CN115112899B - Use of reagent and/or system for detecting carboxypeptidase A4 in preparation of malignant pleural effusion screening product - Google Patents
Use of reagent and/or system for detecting carboxypeptidase A4 in preparation of malignant pleural effusion screening product Download PDFInfo
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- 102100030621 Carboxypeptidase A4 Human genes 0.000 title claims abstract description 50
- 101710097783 Carboxypeptidase A4 Proteins 0.000 title claims abstract description 50
- 206010026673 Malignant Pleural Effusion Diseases 0.000 title claims abstract description 41
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 38
- 238000012216 screening Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 208000002151 Pleural effusion Diseases 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 238000002965 ELISA Methods 0.000 claims description 6
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 4
- 238000001262 western blot Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 4
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
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- 201000011510 cancer Diseases 0.000 description 3
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- 238000000034 method Methods 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 206010008479 Chest Pain Diseases 0.000 description 2
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- 102000004196 processed proteins & peptides Human genes 0.000 description 2
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- 102000003839 Human Proteins Human genes 0.000 description 1
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- 102000006166 Metallocarboxypeptidases Human genes 0.000 description 1
- 108030000089 Metallocarboxypeptidases Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
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- 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
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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Abstract
The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a reagent and/or a system for detecting carboxypeptidase A4 in preparation of a malignant pleural effusion screening product. The invention discovers that the carboxypeptidase A4 level in the pleural effusion of the patient with malignant pleural effusion is obviously higher than that of the patient with benign pleural effusion for the first time. Therefore, the reagent for detecting the carboxypeptidase A4 is used for preparing the malignant pleural effusion screening kit, so that the malignant pleural effusion can be effectively screened.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a reagent and/or a system for detecting carboxypeptidase A4 in preparation of a malignant pleural effusion screening product.
Background
Malignant pleural effusion refers to the phenomenon of excessive accumulation of fluid in the pleural cavity due to invasion of malignant tumor cells into the pleura. This is a common complication of advanced malignancies. Malignant pleural effusion is mostly bloody, is large in quantity and difficult to control, and often causes discomfort such as dyspnea, chest distress and chest pain of patients. Patient prognosis is often poor, with average survival time of only months after diagnosis. Thus, early diagnosis and screening of malignant pleural effusion is of great importance to patients.
Screening of malignant pleural effusion refers to the examination of those individuals who develop pleural effusion, and the detection of malignant pleural effusion in time without any clear indication of whether the patient is suffering from cancer. If a malignant pleural effusion molecular marker in the pleural effusion can be found, the malignant pleural effusion molecular marker is used for prompting a clinician to take relevant therapeutic measures or decisions on a patient in early stage and has important significance.
Carboxypeptidase A4 (CPA 4, uniprot number: Q9UI 42) is a C-terminal residue belonging to the family of metallocarboxypeptidases that cleave polypeptides and proteins with the participation of zinc ions. CPA4 lacks a transmembrane domain, a soluble extracellular protein, which functions biologically in the extracellular environment. There are studies showing that CPA4 has potential as a diagnostic marker for lung cancer (Yu Zhuo, preliminary study of CPA4 expression, biological function and molecular mechanism in liver cancer).
At present, no report is available on the relation between carboxypeptidase A4 and malignant pleural effusion.
Disclosure of Invention
The invention aims to provide a novel malignant pleural effusion marker and application of a detection reagent and/or a detection system of the marker in preparation of malignant pleural effusion screening products.
The technical scheme of the invention comprises the following steps:
Use of a reagent and/or system for detecting carboxypeptidase A4 in the preparation of a malignant pleural effusion screening product.
Preferably, the reagent for detecting the carboxypeptidase A4 is a reagent for an enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.
Preferably, the reagent for detecting the carboxypeptidase A4 is a western blot reagent.
Preferably, the reagent for detecting carboxypeptidase A4 is a reagent for a protein chip detection method.
Preferably, the system for detecting carboxypeptidase A4 is an LC-MS system.
Preferably, the reagent and/or system for detecting carboxypeptidase A4 is a reagent and/or system for detecting carboxypeptidase A4 in human pleural effusion.
The invention also provides a malignant pleural effusion screening system which comprises a liquid chromatography-mass spectrometry detection device and a reagent for cracking carboxypeptidase A4.
The invention also provides a malignant pleural effusion screening kit which comprises a reagent for detecting carboxypeptidase A4.
Preferably, the reagent for detecting the carboxypeptidase A4 is a reagent for an enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.
Preferably, the reagent for detecting the carboxypeptidase A4 is a western blot reagent.
Preferably, the reagent for detecting carboxypeptidase A4 is a reagent for a protein chip detection method.
The key point of the invention is that the content of the carboxypeptidase A4 in the human pleural effusion is determined to be obviously related to the risk of malignant pleural effusion, so that the risk of malignant pleural effusion can be judged by detecting the content of the carboxypeptidase A4 in the human pleural effusion, as for the specific detection of the carboxypeptidase A4 in the human pleural effusion, various means disclosed by the prior art can be adopted, and the embodiment of the invention specifically adopts the DIA technology for detection, but is not limited to the means, and any method capable of detecting the content of the carboxypeptidase A4 can be used for screening malignant pleural effusion.
The invention provides a novel malignant pleural effusion screening marker and a novel malignant pleural effusion screening product, which can realize effective screening of malignant pleural effusion, for example, under the condition that a patient is found to have pleural effusion but cancer is not diagnosed, the risk of the patient belonging to malignant pleural effusion can be evaluated through detection of the pleural effusion. The invention can use pleural effusion as a detection sample, and has low harm to patients. The invention has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to the following embodiments. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 shows the protein marker content in pleural effusions of different patients in example 1;
FIG. 2 is an ROC curve showing the relationship between protein markers and malignant pleural effusion in example 1.
Detailed Description
EXAMPLE 1 carboxypeptidase A4 in pleural effusion in relation to malignant pleural effusion
1. Clinical data
15 Patients with malignant pleural effusion and 15 patients with benign pleural effusion were randomly selected, and the basic information is as follows:
The pleural effusion of the aforementioned patients was taken, and the expression level of carboxypeptidase A4 (CPA 4) therein was examined for ROC analysis.
2. Detection method
Principle (one)
The principle of the DIA technology is that all parent ions and fragmented child ions in each acquisition window are subjected to full scanning at high speed and in a circulating mode during mass spectrum data acquisition, and secondary fragment rescanning is performed after screening.
(II) reagent
(III) detection step
1) The pleural effusion samples were resuspended in lysis buffer containing 8M urea, 100mM Tris-HCl (pH 7.5), 100mM DTT and protease inhibitor. Samples were homogenized using a tissue disruptor and sonicated at 60W f 5 min f 5 s f, then 10 s disrupted, then centrifuged at 15 min f 18000 x g,4 ℃. The supernatant was collected and the protein concentration in the lysate was estimated using BCA protein detection kit (Beyotime institute of biotechnology, china).
2) Protein catalysis was performed using FASP (filtration aid sample). Briefly, proteins were extracted from the ultrafiltration filtrate (30 kDa cut-off, sartorius, germany) with 100 μl UA buffer (8M urea, 150 mM Tris-HCl, ph8.0, 10 mM IAM) and incubated for 30 min. The sample was washed twice with 100. Mu.l UB buffer (8M urea, 150 mM Tris-HCl, pH 8.0) to a filter unit. Protein suspensions (100. Mu.l ABC and trypsin (Promega, USA)) in the filtrate tubes were enzymatically catalyzed after centrifugation with 12000 g for 10 min and incubated at 37℃for 18 h. The filtrate was centrifuged at 12000 g for 10 minutes and then used for LC-MS analysis.
3) Peptides were isolated using the EASY nano LC 1000 system (Thermo FISHER SCIENTIFIC, ma) and PepMap C18 (Thermo FISHER SCIENTIFIC,3 μm,100 a, 75 μm x, 15 cm, ma) and launched into a Q-Exactive HF tandem mass spectrometer of Thermo FISHER SCIENTIFIC, ma. Solvent a was 0.1% formic acid in water, while solvent B was 0.1% formic acid in 98% acetonitrile. For each injection, 3 μl was loaded and eluted using a 60 min gradient from 5% to 20% b, and rapidly assessed to 32% b by a linear gradient over the next 10 minutes, then increased to 90% b over 10 minutes. Finally, in the next 1 minute, B was restored to 5% and equilibrated for 10 minutes before the next injection. For quantitative samples, thermo Q-Exactive HF was configured to obtain a 55X 16m/z diameter spectrum (16 m/z precursor isolation window with resolution 30000, AGC target 1e5, maximum injection time 55 ms). The precursor spectra were collected at 120000 resolution (350-1500 m/z) to achieve the AGC target of 3e 6. The maximum implantation time was set to 50 ms.
4) DIA data was analyzed using Spectronaut Pulsar with DIRECTDIA method. MS/MS data were searched based on human protein sequences downloaded from Uniprot database (2018.720386 version entry), set as follows: enzyme: trypsin/P; maximum number of leaky splits: 2; fixing and modifying: aminomethyl (C); variable modification: oxidation (M) and acetyl (protein N-terminal); precursor mass tolerance: 20 ppm; fragment mass tolerance: 0.05Da.
5) Statistical analysis of the efficacy of CPA4 (carboxypeptidase A4) in diagnosing malignant pleural effusions was performed in R (version 3.5.0) software, and ROC curves were plotted using the metaX package of software.
3. Results
CPA4 content in pleural effusions of two groups of patients is shown in figure 1, CPA4 content in pleural effusions of patients with malignant pleural effusions is 369004 +/-339138 (10763-1423977), CPA4 content in pleural effusions of patients with benign pleural effusions is 53592 +/-85856 (4141-302295). Significant differences in CPA4 levels in the pleural effusions were seen in the two groups of patients (p < 0.05). CPA4 diagnosis of malignant pleural effusion the ROC curve for CPA4 was 92% by ROC analysis as shown in FIG. 2.
From the above results, it is clear that the difference between the detection level of carboxypeptidase A4 in the pleural effusion of patients with malignant pleural effusion and those with non-malignant pleural effusion is significant, and the purpose of screening malignant pleural effusion can be achieved by detecting the detection level of carboxypeptidase A4 in the pleural effusion.
The product of the invention can screen the risk of malignant pleural effusion of the people to be detected by detecting the level of carboxypeptidase A4 in the pleural effusion: if the level of carboxypeptidase A4 is high, the risk of developing malignant pleural effusion is high, and if the level of carboxypeptidase A4 is low, the risk of developing malignant pleural effusion is low. Can be used for the auxiliary diagnosis of clinical malignant pleural effusion, provides effective basis for patients to take relevant therapeutic measures or decisions, and has good clinical application prospect.
Claims (6)
1. Use of a reagent and/or system for detecting carboxypeptidase A4 in the preparation of a malignant pleural effusion screening product, characterized in that: the assay is to detect the carboxypeptidase A4 content of the pleural effusion.
2. The use according to claim 1, wherein the reagent for detecting carboxypeptidase A4 is a reagent for an enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.
3. The use according to claim 1, wherein the reagent for detecting carboxypeptidase A4 is a western blot reagent.
4. The use according to claim 1, wherein the reagent for detecting carboxypeptidase A4 is a reagent for a protein chip detection method.
5. The use according to claim 1, wherein the system for detecting carboxypeptidase A4 is an LC-MS system.
6. The use according to any one of claims 1 to 5, wherein the reagent and/or system for detecting carboxypeptidase A4 is a reagent and/or system for detecting carboxypeptidase A4 in human pleural effusion.
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| CN113186292A (en) * | 2021-05-27 | 2021-07-30 | 四川大学华西医院 | Lung cancer diagnostic kit based on gene methylation in lung tissue |
| CN113917147A (en) * | 2021-09-22 | 2022-01-11 | 核工业四一六医院 | Application of PEFL-1 detection reagent in preparation of lung cancer screening kit |
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| US20040077001A1 (en) * | 2002-08-02 | 2004-04-22 | Millennium Pharmaceuticals Inc. | Use for carboxypeptidase-A4 in the diagnosis and treatment of metabolic disorders |
| US20150178462A1 (en) * | 2011-08-29 | 2015-06-25 | Cardiodx, Inc. | Methods and Compositions for Determining Smoking Status |
| US20180126003A1 (en) * | 2016-05-04 | 2018-05-10 | Curevac Ag | New targets for rna therapeutics |
| US20210253987A1 (en) * | 2020-02-19 | 2021-08-19 | The Regents Of The University Of California | Method and device for early cancer screening |
| WO2021207550A1 (en) * | 2020-04-08 | 2021-10-14 | Remix Therapeutics Inc. | Compounds and methods for modulating splicing |
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| CN113186292A (en) * | 2021-05-27 | 2021-07-30 | 四川大学华西医院 | Lung cancer diagnostic kit based on gene methylation in lung tissue |
| CN113917147A (en) * | 2021-09-22 | 2022-01-11 | 核工业四一六医院 | Application of PEFL-1 detection reagent in preparation of lung cancer screening kit |
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