L.

Liquid phase conditions 2: mobile phase A: ammonium formate + formic acid adjusted ph4.5 ultrapure water solution, mobile phase B: acetonitrile: methanol: isopropanol-8: 1:1, column temperature: 40 ℃, column: acquity BEH C18(2.1 × 50mm,1.7 μm), sample size: 5 μ L.

Liquid phase conditions 3: mobile phase A: ammonium trifluoroacetate adjusted pH3 ultra pure water solution, mobile phase B: acetonitrile: methanol: isopropanol-8: 1:1, column temperature: 40 ℃, column: acquity BEH C18(2.1 × 50mm,1.7 μm), sample size: 5 μ L.

Instruments and reagents: acetonitrile (chromatographically pure grade), methanol (chromatographically pure grade), isopropanol (chromatographically pure grade), ammonium acetate (chromatographically pure grade), acetic acid (chromatographically pure grade), manual or automatic pipettes (10-200. mu.L, 100. mu.L) are all commercially available.

Liquid phase conditions: mobile phase A: 77mg ammonium acetate +250 μ L acetic acid adjusted pH3-4.5 ultrapure water solution, mobile phase B: acetonitrile: methanol: isopropanol-8: 1:1, column temperature: 40 ℃, column: acquity BEH C18(2.1 × 50mm,1.7 μm), sample size: 5 μ L. The procedure for high performance liquid chromatography is shown in table 1 below.

TABLE 1 high Performance liquid chromatography procedure

Time (minutes)	Flow rate (mL/min)	Mobile phase A	Mobile phase B	Curve
						0	0.5	95	5	6
0.5	0.5	70	30	6
					2.0	0.5	5	95	6
4.0	0.5	5	95	6
					4.1	0.5	95	5	6
5.0	0.5	95	5	6

Setting mass spectrum conditions: as shown in table 2, an electrospray ionization source (ESI) was selected, an appropriate ion scanning mode was selected according to the response of the compound, and a multi-reaction monitoring method (MRM) acquisition mode in a positive ion mode was employed to set parameters of the multi-reaction monitoring mode; the scanning time is 0.4-4 min. Respectively injecting standard solutions into ion sources in a semi-automatic sample injection mode, selecting corresponding parent ion peaks, performing secondary mass spectrometry on child ions of the parent ion peaks to obtain fragment ion information, and establishing the MRM mass spectrometry detection method of the compound.

TABLE 2 MRM Mass Spectrometry detection of bilirubin and its metabolitesNumber of

Serial number	Name of Compound	Parent ion (m/z)	Ionic acid (m/z)	CV	CE
						1	Free bilirubin	585.3	299.2	50	20
2	Bilirubin monoglucuronide ester	761.5	299.2	50	40
						3	Bilirubin diglucuronate	937.4	299.2	78	50
4	Biliverdin	583.25	297.17	60	34
						5	Taurine bilirubin	799.35	406.2	18	46

In the mass spectrometry process in the scheme, the ion source temperature is 150 ℃, the capillary voltage is 3000V, the desolventizing gas temperature is 500 ℃, the desolventizing gas flow rate is 1000L/h, and the collision gas is argon.

Typical chromatograms for detecting components of bilirubin under the above detection conditions and three different liquid phase conditions are shown in the accompanying figures 1, 2 and 3.

Preparation of Standard solutions

The above liquid phase condition 1 was adopted.

(1) Preparing a free bilirubin standard solution: weighing appropriate amount of free bilirubin, adding chloroform, and preparing into mother liquor with concentration of 1 mg/mL.

(2) Preparing a bilirubin monoglucuronic acid ester standard solution: weighing a proper amount of bilirubin monoglucuronic acid ester, adding 50% methanol solution, and preparing a mother liquor with the concentration of 1 mg/mL.

(3) Preparing a bilirubin digluconate standard solution: weighing a proper amount of bilirubin diglucuronate, adding 50% methanol solution, and preparing mother liquor with the concentration of 1 mg/mL.

(4) Preparing a biliverdin standard solution: weighing appropriate amount of biliverdin, adding methanol, and making into mother liquor with concentration of 1 mg/mL.

(5) Mixing standard mother liquor: a mixed standard substance is prepared by using methanol containing 1% of BHT, and the concentrations of free bilirubin, bilirubin monoglucuronide, bilirubin diglucoside and biliverdin are all 50 mu mol/L.

(6) Mixing standard solutions: the mixed standard mother liquor is diluted by methanol containing 1 percent of BHT to obtain mixed standard solutions of 20, 4, 1 and 0.2 mu mol/L in sequence.

(7) Diluting the standard mother liquor prepared in the above steps in proportion to prepare a standard solution with the following concentration, sampling volume 1 μ l, and drawing a standard curve by peak area to target content to obtain a standard curve as shown in FIG. 4.

EXAMPLE 2 qualitative and quantitative determination of bilirubin in a blood sample

Sampling: serum, plasma and whole blood samples of 3 test subjects were taken, and each sample was divided into 9 biological samples of 3 samples such as serum 01, serum 02, serum 03 and the like.

And (3) biological sample treatment:

serum 01: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter of a mixture containing 1mg/ml BHT and 200mmol/L ascorbic acid methanol: acetonitrile (volume ratio 1:1) solution, 10 ℃ photophobic 1450rpm oscillation 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Serum 02: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 175mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Serum 03: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 204mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Plasma 01: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 180mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Plasma 02: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 160mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Plasma 03: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 170mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Whole blood 01: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 200mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Whole blood 02: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 206mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

Whole blood 03: taking 20 microliter samples, adding 10 microliter taurine bilirubin as an internal standard, and adding 80 microliter methanol containing 1mg/ml BHT and 190mmol/L ascorbic acid: acetonitrile (1:1) solution, 10 ℃ protected from light and shaking at 1450rpm for 20 minutes. Centrifuge at 18000g for 20 minutes at 4 ℃. Taking 60 microliter of supernatant to a 96-well plate, and taking the supernatant as a detection sample to carry out high performance liquid chromatography tandem mass spectrometry detection.

The sample treated above is injected and detected according to the LC-MS/MS conditions in the embodiment 1; the results of the internal standard calibration and the external standard quantification are shown in the following table 3.

TABLE 3 measurement indexes and measurement results

EXAMPLE 3 qualitative and quantitative determination of bilirubin content in dried blood spots

Dry blood spot sample preparation

The newborn baby collects finger tip blood within 4 days of birth to prepare a dry blood slice sample. The method comprises the following steps:

a) pretreatment of filter paper sheets: and repeatedly soaking a 903 filter paper sheet in an ethanol solution containing 1mg/mL BHT and 200mmol/L ascorbic acid for 2 minutes, taking out and draining, and naturally drying the filter paper sheet in the shade for later use.

b) Preparing: taking a proper test tube, and adding a proper amount of diluent. Connecting a micro suction tube with a latex suction head, and checking whether the joint is air-leakage or not, or taking a disposable micro suction tube (siphon principle), a blood taking needle, 75% ethanol or iodophor, a cotton swab and the like for standby use.

c) Massaging: gently massage the central part with more muscles at the ulnar side of the ring finger tip of the left hand to cause the local tissues to be naturally congested with blood. Avoiding the use of finger sides or fingertips.

d) And (3) disinfection: wiping the blood sampling part with 75% ethanol or iodophor cotton swab, and naturally drying.

e) And (3) needling: fixing the blood sampling part with the left thumb, the index finger and the middle finger to make the skin and subcutaneous tissue tense, and pricking the disposable sterilized blood sampling needle with the right hand from the ulnar side at the finger end with the depth of 2-3mm, and immediately withdrawing the needle.

f) Blood wiping: after the blood had naturally flowed out, the first drop of blood was wiped off with a sterile dry cotton ball (cotton swab).

g) Blood sucking: sucking blood with disposable micropipette or dripping blood on filter paper sheet, and pressing wound with sterile dry cotton ball (cotton swab) to stop bleeding, such as unsmooth blood flow, or slightly pressing with left hand from the distal end of blood sampling part to finger tip to make blood flow out.

h) Patient identification is immediately made after the sample is collected, so that confusion is avoided.

i) Drying in shade for 4 hr, placing into aluminum foil bag, and storing at 4 deg.C.

Dry blood spot sample pretreatment

Taking 3 dried blood spots with a punch sampler, adding 20 microliters of pure water, shaking at 1450rpm for 20min at 10 ℃ with a shaker, adding 10 microliters of internal standard (taurine bilirubin) and 80 microliters of methanol containing 1mg/ml BHT and 200mmol/L ascorbic acid: and (2) continuously oscillating the solution of acetonitrile (1:1) for 20min at 1450rpm and 10 ℃ by using an oscillator, centrifuging the solution for 20min at the centrifugal force of 18000g and 4 ℃, taking 60 microliters of supernatant to a 96-well plate, and injecting the sample.

High performance liquid chromatography-tandem mass spectrometry

The measurement conditions are shown in example 1, the measured contents are shown in table 4 below, and the methodological parameters are shown in table 5.

TABLE 4 measurement indexes and content measurement results

TABLE 5 methodological parameters

Example 4 Stable bilirubin quantitative determination reagent

The invention provides a stable bilirubin quantitative determination reagent, which comprises a bilirubin standard substance, wherein the bilirubin standard substance is distributed in a system containing a first stabilizer and a second stabilizer; the bilirubin comprises one or more of free bilirubin, biliverdin, bilirubin monoglucuronate and bilirubin diglucosuronate. The present example aims to provide a stable bilirubin quantitative determination reagent, and the preparation method and stability thereof were studied.

Preparation of stable bilirubin quantitative determination reagent

Pretreatment of filter paper sheets: the analysis filter paper sheet was repeatedly soaked in an ethanol solution containing 1mg/mL BHT and 200mmol/L ascorbic acid for 2 minutes, taken out and drained, then the filter paper sheet was naturally dried in the shade and stored at 4 ℃.

Preparing a quantitative detection reagent: accurately weighing free bilirubin, biliverdin, bilirubin monoglucuronate and bilirubin biglucuronate, respectively dissolving in ethanol, respectively sucking the solution with disposable micropipette, dripping on filter paper sheet, sun-shielding and drying in shade for 4 hr, placing in aluminum foil bag, and storing at 4 deg.C.

Stability study: using the measurement conditions of example 1, the samples left for the following days were quantitatively examined, and the results are shown in Table 6.

TABLE 6

Example 5 bilirubin level detection kit

The invention provides a kit for quantitatively detecting bilirubin, wherein detection indexes of the kit comprise the content of any one or more of free bilirubin, biliverdin, bilirubin monoglucuronate and bilirubin biglucuronate or the total content of the free bilirubin, the biliverdin monoglucuronate and the bilirubin biglucuronate. The present embodiment aims to exemplarily provide a bilirubin level detection kit.

Comprising a bilirubin standard dispensed in a system comprising a first stabilizer and a second stabilizer; the bilirubin comprises one or more of free bilirubin, biliverdin, bilirubin monoglucuronate and bilirubin diglucosuronate. The present example aims to provide a stable reagent for quantitatively detecting bilirubin, and studies the preparation method and stability thereof.

The composition of the kit of this example is shown in Table 7.

TABLE 7

Name (R)	Principal Components
		Quality control product	Filter paper containing bilirubin standard product
Internal standard	Taurine bilirubin standard
		Internal standard diluent	Methanol-acetonitrile solution containing 2, 6-di-tert-butyl-4-methylphenol

The filter paper containing the bilirubin standard is prepared according to the following method:

the analysis filter paper sheet was repeatedly soaked in an ethanol solution containing 1mg/mL BHT and 200mmol/L ascorbic acid for 2 minutes, taken out and drained, then the filter paper sheet was naturally dried in the shade and stored at 4 ℃. Accurately weighing free bilirubin, biliverdin, bilirubin monoglucuronate and bilirubin biglucuronate, respectively dissolving in ethanol, respectively sucking the solution with disposable micropipette, respectively dripping on filter paper sheet, drying in shade for 4 hr, placing in aluminum foil bag, and storing at 4 deg.C.

Example 6 neonatal biliary atresia screening

The technical scheme provided by the invention makes a bilirubin quantitative detection method possible. The bilirubin content detection method and the bilirubin content detection kit can be used for respectively or simultaneously detecting several kinds of bilirubin and quantitatively detecting free bilirubin, biliverdin, bilirubin monoglucuronate and bilirubin diglucosuronate for any detection purpose. It can be applied to diagnosis, risk assessment and the like related to medical treatment, and can be used for diagnosis, risk assessment and drug treatment effect assessment of any disease or symptom sensitive to the detection indexes or one or more of the detection indexes.

The technical scheme of the invention is applied to screening of biliary tract occlusion of a newborn for explanation; the same applies to the screening, diagnosis, risk assessment and monitoring of the effect of therapeutic drugs of other diseases or conditions that may lead to a change in the bilirubin component. For example, the disease or condition is selected from neonatal biliary atresia, infantile hepatitis syndrome, cytomegalovirus hepatitis, choledocystis, alpha 1 antitrypsin deficiency disease, Alagille syndrome, cystic fibrosis, total parenteral nutrition-related cholestasis, and the like.

This example demonstrates the study of the content of bilirubin and its metabolites in blood samples from normal neonates and biliary atresia newborns. In this example, 39 clinical samples of neonatal biliary atresia patients and 300 normal neonatal controls were collected, and the blood samples used were dried blood spots prepared within 4 days of birth.

Dry blood spot sample preparation

Dry blood spot sample pretreatment

High performance liquid chromatography-tandem mass spectrometry

The measurement conditions are as shown in example 1. The measurement result shows that the average value of bilirubin monoglucuronate in biliary atresia infants is 28 mu mol/L, the maximum value is 82 mu mol/L, and the minimum value is 2.9 mu mol/L; in normal infants, bilirubin monoglucuronate had a mean value of 1.25. mu. mol/L, a maximum value of 3.10. mu. mol/L and a minimum value of 0.02. mu. mol/L. The bilirubin monoglucuronide ester is taken as a diagnosis index, and when the cutoff value is 2.5 mu mol/L, the sensitivity of the bilirubin monoglucuronide ester for diagnosing biliary tract occlusion is 100 percent, and the specificity is 95.2 percent. Therefore, the up-regulation of bilirubin monoglucuronide is expected to be a characteristic detection index of neonatal biliary atresia, as shown in figures 5 and 6.

FIG. 6 is a set of scatter plots showing the difference in bilirubin monoglucuronide levels in biliary atresia neonate and normal neonate test samples.

Example 7 differentiation of patients with biliary atresia from other cholestatic disorders

In this example, serum samples of 26 biliary atresia newborns and 40 cholestasis newborns caused by other diseases were collected, and the content of bilirubin monoglucuronide in the serum samples was determined by the HPLC-MS method in example 1. The detection result shows that the content of bilirubin monoglucuronide in the infant with biliary atresia is obviously higher than that of other cholestatic infants (figure 5, P is less than 0.0001); the area under the ROC curve reaches 0.90 (95% CI is 0.83-0.97), the cutoff value is more than 75 mu mol/L, the sensitivity reaches 84.6% (65.1% -95.6%) and the specificity reaches 82.5% (67.2% -92.7%) according to the detection conditions in example 1, so that bilirubin monoglucuronide can be used as a diagnostic marker for preliminarily distinguishing biliary atresia and other infant cholestasis.

EXAMPLE 8 bilirubin monoglucuronate as diagnostic marker for the preparation of a diagnostic tool

The above examples show that the quantitative detection value of bilirubin monoglucuronide can be used as an index to distinguish normal newborns, newborns with biliary atresia, and newborns with bile stagnation disease caused by other reasons. Based on the creative research results, the purpose of this example is to exemplarily provide an application approach of bilirubin monoglucuronide content as a detection index for early screening and diagnosis of clinical neonatal biliary tract occlusion. After the newborn baby is born, if the screening of biliary tract occlusion can be rapidly, conveniently, efficiently and relatively accurately carried out, the method has very important significance for rapidly taking further medical measures for high-risk infant patients.

For this reason, the inventors propose that bilirubin monoglucuronide can be used as a diagnostic marker for preparing an appropriate tool for determining the biliary atresia risk of a test subject based on the purpose of application, wherein bilirubin monoglucuronide is used as the diagnostic marker, and the expression level of bilirubin monoglucuronide is used as the determination level. The kit may be a diagnostic product or an analytical test product, and as a specific example, this embodiment provides a kit as an example of a diagnostic product, which comprises a reagent for quantitatively detecting bilirubin monoglucuronide and optionally a reagent for quantitatively detecting other metabolic components of bilirubin, and further comprises a reagent for processing a blood sample of a subject, and further comprises a detection reagent for an hplc-tandem mass spectrometry detection method, such as an internal standard and an internal standard diluent, and a mobile phase reagent for hplc.

The inventor explores a preferable scheme, and the kit takes dry blood spots as detection samples. The dry blood spot sampling is convenient, is beneficial to the screening of biliary atresia of newborn infants after birth and is further beneficial to the large-area popularization of the biliary atresia screening of newborn infants in the early and early stages after birth. On the other hand, even if resampling is required, since the preparation technology of dried blood spots is mature, sampling is convenient, and the preparation method is less or almost non-invasive to newborn infants, even when repeated detection is required, and is particularly advantageous.

The kit typically includes at least one container for holding the standard. The container may be single-compartment or multi-compartment. For example, the container may be a multi-well plate (e.g., a 96-well plate), or other similar container. In some kits, this container is also suitable for use of the diagnostic marker in an assay. In some kits, the container can be used for standard detection or instrumental analysis of biomarkers in a sample to be tested, such as high performance liquid chromatography tandem mass spectrometry.

The following kit is taken as one of the specific embodiments:

TABLE 8 kit composition

EXAMPLE 9 use of the kit

The invention further researches and verifies the application of the diagnostic kit.

In this example, the inventors collected dried blood spots prepared within 4 days of birth of 30 clinically confirmed neonatal biliary atresia patients and 562 normal neonates according to the method of example 1, and detected the content of bilirubin monoglucuronide in the dried blood spots by using the reagent kit of example 3 in combination with the detection method of example 1.

The results are shown in Table 9 below, where cutoff > 2.5. mu. mol/L (FIG. 7A) was taken, the sensitivity reached 100% (88.43% -100%) and the specificity reached 98.74% (97.42% -99.49%) (FIG. 7B). It can be seen that the content of bilirubin monoglucuronide in the dried blood spots of the newborn can be used as a marker for evaluating the biliary atresia risk of the newborn, and the kit in the embodiment can be used for evaluating the biliary atresia risk of the newborn.

TABLE 9

Cutoff value (cutoff value)	Sensitivity (95% CI)	Specificity (95% CI)
			>2.5μmol/L	100％(88.43％-100％)	98.74％(95.42％-99.49％)

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. Use of bilirubin monoglucuronide as a diagnostic marker in the manufacture of a diagnostic device for assessing the risk of neonatal biliary atresia, characterized in that said diagnostic device carries out a quantitative detection of bilirubin monoglucuronide.

2. The use of claim 1, wherein the diagnostic device is selected from the group consisting of a medical device, a kit, a strip, and a test device.

3. The use according to claim 1, wherein the diagnostic device is a neonate subject to be tested, and a blood sample from the subject is used for testing; preferably, the blood sample is selected from the group consisting of whole blood, plasma, serum and dried blood spots of a subject.

4. The use of claim 1, wherein the quantitative detection method is liquid chromatography-tandem mass spectrometry; preferably, the liquid chromatography is selected from the group consisting of high performance liquid chromatography, ultra high performance liquid chromatography, and nanoliter liquid chromatography; preferably, the liquid chromatography is selected from high performance liquid chromatography and the liquid chromatography column is selected from C8 and C18 silica gel packed columns.

5. The use according to claim 7, wherein the liquid chromatography-tandem mass spectrometry detection method is characterized in that the composition of a mobile phase of the liquid chromatography is as follows:

the water phase A is selected from ultrapure water with pH value adjusted to 3-4.5 by a pH value adjusting agent;

the organic phase B is selected from one or a combination of more of acetonitrile, ethanol, methanol, propylene glycol and isopropanol;

preferably, the pH adjusting agent is selected from ammonium acetate-acetic acid, ammonium formate-formic acid, trifluoroacetic acid and trichloroacetic acid; preferably, the pH adjusting agent is an ammonium acetate/acetic acid buffer system;

preferably, the organic phase B is selected from one or a combination of more of acetonitrile, methanol and isopropanol; preferably, the organic phase B comprises acetonitrile, methanol and isopropanol.

6. The use according to any one of claims 5 to 12, wherein the liquid chromatography-tandem mass spectrometry detection method is selected from quadrupole mass spectrometry, time-of-flight mass spectrometry, ion hydrazine mass spectrometry and high resolution orbital hydrazine mass spectrometry; preferably, the condition of the mass spectrum and the setting of the qualitative and quantitative mass spectrum detection mode comprise: selecting an electrospray ionization source (ESI) to select an ion scanning mode based on a response to detecting a target compound; and selecting a multi-reaction monitoring method (MRM) and setting a multi-reaction monitoring mode parameter.

7. Use according to any one of claims 1 to 14, wherein the diagnostic device is adapted to assess the risk of biliary atresia in the newborn, optionally in combination with other measures of the subject, selected from the group consisting of free bilirubin, bilirubin diglucuronate, biliverdin, gamma glutamyltransferase, MMP-7 and one or more other measures which are directly or indirectly diagnostic of biliary atresia in the newborn for the purpose of said use.

8. The kit for evaluating the neonatal biliary atresia risk is characterized by comprising a bilirubin monoglucuronide quantitative detection reagent; preferably, the quantitative detection reagent comprises a bilirubin monoglucuronate quality control product and an internal standard product; preferably, the bilirubin monoglucuronide quality control product is a bilirubin monoglucuronide standard product which is distributed in a system containing a first stabilizer and a second stabilizer; preferably, the first stabilizer is one or a combination of more of Butyl Hydroxy Anisole (BHA), dibutyl hydroxy toluene (BHT), Propyl Gallate (PG) and tert-butyl hydroquinone (TBHQ); preferably, the second stabilizer is selected from ascorbic acid; preferably, the system of the first stabilizer and the second stabilizer is an organic solvent dispersion system containing dibutyl hydroxy toluene (BHT) and ascorbic acid; preferably, the organic solvent of the organic solvent dispersion system is selected from one or more of methanol, ethanol, acetone, propylene glycol and acetonitrile.

9. The kit of claim 18, wherein the system is a solid system comprising a solid support; preferably, the solid support is a filter paper sheet selected from the group consisting of analytical filter paper, qualitative analytical filter paper, and slow quantitative ashless filter paper; preferably, when the filter paper sheet is used as a solid carrier, the filter paper sheet is pretreated by the following steps: soaking in organic solvent dispersion system containing first stabilizer and second stabilizer, and drying in the shade.

10. A method of use of a kit according to any one of claims 16 to 25 for assessing the risk of neonatal biliary atresia, comprising,

(1) obtaining a test sample by taking a newborn infant as a detection object;

(3) analyzing the expression level of the biomarker for risk assessment, wherein the analysis can be compared with a cut-off value of a quantitative detection method, and the risk assessment conclusion can be used for assessing the high and low biliary atresia risk of a subject;

preferably, in the step (3) risk assessment, the neonatal biliary atresia risk is optionally assessed in combination with other test indicators of the test subject, the other test indicators being selected from one or more of free bilirubin, bilirubin diglucuronate, biliverdin, gamma-glutamyltransferase, MMP-7, and other indicators having a diagnostic ability directly or indirectly for neonatal biliary atresia;

preferably, the quantitative detection method is the detection method of any one of claims 1 to 7;

preferably, the test sample is selected from the group consisting of whole blood, plasma, serum and dried blood spots of a subject.

11. A method of assessing risk of biliary atresia in a newborn, comprising:

(1) obtaining a test sample by taking a newborn infant as a detection object;

(3) analyzing the expression level of the diagnostic marker for risk assessment, wherein the analysis can be compared with a cutoff value (cutoff value) of a quantitative detection method, and the conclusion of the risk assessment can be used for assessing the high or low biliary atresia risk of the subject;

(4) classifying the test subjects into a high risk group and a low risk group according to the assessment conclusion, wherein the high risk group suggests that further clinical diagnosis is necessary to determine whether the test subjects have biliary atresia;

preferably, the cutoff value of the quantitative determination method in step (3) is a value determined by statistical analysis of the biomarker level in the healthy neonate test sample and the corresponding biomarker level in the biliary atresia neonate test sample in advance by using the quantitative determination method in step (2);

preferably, the test sample is selected from whole blood, plasma, serum or dried blood spots;

preferably, the quantitative detection method is selected from the detection methods as claimed in any one of claims 1 to 7.

12. A device for predicting the risk of neonatal biliary atresia using the expression level of bilirubin monoglucuronate, comprising:

(1) a module for receiving a test sample of a test subject;

(2) a module for detecting data on the expression level of a diagnostic marker; wherein the diagnostic markers comprise at least bilirubin monoglucuronate, optionally in combination with free bilirubin, bilirubin diglucuronate, biliverdin, gamma glutamyltransferase, MMP-7 and other clinical indicators of diagnostic capacity for biliary atresia in the test sample;

a module for generating a risk score based on the expression levels of the diagnostic markers input to a database comprising control expression profiles associated with the test sample and the test method; the control expression profile is obtained in advance according to the detection sample and the detection method, and can be expressed as a cutoff value of the detected diagnostic marker; the risk assessment is carried out by comparing the expression level of the biomarkers in the test sample with a cut-off value of a high performance liquid chromatography tandem mass spectrometry as a detection method, and the detection object is considered to have high risk of biliary atresia if the cut-off value is higher than the cut-off value.