CN114381529B - Application of ACTR10 and CA125 combination in ovarian cancer detection and kit - Google Patents
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Abstract
The invention discloses application of ACTR10 and CA125 in combination in ovarian cancer detection and a kit, and relates to the field of medical diagnosis. The invention discloses preparation of a kit for combined use of ACTR10 and CA125 in ovarian cancer auxiliary diagnosis and/or ovarian cancer risk screening, and meanwhile, the sensitivity and specificity of diagnosis are improved, and the effectiveness of ovarian cancer diagnosis is improved to a higher level.
Description
Technical Field
The invention relates to the field of medical diagnosis, in particular to application of ACTR10 and CA125 in combination in ovarian cancer detection and a kit.
Background
Ovarian cancer is currently the most deaths of gynecological tumors, with a 5-year survival rate of approximately 20% in advanced patients, and greater than 90% when patients are detected and treated early in their life. Early ovarian cancer detection by screening is an important way to reduce the mortality rate of the disease. Tumor antigen 125 (abbreviated as CA125) and vaginal ultrasound imaging examinations are currently the most commonly used means of prescreening ovarian cancer. However, CA125 has no significant effect in the early diagnosis of ovarian cancer, and almost 50% of early ovarian cancer patients (stage I) have no significant increase in CA 125. In addition, an increase in CA125 is accompanied by false positive results, such as in other common gynecological diseases and also in tumors (e.g., bladder and liver cancer). Although vaginal ultrasound can image the ovaries and surrounding organs, it is still difficult to distinguish pre-menopausal ovarian tumors from functional cysts. In addition, a plurality of clinical random control experiments prove that the early screening of ovarian cancer by simultaneously using a tumor marker CA125 and vaginal ultrasound does not obviously reduce the death rate of the tumor. Therefore, health management and clinical need for early diagnosis of ovarian cancer in women is for a more desirable methodology for early screening. A more desirable methodology should have certain essential features, such as ease of operation, cost-effectiveness, and high sensitivity and specificity as clinically proven.
Episomal dna (cfdna) is also currently attempted for early diagnosis of ovarian cancer. Compared to healthy individuals, the cfDNA content of epithelial ovarian cancer patients is significantly higher than that of healthy individuals. However, the sensitivity of cfDNA detection of early stage ovarian cancer is only 55%. The cfDNA content of ovarian cancer patients also increased with tumor progression with a diagnostic sensitivity of 88.9%. Since cfDNA concentrations are elevated in the circulatory system of many tumor patients, diagnosis of early stage ovarian cancer with cfDNA concentrations is not specific.
In addition to cfDNA, multiple studies have shown that circulating mirnas are specifically expressed in ovarian cancer. Compared with healthy individuals, the expression level of free let-7 in blood of ovarian cancer patients is reduced, and the expression level of miR-205 is increased. However, multiple studies find that the expression of miR-205 is increased, and similar situations are found in the blood of other tumor patients, such as endometrial cancer and squamous cell lung cancer. Because cfDNA and free miRNA have no tissue specificity, the emphasis on early screening and diagnosis of ovarian cancer is shifted to the detection of extracellular vesicle-derived nucleic acid and protein.
Extracellular vesicles are secreted by cells and can circulate in body fluids such as blood and urine. The vesicles carry signal molecules derived from blast cells, and the vesicles secreted by tumor cells can transport important signal molecules in the microenvironment of tumorigenesis, so that the tumor progression and metastasis are promoted. Therefore, the detection of vesicle signal molecules for early screening and diagnosis of tumors is the focus of scientific research and industrial transformation at present.
The prior art scheme for diagnosing ovarian cancer by separating total vesicles or specific types of vesicles in serum and plasma and detecting the expression amount of miRNA in the vesicles is based on the vesicles.
However, the diagnostic sensitivity and specificity of such technical solutions are not ideal and still need to be improved.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide application of ACTR10 and CA125 in combination in ovarian cancer detection and a kit. According to the invention, the ACTR10 and CA125 are combined for the first time to be used as a marker group for diagnosing the ovarian cancer, and the sensitivity and specificity of diagnosis are unexpectedly improved at the same time, so that the effectiveness of diagnosis of the ovarian cancer is improved to a higher level.
The invention is realized by the following steps:
in a first aspect, the present invention provides the use of a detection reagent for a marker panel comprising ACTR10 and CA 125; the kit is used for auxiliary diagnosis of ovarian cancer and/or screening ovarian cancer risk.
ACTR10 protein (actin-related protein 10) is involved in a variety of cellular functions, including transport of endoplasmic reticulum to the golgi apparatus, centripetal movement of lysosomes and endosomes, spindle formation, chromosomal movement, nuclear localization, and axonogenesis. ACTR10 is a subunit of the dynactin complex. Several studies have shown that dynactin binds to the domain of membrane vesicles (e.g., golgi or late endosome), but there have been no reports of biological relevance of ACTR10 to ovarian cancer.
According to the invention, the ACTR10 and the CA125 are creatively combined to be used as a marker group for auxiliary diagnosis of ovarian cancer and/or risk screening of ovarian cancer, so that the diagnosis sensitivity and specificity can be improved to a higher level, and the effectiveness of diagnosis of ovarian cancer is greatly improved.
Optionally, in some embodiments, the kit comprises instructions for use bearing an ovarian cancer aided diagnosis model and/or an ovarian cancer risk screening model;
wherein the ovarian cancer auxiliary diagnosis model comprises the following steps:
OCS1=K1×CCA125-K2×CACTR10(ii) a Wherein the value of K1 is 0.3-0.4, the value of K2 is 0.6-0.7, and OCS1 is the threshold value of a diagnostic model;
the ovarian cancer risk screening model comprises the following steps:
OCS2=K3×CACTR10–K4×CCA125(ii) a Wherein the value of K3 is 0.7-0.8, the value of K4 is 0.4-0.5, and OCS2 is the screening model threshold;
in the above two models, CCA125Concentration values, C, representing CA125 of the subjectACTR10Concentration values representing subject ACTR 10.
Alternatively, in some embodiments, K1 may take on a value in the range of any one or between any two of 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, and 0.40.
Optionally, in some embodiments, K1 is 0.38.
Alternatively, in some embodiments, K2 may take on a value in the range of any one or between any two of 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, and 0.70.
Optionally, in some embodiments, K2 has a value of 0.66.
Alternatively, in some embodiments, K3 may take on any one or a range between any two of 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, and 0.80.
Optionally, in some embodiments, the value of K3 is 0.78.
Alternatively, in some embodiments, K4 may take on a value in the range of any one or between any two of 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, and 0.50.
Optionally, in some embodiments, K4 takes on a value of 0.41.
Alternatively, in some embodiments, the concentration of CA125 reflects the concentration of CA125 in the plasma of the subject in U/ml; the concentration of ACTR10 reflects the concentration of ACTR10 in U/ml in plasma vesicles of the subject.
Alternatively, in some embodiments, the ovarian cancer is stage I ovarian cancer.
Optionally, in some embodiments, the detection reagent includes, but is not limited to, any one of the following methods suitable for effecting detection of the marker panel: chemiluminescence method, flow fluorescence method, single molecule array method, enzyme linked immunosorbent assay, colloidal gold, electrochemical luminescence and quantum dot technology method.
In a second aspect, the invention provides a system for assisted diagnosis of ovarian cancer and/or for screening for ovarian cancer risk, the system comprising an information acquisition module, a calculation module and a diagnosis module;
the information acquisition module is used for executing operation of acquiring detection information of the subject, wherein the detection information comprises concentration information of CA125 and concentration information of ACTR 10;
the calculation module is used for executing the operation of substituting the detection information into a calculation model to calculate a threshold value;
the diagnosis module is used for executing the operation of judging the health condition of the subject according to the threshold value and the concentration information of the CA 125.
Optionally, in some embodiments, the computational model is an ovarian cancer aided diagnosis model or an ovarian cancer risk screening model;
wherein, the ovarian cancer auxiliary diagnosis model is as follows:
OCS1=K1×CCA125-K2×CACTR10(ii) a Wherein, K1 takes the value of 0.3-0.4, K2 takes the value of 0.6-0.7, and OCS1 is the threshold value of the diagnosis model;
the ovarian cancer risk screening model is as follows:
OCS2=K3×CACTR10–K4×CCA125(ii) a Wherein the value of K3 is 0.7-0.8, the value of K4 is 0.4-0.5, and OCS2 is the screening model threshold;
in the above two models, CCA125Concentration values, C, representing CA125 of the subjectACTR10Concentration values representing subject ACTR 10.
Alternatively, in some embodiments, K1 may take on any one or a range between any two of 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, and 0.40.
Optionally, in some embodiments, K1 has a value of 0.38.
Alternatively, in some embodiments, K2 may take on any one or a range between any two of 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, and 0.70.
Optionally, in some embodiments, K2 has a value of 0.66.
Alternatively, in some embodiments, K3 may take on a value in the range of any one or between any two of 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, and 0.80.
Optionally, in some embodiments, the value of K3 is 0.78.
Alternatively, in some embodiments, K4 may take on a value in the range of any one or between any two of 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, and 0.50.
Optionally, in some embodiments, K4 takes on a value of 0.41.
Alternatively, in some embodiments, the concentration of CA125 reflects the concentration of CA125 in the plasma of the subject in U/ml; the concentration of ACTR10 reflects the concentration of ACTR10 in U/ml in plasma vesicles of the subject.
Optionally, in some embodiments, when the subject is a subject with gynecological symptoms, the calculation model is an ovarian cancer aided diagnosis model, and the diagnosis module judges according to the following conditions:
if CCA12535 or more and 4.7 or more and OCS1 or 27.8 or CCA125(ii) < 35 and-11 < OCS1 < 10.3, then the subject is judged to be stage I ovarian cancer.
The ovarian cancer auxiliary diagnosis model subjects are usually gynecological outpatients and have nonspecific symptoms which can describe clear gynecological symptoms, such as patient complaints, such as anorexia, abdominal distension, abdominal pain or emaciation.
Optionally, in some embodiments, when the subject has no significant gynecological symptoms, the computational model is an ovarian cancer risk screening model, and the diagnosis module determines the following conditions:
if CCA125(ii) < 35 and-10.4 < OCS2 < 12.8, then the subject is judged to be stage I ovarian cancer. If CCA125If the model is more than or equal to 35, the model is not applicable.
The subjects in the ovarian cancer risk screening model are generally women with routine physical examination, have no obvious gynecological symptoms, and have a CA125 concentration index of less than 35U/ml.
Subjects with CA125 concentrations less than 35U/ml often also present with stage I ovarian cancer patients, resulting in false negatives relying on only a single indicator of CA 125. According to the invention, the ACTR10 is combined with CA125, so that the ovarian cancer diagnosis accuracy can be improved. Optionally, in some embodiments, the diagnostic system further comprises a result display module for displaying the diagnostic conclusion drawn by the diagnostic module.
Optionally, in some embodiments, the result display module displays the diagnosis result by means of screen display, voice broadcast or printing.
In a third aspect, the invention provides a kit for auxiliary diagnosis of ovarian cancer and/or for screening for ovarian cancer risk, comprising detection reagents for a marker panel comprising ACTR10 and CA 125.
Optionally, in some embodiments, the kit comprises instructions for use bearing an ovarian cancer aided diagnosis model and/or an ovarian cancer risk screening model;
wherein the ovarian cancer auxiliary diagnosis model comprises the following steps:
OCS1=K1×CCA125-K2×CACTR10(ii) a Wherein, K1 takes the value of 0.3-0.4, K2 takes the value of 0.6-0.7, and OCS1 is the threshold value of the diagnosis model;
the ovarian cancer risk screening model is as follows:
OCS2=K3×CACTR10–K4×CCA125(ii) a Wherein the value of K3 is 0.7-0.8, the value of K4 is 0.4-0.5, and OCS2 is the screening model threshold;
in the above two models, CCA125Concentration values, C, representing CA125 of the subjectACTR10Concentration values representing subject ACTR 10.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the results of chemiluminescence assay for ACTR10 in stage I ovarian cancer patients and non-ovarian cancer individuals as described in example 1.
FIG. 2 shows the results of flow-based fluorescence assays for ACTR10 expression in stage I ovarian cancer patients and non-ovarian cancer individuals as described in example 1.
FIG. 3 shows the results of the chemiluminescence assay for the expression of ACTR10 in different cancer patients in example 2.
FIG. 4 is a ROC curve obtained by using the ovarian cancer auxiliary diagnosis model in 100 gynecological outpatients in example 3.
FIG. 5 is a ROC curve obtained by applying the ovarian cancer risk screening model to 200 women in health examination in example 4.
Fig. 6 is a detection flowchart of ACTR 10.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Differential detection of ACTR10 expression in stage I ovarian cancer patients and non-ovarian cancer individuals
ACTR10 can be used to detect differences in expression between stage I ovarian cancer patients and non-ovarian cancer individuals using different detection methods.
ACTR10 concentration was measured by chemiluminescence, and the results are shown in fig. 1: the mean for stage I ovarian cancer patients was 16.2U/ml (i.e., 16.2U ACTR10 protein per ml plasma), the mean for benign ovarian disease patients was 33.3U/ml, and the p-value for both statistical tests was less than 0.0001. The mean value for healthy individuals was 39.01U/ml, and the statistically tested p-values for the group of stage I ovarian cancer patients and the group of healthy individuals were less than 0.0001.
ACTR10 concentration was detected by flow fluorescence method and the results are shown in fig. 2: the mean value for stage I ovarian cancer patients was 15.8U/ml, the mean value for benign ovarian disease patients was 26.6U/ml, and the p-value for both groups of statistical tests was less than 0.0001. The mean value of healthy individuals was 33.99U/ml, and the statistically examined p-values of the group of patients with stage I ovarian cancer and the group of healthy individuals were less than 0.0001.
The different detection methods all show that the ACTR10 has expression difference between the patients with stage I ovarian cancer and the individuals without ovarian cancer.
Example 2
Detection of expression in ACTR10 different cancer patients
ACTR10 has specific expression in plasma vesicles from ovarian cancer patients. In the prior art, the method for detecting a plurality of miRNAs in vesicles affects the specificity of ovarian cancer diagnosis. The same miRNA has the condition of increasing expression quantity in various tumors.
This example detects ACTR10 in different cancer patients by chemiluminescence, and the results are shown in fig. 3: ACTR10 did not show significant differences in plasma vesicles from other tumor patients or benign patients common to women; there was no significant difference in ACTR10 concentrations in breast cancer, benign cystic hyperplasia of the breast, cervical cancer, benign leiomyoma of the cervix, and corresponding healthy individuals, with the mean values for each group being: 32.65, 32.32, 33.37, 30.94, 35.73U/ml.
Example 3
ACTR10 is used in combination with CA125 for the auxiliary diagnosis of early stage ovarian cancer.
100 gynecological outpatients were enrolled. The standard of group entry is that CA125 is 35U to 100U/ml, discomfort is caused in the abdomen, ovarian space occupying lesion is prompted by vaginal ultrasound, and the like, but laparoscopy is not performed. ACTR10 in the 100 female plasma vesicles was detected by a chemiluminescence platform and combined with a CA125 value to be substituted into an ovarian cancer auxiliary diagnosis model: OCS1=0.38 × CCA125-0.66×CACTR10，CCA125Represents the concentration value of CA125 in the plasma of the subject,CACTR10represents the concentration value of ACTR10 in plasma vesicles of the subject; and (3) judging the mode: if CCA12535 or more (i.e. the concentration of CA125 is 35U/ml or more) and 4.7 < OCS1 < 27.8, or CCA125(ii) < 35 and-11 < OCS1 < 10.3, the subject is judged to be stage I ovarian cancer.
Based on whether 100 model results ranged from 4.7 to 27.8, results were obtained as to whether women meeting the inclusion criteria had stage I ovarian cancer. According to the ovarian cancer diagnosis and treatment guidelines, 100 patients are followed up until clear clinical diagnosis results are obtained. And comparing the clinical diagnosis result with the ovarian cancer auxiliary diagnosis model result, and analyzing to obtain an ROC curve, which is shown in figure 4.
From the curve, it can be seen that the indexes of the single CA125 or the single ACTR10 are limited for auxiliary diagnosis of stage I ovarian cancer, and the sensitivity of auxiliary diagnosis of stage I ovarian cancer by combining the ACTR10 and the CA125 through an auxiliary diagnosis model of ovarian cancer reaches 94.7%, meanwhile, the corresponding specificity reaches 85.9%, and the comprehensive accuracy reaches 88.5%.
Example 4
ACTR10 is used in combination with CA125 for risk management in ovarian cancer screening. 200 women with the group age of 30-60 years are tested, and the CA125 concentration test result is less than 35U/ml.
The ACTR10 in the 200 female plasma vesicles is detected by using a chemiluminescence platform, and combined with a CA125 value, the value is substituted into an ovarian cancer risk screening model: OCS2=0.78 × CACTR10–0.41×CCA125；CCA125Represents the concentration value, C, of CA125 in the plasma of the subjectACTR10Represents the concentration value of ACTR10 in plasma vesicles of the subject; and (3) judging the mode: if C CA125(ii) < 35 and-10.4 < OCS2 < 12.8, then the subject is judged to be stage I ovarian cancer. If the concentration of CA125 is more than or equal to 35U/ml, the model is not applicable.
Based on whether the 200 model results ranged from-10.4 to 12.8, results were obtained as to whether asymptomatic women at risk for stage I ovarian cancer were obtained. Women who indicate risks are followed up for 6 months to have clear clinical diagnosis results, such as no ovarian diseases, benign ovarian lesions and stage I ovarian cancer. The results of the clinical diagnosis were compared with the results of the OCS2 model at 6 months ago, and the ROC curve was analyzed, as shown in fig. 5.
From the curve, the indexes of CA125 alone or ACTR10 alone are limited when being used for screening the ovarian cancer of stage I, while the sensitivity of screening the ovarian cancer of stage I through the OCS2 model is up to 96.6%, the corresponding specificity is more up to 90.7%, and the comprehensive accuracy is up to 94.2%.
Comparative example 1
Prior document 1 (Exosomal microRNAs as tumor markers in epithelial ovarian cancer) shows that four plasma total vesicle-derived miRNAs (miR-21, miR-100, miR-200b and miR-320) can be used for early diagnosis of ovarian cancer. By means of Taqman microRNA array and Taqman real time PCR, miR-200b is found and determined to have the highest sensitivity of 64% and the highest specificity of 86%, and the diagnosis effectiveness of the miR-200b on stage I ovarian cancer is only 50% higher than that of the current ovarian cancer marker CA 125.
Comparative example 2
The existing document 2 (Serum exosomal miRNA-145 and miRNA-200c as a promoting biomarkers for a predicting diagnosis of ovarian cancer) shows that the miRNAs (miR-145 and miR-200c) in the Serum vesicle are highly expressed in patients with high-grade ovarian cancer, the two miRNAs are independently detected by a Taqman fluorescence PCR method, or are respectively detected by combining with an existing ovarian cancer marker CA125 for diagnosis, so that the sensitivity can be improved, and the diagnosis sensitivity of the CA125+ miR-145+ miR-200c combination reaches 100% but the specificity is only 55%. The combination of the serum vesicle miRNA and CA125 for ovarian cancer diagnosis can improve the diagnosis effectiveness. However, the research not only diagnoses the ovarian cancer of stage I, but also comprises more patients with ovarian cancer of stages II to IV, and the early diagnosis cannot be effectively realized.
In conclusion, the current problem of using vesicle methodology for ovarian cancer diagnosis is limited to the detection of miRNA, and neither single miRNA or multiple miRNA nor combined diagnosis with the existing ovarian cancer marker CA125 can achieve sensitivity and specificity exceeding 85%. In the prior art, ovarian cancer and non-ovarian cancer are identified, and the differential diagnosis of stage I ovarian cancer and non-ovarian cancer is not aimed, and in addition, a plurality of miRNA used for ovarian cancer diagnosis in the prior art have higher expression level in other tumors, so that the specificity of ovarian cancer diagnosis is influenced.
Through the examples and the comparative examples, the ACTR10 and the CA125 are combined for the first time, so that the differential diagnosis of early ovarian cancer (stage I) and benign ovarian diseases and early ovarian cancer (stage I) and healthy individuals is realized, and the auxiliary diagnosis of ovarian cancer and the risk management of ovarian cancer of healthy individuals in clinic are realized;
the sensitivity and specificity of the kit exceed 85%, so that the effectiveness of ovarian cancer diagnosis is greatly improved, and the kit has excellent diagnosis accuracy;
different from the prior art that only miRNA in the vesicle can be detected by PCR, ACTR10 protein detection in the invention can be realized by a plurality of detection platforms such as chemiluminescence, flow fluorescence, single molecule array and the like, thereby ensuring that a plurality of methods are clinically used for diagnosing individuals with ovarian cancer risks.
The detection of the ACTR10 protein in the above example was performed with reference to the following steps, the flow chart of which is shown in fig. 6:
1. separating 4 ml venous blood by using an EDTA-Na anticoagulation tube, standing for no more than 4 hours at room temperature, and standing for no more than 12 hours in a 4-DEG refrigerator.
2. The anticoagulation tube was centrifuged at 3000 Xg for 15 minutes at 4 ℃ and the upper layer of the anticoagulation tube, about 2 ml of plasma, was separated.
3. The plasma was centrifuged at 10000 Xg for 15 minutes at 4 ℃ and the upper layer of approximately 1.8 ml of plasma was retained.
4. Separating the total plasma vesicles by ultracentrifugation, exclusion chromatography or commercial kit method, wherein the separated vesicles can be directly subjected to protein extraction or stored in a refrigerator at-80 degrees for a long time:
4.1 ultracentrifugation method
4.1.1 Add 1.8 ml of plasma from 3 to 4 ml of 2.2 ml of phosphate buffer, centrifuge at 100000 Xg for 60 minutes at 4 ℃, remove the upper layer of about 3.6 ml of solution, and add 1 ml of phosphate buffer to resuspend the pellet at the bottom of the centrifuge tube;
4.1.2 the resuspended solution was centrifuged again at 100000 Xg at 4 ℃ for 60 minutes, the supernatant was removed and only the pellet was retained, and 200. mu.l of phosphate buffer was added to resuspend the pellet at the bottom of the centrifuge tube to obtain small extracellular vesicles.
4.2 exclusion chromatography
4.2.1 2 ml of 2 plasma was filtered through a 0.8 micron filter and the filtered plasma was centrifuged at 10000 Xg for 15 minutes at 4 ℃ leaving approximately 1.5 ml of plasma in the upper layer;
4.2.2 Add 1.5 ml of plasma to the activated column and continue to add phosphate buffer to 10 ml, collect 3.5 to 5 ml of solution passing through the column with a centrifuge tube, this volume fraction of solution being small extracellular vesicles.
4.3 commercial kit method
4.3.1 the principle of plasma vesicle separation by commercial kit method is that hydrophilic polymer is incubated with vesicle in plasma, during incubation, the hydrophilicity of polymer reduces the solubility of vesicle in solution significantly, which results in aggregation of vesicle in plasma, and then the aggregated vesicle is precipitated by centrifugation. Thermofeisher (Kit name: Total Exosome isolation Kit (from Plasma), cat # 4484450), SBI, Norgen and other reagent suppliers all have Plasma vesicle isolation kits, and the present invention is exemplified by Serum/Plasma vesicle isolation Kit of Norgen (Kit name: Plasma/Serum Exosome Purification Mini Kit, cat # 57400).
4.3.2 Add 1.8 ml of plasma from 3 to 2.2 ml of phosphate buffer, mix well to 4 ml solution, and add 100. mu.l of ExoC buffer;
4.3.3 to the mixed 4.1 ml solution, 200. mu.l of SlurryE solution was added and vortexed for 10 seconds, and the solution was allowed to stand vertically for 5 minutes;
4.3.4 the solution after standing was vortexed again for 10 seconds and centrifuged at 1500 Xg for 2 minutes at room temperature to remove the supernatant until the precipitate remained;
4.3.5 Add 200. mu.l ExoR buffer to the pellet and vortex for 10 seconds, then let stand at room temperature for 5 minutes, and vortex again for 10 seconds, and centrifuge at 300 Xg for 2 minutes at room temperature;
4.3.6 solution is added to a 0.2 micron filter and the solution passing through the filter is collected by centrifugation at 5000 Xg for 1 minute at room temperature, which is a small extracellular vesicle.
5. To each ml of vesicle solution, 200. mu.l of RIPA lysate was added, and the solution was mixed by pipette washing and the tube containing the solution was placed on ice for 15 minutes for protein extraction.
6. The extracted solution was centrifuged at 10000 Xg at 4 ℃ for 15 minutes, and the supernatant solution in which the vesicle proteins were present was retained. The protein solution can be directly used for downstream protein analysis or stored in a refrigerator at-80 ℃ for a long time.
7. The ACTR10 is detected by chemiluminescence, flow fluorescence or single molecule array methods:
7.1 chemiluminescence method, commercially available full-automatic chemiluminescence immunoassay analyzer can detect ACTR10 protein, taking model No. 2000SR as an example in the invention.
7.1.1, performing routine inspection before starting the instrument, starting the instrument after confirming no error and confirming the state of the instrument;
7.1.2 loading reagents required by immune reaction, including separating medium, reaction substrate, reaction cup and sample needle cleaning solution;
7.1.3 before reaction, carrying out calibration test and quality control test on the instrument, and carrying out sample detection after confirming no error;
7.1.4 detecting the vesicle protein sample, putting the sample to be detected into a sample frame, putting the sample frame into a sample putting area, inputting the positions of the first sample and the last sample, clicking after confirmation, waiting for the instrument to read the reaction value and exporting.
7.2 flow-type fluorescence method, ACTR10 protein can be detected by commercially available mainstream fluorometers, and Luminex200 is taken as an example in the present invention.
7.2.1 opening the host, the XY platform, the SD and the matched computer, opening a Luminex control program, and performing instrument self-check to determine whether the system is normal;
7.2.2 executing an instrument calibration program, adding a calibration reagent, pure water and calibration microspheres at the designated position of the program, executing the calibration program and storing the result;
7.2.2 after the instrument calibration is finished, opening a bin, placing a copper plate, heating, placing a hybridization plate on the copper plate, and setting a reagent retention program file required for detecting ACTR 10;
7.2.3 Add template for analysis of ACTR10 protein and derive the raw fluorescence values and the calibrated concentration values calculated sequentially.
7.3 it should be noted that in addition to the above two methods, common protein detection technology platforms can also measure the concentration of ACTR10, including but not limited to enzyme-linked immunosorbent assay, colloidal gold assay, electrochemiluminescence, and quantum dot technology.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The application of the detection reagent of the marker group in the preparation of the kit is characterized in that the marker group comprises ACTR10 and CA 125; the kit is used for auxiliary diagnosis of ovarian cancer and/or screening ovarian cancer risk.
2. The use of claim 1, wherein the kit comprises instructions for use bearing an ovarian cancer aided diagnosis model and/or an ovarian cancer risk screening model;
Wherein the ovarian cancer auxiliary diagnosis model comprises the following steps:
OCS1=K1×CCA125-K2×CACTR10(ii) a Wherein the value of K1 is 0.3-0.4, the value of K2 is 0.6-0.7, and OCS1 is the threshold value of a diagnostic model;
the ovarian cancer risk screening model is as follows:
OCS2=K3×CACTR10–K4×CCA125(ii) a Wherein the value of K3 is 0.7-0.8, the value of K4 is 0.4-0.5, and OCS2 is the screening model threshold;
at the two sidesIn the model, CCA125Concentration values, C, representing CA125 of the subjectACTR10Concentration values representing subject ACTR 10.
3. The use of claim 2, wherein the concentration of CA125 reflects the concentration of CA125 in the plasma of the subject in U/ml; the concentration of ACTR10 reflects the concentration of ACTR10 in U/ml in plasma vesicles of the subject.
4. The use of any one of claims 1 to 3, wherein the ovarian cancer is stage I ovarian cancer.
5. The use according to any one of claims 1 to 3, wherein the detection reagent is adapted to any one of the following methods for effecting detection of the marker panel: chemiluminescence method, flow fluorescence method, single molecule array method, enzyme linked immunosorbent assay, colloidal gold, electrochemical luminescence and quantum dot technology method.
6. A system for assisted diagnosis of ovarian cancer and/or for screening for ovarian cancer risk, the system comprising an information acquisition module, a calculation module, and a diagnosis module;
The information acquisition module is used for executing operation of acquiring detection information of the subject, wherein the detection information comprises concentration information of CA125 and concentration information of ACTR 10;
the calculation module is used for executing the operation of substituting the detection information into a calculation model to calculate a threshold value; the calculation model is an ovarian cancer auxiliary diagnosis model or an ovarian cancer risk screening model;
wherein, the ovarian cancer auxiliary diagnosis model is as follows:
OCS1=K1×CCA125-K2×CACTR10(ii) a Wherein, K1 takes the value of 0.3-0.4, K2 takes the value of 0.6-0.7, and OCS1 is the threshold value of the diagnosis model;
the ovarian cancer risk screening model is as follows:
OCS2=K3×CACTR10–K4×CCA125(ii) a Wherein the value of K3 is 0.7-0.8, the value of K4 is 0.4-0.5, and OCS2 is the screening model threshold;
in the above two models, CCA125Concentration values, C, representing CA125 of the subjectACTR10Concentration values representative of subject ACTR 10; the concentration of CA125 reflects the concentration of CA125 in the plasma of the subject in U/ml; the concentration of ACTR10 reflects the concentration of ACTR10 in U/ml in plasma vesicles of the subject
The diagnosis module is used for executing the operation of judging the health condition of the subject according to the threshold value and the concentration information of CA 125;
when the subject is a subject with gynecological symptoms, the calculation model is an ovarian cancer auxiliary diagnosis model, and the diagnosis module judges according to the following conditions:
If CCA12535 or more and 4.7 or more and OCS1 or 27.8 or CCA125(ii) < 35 and-11 < OCS1 < 10.3, then the subject is judged to be stage I ovarian cancer;
when the subject is a subject without obvious gynecological symptoms, the calculation model is an ovarian cancer risk screening model, and the diagnosis module judges according to the following conditions:
if CCA125(ii) < 35 and-10.4 < OCS2 < 12.8, then the subject is judged to be stage I ovarian cancer.
7. The system of claim 6, further comprising a result display module for displaying a diagnosis conclusion drawn by the diagnosis module.
8. The system of claim 7, wherein the result display module displays the diagnosis result by means of screen display, voice broadcast or printing.
9. A kit for assisted diagnosis of ovarian cancer and/or for screening for ovarian cancer risk, comprising detection reagents for a marker panel comprising ACTR10 and CA 125.
10. The kit of claim 9, wherein the kit comprises instructions for use bearing an ovarian cancer aided diagnosis model and/or an ovarian cancer risk screening model;
Wherein, the ovarian cancer auxiliary diagnosis model is as follows:
OCS1=K1×CCA125-K2×CACTR10(ii) a Wherein, K1 takes the value of 0.3-0.4, K2 takes the value of 0.6-0.7, and OCS1 is the threshold value of the diagnosis model;
the ovarian cancer risk screening model is as follows:
OCS2=K3×CACTR10–K4×CCA125(ii) a Wherein the value of K3 is 0.7-0.8, the value of K4 is 0.4-0.5, and OCS2 is the screening model threshold;
in the above two models, CCA125Concentration values, C, representing CA125 of the subjectACTR10Concentration values representing subject ACTR 10.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103575891A (en) * | 2012-08-03 | 2014-02-12 | 河南生生医疗器械有限公司 | Kit for comprehensively detecting HE4 and CA125 and application of kit |
| CN106442993A (en) * | 2016-09-10 | 2017-02-22 | 天津大学 | Preparation method of enzyme-linked immunosorbent assay kit for detecting ovarian cancer tumor marker CA125 based on trypsin fluorogenic substrate |
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| US8288110B2 (en) * | 2006-12-04 | 2012-10-16 | Perkinelmer Health Sciences, Inc. | Biomarkers for detecting cancer |
| BR112013032232A2 (en) * | 2011-06-16 | 2016-09-20 | Caris Life Sciences Switzerland Holdings S A R L | Cancer characterization method using nucleic acid biomarker |
| US20150267259A1 (en) * | 2012-10-12 | 2015-09-24 | Agency For Science, Technology And Research | Method of prognosis and stratification of ovarian cancer |
| CN204514921U (en) * | 2015-04-28 | 2015-07-29 | 殷晓燕 | A kind of analytical equipment for detecting advanced ovarian cancer Peripheral Blood CA125 |
| CN108008132B (en) * | 2017-12-04 | 2020-05-08 | 北京惠中医疗器械有限公司 | Kit for combined detection of ovarian cancer tumor markers HE4 and CA125 and preparation method and application thereof |
| CN111521807B (en) * | 2020-07-02 | 2020-10-27 | 北京信诺卫康科技有限公司 | Spondin1 and CA125 combined used as early ovarian cancer biomarker and kit |
| CN111735949B (en) * | 2020-07-17 | 2023-07-21 | 北京信诺卫康科技有限公司 | Wnt7a and CA125 combined as early ovarian cancer biomarker and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103575891A (en) * | 2012-08-03 | 2014-02-12 | 河南生生医疗器械有限公司 | Kit for comprehensively detecting HE4 and CA125 and application of kit |
| CN106442993A (en) * | 2016-09-10 | 2017-02-22 | 天津大学 | Preparation method of enzyme-linked immunosorbent assay kit for detecting ovarian cancer tumor marker CA125 based on trypsin fluorogenic substrate |
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