CN115369101A - Marker combination for liver disease detection and application thereof - Google Patents
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Abstract
The invention discloses a marker combination for liver disease detection and application thereof, belonging to the technical field of biological detection. The marker combination for liver disease detection comprises at least one of the following components: chitinase 3-like protein 1, alpha-fetoprotein heteroplasmon, or aberrant prothrombin. The invention also verifies the application of the CHI3L1, AFP-L3 and DCP combination, namely the CA3D model in diagnosing liver cancer, and in addition, the CA3D model also greatly improves the specificity, sensitivity and accuracy of diagnosis, the specificity can reach 0.927, the sensitivity can reach 0.933, and the accuracy can reach 91.2%. Therefore, the CA3D model can be used for diagnosing liver cancer and contributes to improving the specificity, sensitivity and accuracy of liver cancer diagnosis.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a marker combination for liver disease detection and application thereof.
Background
Primary liver cancer, liver cancer for short, is a common malignant tumor of the digestive system worldwide. According to new data published by GLOBOCAN 2018, the number of new annual liver cancer cases reaches 84.1 ten thousand, and the new annual liver cancer cases reside in the 6 th position of malignant tumor, and 78.2 ten thousand dead cases reside in the 2 nd position of malignant tumor. The pathological type of the primary liver cancer is hepatocellular carcinoma (HCC) which accounts for 85% -90%. If it is found early, liver cancer can be cured by surgical resection, orthotopic liver transplantation, or ablative procedures such as microwave or radiofrequency ablation. However, more than 70% of liver cancers have been diagnosed as advanced disease and are not amenable to curative treatment, with median survival typically between 1-3 years. Therefore, a method for diagnosing liver cancer as early as possible is urgently required.
The current liver cancer early screening strategy comprises the detection of a plurality of markers, and the liver cancer is diagnosed by detecting the content of the markers in a patient body. Serum alpha-fetoprotein (AFP) is one of markers for liver cancer detection, and clinically, AFP positivity means that AFP is more than or equal to 400ng/mL, chronic or active hepatitis, liver cirrhosis, testis or ovary embryonic-derived tumors, pregnancy and the like are excluded, and liver cancer is highly suspected. However, the false negative is high in the detection of small liver cancer and early liver cancer, and the false negative is not ideal as an early screening index of liver cancer. Therefore, AFP is no longer used as a necessary index for screening and diagnosing liver cancer by the American society for research and liver disease (AASLD) and the European society for liver disease (EASL). The alpha fetoprotein heteroplasmon AFP-L3 is specific to liver cancer cells and is correspondingly increased along with the increase of canceration degree, so that the percentage of AFP-L3 in AFP (AFP-L3%) is commonly used as a detection index of primary liver cancer, and small liver cancer with the diameter of less than 2cm can be found 4.0 +/-4.9 months ahead of the imaging. Abnormal prothrombin (DCP, also known as PIVKA-II) is an abnormal prothrombin specifically produced in association with liver cancer. As a supplement of AFP, DCP has a certain diagnostic value for liver cancer negative in AFP, and has entered a clinical application stage as a liver cancer tumor marker. Research reports that the sensitivity and specificity of the DCP for diagnosing early liver cancer are respectively 64% and 89% and the accuracy is 86.3% when the DCP is more than or equal to 40 mAU/mL.
For example, chinese patent CN114487426A discloses a combined marker for predicting liver cancer risk of a male hepatitis b virus infected person and applications thereof, including at least one of γ -glutamyl transpeptidase, platelet, leukocyte, albumin and liver cirrhosis index, and alpha-fetoprotein and abnormal prothrombin. Chinese patent CN114592066A discloses a novel multi-target liver cancer early detection combined marker and application thereof, and finds a series of novel methylation sites which can efficiently distinguish liver cancer and hepatitis patients from high-depth whole genome methylation data of liver cancer tissues, tissues beside the cancer and hepatitis patient samples, so that the combined marker can be used for efficiently detecting early liver cancer; the novel gene methylation site is combined with other liver cancer detection markers (such as AFP, AFP-L3 and DCP), so that the AUC value can be further improved, and the sensitivity and specificity of early liver cancer screening can be improved. However, even in the case of a method involving the joint detection of a plurality of markers, there is a certain lack of specificity and sensitivity, and therefore, further studies are required for a novel detection method or detection product for detecting a marker and a higher specificity and sensitivity.
Disclosure of Invention
The invention aims to provide a universal marker combination suitable for liver cancer caused by various etiological factors. In the process of detecting the liver cancer by using the marker combination, the accuracy, specificity and sensitivity of detection can be effectively improved. Liver cancer can be diagnosed as early as possible and related treatments can be performed as early as possible.
In order to achieve the purpose of the invention, the following technical scheme is adopted.
A polypeptide composition comprising chitinase 3-like protein 1 (CHI 3L 1) and at least one of the following polypeptides:
alpha-fetoprotein (AFP), alpha-fetoprotein heteroplasmon (AFP-L3), or abnormal prothrombin (DCP).
Chitinase 3-like protein 1 (CHI 3L 1), also known as YKL-40, is a newly discovered inflammation marker, participates in the formation of fibrosis, and has important clinical value. Experiments prove that CHI3L1 has the function of being used as a marker for diagnosing liver cancer and can detect liver cancer patients with triple negative of AFP, AFP-L3 and DCP. Therefore, the accuracy of detection is improved, and the early detection and the early treatment of the liver cancer are facilitated.
In a preferred embodiment, the concentration of each polypeptide in the composition is:
chitinase 3-like protein 1 > 72.68ng/mL, alpha fetoprotein > 6.011ng/mL, alpha fetoprotein heteroplasmon > 0.83ng/mL or abnormal prothrombin > 33.96ng/mL.
In a preferred embodiment, the source of the composition comprises: whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid or ascites fluid.
In a preferred embodiment, the concentration of each polypeptide in the serum of the above composition is:
chitinase 3-like protein 1 > 72.68ng/mL, alpha fetoprotein > 6.011ng/mL, alpha fetoprotein heteroplasmon > 0.83ng/mL or aberrant prothrombin > 33.96ng/mL.
In a preferred embodiment, the polypeptide composition is chitinase 3-like protein 1, alpha-fetoprotein heteroplasmons and aberrant prothrombin.
It is another object of the present invention to provide a nucleic acid molecule composition comprising a DNA encoding chitinase 3-like protein 1 and at least one of the following nucleic acid molecules:
DNA encoding alpha-fetoprotein; or
DNA encoding an alpha-fetoprotein heteroplasmon; or
DNA encoding abnormal prothrombin.
In a preferred embodiment, the above-mentioned nucleic acid molecule composition further comprises cDNA encoding chitinase 3-like protein 1 and at least one of the following nucleic acid molecules:
a cDNA encoding an alpha-fetoprotein; or
A cDNA encoding an alpha-fetoprotein heteroplasmon; or
A cDNA encoding abnormal prothrombin.
In a preferred embodiment, the nucleic acid molecule composition further comprises at least one of:
RNA molecules transcribed from the above DNAs;
an RNA molecule capable of reverse transcription to obtain the above cDNA.
The nucleic acid molecule composition disclosed by the application can be used as a gene marker for detecting liver cancer, and whether a patient has the liver cancer or not can be judged by measuring the expression level of the nucleic acid molecule composition in a human body.
In a preferred embodiment, the source of the nucleic acid molecule composition comprises at least one of: whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid or ascites.
The invention also discloses the application of the nucleic acid molecule composition, which comprises at least one of the following components:
the use in the preparation of a product for diagnosing liver cancer;
the application of the product in improving the sensitivity of liver cancer diagnosis of products for diagnosing liver cancer;
the application of the product in improving the specificity of liver cancer diagnosis of a product for diagnosing liver cancer;
the application in preparing products for improving the sensitivity of liver cancer diagnosis;
the application in preparing products for improving the specificity of liver cancer diagnosis;
the application of the compound in preparing products for screening liver cancer;
the use in the preparation of a product for assessing the risk of liver cancer;
the application of the polypeptide in preparing products for liver cancer prognosis evaluation;
use in the manufacture of a product for the diagnosis of cirrhosis of the liver;
it is a further object of the present invention to provide a use of a polypeptide composition comprising at least one of:
use in the manufacture of a product for diagnosing liver cancer;
the application of the product in improving the sensitivity of liver cancer diagnosis of products for diagnosing liver cancer;
the application of the product in improving the specificity of liver cancer diagnosis of a product for diagnosing liver cancer;
the application in preparing products for improving the sensitivity of liver cancer diagnosis;
the application in preparing products for improving the specificity of liver cancer diagnosis;
the application of the compound in preparing products for screening liver cancer;
the use in the preparation of a product for assessing the risk of liver cancer;
the application of the compound in preparing products for prognosis evaluation of liver cancer;
use in the manufacture of a product for the diagnosis of cirrhosis of the liver;
wherein the polypeptide composition comprises chitinase 3-like protein 1 and at least one of the following polypeptides:
alpha-fetoprotein, an alpha-fetoprotein heteroplasmon, or abnormal prothrombin.
In a preferred embodiment, the above sensitivity is > 90%.
In a preferred embodiment, the above specificity is > 90%.
Still another object of the present invention is to provide a kit for detecting liver diseases, comprising a reagent for detecting the content of chitinase 3-like protein 1 and at least one of the following reagents:
a reagent for detecting the content of alpha-fetoprotein; or
A reagent for detecting the content of alpha-fetoprotein heteroplasmons; or
A reagent for detecting abnormal prothrombin content.
In a preferred embodiment, the above reagent further comprises a substance for detecting the polypeptide by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, co-immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR or biotin-avidin.
In a preferred embodiment, the sensitivity of the kit for diagnosing liver cancer is more than 90%.
In a preferred embodiment, the specificity of the kit for diagnosing liver cancer is greater than 90%.
In a preferred embodiment, the kit is an enzyme-linked immunoassay detection kit.
In a preferred embodiment, the enzyme-linked immunoassay detection kit comprises an anti-chitinase 3-like protein 1 antibody and at least one of the following reagents:
anti-alpha-fetoprotein antibodies; or
And anti-alpha-fetoprotein heteroplasmon antibodies; or
Anti-aberrant prothrombin antibodies;
the antibody is a monoclonal antibody or a polyclonal antibody.
In a preferred embodiment, the enzyme-linked immunoassay kit comprises the following reagents:
an anti-human chitinase 3-like protein 1 antibody marked by horseradish peroxidase, a rat anti-chitinase 3-like protein 1 monoclonal antibody and a goat anti-chitinase 3-like protein 1 polyclonal antibody marked by horseradish peroxidase;
and at least one set of the following:
a first group:
a horse radish peroxidase-labeled anti-human alpha fetoprotein antibody, a rat anti-alpha fetoprotein monoclonal antibody and a horse radish peroxidase-labeled goat anti-alpha fetoprotein polyclonal antibody;
second group:
the kit comprises an anti-human alpha-fetoprotein heteroplasmon antibody marked by horseradish peroxidase, a rat anti-alpha-fetoprotein heteroplasmon monoclonal antibody and a goat anti-alpha-fetoprotein heteroplasmon polyclonal antibody marked by horseradish peroxidase;
third group:
the kit comprises a horse radish peroxidase-labeled anti-human abnormal prothrombin antibody, a rat anti-abnormal prothrombin monoclonal antibody and a horse radish peroxidase-labeled goat anti-abnormal prothrombin polyclonal antibody.
In a preferred embodiment, the sensitivity of the kit for diagnosing liver cancer is greater than 90%.
In a preferred embodiment, the specificity of the kit for diagnosing liver cancer is more than 90%.
In a preferred embodiment, the kit is a colloidal gold assay kit.
In a preferred embodiment, the enzyme-linked immunoassay detection kit comprises an anti-chitinase 3-like protein 1 antibody and at least one of the following reagents:
anti-alpha-fetoprotein antibodies; or
And anti-alpha-fetoprotein heteroplasmon antibodies; or
Anti-aberrant prothrombin antibodies;
in a preferred embodiment, the colloidal gold assay kit comprises the following reagents:
a murine anti-chitinase 3-like protein 1 antibody and a colloidal gold-labeled goat anti-chitinase 3-like protein 1 antibody;
and at least one set of the following:
a first group:
a mouse anti-alpha fetoprotein antibody and a sheep anti-alpha fetoprotein antibody marked by colloidal gold;
second group:
a mouse anti-alpha fetoprotein heterogeneous antibody and a sheep anti-alpha fetoprotein heterogeneous antibody marked by colloidal gold;
third group:
murine anti-aberrant prothrombin antibodies and colloidal gold-labeled goat anti-aberrant prothrombin antibodies.
In a preferred embodiment, the sensitivity of the kit for diagnosing liver cancer is more than 90%.
In a preferred embodiment, the specificity of the kit for diagnosing liver cancer is more than 90%.
In a preferred embodiment, the kit is a magnetic particle chemiluminescence detection kit.
In a preferred embodiment, the enzyme-linked immunoassay detection kit comprises an anti-chitinase 3-like protein 1 antibody and at least one of the following reagents:
anti-alpha-fetoprotein antibodies; or
And anti-alpha-fetoprotein heteroplasmon antibodies; or
Anti-aberrant prothrombin antibodies;
in a preferred embodiment, the colloidal gold assay kit comprises the following reagents:
a biotin-labeled mouse anti-chitinase 3-like protein 1 monoclonal antibody and an acridinium ester-labeled sheep anti-chitinase 3-like protein 1 polyclonal antibody;
and at least one set of reagents from the group consisting of:
a first group:
a mouse anti-alpha fetoprotein monoclonal antibody marked by biotin and a sheep anti-alpha fetoprotein polyclonal antibody marked by acridinium ester;
second group:
a biotin-labeled murine anti-alpha fetoprotein heteroplasmon monoclonal antibody and an acridinium ester-labeled ovine anti-alpha fetoprotein heteroplasmon polyclonal antibody;
third group:
biotin-labeled murine anti-abnormal prothrombin monoclonal antibodies and acridinium ester-labeled ovine anti-abnormal prothrombin polyclonal antibodies.
In a preferred embodiment, the sample detected by the kit comprises:
whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid or ascites.
In a preferred embodiment, the sample detected by the kit is serum.
In a preferred embodiment, the kit has a shelf life of at least 1 year.
In a preferred embodiment, the sensitivity of the kit for diagnosing liver cancer is more than 90%.
In a preferred embodiment, the specificity of the kit for diagnosing liver cancer is more than 90%.
It is a further object of the present invention to provide a composite combination comprising at least one of:
an antibody-chitinase 3-like protein 1 complex; or
Antibody-alpha fetoprotein complexes; or
Antibody-alpha fetoprotein heteroplasmon complex; or
Antibody-aberrant prothrombin complexes.
In a preferred embodiment, the composite combination comprises:
antibody-chitinase 3-like protein 1 complexes and antibody-alpha fetoprotein heteroplasmon complexes.
In a preferred embodiment, the composite combination comprises:
antibody-chitinase 3-like protein 1 complexes and antibody-abnormal prothrombin complexes.
In a preferred embodiment, the composite combination comprises:
antibody-chitinase 3-like protein 1 complex, antibody-alpha fetoprotein heteroplasmon complex, and antibody-aberrant prothrombin complex.
In a preferred embodiment, the antibody comprises a detection marker comprising at least one of:
an enzyme; or
Biotin; or
Streptavidin; or
A fluorescent protein; or
A chemiluminescent label; or
Colloidal gold.
In a preferred embodiment, the enzyme comprises horseradish peroxidase.
In a preferred embodiment, the chemiluminescent label comprises an acridinium ester.
In a preferred embodiment, the use of the above-described composite comprises at least one of:
the application in preparing products for improving the sensitivity of liver cancer diagnosis;
the application in preparing products for improving the specificity of liver cancer diagnosis;
the application of the product in improving the sensitivity of liver cancer diagnosis of products for diagnosing liver cancer;
the application of the product in improving the specificity of liver cancer diagnosis in the product for diagnosing liver cancer.
In a preferred embodiment, the sensitivity is > 90%.
In a preferred embodiment, the above specificity is > 90%.
The invention also discloses an application of the chitinase 3-like protein 1, which comprises at least one of the following components:
the use in the preparation of a product for diagnosing liver cancer;
the application of the product in improving the sensitivity of liver cancer diagnosis of products for diagnosing liver cancer;
the application of the product in improving the specificity of liver cancer diagnosis of a product for diagnosing liver cancer;
the application in preparing products for improving the sensitivity of liver cancer diagnosis;
the application in preparing products for improving the specificity of liver cancer diagnosis;
the application of the compound in preparing products for screening liver cancer;
the use in the preparation of a product for assessing the risk of liver cancer;
the application of the polypeptide in preparing products for liver cancer prognosis evaluation;
use in the preparation of a product for the diagnosis of AFP, AFP-L3 and DCP triple negative liver cancer patients;
the application in constructing liver cancer diagnosis model.
The chitinase 3-like protein 1 has great potential in the aspect of liver cancer detection, and can effectively improve the specificity, sensitivity and accuracy of diagnosis. And in addition, when the alpha fetoprotein, an alpha fetoprotein heteroplasmon or abnormal prothrombin can not be diagnosed, the chitinase 3-like protein 1 can be used as a marker to accurately diagnose liver cancer, so that the liver cancer patient can be diagnosed as soon as possible, and the treatment success rate and the survival rate are improved.
In a preferred embodiment, the method for constructing the liver cancer diagnosis model comprises at least one of the following models:
CHI3L1-AFPL3 model; or
CHI3L1-AFP model; or
CHI3L1-DCP model; or
CA3D model.
In a preferred embodiment, the model includes at least one of:
CHI3L1-AFPL3 model:
CHI3L1-AFPL3 score =40.5887 x AFP-L3+0.0246 x CHI3L1-27.4235;
CHI3L1-AFP model:
CHI3L1-AFP score =0.1047 × AFP +0.0238 × CHI3L1-26.5124-3.4024;
CHI3L1-DCP model:
CHI3L1-DCP score =0.0270 × DCP +0.0240 × CHI3L1-3.4573;
CA3D model:
CA3D score =0.0129 × AFP +37.2319 × AFP-L3+0.0259 × DCP +0.0237 × CHI3L1-26.5124;
in the above formula:
AFP is the content (ng/mL) of AFP in a tested sample;
AFP-L3 is the content (ng/mL) of AFP-L3 in a tested sample;
the content (ng/mL) of the DCP in a tested sample;
CHI3L1 is the content (ng/mL) of CHI3L1 in the sample to be tested.
The diagnosis model has the characteristics of high accuracy, strong specificity and high sensitivity, is favorable for detecting the liver cancer which is difficult to detect, and can help patients to diagnose the liver cancer as soon as possible, thereby increasing the probability of cure and survival.
In a preferred embodiment, the criteria for determining liver cancer are: the test patient score was higher than the median of the control population score.
In a preferred embodiment, the sensitivity of the diagnostic model for diagnosing liver cancer is > 90%.
In a preferred embodiment, the specificity of the diagnosis model for liver cancer is > 90%.
In a preferred embodiment, the construction method for constructing the liver cancer diagnosis model comprises the following steps:
step 1: extracting body fluid from control population and liver cancer patient respectively;
and 2, step: determining the level of the marker in the body fluid of the patient;
and step 3: carrying out statistical analysis on the content of the marker;
and 4, step 4: and constructing a model according to the statistical analysis result.
In a preferred embodiment, the body fluid comprises at least one of: whole blood, serum, plasma, urine, saliva, sweat, cerebrospinal fluid or ascites.
In a preferred embodiment, the body fluid is serum.
In a preferred embodiment, the control population includes healthy population or other liver disease patients without liver cancer.
In a preferred embodiment, the marker comprises at least one of:
chitinase 3-like protein 1, alpha-fetoprotein heteroplasmon, or abnormal prothrombin.
In a preferred embodiment, the marker includes:
chitinase 3-like protein 1, alpha-fetoprotein heteroplasmon, and abnormal prothrombin; the model containing this marker combination is called CA3D model.
In a preferred embodiment, the variables of the statistical analysis include at least one of:
chitinase 3-like protein 1 content, alpha-fetoprotein heteroplasmon content or abnormal prothrombin content.
In a preferred embodiment, the variables of the statistical analysis further include patient gender, age, platelet count, total bilirubin.
In a preferred embodiment, the statistical analysis comprises regression analysis.
In a preferred embodiment, regression analysis uses a logistic regression model and draws ROC curves for analysis, and specific parameters can be determined based on sensitivity preference (finding more cancer patients) or specificity preference (reducing false positives) as required; and forming a scoring formula according to the analysis result.
In a preferred embodiment, the number of samples of the control population and the liver cancer patient required to construct the model is not less than 30, respectively.
In a preferred embodiment, the number of samples of the control population and the liver cancer patient required to construct the model is not less than 80, respectively.
In a preferred embodiment, the criteria for determining liver cancer are: the test patient score was higher than the median of the healthy population score.
Compared with the prior art, the invention has the beneficial effects that:
the invention verifies the application of CHI3L1 as a marker in diagnosing liver cancer, and also verifies the application of the combination of CHI3L1 and AFP, AFP-L3 or DCP in diagnosing liver cancer, and the combination of CHI3L1 and any one of the markers of AFP, AFP-L3 or DCP can improve the specificity, sensitivity and accuracy of diagnosis; CHI3L1 can also detect liver cancer patients with triple negative of AFP, AFP-L3 and DCP, and avoid the liver cancer patients from being missed. And CHI3L1 can also detect liver cancer patients with triple negative of AFP, AFP-L3 and DCP, and is favorable for patients to diagnose liver cancer as soon as possible and receive timely treatment. The invention also verifies the application of the CHI3L1, AFP-L3 and DCP combination, namely the CA3D model in diagnosing liver cancer, and in addition, the CA3D model also greatly improves the specificity, sensitivity and accuracy of diagnosis, the specificity can reach 0.927, the sensitivity can reach 0.933, and the accuracy can reach 91.2%. Therefore, the CA3D model can be used for diagnosing liver cancer and contributes to improving the specificity, sensitivity and accuracy of liver cancer diagnosis. Therefore, based on the CA3D model, the further developed detection reagent and detection kit also have accurate prompt effect on liver cancer with slight clinical symptoms and difficult detection, and have extremely high medical and practical values. Moreover, the model for diagnosing the liver cancer has the characteristics of high accuracy, strong specificity and high sensitivity, is favorable for detecting the liver cancer which is difficult to detect, and can help patients to diagnose the liver cancer as early as possible, thereby increasing the probability of cure and survival.
Drawings
FIG. 1 is a ROC curve for each marker.
Detailed Description
The exemplary embodiments will be described herein in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure.
The experimental procedures in the following examples are, unless otherwise specified, either conventional or according to the manufacturer's recommendations. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Verifying that the serum levels of four biomarkers, namely chitinase 3-like protein 1 (CHI 3L 1), alpha-fetoprotein (AFP), alpha-fetoprotein heteroplasmon (AFP-L3) and abnormal prothrombin (DCP) of liver cancer patients and normal people are different
Liver cancer (HCC) patients and normal persons were selected to be divided into HCC group and healthy group, and serum levels of CHI3L1, AFP-L3 and DCP were measured using the kit.
The detection method is as follows (taking detection of CHI3L1 as an example, the used kit is produced by Hangzhou general biotechnology limited, and the detection method of other markers can refer to the detection method of CHI3L 1):
the kit comprises:
1. chitinase 3-like protein 1 microporous enzyme-labeled plate: 8 holes are multiplied by 12 strips; rat anti-chitinase 3-like protein 1 monoclonal antibody is coated in the micropores, and 1 sample can be detected in each micropore; sealing with aluminum foil bag, and containing desiccant;
2. chitinase 3-like protein 1 detection antibody: 1 vial × 11mL; horse radish peroxidase labeled goat anti-chitinase 3-like protein 1 polyclonal antibody is stored in an enzyme-linked diluent containing bovine serum albumin; amount added per well: 100uL;
3. chitinase 3-like protein 1 sample diluent: 1 bottle × 50mL; the component is phosphate buffer solution containing bovine serum albumin, which is used for diluting a sample before detection;
4. color developing agent: 1 vial × 11mL; a buffer containing 3,3', 5' -tetramethylbenzidine; required amount per well: 100uL; 5. washing solution (30 ×): 1 bottle × 30mL; diluting by 30 times;
6. stopping liquid: 1 vial × 6mL;1 mol/L dilute sulfuric acid solution, and the required amount per hole: 50uL;
7. self-adhesive sheet: 2, slicing;
8. chitinase 3-like protein 1 calibrator: and 6 bottles of phosphate buffer containing recombinant human chitinase 3-like protein 1 and bovine serum albumin, wherein each bottle is 0.50mL.
The chitinase 3-like protein 1 concentration in the calibrator is shown in table 1:
TABLE 1 concentration of chitinase 3-like protein 1 in calibrator
| Calibration article |
| 0 | 1 | 2 | 3 | 4 | 5 | |
| CHI3L1 concentration (ng/mL) | 0 | 1 | 2 | 4 | 8 | 16 |
The specific detection method comprises the following steps:
1. the kit components were equilibrated to room temperature.
2. And preparing a washing solution with working concentration (diluted by 20 times by using ultrapure water for later use).
The calibrator and the quality control material are fully dissolved according to 0.50 mL/piece, and are placed for more than 15 minutes at room temperature. The CHI3L1 calibrator and the quality control material are dissolved by deionized water.
3. Diluting the sample: serum samples for CHI3L1 detection were diluted with CHI3L1 sample diluent (500-fold dilution is recommended). If the concentration exceeds the detection range, the dilution factor is selected as appropriate. (samples are recommended to be diluted in 96-well plates, taking care of the amount of dilution provided by the kit.)
4. According to the experimental requirements, a certain amount of CHI3L1 enzyme label plate is selected and marked. The used microporous plate strips are stably placed on the bracket and numbered according to the needs of the experiment. It is recommended that the microplate be clearly marked before the beginning of aspiration of the calibrator, quality control and patient samples in order to unambiguously distinguish the position of each sample during and after the test.
5. Adding the calibrator, the quality control material and the diluted sample at a concentration of 100 mu L per well, making multiple wells for the calibrator and the quality control material, and lightly tapping the ELISA plate to mix uniformly.
6. After the enzyme label plate was covered with a pressure sensitive adhesive sheet, the plate was incubated at 37 ℃ for 60 minutes.
7. Taking out the enzyme label plate, tearing off the non-dry film, discarding the liquid in the hole, adding liquid into the working concentration cleaning solution prepared in the step 2 according to 200 mu L/hole, standing for 30-60 seconds, discarding the cleaning solution, patting dry on clean absorbent paper, and washing for 4 times. If a plate washing machine is adopted for washing plates, a washing program is selected for 5 times, liquid is added into each hole of 300 mu L, the retention time of the washing liquid with the working concentration in the micropores is 30-60 seconds each time, the washing liquid is completely sucked each time without residue, and the washing liquid is patted dry on clean absorbent paper after washing.
8. Adding corresponding detection antibody at 100 μ L/hole, covering the ELISA plate with adhesive sticker, and incubating at 37 deg.C for 60 min.
9. Operation 7 is repeated.
10. Preparing working concentration HRP-labeled avidin: HRP-labeled avidin (100X) was mixed with HRP-labeled avidin dilution at 99 ℃ and 100. Mu.L/well, and the microplate was covered with a sticker and incubated at 37 ℃ for 30 minutes.
11. Operation 7 is repeated.
12. Color-developing agent A: developer B is mixed according to the volume ratio of 1.
13. And (3) adding the color developing agent prepared in the step (12) into a reaction hole, reacting at 100 mu L/hole for 15 minutes in the dark at room temperature.
14. Add 50. Mu.L/well stop solution into all wells, tap the ELISA plate, and mix them well.
15. The enzyme-linked immunosorbent assay takes the wavelength of 450nm and reads the OD value.
And (4) calculating a result: the concentration of the specimen to be measured was calculated by inputting the calibration standard CHI3L1 of each calibrator using a microplate reader with a built-in data calculation program. The standard curve fitting mode uses a four-parameter mathematical model fitting, and requires that the correlation coefficient of the standard curve should be more than 0.9900.
The results of the measurements are shown in tables 2 and 3.
TABLE 2 health group test results
| Number of samples | Mean serum concentration (ng/mL) | 95% CI | Median serum concentration (ng/mL) | 95% CI | |
| AFP | 551 | 3.714 | 3.374-4.054 | 2.683 | 2.542-2.805 |
| AFP-L3 | 551 | 0.6 | 0.600-0.600 | 0.6 | 0.600-0.600 |
| DCP | 551 | 21.124 | 19.843-22.405 | 19.35 | 18.630-20.420 |
| CHI3L1 | 551 | 62.724 | 59.389-66.058 | 52.18 | 49.761-55.319 |
TABLE 3 detection results of liver cancer group
| Number of samples | Mean serum concentration (ng/mL) | 95% CI | Median serum concentration (ng/mL) | 95% CI | |
| AFP | 812 | 428.24 | 393.242-463.238 | 80.625 | 51.792-136.519 |
| AFP-L3 | 711 | 405.165 | 252.254-558.077 | 4.486 | 3.257- 6.226 |
| DCP | 812 | 3973.541 | 3503.966-4443.115 | 305 | 219.707-414.468 |
| CHI3L1 | 812 | 205.03 | 194.699-215.361 | 156.245 | 146.400-165.487 |
As can be seen from tables 2 and 3, the serum levels of the four biomarkers CHI3L1, AFP-L3 and DCP in the HCC group were significantly higher than in the healthy group (P < 0.001). The mean value of CHI3L1 in the liver cancer group is 205.03ng/mL, and the mean value of the normal population is 62.724 ng/mL; the average value of AFP in the liver cancer group is 428.24 ng/mL, and the average value of the normal population is 3.714ng/mL; AFP-L3 is 405.165 in a liver cancer group mean value and 0.6 in a normal population mean value; DCP had a mean value of 3973.541 in the liver cancer group and 19.35 in the normal group. There were significant differences between groups (P < 0.001). Therefore, the markers CHI3L1, AFP-L3 and DCP can be used as the markers for detecting liver cancer.
Example 2
Verifying the capability of CHI3L1, AFP-L3 and DCP in detecting liver cancer
Based on the serum levels of the markers in example 1, receiver Operating Characteristic (ROC) curves were constructed to further evaluate the ability of the markers CHI3L1, AFP-L3 and DCP to identify patients with liver cancer and healthy populations.
The ROC curve reflects the balance between sensitivity and specificity, the area under the ROC curve is an important test accuracy index, and the larger the area under the ROC curve is, the higher the diagnostic value of the test is.
Sensitivity (true positive rate): actually, the percentage of HCC patients correctly judged according to the experimental standard is that the larger the sensitivity is, the better the sensitivity is, and the ideal sensitivity is 100%.
Specificity (true negative rate): the percentage of patients who are actually not HCC and are correctly judged as non-HCC according to the experimental standard is that the greater the specificity is, the better the specificity is, and the ideal specificity is 100%.
The ROC analysis results are shown in fig. 1 and table 4.
TABLE 4 ROC analysis results
| Markers or combinations | Sensitivity of the probe | Specificity of | AUC | AUC（95CI%,%） | Threshold (ng/mL) | Accuracy (%) | Kappa |
| CHI3L1 | 0.861 | 0.825 | 0.906 | 89.9-92.4 | 72.68 | 80.85 | 0.665 |
| AFP | 0.75 | 0.955 | 0.878 | 85.9-89.2 | 6.011 | 79.31 | 0.498 |
| DCP | 0.74 | 0.978 | 0.827 | 80.4-85.0 | 33.96 | 82.1 | 0.611 |
| AFPL3 | 0.625 | 0.998 | 0.812 | 78.9-83.4 | 0.83 | 76.15 | 0.514 |
| AFP-AFPL3-DCP | 0.878 | 0.958 | 0.923 | 90.7-93.9 | 1.647 | 88.11 | 0.791 |
| AFP-AFPL3-DCP-CHI3L1 | 0.933 | 0.927 | 0.977 | 96.9-98.4 | 3.971 | 91.2 | 0.845 |
ROC analysis showed that AUC and Kappa values for HCC diagnosis using CHI3L1 were 0.906 and 0.665, respectively, and the optimal cut-off value for the data set was 72.68ng/mL, exceeding the performance of any of the three single biomarkers. The combination of CHI3L1 with AFP, AFP-L3 and DCP further improved performance with AUC and Kappa values of 0.977 and 0.845, respectively. The combination of CHI3L1 and AFP, AFP-L3 and DCP, namely a CA3D model can be used for diagnosing liver cancer, and the CA3D model greatly improves the sensitivity and specificity of detection.
The analysis result of the embodiment 2 shows that the detection reagent and the detection kit which are further developed based on the marker combination have a relatively accurate prompting function on the liver cancer with slight clinical symptoms and difficult detection, and have extremely high medical and practical values.
Example 3
Verification of capability of CHI3L1 in improving liver cancer detection accuracy
Further ROC analysis of CHI3L1 with AFP, AFP-L3 or DCP gave the results shown in Table 5.
TABLE 5 ROC analysis results
| Marker combination | AUC | Accuracy (%) |
| CHI3L1-AFP | 0.954 | 87.75 |
| CHI3L-DCP | 0.943 | 87.67 |
| CHI3L-AFP-L3 | 0.948 | 87.32 |
Comparing the data in Table 5 with the data for the single marker in Table 4, any combination with CHI3L1 significantly improved AUC, with 0.954 AUC for CHI3L1-AFP, 0.943 AUC for CHI 3L-AFP-DCP, and 0.948 AUC for CHI 3L-AFP-L3. The combination of CHI3L1 with AFP, AFP-L3 and DCP further improved performance with AUC and accuracy of 0.977 and 91.2%, respectively. Indicating that CHI3L1 has the ability to improve detection accuracy, sensitivity and specificity.
In addition, the serum level of CHI3L1 was measured in HCC cases triple negative for AFP, AFP-L3 and DCP using CHI3L1 as a marker, with 72.68ng/mL as a cutoff value, and the results shown in Table 6 were obtained.
TABLE 6 detection results of CHI3L1 on AFP, AFP-L3 and DCP triple-negative HCC cases
| Number of samples | Detection rate（%） | |
| Total HCC cases | 847 | - |
| AFP, AFP-L3, DCP triple negative and CHI3L1>72.68（ng/mL） | 107 | 12.63 |
As can be seen from Table 6, CHI3L1 helped to identify 107 (12.63%) cases of AFP, AFP-L3, DCP triple-negative HCC among 847, further indicating that CHI3L1 has the ability to improve detection accuracy, sensitivity and specificity, and is able to detect patients whose symptoms are not obvious or are not easily detected, which are easily missed.
The analysis result of the embodiment 3 shows that the detection reagent and the detection kit further developed based on the marker combination have more accurate prompt effect on liver cancer with slight clinical symptoms and difficult detection or AFP, AFP-L3 and DCP triple-negative liver cancer, and have extremely high medical and practical values.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
Example 4
Diagnosis of liver cancer by liver cancer diagnosis model
1. Construction of models
Step 1: respectively extracting blood from healthy people and liver cancer patients, and separating serum;
and 2, step: measuring the contents of chitinase 3-like protein 1, alpha-fetoprotein heteroplasmon and abnormal prothrombin in the serum of a patient;
and step 3: performing statistical analysis on the content of the marker by using a logistic regression model, drawing an ROC curve, and calculating the area under the curve;
and 4, step 4: the following model was constructed from the statistical analysis results:
CHI3L1-AFPL3 model:
CHI3L1-AFPL3 score =40.5887 x AFP-L3+0.0246 x CHI3L1-27.4235;
CHI3L1-AFP model:
CHI3L1-AFP score =0.1047 × AFP +0.0238 × CHI3L1-26.5124-3.4024;
CHI3L1-DCP model:
CHI3L1-DCP score =0.0270 × DCP +0.0240 × CHI3L1-3.4573;
CA3D model:
CA3D score =0.0129 × AFP +37.2319 × AFP-L3+0.0259 × DCP +0.0237 × CHI3L1-26.5124;
in the above formula:
AFP is the content (ng/mL) of AFP in a tested sample;
AFP-L3 is the content (ng/mL) of AFP-L3 in a tested sample;
the content (ng/mL) of the DCP in a tested sample;
CHI3L1 is the content (ng/mL) of CHI3L1 in the sample tested.
2. Case diagnosis
Taking the patient with the number GX121 as an example, the patient has already diagnosed cirrhosis before the liver cancer is diagnosed;
2020.8.5 serum levels of CHI3L1, AFP-L3 and DCP were determined in the patients at the time of enrollment and the results are shown in Table 7:
TABLE 7 patient marker detection results
| CHI3L1（ng/mL） | AFP-L3（ng/mL） | AFP（ng/mL） | AFP-L3%（ng/mL） | DCP（ng/mL） | Age (age) | Sex |
| 402.69 | 3.58 | 9.075 | 39.521 | 10.98 | Age 51 years old | For male |
As can be seen from the table, the AFP of the patient is less than 20ng/mL, the DCP is less than 40ng/mL, and the AFP and the DCP are all lower than the existing liver cancer diagnosis standard and are difficult to be diagnosed according to the existing liver cancer diagnosis standard; since patient CHI3L1 was as high as 402.69, suggesting a risk of developing liver cancer, the CA3D score of the patient was calculated to be 116.7 using the CA3D model for evaluation. The CA3D scores of liver cirrhosis patients (84 patients) are evaluated at the same time, the median value of the CA3D scores of the liver cirrhosis patients is 0.53, and the CA3D score of the GX121 patient is far higher than the median value, so that liver cancer can be diagnosed.
In fact, the patient does not confirm liver cancer according to the CA3D model in 2020.8.5, but does not confirm liver cancer until 2022.1.3 according to the existing liver cancer diagnosis standard. The CA3D can diagnose the patient as liver cancer 4 months in advance 1 year, has the capability of identifying the liver cancer which is difficult to detect, and has value in early diagnosis of the liver cancer.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A polypeptide composition comprising chitinase 3-like protein 1 and at least one of the following polypeptides:
alpha-fetoprotein, an alpha-fetoprotein heteroplasmon, or aberrant prothrombin.
2. The polypeptide composition of claim 1, wherein the concentration of each polypeptide in the composition is:
chitinase 3-like protein 1 > 72.68ng/mL, alpha fetoprotein > 6.011ng/mL, alpha fetoprotein heteroplasmon > 0.83ng/mL or aberrant prothrombin > 33.96ng/mL.
3. A nucleic acid molecule composition comprising DNA encoding chitinase 3-like protein 1 and at least one of the following nucleic acid molecules:
DNA encoding alpha-fetoprotein; or
DNA encoding an alpha-fetoprotein heteroplasmon; or
DNA encoding abnormal prothrombin.
4. The nucleic acid molecule composition of claim 3, further comprising cDNA encoding chitinase 3-like protein 1 and at least one of the following nucleic acid molecules:
a cDNA encoding an alpha-fetoprotein; or
A cDNA encoding an alpha-fetoprotein heteroplasmon; or
A cDNA encoding abnormal prothrombin.
5. The nucleic acid molecule composition of claim 3, further comprising at least one of:
an RNA molecule transcribed from the nucleic acid molecule composition of claim 3; or
An RNA molecule capable of reverse transcription to obtain the nucleic acid molecule composition of claim 4.
6. Use of a polypeptide composition comprising at least one of:
the use in the preparation of a product for diagnosing liver cancer;
the application of the product in improving the sensitivity of liver cancer diagnosis of products for diagnosing liver cancer;
the application of the product in improving the specificity of liver cancer diagnosis of a product for diagnosing liver cancer;
the application in preparing products for improving the sensitivity of liver cancer diagnosis;
the application in preparing products for improving the specificity of liver cancer diagnosis;
the application of the compound in preparing products for screening liver cancer;
the use in the preparation of a product for assessing the risk of liver cancer;
the application of the compound in preparing products for prognosis evaluation of liver cancer;
use in the manufacture of a product for the diagnosis of cirrhosis of the liver;
wherein the polypeptide composition comprises chitinase 3-like protein 1 and at least one of the following polypeptides:
alpha-fetoprotein, an alpha-fetoprotein heteroplasmon, or abnormal prothrombin.
7. Use according to claim 6, wherein the sensitivity is > 90%.
8. Use according to claim 6, wherein the specificity is > 90%.
9. A kit for detecting liver diseases, which comprises a reagent for detecting the content of chitinase 3-like protein 1 and at least one of the following reagents:
a reagent for detecting the alpha-fetoprotein content; or
A reagent for detecting the content of alpha-fetoprotein heteroplasmons; or
A reagent for detecting abnormal prothrombin content.
10. The kit of claim 9, wherein the reagents further comprise a substance for detecting the polypeptide by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, co-immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin.
11. The kit of claim 9, wherein the kit has a sensitivity of > 90% for diagnosing liver cancer.
12. The kit of claim 9, wherein the kit diagnoses liver cancer with a specificity of > 90%.
13. A composite combination comprising at least one of:
an antibody-chitinase 3-like protein 1 complex; or
Antibody-alpha fetoprotein complex; or
Antibody-alpha fetoprotein heteroplasmon complex; or
Antibody-aberrant prothrombin complexes.
14. The complex of claim 13, wherein the antibody comprises a detectable label comprising at least one of:
an enzyme; or
Biotin; or
Streptavidin; or
A fluorescent protein; or
A chemiluminescent label; or
Colloidal gold.
15. Use of a complex according to claim 13, comprising at least one of:
the application in preparing products for improving the sensitivity of liver cancer diagnosis;
the application in preparing products for improving the specificity of liver cancer diagnosis;
the application of the product in improving the sensitivity of liver cancer diagnosis of products for diagnosing liver cancer;
the application of the product in improving the specificity of liver cancer diagnosis in the product for diagnosing liver cancer.
16. Use of chitinase 3-like protein 1, comprising at least one of:
use in the manufacture of a product for diagnosing liver cancer;
the application of the product in improving the sensitivity of liver cancer diagnosis of products for diagnosing liver cancer;
the application of the product in improving the specificity of liver cancer diagnosis of a product for diagnosing liver cancer;
the application in preparing products for improving the sensitivity of liver cancer diagnosis;
the application in preparing products for improving the specificity of liver cancer diagnosis;
the application of the compound in preparing products for screening liver cancer;
the use in the preparation of a product for assessing the risk of liver cancer;
the application of the polypeptide in preparing products for liver cancer prognosis evaluation;
use in the preparation of a product for the diagnosis of AFP, AFP-L3 and DCP triple negative liver cancer patients;
the application in constructing a liver cancer diagnosis model.
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| CN117368494A (en) * | 2022-12-09 | 2024-01-09 | 元典(天津)新技术有限公司 | Kit for detecting liver fibrosis and liver cirrhosis |
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| CN116559107B (en) * | 2023-04-27 | 2024-08-20 | 中国人民解放军海军军医大学 | System for diagnosing early liver cancer based on exosome infrared spectrum characteristics |
| CN118604339A (en) * | 2024-05-09 | 2024-09-06 | 深圳市第二人民医院(深圳市转化医学研究院) | Application of SNRPA protein detection reagent in the preparation of liver cancer diagnosis kit |
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| CN101587125B (en) * | 2008-05-21 | 2013-07-24 | 林标扬 | High expression cancer marker and low expression tissue organ marker kit |
| US20110311450A1 (en) * | 2008-12-08 | 2011-12-22 | Zurit Levine | Polypeptides and polynucleotides, and uses thereof as a drug target for producing drugs and biologics |
| BR122020001802B1 (en) * | 2010-10-22 | 2021-05-18 | T2 Biosystems, Inc | methods for detecting the presence of a pathogen in a whole blood sample and for detecting an analyte in a sample |
| CN105092855B (en) * | 2014-05-23 | 2017-10-03 | 杭州普望生物技术有限公司 | A kind of kit detected for liver fibrosis and hepatic sclerosis |
| CN110687288B (en) * | 2019-04-28 | 2022-09-23 | 中山大学肿瘤防治中心 | A kind of multiplex detection kit for rapid detection of liver disease and preparation method |
| CN110488006A (en) * | 2019-09-26 | 2019-11-22 | 天津华科泰生物技术有限公司 | A kind of immunochromatographydetection detection card and preparation method thereof of 3 sample albumen 1 of quick detection chitinase |
| CN112331333A (en) * | 2020-11-02 | 2021-02-05 | 高春芳 | Method for establishing liver cancer diagnosis model based on liver cancer triple-joint inspection |
| CN214453182U (en) * | 2020-12-17 | 2021-10-22 | 北京大学深圳医院 | Multiplex Test Kit for Rapid Detection of Liver Disease |
| CN112526134A (en) * | 2020-12-24 | 2021-03-19 | 北京世纪沃德生物科技有限公司 | Chitinase 3-like protein 1 determination kit |
| CN113484517B (en) * | 2021-07-05 | 2024-06-25 | 川北医学院附属医院 | Biomarker for diagnosing early hepatocellular carcinoma and construction method of diagnosis mode |
| CN114414802A (en) * | 2021-09-23 | 2022-04-29 | 湖南莱拓福生物科技有限公司 | Early diagnosis kit based on four combined detections of liver cancer and application |
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| CN117368494A (en) * | 2022-12-09 | 2024-01-09 | 元典(天津)新技术有限公司 | Kit for detecting liver fibrosis and liver cirrhosis |
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