CN114518455A - Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis - Google Patents

Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis Download PDF

Info

Publication number
CN114518455A
CN114518455A CN202210055678.4A CN202210055678A CN114518455A CN 114518455 A CN114518455 A CN 114518455A CN 202210055678 A CN202210055678 A CN 202210055678A CN 114518455 A CN114518455 A CN 114518455A
Authority
CN
China
Prior art keywords
lipocalin
bile
product
malignant biliary
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210055678.4A
Other languages
Chinese (zh)
Inventor
秦文昊
夏明星
胡冰
饶春美
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University
Original Assignee
Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University filed Critical Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University
Priority to CN202210055678.4A priority Critical patent/CN114518455A/en
Publication of CN114518455A publication Critical patent/CN114518455A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of a product for diagnosing the malignant biliary stenosis, which belongs to the field of medical diagnosis, and the Lipocalin-2 is used as a novel biomarker to detect the protein level of the Lipocalin-2 in bile so as to diagnose the malignant biliary stenosis and improve the sensitivity and specificity of the diagnosis of the malignant biliary stenosis. Compared with the traditional diagnosis method, the technology has the advantages of timeliness, specificity and sensitivity in diagnosing the malignant biliary stricture, so that a patient can confirm the nature of lesion as early as possible, and a corresponding treatment scheme is adopted. In addition, bile is used as a detection sample, is easy to obtain in the ERCP operation process, does not need invasive operation, and does not bring additional risk to patients; and the Lipocalin-2 detection method needs less cost and has lower cost.

Description

Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis
Technical Field
The invention belongs to the field of medical diagnosis, and particularly relates to application of bile Lipocalin-2 in diagnosis of malignant bile duct stenosis and preparation of a product for diagnosing the malignant bile duct stenosis.
Background
The symptoms such as jaundice caused by bile duct stenosis are often the direct reasons for the patients to see a doctor, and the causes of the symptoms include benign and malignant diseases, wherein the benign stenosis includes primary sclerosing cholangitis, chronic pancreatitis, cholelithiasis, biliary tract injury and the like, and the malignant stenosis includes malignant tumors of the liver, gallbladder and pancreatic system and other metastases, and bile duct cancer and pancreatic cancer are the most common. The biliary pancreatic malignant tumor is latent in disease, most of the biliary pancreatic malignant tumors belong to advanced tumor when clinical symptoms appear, the surgical resection rate is low, the effect of conventional chemoradiotherapy is poor, and the overall prognosis is very poor.
The property of bile duct stenosis is important to be determined at an early stage, and although the sensitivity of the serum tumor marker CA19-9 in diagnosing malignant bile duct stenosis reaches about 80%, the false positive rate is high. Endoscopic Retrograde Cholangiopancreatography (ERCP) is a diagnostic procedure often used earlier to obtain detached cell or tissue biopsy samples for pathological diagnosis, in addition to cholangiography. However, the positive rate of traditional pathological diagnosis is low, only about 30%, so that most clinical patients cannot obtain accurate diagnosis in time, and the treatment time is delayed. Therefore, there is an urgent need to find new markers with high sensitivity and specificity to improve the early diagnosis rate of malignant biliary stricture, and further improve the surgical resection rate and prognosis of the pancreatic cancer.
Patients with bile duct stenosis often need to perform Endoscopic Retrograde Cholangiopancreatography (ERCP) treatment, bile samples can be easily obtained through the ERCP, the bile can directly contact with malignant bile duct tumors, and theoretically, compared with serum, bile juice has the advantage that the content of tumor-related proteins is richer, especially in the early stage of diseases. Therefore, molecular diagnosis of malignant biliary stricture using biomarkers in bile has become a hotspot of recent research. To date, a number of biomarkers have been identified in bile, such as ApoA-I, actin-1, S100a9, CEAM6 and MCM5, but only a few of these markers have been incorporated into routine clinical practice. This may be due to inconsistencies in patient recruitment criteria, as well as biological variability concerns for individual markers.
Lipocalin-2 protein belongs to the Lipocalin family. The physiological function of this family of proteins is to transport small hydrophobic molecules such as lipids, steroid hormones, and retinoic acid. Lipocalin-2 is a neutrophil gelatinase-associated lipid carrier protein that functions in innate immunity by sequestering siderophores to limit bacterial growth. Earlier studies showed that Lipocalin-2 in blood and urine is an early biomarker of acute kidney injury. While Lipocalin-2 is also a key factor in the pathophysiological process of tumors, a recent meta-analysis report suggests that the measurement of Lipocalin-2 in plasma and urine may be helpful in predicting the prognosis of colorectal and breast cancers. At present, no report is found on the diagnosis of malignant biliary stenosis by Lipocalin-2 in bile and plasma.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide an application of bile Lipocalin-2 in diagnosis of malignant biliary stricture and preparation of a product for diagnosing malignant biliary stricture.
The invention provides an application of bile Lipocalin-2 in diagnosis of malignant biliary stricture.
The invention provides an application of bile Lipocalin-2 in preparation of a product for diagnosing malignant bile duct stenosis.
In the application of the bile Lipocalin-2 provided by the invention in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: wherein the product is used for detecting the Lipocalin-2 level in the bile of a patient to diagnose malignant biliary stenosis.
In the application of the bile Lipocalin-2 provided by the invention in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: wherein the product is a protein chip.
In the application of the bile Lipocalin-2 provided by the invention in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: wherein, the application includes: firstly, carrying out bile pretreatment, and then detecting the Lipocalin-2 level in the pretreated bile by using a protein chip, wherein the bile pretreatment comprises the following steps: the bile was harvested by ERCP, added with Tween 20 and protease inhibitor at a ratio of 0.1%, centrifuged at 14000rpm for 10 minutes at 4 ℃ and the lipid was removed.
In the application of the bile Lipocalin-2 provided by the invention in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: wherein, the product is an enzyme-linked immunosorbent assay kit.
In the application of the bile Lipocalin-2 in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: wherein, the application is to detect the Lipocalin-2 level in bile by using an enzyme-linked immunosorbent assay kit and an ELISA method.
In the application of the bile Lipocalin-2 provided by the invention in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: wherein the product is Lipocalin-2 colloidal gold test paper.
In the application of the bile Lipocalin-2 provided by the invention in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: the preparation process of the Lipocalin-2 colloidal gold test paper comprises the following steps: preparing a gold label pad, wherein the gold label pad comprises a PVC (polyvinyl chloride) base plate, a colloidal gold pad, a sample pad, an NC (numerical control) film and absorbent paper, the sample pad, the colloidal gold pad, the NC film and the absorbent paper are sequentially arranged on the PVC base plate from left to right, and adjacent components are partially overlapped; connecting the colloidal gold particles with the Lipocalin-2 antibody to prepare a colloidal gold-labeled Lipocalin-2 antibody, and arranging the colloidal gold-labeled Lipocalin-2 antibody on the gold-labeled pad in a spraying or soaking mode; and fixedly coating a Lipocalin-2 antibody at the middle position of the NC membrane, and coating a goat anti-mouse polyclonal antibody at the right end of the NC membrane to obtain the Lipocalin-2 colloidal gold detection test paper.
In the application of the bile Lipocalin-2 provided by the invention in preparing a product for diagnosing malignant bile duct stenosis, the bile Lipocalin-2 also has the following characteristics: wherein the size of the colloidal gold particles is 20 nm-40 nm.
Action and Effect of the invention
According to the application of the bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of products for diagnosing the malignant biliary stenosis, the diagnosis of the malignant biliary stenosis can be carried out by detecting the protein level of the Lipocalin-2 in the bile by using the Lipocalin-2 as a novel biomarker, so that the sensitivity and the specificity of the diagnosis of the malignant biliary stenosis are improved. Compared with the traditional diagnosis method, the technology has the advantages of timeliness, specificity and sensitivity in diagnosing the malignant biliary stricture, so that a patient can confirm the nature of lesion as early as possible, and a corresponding treatment scheme is adopted.
In addition, bile is used as a detection sample, is easy to obtain in the ERCP operation process, does not need invasive operation, and does not bring additional risk to patients; and the Lipocalin-2 detection method needs less cost and has lower cost.
Drawings
FIG. 1 is a graph showing the results of detection of a protein chip in example 1 of the present invention;
FIG. 2 is a graph showing the result of quantifying the fluorescence intensity of the protein chip in example 1 of the present invention;
FIG. 3 is a graph showing the results of ELISA detection of the protein level of bile juice Lipocalin-2 in example 2 of the present invention;
FIG. 4 is a ROC analysis curve according to the level of bile Lipocalin-2 protein in example 2 of the present invention.
Detailed Description
In order to make the technical means, creation characteristics, achievement purposes and effects of the invention easy to understand, the following embodiments will specifically describe the application of bile Lipocalin-2 in the diagnosis of malignant bile duct stenosis and the preparation of products for diagnosing malignant bile duct stenosis with reference to the attached drawings.
< example 1>
Example 1 provides an application of bile Lipocalin-2 in preparing a product for diagnosing malignant biliary stricture.
The product of example 1 was a protein chip, available from RayBiotech Inc. (G-Series Human cytokine antibody array 440; RayBiotech Inc., Norcross, GA, USA), for detecting the level of Lipocalin-2 protein in a patient's bile.
The protein chip is used for respectively detecting the Lipocalin-2 level in the bile of 6 patients with malignant biliary stricture and benign biliary stricture, and the specific process is as follows:
1. bile pretreatment
Bile was harvested by ERCP, Tween 20 and protease inhibitor (Sigma-Aldrich, St. Louis, Missouri, USA) were added at a ratio of 0.1%, centrifuged at 14000rpm for 10 minutes at 4 ℃ and washed with CleanAsciteTM(Biotech Support Group, NJ, USA) removes lipids.
2. Protein chip detection
1) The chip membrane was blocked with 5% fetal bovine albumin solution and incubated with 10-x PBS diluted bile for 2 hours at room temperature.
2) Wash 3 times with 1 XTSST for 5 minutes each.
3) The anti-cytokine mixture bound to biotin (provided in the kit) was incubated for 2 hours.
4) After washing 3 times with 1 × TBST for 5 minutes each, unbound antibody was removed,
5) the chip membrane was incubated with horseradish peroxidase conjugated streptavidin for 2 hours at RT.
6) Wash 3 times with 1 XTSST for 5 minutes each.
7) Fluorescent signal (Cy 3; 555nm excitation) were scanned and recorded with an InnoScan 300 microarray scanner (Innopsys, Parcd' activity instruments, 31390 carbon-France).
FIG. 1 is a graph showing the results of detection of a protein chip in example 1 of the present invention; FIG. 2 is a graph showing the results of quantifying the fluorescence intensity of the protein chip in example 1 of the present invention.
As shown in fig. 1 and 2, the bile Lipocalin-2 protein level was significantly higher in the malignant stenosis group than in the benign stenosis group of the bile duct.
< example 2>
Embodiment 2 provides an application of bile Lipocalin-2 in preparation of a product for diagnosing malignant bile duct stenosis.
The product of example 2 is an enzyme-linked immunosorbent assay (ELISA) kit, purchased from Raybiotech. The bile of 20 bile duct malignant stenosis and benign stenosis patients is detected by using the ELISA kit and the ELISA method, and the detection process is as follows:
elisa test plate coating process:
lipocalin-2 antibody was diluted to 200. mu.g/ml with coating diluent, 100. mu.l per well, placed in a wet box, incubated in a refrigerator at 4 ℃ for 24h, and excess antibody was discarded.
2. And (3) standard product configuration:
preparing a standard substance: the recombinant human Lipocalin-2 protein was diluted to 1. mu.g/ml, 500ng/ml, 250ng/ml, 125ng/ml, 50ng/ml, 15ng/ml, 5ng/ml, and a zero-concentration blank control tube was set.
3. And (3) sealing the enzyme-labeled reaction hole:
filling 5% fetal calf serum in the reaction holes, removing bubbles in each hole by shaking, sealing in a 37 deg.C incubator for 40min, and washing 3 times each for 3min after sealing.
The washing method comprises sucking dry reaction solution with pipette, filling the plate hole with washing solution, standing for 2min, slightly shaking, pouring off the solution, and patting on absorbent paper. Number of washes 3 times
4. Adding a sample to be detected and a standard substance:
adding bile into enzyme-labeled reaction well, adding 100 μ l of each sample, incubating in 37 deg.C incubator for 40-60min, washing with washing solution for 3 times, and 3min each time.
5. Adding an enzyme-labeled antibody:
10. mu.g/ml of horseradish peroxidase-labeled Lipocalin-2 antibody was added to the reaction wells, incubated at 37 ℃ for 60min, and 100. mu.l was added to each well, and washed as before.
6. Adding substrate solution (prepared as used):
adding 100 μ l of horseradish peroxidase substrate into each well, placing at 37 deg.C in dark for 3-5 min, adding stop solution, and developing.
7. And (3) terminating the reaction:
the reaction was stopped by adding 50. mu.l of stop solution to each well and the results were measured within 20 min.
8. And (5) judging a result:
and reading the fluorescence value at a proper wavelength by using a microplate reader, calculating a standard curve, and calculating the concentration of the sample Lipocalin-2 according to a standard curve formula.
9. The statistical method comprises the following steps:
lipocalin-2 efficacy in diagnosing malignant biliary stricture was analyzed by receiver operating characteristic curve (ROC) analysis, Cut-off value was selected by the Johnson index method, and the software used was SPSS 21.0.
FIG. 3 is a graph showing the results of ELISA detection of the protein level of bile juice Lipocalin-2 in example 2 of the present invention. FIG. 4 is a ROC analysis curve according to the level of bile Lipocalin-2 protein in example 2 of the present invention.
As shown in fig. 3 and 4, the ELISA test results show that: the Lipocalin-2 level in bile of a patient with malignant biliary stricture is obviously higher than that in a benign biliary stricture group, the diagnosis efficiency of the Lipocalin-2 in the bile on the malignant biliary stricture is realized by utilizing a receiver operating characteristic curve (ROC), the area under the curve (AUC) is 0.948, the Cut-off value is 36.79ng/ml by calculating a Youden index (Youden index), the upper limit of a reference value is taken as the upper limit, and the sensitivity and the specificity of the Lipocalin-2 for diagnosing the malignant biliary stricture are 90% and 95%.
< example 3>
Embodiment 3 provides an application of bile Lipocalin-2 in preparing a product for diagnosing malignant bile duct stenosis.
The product in example 3 was Lipocalin-2 colloidal gold test paper. The preparation process of the Lipocalin-2 colloidal gold test paper is as follows:
1. preparing a gold label pad, wherein the gold label pad comprises a PVC bottom plate, a colloidal gold pad, a sample pad, an NC film and absorbent paper, the sample pad, the colloidal gold pad, the NC film and the absorbent paper are sequentially arranged on the PVC bottom plate from left to right, and the adjacent components are partially overlapped.
2. And connecting the colloidal gold particles with the Lipocalin-2 antibody to prepare a colloidal gold-labeled Lipocalin-2 antibody, and arranging the colloidal gold-labeled Lipocalin-2 antibody on the gold label pad in a spraying or soaking mode.
3. And fixedly coating a Lipocalin-2 antibody at the middle position of the NC membrane, and coating a goat anti-mouse polyclonal antibody at the right end of the NC membrane to obtain the Lipocalin-2 colloidal gold detection test paper.
The qualitative detection of the bile is carried out by using the Lipocalin-2 colloidal gold test paper and the colloidal gold method, and the detection process is as follows:
2-20 μ l of bile sample was added to the test well of the sample pad, 10-50 μ l of sample diluent was added, and the sample diluent and sample were chromatographically shifted to the right. The sample diluent consists of Tween 20, protease inhibitor and the like.
And (5) judging a result: the positive result of Lipocalin-2 is that red and wine red appear at the test line (T line), and red and wine red appear at the quality control line (C line); the negative result of Lipocalin-2 is that no color reaction is generated at the test line (T line), and red and wine red appear at the quality control line (C line); and (3) invalid detection: there was no color reaction at the control line (line C).
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
For example, in the above example 1, the product for diagnosing malignant biliary stricture is a protein chip; in the above example 2, the product for diagnosing malignant biliary stricture is an ELISA kit; in the above example 3, the product for diagnosing malignant biliary stricture is Lipocalin-2 colloidal gold test paper; in practical application, the product for diagnosing the malignant bile duct stenosis can also be other products such as a protein immunoblotting kit and the like.

Claims (10)

1. An application of bile Lipocalin-2 in diagnosing malignant biliary stricture is disclosed.
2. An application of bile Lipocalin-2 in preparing the product for diagnosing the malignant biliary stricture is disclosed.
3. Use of bile Lipocalin-2 according to claim 1 for the preparation of a product for the diagnosis of malignant biliary strictures, characterized in that:
wherein the product is used for detecting Lipocalin-2 levels in bile of a patient for diagnosing malignant biliary strictures.
4. Use of bile Lipocalin-2 according to claim 3 in the preparation of a product for diagnosing malignant biliary stricture, characterized in that:
wherein, the product is a protein chip.
5. The use of bile Lipocalin-2 according to claim 4 in the preparation of a product for diagnosing malignant biliary stricture, characterized in that:
wherein the application comprises: firstly, bile pretreatment is carried out, then the protein chip is used for detecting the Lipocalin-2 level in the pretreated bile,
the bile pretreatment comprises the following steps: the bile was harvested by ERCP, added with Tween 20 and protease inhibitor at a ratio of 0.1%, centrifuged at 14000rpm for 10 minutes at 4 ℃ and the lipid was removed.
6. Use of bile Lipocalin-2 according to claim 3 in the preparation of a product for diagnosing malignant biliary stricture, characterized in that:
wherein, the product is an enzyme-linked immunosorbent assay kit.
7. The use of bile Lipocalin-2 according to claim 6 in the preparation of a product for diagnosing malignant biliary stricture, characterized in that:
wherein, the application is to detect the Lipocalin-2 level in bile by using the ELISA kit and an ELISA method.
8. Use of bile Lipocalin-2 according to claim 3 in the preparation of a product for diagnosing malignant biliary stricture, characterized in that:
wherein the product is Lipocalin-2 colloidal gold test paper.
9. Use of bile Lipocalin-2 according to claim 8 in the preparation of a product for diagnosing malignant biliary stricture, characterized in that:
wherein the preparation process of the Lipocalin-2 colloidal gold test paper is as follows:
preparing a gold label pad, wherein the gold label pad comprises a PVC base plate, a colloidal gold pad, a sample pad, an NC (numerical control) membrane and absorbent paper, the sample pad, the colloidal gold pad, the NC membrane and the absorbent paper are sequentially arranged on the PVC base plate from left to right, and adjacent components are partially overlapped;
connecting colloidal gold particles with a Lipocalin-2 antibody to prepare a colloidal gold-labeled Lipocalin-2 antibody, and arranging the colloidal gold-labeled Lipocalin-2 antibody on the gold label pad in a spraying or soaking mode;
and fixedly coating a Lipocalin-2 antibody in the middle of the NC membrane, and coating a goat anti-mouse polyclonal antibody at the right end of the NC membrane to obtain the Lipocalin-2 colloidal gold detection test paper.
10. Use of bile Lipocalin-2 according to claim 9 in the preparation of a product for diagnosing malignant biliary stricture, characterized in that:
wherein the size of the colloidal gold particles is 20 nm-40 nm.
CN202210055678.4A 2022-01-18 2022-01-18 Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis Pending CN114518455A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210055678.4A CN114518455A (en) 2022-01-18 2022-01-18 Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210055678.4A CN114518455A (en) 2022-01-18 2022-01-18 Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis

Publications (1)

Publication Number Publication Date
CN114518455A true CN114518455A (en) 2022-05-20

Family

ID=81596528

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210055678.4A Pending CN114518455A (en) 2022-01-18 2022-01-18 Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis

Country Status (1)

Country Link
CN (1) CN114518455A (en)

Similar Documents

Publication Publication Date Title
JP6096813B2 (en) Multi-biomarker set for breast cancer diagnosis, detection method thereof, and breast cancer diagnosis kit including antibody thereto
WO2022083673A1 (en) Biomarker for esophageal cancer, and use thereof
JP2016519285A (en) Methods and compositions for detecting pancreatic cancer
US20080113389A1 (en) Detecting the Presence of Pyruvate Kinase Isoenzyme in Feces
JP2008509411A (en) Diagnosis method of liver fibrosis
EP1678503A2 (en) Specific method for cancer detection
CN110187109B (en) Autoantibody joint detection ELISA kit for early screening of cardia adenocarcinoma
US20120295288A1 (en) Serological marker for detecting pancreatic cancer and a method for using the serological marker
KR20110069618A (en) Kit and method for diagnosis of prostate cancer
CN110187111B (en) ELISA kit for screening early cardiac cancer
CN102803968B (en) Esophageal cancer marker
CN113777311A (en) ELISA kit for auxiliary diagnosis of esophageal squamous cell carcinoma
Sánchez-Carbayo et al. Urinary tissue polypeptide-specific antigen for the diagnosis of bladder cancer
CN114518455A (en) Application of bile Lipocalin-2 in diagnosis of malignant biliary stenosis and preparation of product for diagnosing malignant biliary stenosis
CN112129954B (en) Application of MMP7, CTSE or LAMC2 protein in preparation of intrahepatic cholangiocellular carcinoma diagnostic reagent
KR101311717B1 (en) Protein marker melanotransferrin for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same
CN115372616A (en) Gastric cancer related biomarker and application thereof
CN114414814A (en) Application of bile P-cadherin in diagnosis of malignant bile duct stenosis and preparation of product for diagnosing malignant bile duct stenosis
CN113671180A (en) Application of PAIP1 autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma
KR101311718B1 (en) Protein marker RPE-spondin for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same
CN117607443B (en) Biomarker combinations for diagnosing breast cancer
US20080153119A1 (en) Detecting the presence of pyruvate kinase isoenzyme in feces
JP4795353B2 (en) Use of carbamoyl phosphate synthase 1 (CPS1) as a humoral biomarker for the diagnosis of tumor diseases and chronic inflammatory bowel disease
CN118376789B (en) Detection kit for diagnosing lung cancer and application thereof
CN118243928B (en) Detection kit for diagnosing colorectal cancer and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination