CN114487418A - Application of TGFb RIII protein as gastric cancer serum biomarker - Google Patents
Application of TGFb RIII protein as gastric cancer serum biomarker Download PDFInfo
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- CN114487418A CN114487418A CN202210143790.3A CN202210143790A CN114487418A CN 114487418 A CN114487418 A CN 114487418A CN 202210143790 A CN202210143790 A CN 202210143790A CN 114487418 A CN114487418 A CN 114487418A
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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
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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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
- 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
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- 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
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
Abstract
The application provides an application of TGFb RIII protein as a gastric cancer serum biomarker, and the application has the advantages that: provides an application of TGFb RIII protein as a gastric cancer serum biomarker.
Description
Technical Field
The application relates to application of TGFb RIII protein as a serum biomarker of gastric cancer.
Background
Currently, the screening mode of gastric cancer is mainly upper gastrointestinal endoscopy and the like. However, the upper gastrointestinal endoscopy requires advanced instruments and equipment and special operators, has high technical requirements, high cost, painful subjects and poor compliance, and is not suitable for repeated examination and crowd census. In addition, there are other methods for screening gastric cancer or its precursor lesions, such as Helicobacter pylori (Hp) detection, serum Pepsinogen (PG) detection, gastrin (gastin-17, G-17) detection, etc., but further research is still needed due to high false positive rate and low sensitivity.
Biomarkers refer to biomolecules used for disease diagnosis, risk assessment, and prognosis. Through the proteomic analysis of plasma/serum and urine of different cancer patients, a series of protein markers related to the occurrence and development of tumors are discovered, and the markers can be used for screening, diagnosis and prognosis monitoring of cancers. The lack of biomarkers, especially non-invasive biomarkers, clinically used for gastric cancer diagnosis is of great significance.
Disclosure of Invention
The application aims to provide application of TGFb RIII protein as a gastric cancer serum biomarker.
The purpose of the application is realized by the following technical scheme:
in a first aspect of the application, the use of TGFb RIII protein as a serum biomarker for gastric cancer is disclosed.
Further, the TGFb RIII protein is a human TGFb RIII protein.
In a second aspect of the application, a gastric cancer serum biomarker is disclosed, comprising a TGFb RIII protein.
The alternatives of the present application can be either as independent solutions or in combination with each other. The structure in any aspect of the present application may be used as an independent technical aspect or may be combined with other technical aspects.
The application has the advantages that: provides an application of TGFb RIII protein as a gastric cancer serum biomarker.
Drawings
FIG. 1 shows the TGFb RIII protein content and variability in the samples of example 1 of the present application;
FIG. 2 is a ROC curve of TGFb RIII protein of healthy-advanced gastric cancer patients in example 1 of the present application;
FIG. 3 shows TGFb RIII protein content and inter-group variability in example 2 of the present application;
FIG. 4 is a ROC curve of TGFb RIII protein of healthy-gastric cancer patients in example 2 of the present application.
Detailed Description
The term "ROC curve" or "ROC graph" as used herein refers to a graphical curve that shows the variation of the performance of a binary classifier system as a function of its discrimination threshold. The curve is created by plotting a true positive rate versus a false positive rate at various threshold settings. The true positive rate is also referred to as sensitivity. False positive rate was calculated as 1-specificity. Thus, the ROC curve is a graphical display of true positive versus false positive rates (sensitivity vs (1-specificity)) over a range of cut-off values and a way to select the optimal cut-off value for clinical use. Accuracy is expressed as the area under the ROC curve (AUC) and provides a useful parameter for comparing test performance. An AUC of approximately 1 indicates that the test is highly sensitive and highly specific, while an AUC of approximately 0.5 indicates that the test is neither sensitive nor specific.
The term "statistical difference" or "statistical significance" when referring to the difference between a test sample and a control sample or reference sample relates to the case where the probability of identity is less than 5% (e.g. p <0.05) for each group when using appropriate statistical analysis. In other words, the probability of obtaining the same result on a completely random basis is less than 5 out of 100 attempts. The person skilled in the art knows how to select an appropriate statistical analysis. An appropriate statistical analysis is typically determined based on whether the variables under study have a normal distribution (e.g., by using the Kolmogorov-Smirnov test) and whether homologies exist (e.g., using the Levene test). Preferably, in the presence of normal distribution and homoscedasticity, a parametric model such as the t-test or the ANOVA test is used; and in the event that at least one of these two requirements is not met, a non-parametric model, such as the MannWhitney U test or the Kruskal-Wallis test, is typically used.
The TGFb RIII protein in this application is a human protein TGFb RIII (type III transformation growth factor beta receptor, also known as betaglycan) encoded by the TGFb RIII gene on chromosome 1p31-32, which consists of 16 exons, there are two promoters, one proximal and one distal, which produce two mrnas. TGFb RIII is an 851 amino acid transmembrane proteoglycan comprising a large 766 amino acid extracellular domain, a hydrophobic transmembrane domain, and a short 42 amino acid cytoplasmic domain. TGFb RIII is expressed on the cell surface as a non-covalently linked homodimer.
In order to make the technical solutions of the present application better understood, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1:
firstly, obtaining a serum sample
By means of recruitment, a total of 330 serum samples of subjects were obtained, of which 39 healthy persons, 128 cases of early cancer, 80 cases of advanced gastric cancer, 13 cases of gastritis, 22 cases of liver cancer, 32 cases of colorectal cancer and 19 cases of breast cancer.
Second, serum detection
In this example, serum samples of each subject were tested using a QAH-cure kit comprising a quantitative Chip (quantum Arxax Glass Chip), a Sample Diluent (Sample Diluent), 20 × Wash solution I (20X Wash Buffer I), 20 × Wash solution II (20X Wash Buffer II), a standard mixture (lysostabilized standard mix), a Detection antibody mixture (Detection antibody cocktail), Cy 3-Streptavidin (Cy3equivalent dye-conjugated Streptavidin), a Slide washer and Dryer (Slide washer/Dryer), a seal (additive separator); the specific detection method comprises the following steps:
s1 quantitative chip drying
Taking out the quantitative chip from the box, standing at room temperature for 20-30min, opening the packaging bag, uncovering the sealing strip, and then drying the quantitative chip in a dryer or at room temperature for 1-2 hours.
S2 configuration of standard product
S21, adding 500. mu.L of the sample diluent to the vial of the standard mixture, and redissolving the standard mixture. Before opening the vial, the vial was rapidly centrifuged and gently blown up and down to dissolve the powder, and the vial was labeled Std 1.
S22, labeling 6 clean centrifuge tubes separately as Std2, Std3, Std4, Std5, Std6, Std7, and adding 200. mu.L of sample diluent to each centrifuge tube.
S23, drawing 100 μ L of sample diluent from the centrifuge tube marked Std1, adding the sample diluent into the centrifuge tube marked Std2, mixing gently, drawing 100 μ L of sample diluent from the centrifuge tube marked Std2, adding the sample diluent into the centrifuge tube marked Std3, and so on, and performing gradient dilution to the centrifuge tube marked Std7 to obtain a standard solution.
S24, draw 100. mu.L of the standard solution from the tube labeled Std7 to a new tube labeled CNTRL as a negative control.
S3 operation flow of quantitative chip
S31, adding 100 mu L of sample diluent into each hole of the quantitative chip, incubating for 1h on a shaking bed at room temperature, and sealing the quantitative chip.
S32, the sample dilution in each well was aspirated, 80. mu.L of standard solution was added to the wells along with the serum sample (2-fold dilution of sample) from the centrifuge tube labeled CNTRL, and incubated overnight at 4 ℃.
S33, washing a quantitative chip (slide glass), wherein a Thermo Scientific Wellwash Versa chip plate washing machine is used in the embodiment and is divided into two steps, washing is firstly carried out by 1 × washing liquid I, 250 μ L of 1 × washing liquid I is washed for 10 times, each time the washing is carried out for 10S, the shaking intensity is selected to be high, and 20 × washing liquid I is diluted by deionized water. Then the channel was washed with 1 × washing solution II, 250 μ L of 1 × washing solution II per well for 6 times with 10s shaking each time, with high shaking intensity, and 20 × washing solution II was diluted with deionized water.
S34 incubation of detection antibody mixture
The vial containing the detection antibody mixture was centrifuged, then 1.4ml of sample diluent was added, mixed well and then centrifuged rapidly again, then 80. mu.L of detection antibody mixture was added to each well and incubated on an RT shaker for 2 hours.
S35, cleaning and synchronizing with S33.
Incubation of S36, Cy 3-streptavidin
Centrifuging the vial containing Cy 3-streptavidin, then adding 1.4ml of sample diluent, mixing well, then quickly centrifuging again, adding 80. mu.L of Cy 3-streptavidin to each well, wrapping the quantitative chip (slide) with aluminum foil paper, incubating in the dark, and incubating on an RT shaker for 1 hour.
S37, cleaning and synchronizing with S33.
S38 fluorescence detection
Scanning the signal with a laser scanner using either Cy3 or green channel (excitation frequency 532nm)
In this embodiment, an InnoScan 300 is adopted, and the model of the apparatus is: innoscan 300Microarray Scanner; the manufacturer is Innopsys; the producing area: parc d' Activies ActiveStre; 31390 Carbonne-France; scanning parameters are as follows: WaveLengh: 532 nm; resolution: 10 μm.
S39, data analysis is carried out by adopting data analysis software of QAH-CUST, and the content of TGFb RIII protein in each serum sample is obtained.
Third, experimental results
As shown in fig. 1, the level of TGFb RIII protein in the advanced gastric cancer group was increased and was statistically significant from the other groups (wherein "×" between the healthy group and the advanced gastric cancer group in fig. 1 indicates a statistical difference P value of less than 0.001), and further analysis of the data was performed, and the results obtained by ROC Curve (Receiver Operating charateristic Curve) are shown in fig. 2, and AUC (Area Under ROC Curve, which is the Area covered by the ROC Curve, the larger the AUC, the better the effect of distinguishing the two groups) of TGFb RIII protein in the advanced patients was 0.797.
Example 2:
to further demonstrate the reliability of the results, 60 additional subjects were enrolled: healthy 10 cases, early cancer 10 cases, and late cancer 40 cases. After collection of the subject sera, an Elisa test was performed.
In this example, serum samples from each subject were tested using a TGFb RIII assay kit to obtain TGFb RIII protein content in each serum sample, which kit comprises the following components:
TGFb RIII Microplate (Item A) 96-well plate coated with anti-Human TGFb RIII antibody
Wash Buffer Concentrate (20X) (Item B) 25ml of 20X concentrated Wash
Standards (item C) recombinant Human TGFb RIII standards
Assay Diluent (Item E2), 15ml,5 Xconcentrate, for dilution of standards and samples
Detection Antibody TGFb RIII (Item F): Biotin-labeled anti-TGFb RIII Antibody
HRP-Streptavidin concentrate (Item G) 200. mu.L 200 Xconcentrated HRP-Streptavidin
TMB One-Step Substrate Reagent(Item H):12ml of 3,3’,5,5’-tetramethylbenzidine(TMB)
Stop Solution (Item I) 8ml of 0.2M sulfuric acid
First, reagent preparation
1) Equilibrating the kit and serum sample to room temperature (18-25 ℃);
2) serum sample dilution 2 times loading according to preliminary experiment
3) Diluting the diluent (Item E2) with deionized water by 5 times for later use;
4) preparing a standard product (Item C), namely centrifuging the tube filled with the standard product (Item C), adding 400 mu L of 1x diluent (Item E2) into the tube filled with the standard product (Item C), and uniformly mixing to obtain a standard product stock solution of 50000 pg/ml; sucking 100 μ L of the stock solution of the standard, adding into a centrifuge tube containing 400 μ L of 1 × diluent (Item E2), and mixing, wherein the label is STD 1; preparing 7 1.5ml small centrifuge tubes, adding 300. mu.L of 1 Xdiluent (Item E2) as a buffer solution to each of the 7 tubes, and labeling the 7 1.5ml small centrifuge tubes as STD2, STD3, STD4, STD5, STD6, STD7 and STD8 in this order; then a 10000pg/ml stock solution of standard in STD1 was diluted in a gradient: extracting 200 μ L of 10000pg/ml stock solution (STD 1) into STD2 vial, mixing, extracting 200 μ L of solution from STD2 vial, adding into STD3 vial, mixing, and sequentially until STD7 is prepared; STD8 is 300. mu.L of 1 Xdilution (Item E2) which is 0pg/ml of standard product;
5) dilution of concentrated wash (Item B): diluting the concentrated washing solution (Item B) with deionized water by 20 times to obtain a washing buffer solution for later use;
6) centrifuging TGFb RIII detection antibody (Item F), adding 100L 1x diluent (Item E2) for fully dissolving, gently blowing up and down by using a pipette, and diluting by 80 times with 1x diluent (Item E2) for later use;
7) centrifuging HRP-streptavidin (Item G), and then diluting with 1 Xdiluent (Item E2) for 200 times for later use;
second, the operation steps
1) After the antibody-coated ELISA plate (Item A) was equilibrated to room temperature, 100. mu.L of the prepared stock solution of standard (STD7) and serum sample was added to the corresponding well, the whole plate was sealed with a sealing plate, and incubated overnight at 4 ℃;
2) adding the prepared 1x washing buffer solution to a plate washing machine, and washing the plate strip for 4 times by using the plate washing machine, wherein 300 mu L of washing buffer solution is added into each hole during washing;
3) after the plate is cleaned, 100 mu L of prepared TGFb RIII detection antibody (Item F) is added into each hole, and the plate is incubated for 1h at room temperature;
4) cleaning in the same step 2);
5) adding 100 mu L of prepared HRP-streptavidin (Item G) into each hole, and incubating for 45min at room temperature;
6) cleaning in the same step 2);
7) adding 100 μ L of TMB developing solution (Item H) into each well, and incubating for 30min at room temperature in the dark;
8) add 50. mu.L of stop buffer (Item I) to each well and read immediately on a microplate reader (450 nm).
9) Concentration values were calculated using sigmaplot 12.0 software.
The results obtained are shown in fig. 3, and it can be seen that in 3 populations, the expression level of TGFb RIII protein has statistical difference (wherein "×" between healthy group and early gastric cancer group in fig. 3 indicates that the P value of statistical difference is less than 0.01, "×" between early gastric cancer group and late gastric cancer group indicates that the P value of statistical difference is less than 0.01, and "×" between healthy group and late gastric cancer group indicates that the P value of statistical difference is less than 0.001), and ROC graph of gastric cancer population and healthy population is further plotted, wherein AUC value for differentiating gastric cancer from healthy population can reach 0.984, and serum protein concentration with serum value greater than 5752.909pg/ml can be used as a critical value for differentiating two populations, i.e., when serum is detected by the elpgisa kit, the population with TGFb RIII expression level higher than 5752.909/ml is gastric cancer population.
Any embodiment of the present application may be used as an independent technical solution, or may be combined with other embodiments. All patents and publications mentioned in the specification of the present application are indicative of the levels of those skilled in the art to which the present application pertains and as may be applied. All patents and publications cited herein are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The present application may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described, but it is recognized that various modifications and changes may be made within the scope of the claims and the application. It is understood that the embodiments described herein are examples of some implementations and features in some embodiments and that modifications and variations can be made by one of ordinary skill in the art in light of the teachings of this disclosure, which are also to be considered as within the purview of this application and the scope of the appended claims and their equivalents.
Claims (5)
1. An application of the immunoglobulin G antibody of TGFb RIII protein in preparing the diagnostic product of serum biomarker of gastric cancer.
2. Use of immunoglobulin G antibodies of TGFb RIII protein according to claim 1 in the preparation of gastric cancer serum biomarker diagnostic products characterized in that: the TGFb RIII protein is a human TGFb RIII protein.
3. Use of immunoglobulin G antibodies of TGFb RIII protein according to claim 1 in the preparation of gastric cancer serum biomarker diagnostic products characterized in that: the diagnostic product is a detection kit.
4. An application of TGFb RIII protein as a gastric cancer serum biomarker.
5. An application of TGFb RIII protein with non-diagnosis purpose as gastric cancer serum biomarker in gastric cancer judgment.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210143790.3A CN114487418A (en) | 2022-02-17 | 2022-02-17 | Application of TGFb RIII protein as gastric cancer serum biomarker |
| JP2022031344A JP7133159B1 (en) | 2022-02-17 | 2022-03-01 | Methods of Using TGFb RIII Protein as a Gastric Cancer Serum Biomarker |
| CN202210653139.0A CN115032394A (en) | 2022-02-17 | 2022-06-09 | Gastric cancer serum biomarker and application thereof |
| CN202210650870.8A CN114994320A (en) | 2022-02-17 | 2022-06-09 | Gastric cancer serum biomarker group and application thereof |
| CN202210684890.7A CN114966029A (en) | 2022-02-17 | 2022-06-13 | Application of TGFbRIII protein as gastric cancer serum biomarker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202210143790.3A CN114487418A (en) | 2022-02-17 | 2022-02-17 | Application of TGFb RIII protein as gastric cancer serum biomarker |
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| CN202210143790.3A Withdrawn CN114487418A (en) | 2022-02-17 | 2022-02-17 | Application of TGFb RIII protein as gastric cancer serum biomarker |
| CN202210684890.7A Withdrawn CN114966029A (en) | 2022-02-17 | 2022-06-13 | Application of TGFbRIII protein as gastric cancer serum biomarker |
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| CN202210684890.7A Withdrawn CN114966029A (en) | 2022-02-17 | 2022-06-13 | Application of TGFbRIII protein as gastric cancer serum biomarker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005304497A (en) * | 2004-03-25 | 2005-11-04 | Joji Inasawa | Method for detecting cancer using specified cancer-related gene and method for inhibiting the cancer |
| EP2083088A3 (en) * | 2005-04-07 | 2009-10-14 | Novartis Vaccines and Diagnostics, Inc. | Cancer-related genes |
| EP2411508B1 (en) * | 2009-03-27 | 2013-03-20 | Wyeth LLC | Tumor-initiating cells and methods for using same |
| CN111996257A (en) * | 2020-09-07 | 2020-11-27 | 复旦大学附属肿瘤医院 | Gastric cancer detection panel based on next-generation sequencing technology and application thereof |
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- 2022-02-17 CN CN202210143790.3A patent/CN114487418A/en not_active Withdrawn
- 2022-03-01 JP JP2022031344A patent/JP7133159B1/en active Active
- 2022-06-13 CN CN202210684890.7A patent/CN114966029A/en not_active Withdrawn
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| JP2023120118A (en) | 2023-08-29 |
| CN114966029A (en) | 2022-08-30 |
| JP7133159B1 (en) | 2022-09-08 |
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Application publication date: 20220513 |