CN113325183B - Kit for differential diagnosis of EM/FEM - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a reagent for identifying ovarian endometriosis/other benign tumors of an ovary and application thereof. The invention provides the use of reagents for detecting the level of biomarkers comprising CA125, SELL, LILRA3, IGKC and/or DSC2 in a sample from a subject for the manufacture of a differential diagnostic product for the identification of endometriosis ovaries/other benign tumours of the ovaries. The invention provides a marker for differential diagnosis of endometriosis ovaries/other benign tumors of ovaries, CA125, SELL, LILRA3, IGKC and/or DSC2, which can be used for preparing a detection kit for differential diagnosis of EM/FEM and the like, and has good differential specificity and high sensitivity. The kit can improve the accuracy of clinical EM/FEM differential diagnosis, so that patients can obtain more accurate treatment measures and better treatment effect.

Description

Kit for differential diagnosis of EM/FEM

Technical Field

The invention relates to the technical field of biology, in particular to a kit for differential diagnosis of ovarian endometriosis/other benign tumors of the ovary.

Background

Endometriosis (endometriosis) is a common benign disease of the gynaecology, characterized by functional endometrial glandular and interstitial growth outside the uterine cavity. One patient will have endometriosis approximately every 10 women of childbearing age. However, in women seeking infertility assessment, the incidence is about 50% and disease progression is more likely to occur; the incidence in pain patients is as high as 70%. Endometriosis is a benign disorder in tissue morphology, but may exhibit tumor-like characteristics in biological behavior, such as invasion, implantation, and distant metastasis. Clinical manifestations of endometriosis are complicated and disturbing the normal life of patients, and serious patients can cause heavy psychological stress. Early detection plays a crucial role in timely and effective treatment, but the diagnostic gold standard of endometriosis is laparoscopic surgery, which brings great difficulty to accurate diagnosis of outpatient common patients. Therefore, the value of noninvasive diagnosis of endometriosis by imaging examination or easily obtained biological specimens (such as blood, urine, saliva, etc.) is not negligible.

Ovarian Endometriosis (EM) is also called as an ovarian chocolate-like cyst, and factor endometrioid tissues are planted on the surface of an ovary to repeatedly bleed to form the cyst, which is one of the most common types of endometriosis. Some EM patients have symptoms such as dysmenorrheal, infertility and chronic pelvic pain, which affect the life quality of the patients, and some EM patients only see a doctor for finding the ovarian tumor. Statistically, approximately 5% to 10% of women receive surgical treatment for suspected ovarian tumors at some time during their lifetime. The EM treatment mode comprises drug treatment and operation treatment, and researchers indicate that the EM-related operation treatment can cause oocyte loss and damage of ovarian tissues to different degrees and can cause reduction of the anti-Mullerian hormone level, so that the operation should be implemented carefully considering the overall condition of a patient, and accurate preoperative diagnosis helps to perform individualized treatment on the patient. Most of the studies only include the heteropathy patients and healthy people as experimental groups and control groups, and related marker studies aiming at EM differential diagnosis are lacked.

Pelvic ultrasound examination, particularly transvaginal ultrasound (TVUS), is an effective method for preliminary diagnosis of EM, but its diagnosis depends on the subjective judgment of the sonographer and the degree of precision of the instrument, so the accuracy is not stable. One Meta analysis compares 19 methods for identifying benign and malignant ovarian tumors before operation, and finds that the accuracy rate of different methods is greatly different, the sensitivity of the most effective method is 93 percent, the specificity is 81 percent, and if EM and other benign tumors need to be distinguished, the difficulty of identifying ovarian tumors is further increased. Serum cancer antigen 125 (CA 125) is a commonly used diagnostic marker for EM differentiation in clinical work, but the accuracy and stability of the marker are still controversial. With the continuous development of high-throughput technology, proteomics technology becomes an important means for finding markers, and a data-independent scanning mode (DIA) is a newer targeted quantitative proteomics mass spectrum data acquisition mode. Currently, EM differential diagnosis biomarkers with high sensitivity, high specificity and high clinical relevance are yet to be discovered.

Disclosure of Invention

In view of the above problems, the present invention aims to provide biomarkers CA125, sel, LILRA3, IGKC and/or DSC2 for differential diagnosis of endometriosis ovaries (EM)/other benign tumors of ovaries (FEM) and applications thereof in preparing products.

In order to realize the purpose, the specific technical scheme of the invention is as follows:

in a first aspect, the invention provides the use of an agent for detecting the level of a biomarker in a sample from a subject in the manufacture of a product for use in the differential diagnosis of endometriosis of the ovary/other benign tumours of the ovary, the biomarkers include CA125, SELL, LILRA3, IGKC, and/or DSC2.

In some embodiments, the CA125, DSC2, and IGKC are expressed at elevated levels in an ovarian endometriosis patient sample compared to a control sample; the SELL and LILRA3 are expressed at reduced levels in a sample from a patient with endometriosis ovaries.

In some embodiments, the control sample is an ovarian other benign tumor sample.

In some embodiments, the reagent for detecting the amount of a biomarker in a sample from a subject comprises a mass spectrometric identification reagent, an antibody or antigen binding fragment thereof.

In some embodiments, the product comprises a kit, chip, or strip.

In some embodiments, the reagents include mass spectrometry identification reagents, antibodies or antigen-binding fragments thereof, primers, and probes.

In a second aspect, the invention provides a test kit for the differential diagnosis of endometriosis ovaries/other benign tumours comprising reagents for detecting the level of CA125, SELL, LILRA3, IGKC and/or DSC2 expression in a sample from a subject.

In some embodiments, the kit comprises reagents for detecting the expression level of CA125 and/or IGKC in a sample from a subject.

In a preferred embodiment, the kit comprises reagents for detecting the expression levels of CA125 and IGKC in a sample from a subject. The AUC of the differential diagnosis EM/FEM of the kit is 0.786, when CA125>0.1ng/ml and IGKC >504.52ng/ml are set to be positive (namely, the diagnosis EM), the sensitivity of the diagnosis is 85.23 percent, and the specificity is 62.22 percent.

In some embodiments, the reagent comprises a mass spectrometry identification reagent, an antibody, or an antigen binding fragment thereof.

In some embodiments, the reagents are detected by Elisa, western Blot, mass spectrometry.

In a third aspect, the present invention provides a differential diagnostic kit for patients with endometriosis <45 years of age and a tumor diameter <5cm and other benign tumors of the ovary, comprising reagents for detecting the amount of CA125 and IGKC in a sample from a subject.

Based on the technical scheme, the invention has the following beneficial effects:

the invention provides a marker for differential diagnosis of EM/FEM, CA125, SELL, LILRA3, IGKC and/or DSC2, which can be used for preparing differential diagnosis kits of EM/FEM and the like, and has good differential specificity and high sensitivity. The kit can improve the accuracy of clinical EM/FEM differential diagnosis, so that patients can obtain more accurate treatment measures and better treatment effect.

Drawings

Figure 1DIA method flow diagram.

FIG. 2 sets of Wen plots, (A) differential protein analysis with EM control; (B) differential protein analysis with OC as control; (C) differential protein analysis with FEM as control.

FIG. 3GO enrichment analysis of EM and FEM group differential proteins; the GO item is represented on the ordinate, the enrichment condition of the differential protein in the corresponding function item is represented on the abscissa, the larger the Value of the-Log 10P-Value is, the more the differential protein is related to the function, and the differential protein of the function can be subjected to expansion analysis.

FIG. 4 concentration differences of different protein markers in serum of patients in EM/FEM/OC group.

FIG. 5 ROC graph of CA125.

FIG. 6 ROC graph of IGKC.

FIG. 7 ROC graph of DSC2

FIG. 8 ROC graph of SELL.

FIG. 9 ROC plot of LILRA 3.

FIG. 10CA125+ SELL + LILRA3+ IGKC + DSC2,5 protein markers combined ROC curve diagram.

FIG. 11CA125 IGKC + DSC2,3 protein marker combination ROC plot.

FIG. 12CA125 IGKC,2 protein marker association ROC plot.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

All materials, reagents and the like in the following examples are commercially available unless otherwise specified.

The present invention utilizes proteomics techniques to explore diagnostic biomarkers for the identification of patients with internal disorders. In the screening stage of protein markers, the inventors performed protein detection on collected serum samples of EM group, FEM group and OC group patients by using DIA technology, and performed differential protein analysis on the detection results, and found out 4 protein markers (SELL, LILRA3, IGKC and DSC 2) with potential differential diagnosis value. Functional analysis of two groups of differential proteins, EM and FEM, has shown that it focuses mainly on metabolic pathways (e.g., various amino acid metabolic pathways) as well as PPAR signaling pathways, AMPK signaling pathways, and Cell Adhesion Molecule (CAMs) pathways.

In the stage of protein marker verification, the inventors selected 5 proteins, namely CA125, SELL, LILRA3, IGKC and DSC2. According to analysis, for diagnosis of EM and FEM, IGKC and CA125 have stronger diagnostic capability for a single marker; in general, the 5 protein marker combinations were most diagnostic. The combination of CA125+ IGKC is more suitable for clinical application.

Currently, the unified accurate measurement standard definition of gynecology and imaging is not available internationally, researchers such as Muzii and the like propose to classify the size of the heterosis cyst by taking the maximum diameter of the heterosis cyst as a boundary, and propose to limit the implementation of an operation to reduce the risk of reducing the iatrogenic ovarian reserve function for young women with the tumor size within 5cm and with the fertility demand.

Based on the above theory, the inventors first grouped patients in 45 years of age, and for the subgroup aged <45 years, analysis was performed only on EM and FEM patients and found that AUC for CA125 was greater. When patients are more than or equal to 45 years old, the IGKC has strong discrimination capability on EM and FEM patients. Also the inventors performed a hierarchical analysis of tumor size, and for EM and FEM, CA125 was more diagnostic for patient populations with tumor diameters <5cm or tumor diameters ≧ 5cm, with AUC of 0.854 and 0.751, respectively. Finally, the inventors analyzed patients with age less than 45 years and tumors less than 5cm and found that CA125+ IGKC was better able to identify ectopic patients in this population. The hierarchical analysis provides reference for individual management of the ectopic patients and assists doctors in making flexible treatment.

Example 1 screening of protein markers associated with EM/OC/FEM

1. Experimental Material

1. Research sample

Three groups of disease type patients who visit the department of obstetrics and gynecology in Beijing cooperative hospital and received surgical treatment in the period from 1 month in 2019 to 1 month in 2020 were collected and included, and the basic conditions of the patients are shown in Table 1.

(1) Ovarian endometriosis group (EM group) 10 cases: the medicine is not used for treating endometriosis related medicines half a year before the operation of a patient.

(2) Ovarian malignancy group (OC group) 10 cases: the pathology is consistent with epithelial ovarian cancer.

(3) Other benign tumor groups of ovary (FEM group) 10 cases: the pathology is excluded from combining endometriosis.

TABLE 1 protein markers basic information for screening patient populations

IQR: a quartile difference;

#: endometriosis staging reference to american fertility association modified staging method (r-AFS);

* : FIGO stage principle of ovarian cancer

2. Sample collection and storage

(1) All patients collected 5ml venous blood through a vacuum procoagulant blood collection tube before operation after admission, placed on a clean laboratory bench test tube rack, and vertically stood for 30min at room temperature (about 25 ℃).

(2) Centrifuge at 1600G for 10min at 4 deg.C, and take the supernatant into an EP tube.

(3) Serum samples were stored in a-80 ℃ freezer for subsequent use.

3. Main experimental reagent and consumable

Chromatographic grade acetonitrile: united states, ABI corporation;

urea, dithiothreitol (DTT), iodoacetamide (IAA), CHAPS: bio-Rad, inc., USA;

tris (Tris): U.S., USB corporation;

ammonia, formic Acid (FA): sigma-Aldrich, USA;

mass-spectrum-grade trypsin: promega corporation, USA;

thiourea: sigma-Aldrich, USA;

protease Inhibitor Cocktail: usa, roche corporation;

10KD ultrafiltration tube, ziptip C18 extraction column: milipore corporation, USA.

4. Main experimental instrument

Microplate reader (Synogen 4): U.S. Thermo corporation;

RIGOL L-3000 high performance liquid chromatography: beijing Puyuan Smart technologies, inc.;

orbitrap Fusion mass spectrometer: U.S. Thermo corporation;

a vacuum drying instrument: U.S. Thermo corporation;

5. reagent preparation

1) Lysis solution

CHAPS was weighed out at 0.1% by volume of 7M urea and 2M thiourea, and 1 Protease Inhibitor Cocktail was taken and mixed with deionized water in a 50mL centrifuge tube to a volume of 50mL.

2)1M DTT

0.154g DTT was weighed and added to 1mL 25mM NH ₄ HCO ₃ Shaking to dissolve completely, and storing at-20 deg.C.

3)1M IAA

0.185g IAA was weighed out and 1mL 25mM NH added ₄ HCO ₃ Shaking to dissolve it completely, mixing immediately before use, and keeping out of the sun.

2. Experimental methods

1. Extraction of sample proteins

Serum proteins were desaturated using a desaturation column (Thermo, cat # A36370) as follows: 13000r/min of a serum sample, centrifuging for 10min, and taking a supernatant; equilibrating the column to room temperature; add 10. Mu.L serum sample; the mixture is inverted and mixed to ensure that the resin is evenly distributed in the serum; incubating on a rotary homogenizer for 10min at room temperature; putting the column into a 2mL collection tube, centrifuging for 2min at 1000 g/min; discarding the chromatographic column containing the resin to obtain the low-abundance protein in serum.

Determination of protein concentration by Bradford method

Preparing a BSA standard solution with a concentration gradient of 0. Mu.g/. Mu.L, 0.025. Mu.g/. Mu.L, 0.125. Mu.g/. Mu.L, 0.25. Mu.g/. Mu.L, 0.5. Mu.g/. Mu.L, 0.75. Mu.g/. Mu.L, 1. Mu.g/. Mu.L, 1.5. Mu.g/. Mu.L with a lysis solution in the kit; respectively adding 10 mu L of standard substance solutions or protein samples with different concentrations into each hole of a 96-hole plate, and preparing 3 parallel multiple holes by using the same sample; adding 300 mu L of Bradford working solution into each hole respectively, and reacting for 5-10min under the condition of keeping out of the light; measuring the absorbance of the sample at 595nm by using an enzyme-labeling instrument; and drawing a concentration/absorbance standard curve according to the concentration of the standard substance and the corresponding absorbance, obtaining a formula for calculating the protein concentration according to the absorbance, and calculating the protein concentration of each sample.

3. Membrane-assisted in-solution proteolytic digestion

1) Reduction of the protein:

adding equal amount of protein into 10KD ultrafiltration tube, adding 1MDTT (making final concentration of DTT 25 mM) according to the volume ratio of total solution volume to 1MDTT of 40

2) Alkylation of proteins:

the reduced sample was mixed with 1MIAA (to give a final IAA concentration of 50 mM) at a ratio of 20% by volume of the total solution to 1M IAA, vortexed, and left at room temperature for 30min in the dark.

3) Cleaning a sample:

add 5 times diluted TEAB 300. Mu.L, centrifuge at 12,000rpm (13,400g) for 10min, discard the bottom solution of the collection tube, repeat 3 times, add 2 times diluted TEAB 300. Mu.L, centrifuge at 12,000rpm (13,400g) for 10min.

4) Cleavage of protein by enzyme in membrane-assisted solution:

the collection tube was replaced with a new one, trypsin was added to the ultrafiltration tube, the total to protein mass ratio was set to 1.

5) Collecting the polypeptide:

the next day, 100. Mu.L of TEAB diluted 2 times was added, centrifugation was carried out at 12,000rpm (13,400g) for 10min, repeated 3 times, and the peptide fragment solution after enzymatic digestion was centrifuged at the bottom of the collection tube.

4. High pH reverse phase C18 chromatography

1) Redissolving the polypeptide sample in solution A (98% ddH) ₂ O,2% acetonitrile, aqueous ammonia to pH 10.0), and equal amounts of each group were mixed.

2) Is taken off line

peptide BEH C18 high performance liquid chromatography column (

3.5 μm,4.6mm × 15 mm), elution buffer B1 was 98.0% acetonitrile, 2.0% deionized water (ph 10.0), and the separation gradient is shown in table 2. The flow rate was 1mL/min and the separation was 46min.

3) Fractions were collected one tube at 1min intervals for a total of 40 fractions. According to 1, 11, 21, 31; 2. 12, 22, 32; 3. 13, 23, 33; 4. 14, 24, 34; the gradient sequence of … … combined the samples into 10-tube components.

4) Placing the collected components in a rotary vacuum drying instrument, vacuum drying, and freezing and storing at-20 ℃ for later use.

TABLE 2 high pH reverse phase chromatographic gradient

Ziptip C18 solid phase extraction

1) C18 solid phase extraction column activation: aspirate 100 μ L of 100% ACN, blow-beat clean, repeat twice.

2) C18 solid phase extraction column equilibrium: pipetting 10. Mu.L of 2% ACN 0.1% FA, washing the residual ACN in the extraction column, pipetting clean, and repeating twice.

3) Sampling: repeatedly sucking and blowing the enzyme-cut polypeptide solution, and repeating for more than 10 times.

4) Cleaning and desalting: pipetting 10 μ L of 2% ACN 0.1% FA, washing the salt in the sample, and repeating five times.

5) And (3) elution: sucking 10 μ L of 50% ACN 0.1% FA, repeatedly blowing 10 times, and collecting the eluate with an EP tube. Repeating the steps again, and combining the eluates.

6) And (3) placing the collected eluent in a rotary vacuum drier, vacuumizing and drying the eluent under the condition of no heating, and storing the eluent in a refrigerator at the temperature of-80 ℃ for later use.

DDA for Signal acquisition

6.1LC-MS/MS scanning

1) The polypeptide fraction was redissolved in 20. Mu.L of 0.1% FA solution, and 2. Mu.L of iRT reagent was added to each fraction.

2) Separation by EASY-nLC liquid phase, low pH reverse phase C18 capillary chromatography (150 μm. Times.150mm, 1.9 μm), phase A99.9% ₂ O and 0.1% FA, B phase 99.9% ACN,0.1% FA, effective elution gradient 3% -35%, total elution time set to 90min, flow rate 0.5 μ L/min. The liquid phase conditions are shown in Table 3.

3) The polypeptide mixture was identified using the Orbitrap Fusion mass spectrometer analysis. Using the high sensitivity mode, the instrument parameters are set to: each full scan is a high-speed signal dependent scan, with a total scan time of 90min. The primary full scan resolution was 60000, the scan range was set to 300-1400m/z, AGC5e5, maximum injection time was 50ms, collision energy was 32%, the secondary scan resolution was 15000, charge state screening (precursors containing +2 to +6 charges), AGC5e 4, maximum injection time was 45ms.

TABLE 3 Low pH reverse phase chromatographic gradient

6.2 data analysis

1) The mass spectra data obtained were collected and retrieved by the Proteome discover (version 2.3.0.523) software, the database was: uniprot _ human _75778_20201211_iRT. Fasta (total number of sequences 73940).

2) The retrieval parameters are: the protein database of the corresponding species, tryptic cleavage, maximum 2 missed cleavage sites, mass errors of parent and fragment ions of 10ppm and 0.02Da respectively, fixed modification as Carbammidomethyl (C), variable modification as Oxidation (M), acetyl (N-terminal). The false positive rate of polypeptides and proteins was set at (FDR) <1.0%, and at least 1 specific polypeptide was identified per protein.

3) And (4) counting the size of the analysis window, wherein the window is set as follows:

TABLE 4DIA Window settings

DIA for signal acquisition

LC-MS/MS scanning

1) The quantified polypeptide was taken at 10. Mu.g/sample, redissolved in 20. Mu.L of 0.1% FA solution, and mixed with 2. Mu.LiRT reagent.

2) Separated by EASY-nLC liquid chromatography, low pH reverse phase C18 capillary chromatography (150 μm. Times.150mm, 1.9 μm) under the conditions described for "low pH reverse phase chromatographic gradient".

3) Modifying a mass spectrum variable window, wherein the scanning time is 90min, and the collision energy is as follows: 32 percent; the primary scanning resolution is set to 60000, and the secondary resolution is 30000; the maximum injection time of the parent ions is 50ms; parent ion scan range: 300-1300m/z; sub-ion scan range: starting from 300m/z; 35 scanning windows are set; the window size is determined according to the number of primary parent ions and is evenly distributed.

8. Searching and building storeroom

1) And searching the library by using Spectronaut Pulsar software based on the Raw file obtained by mass spectrum detection, importing the PD library searching result, and establishing a spectrogram library.

2) The DIA data was imported and matched to the data in the spectrogram library with q value <1.0%, with retention time corrected by iRT reagent. The overall flow of the DIA method is shown in fig. 1:

9. data quality control analysis

The chromatograph-mass spectrometer is precise in structure, and there are many factors that cause systematic errors in sample collection during use, such as: temperature, humidity, degree of cleanliness of the instrument, etc. Quality control is performed in two ways: and (4) carrying out statistics on sample parallelism quality control and Coefficient of Variation (CV) value intervals.

10. Bioinformatics analysis

10.1 differential protein screening

The selection of the different candidate proteins needs to be jointly judged according to the change multiple and the statistical significance. In data analysis, 2 groups of proteins with ratio ≦ (1-2 × SD) or ratio ≧ (1 +2 × SD) between samples, and P value <0.05 were selected as candidate difference proteins.

10.2 differential protein analysis

1) Venn analysis: and analyzing the difference proteins among the groups by using a Wien diagram, and visually displaying the difference proteins by comparing.

The above differential protein analysis was performed by Perseus (version: 1.5.5.1) software.

2) Functional analysis:

the differential expression protein was subjected to GO analysis using the Blast2GO functional annotation module of the OmicsBox (version: 1.3.11) software, and gene enrichment analysis was performed from the Biological Process (BP), molecular Function (MF), and cellular fraction (CC) of the protein.

Pathway analysis was performed for differential proteins using the KEGG (Kyoto Encyclopedia of Genes and Genomes) public database.

3. Results

1. Screening for differential proteins

The screening criteria were: fold Change is more than or equal to 1.4, P after correction is less than 0.05, three groups are compared with each other, and the statistical results of different proteins in each group are shown in Table 5.

TABLE 5 statistical results for differential proteins

In this study, not only are the different proteins present in different amounts in each group, but also different proteins are present in different groups or the same protein is present in different groups at the same time. We performed wien graph analysis on differential proteins between groups, and fig. 2A, 2B and 2C analyzed differential proteins from the perspective of EM, OC and FEM, respectively.

Among them, the inventors compared with the EM group as a control and found 4 overlapping differential proteins, i.e., proteins with protein IDs P14151, A0G2JMY9, P01834 and A0A3B3ISU0, and their corresponding protein names SELL (selectin), LILRA3 (Leukocyte Immunoglobulin-receptor subfamily a member 3, leukocyte Immunoglobulin-like receptor subfamily a member 3), IGKC (Immunoglobulin kappa constant, immunoglobulin light chain kappa) and DSC2 (Desmocollin-2, desmocollin 2), respectively.

2. Functional analysis of differential proteins

2.1 differential protein GO analysis

The results of GO enrichment analysis of EM and FEM differential proteins are shown in fig. 3.

It can be seen that the main focus is on positive regulation of random transport, response to arbitrary transport and regulation of random transport etc. in BP aspect, the main focus is on extrinsic component of organic transport, extrinsic component of gold transport and spatial side of gold transport etc. in CC aspect, and the main focus is on carboxydic ester hydrolases activity, lipase activity and glycoolic binding etc. in MF aspect.

2.2 differential protein KEGG analysis

The two groups of differential proteins of EM and FEM are respectively subjected to KEGG enrichment analysis, and mainly focus on metabolic pathways (such as various amino acid metabolic pathways) and PPAR signal pathways, AMPK signal pathways, cell Adhesion Molecule (CAMs) pathways and the like.

Example 2 protein marker validation

1. Experimental materials

1. Research sample

Serum samples of 203 hospitalized patients who were treated in Beijing coordination with the obstetrics and gynecology department of hospitals from 1 month 2019 to 1 month 2020 were collected for protein marker verification, wherein 88 patients in EM group, 90 patients in FEM group and 25 patients in OC group were obtained, and all the basic information of the included patients is shown in Table 6.

Inclusion criteria for samples:

inclusion criteria were: a. the ultrasonic result shows that the adnexal tumor or pelvic mass, suspicious EM or OC or unknown meaning; b. determined in Beijing in coordination with the operation treatment in hospital.

Exclusion criteria: a. combining other gynecological benign and malignant tumors; b. clinically suspected malignant tumors; c. other systemic malignancies or history of malignancies; d. huai Yunzhe.

TABLE 6 protein markers basic information for screening patient populations

IQR: a four-bit spacing;

#: endometriosis staging reference american birth society modified staging (r-AFS);

* : FIGO stage principle of ovarian cancer

2. Sample collection and storage

The same procedure as in example 1 was followed.

3. Experimental reagent

The human ovarian cancer antigen CA125 kit (product number: SEKH-0527), the human L-Selectin kit (L-Selectin, product number: SEKH-0231), the human leukocyte immunoglobulin-like receptor A3 kit (LILRA 3, product number: SEKH-0528) were purchased from Beijing, solarbio (Beijing Solarouba technologies, ltd.);

human immunoglobulin light chain kappa kit (kappa-IgKC) was purchased from shanghai yu biotechnology limited;

human Desmocollin-2 kit (Desmocollin-2, product No.: ELH-DSC 2) was purchased from RayBiotech, inc., USA.

4. Main instrument and consumable

An automatic plate washing machine (PW-960), shenzhen Shenhuijong (HEALES) science and technology development Limited;

microplate reader (Multiskan GO): U.S. Thermo corporation;

chemiluminescence imaging System (Fusion), france, vilber.

2. ELISA test method

1. Preparation step

(1) And (3) temperature return of the reagent: the kit and the sample to be tested are firstly placed at room temperature (18-25 ℃) 30min before the experiment, and the concentrated washing liquid is placed in a water bath kettle for dissolution at 37 ℃ if crystallization occurs.

(2) Preparing a washing solution: the usage volume of the diluted washing solution is calculated in advance, 20 times of the concentrated washing solution is diluted into 1 time of the application solution by distilled water, and the unused concentrated washing solution is stored in a refrigerator at 4 ℃.

(3) And (3) standard gradient dilution: adding 0.5ml of standard substance/sample diluent (SR 1) into the freeze-dried standard substance, standing for 15 minutes, mixing gently (with a concentration of 20 ng/ml) after the standard substance/sample diluent is completely dissolved, and then diluting by 2 times according to the following concentration: 10. 5, 2.5, 1.25, 0.625, 3.125 and 0ng/ml, wherein 20ng/ml is the highest concentration of the standard curve, and the standard substance/sample diluent (SR 1) is used as the zero point (0 pg/ml) of the standard curve. Subpackaging the redissolved stock solution (20 ng/ml) of the standard substance according to the dosage of one time, and storing in a refrigerator at the temperature of between 20 ℃ below zero and 80 ℃ below zero.

(4) Biotinylated antibody working solution: the amount required for the assay was pre-calculated, and 100 times the antibody concentrate was diluted to 1 time the working solution (mixed well before dilution) with the test diluent (SR 2) and added to the reaction wells within 30 minutes.

(5) Enzyme conjugate working solution: the formulations were prepared in the amounts required for each experiment, and 100-fold dilution of the concentrated enzyme conjugate with enzyme conjugate diluent (SR 3) to 1-fold dilution using working solution (centrifugation before dilution) was used within 30min.

(6) Washing: and (3) throwing off the liquid in the holes of the enzyme-labeled plate, patting the liquid on thick water-absorbent paper, adding 300 mu l/hole of washing liquid into a washing bottle, throwing off the liquid in the holes of the enzyme-labeled plate after standing for 30 seconds, patting the liquid on the thick water-absorbent paper, and washing the plate for 5 times.

2. Experimental procedure

(1) Sample adding: mu.l of the standard and the test sample were added to the reaction well, and the plate was sealed with a membrane and incubated at room temperature (25. + -. 2 ℃) for 120 minutes with shaking. Clapping and washing the plate 4 times.

(2) Adding an antibody: mu.l of biotinylated antibody working solution was added to each reaction well, the plate was sealed with a membrane, and incubated at room temperature (25. + -. 2 ℃) for 60 minutes with shaking. Clapping and washing the plate 4 times.

(3) Adding an enzyme: mu.l of the working solution of the enzyme conjugate was added to each reaction well, and the wells were sealed with a membrane and incubated at room temperature (25. + -. 2 ℃ C.) for 30 minutes with shaking. Clapping and washing the plate 5 times.

(4) Color development: mu.l of chromogenic substrate was added to each reaction well, and the plates were sealed with a membrane and incubated at room temperature (25. + -. 2 ℃) for 10 to 20 minutes with shaking.

(5) And (4) terminating: the reaction was terminated by adding 50. Mu.l of a stop solution to each reaction well.

(6) And (3) detection: the absorbance value (OD value) was measured at a wavelength of 450nm with a microplate reader within 5 minutes of the addition of the stop solution.

3. Determination of differential diagnosis efficiency

Retrieving and recording postoperative histopathological results of patients in a verification group, and comparing the expression conditions of different protein markers of three groups of patients. Drawing a receiver operating characteristic curve (ROC curve for short) by MedCalc software (19.4.1 version), calculating the Area (AUC), sensitivity and specificity under the curve, carrying out binary logistic regression analysis on the jointly detected data to obtain a probability predicted value of joint detection, and drawing the ROC curve by using the predicted value. AUC values were between 1.0 and 0.5. The closer the AUC is to 1, the better the diagnostic effect. AUC has lower accuracy at 0.5-0.7, AUC has certain accuracy at 0.7-0.9, and AUC has higher accuracy at above 0.9. AUC =0.5, indicating that the diagnostic method is completely ineffective and of no diagnostic value. P <0.05 was scored as statistically different.

3. Results

Analysis of Elisa test results

The target protein detected by the Elisa experiment is derived from the screening result of the protein marker in the previous part. 4 overlapping difference proteins (SELL, LILRA3, IGKC and DSC 2) and a clinical common marker CA125 by taking an EM group as a control. The proteins detected in this study were CA125, SELL, LILRA3, IGKC and DSC2.

Comparison of CA125, SELL, LILRA3, IGKC and DSC2 expression concentrations among three groups of EM, FEM and OC samples is shown in fig. 4. For CA125, OC group concentrations were significantly higher than EM group (P < 0.001), whereas EM group concentrations were significantly higher than FEM group (P = 0.002); similarly, for DSC2, OC group concentrations were significantly higher than EM group (P < 0.001), and EM group concentrations were also significantly higher than FEM group (P = 0.010); for IGKC, the difference between the EM and OC concentrations was significant (P = 0.044); for LILRA3, concentration differences between the three groups were not significant (EM group vs. oc group: P =0.704; for SELL, concentration differences between the three groups were also not significant (EM group vs. oc group: P = 0.216.

2. Overall diagnostic capability analysis

The performance of 5 different protein markers and combinations thereof for identifying EM/FEM is shown in table 7 and fig. 5-12.

TABLE 7 diagnostic capabilities of different protein markers and combinations thereof

2.1 ROC Curve analysis of Individual protein markers

For the EM and FEM groups, the inventors performed ROC curve analysis for 5 protein markers, and the results are shown in FIGS. 5-9. The AUC of CA125 differential diagnosis EM and FEM was maximal, followed by IGKC, with no significant difference in diagnostic ability (P = 0.785). LILRA 3is the least diagnostic.

2.2 ROC Curve analysis of protein marker combinations

For the EM and FEM groups, the inventors performed ROC curve analysis on the CA125+ SELL + LILRA3+ IGKC + DSC2, CA125+ IGKC protein marker combinations. The results are shown in fig. 10-12, where the 5 protein combinations differentially diagnosed the highest AUC for EM and FEM, followed by the CA125+ IGKC combination, with no significant difference in diagnostic ability (P = 0.158). The combination CA125+ IGKC + DSC2 had the weakest diagnostic ability.

2.3 layer analysis

2.3.1 stratification analysis by patient age

The inventor divides each patient group into 2 groups with age of 45 as boundary.

The diagnostic capabilities of the individual proteins as well as the combination of proteins for the EM and FEM groups are shown in table 2. The AUC for CA125 was relatively greater when the patient was <45 years of age (0.733), sensitivity 70.51%, specificity 70.97%, while the combination of protein markers did not significantly improve diagnosis;

when the patient is more than or equal to 45 years old, the AUC of the IGKC is 0.900, the sensitivity is 100 percent, and the specificity is 82.14 percent; the AUC for the CA125+ IGKC combination was greater (0.932), but the specificity was reduced. For patients > 45 years old in the EM and OC groups, the AUC for the CA125+ IGKC combination was greater (0.980), with high sensitivity (100%) and specificity (90%) (Table 8).

TABLE 8 expression of different protein markers between different age groups, EM and FEM groups

2.3.2 hierarchical analysis by tumor size

According to the clinical treatment experience, the inventor performs grouping by taking the maximum diameter of the tumor as a boundary, the OC group has fewer sub-groups, and no diagnosis analysis is performed. The diagnostic capabilities of the individual proteins as well as combinations of proteins for the EM and FEM groups of patients are shown in table 9. When the tumor diameter is less than 5cm, the AUC of CA125 is 0.854, the sensitivity is 71.43 percent, the specificity is 85.00 percent, and the combination of protein markers is not obviously improved; when the tumor diameter is more than or equal to 5cm, the CA125 performance is relatively optimal, the AUC is 0.751, the sensitivity is 73.33 percent, and the specificity is 72.86 percent.

TABLE 9 stratification by tumor size, expression of different protein markers

2.3.3 analysis of diagnostic Capacity after Limit conditions

When the EM and FEM groups of patients aged <45 years and with tumors of <5cm diameter were further selected for analysis, the diagnostic power of the individual proteins and combinations of proteins is shown in table 10, with the greater AUC (0.834) and the highest sensitivity but relatively lower specificity for the CA125+ IGKC combination.

TABLE 10 expression of different protein markers for years <45 years old and tumors <5cm, between EM and FEM groups

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (4)

1. Use of an agent for detecting the level of a biomarker comprising DSC2, IGKC, CA125+ SELL + LILRA3+ IGKC + DSC2, CA125+ IGKC + DSC2 or CA125+ IGKC in a sample from a subject in the manufacture of a product for the differential diagnosis of endometriosis of the ovary and other benign tumours of the ovary.

2. The use of claim 1, wherein the product comprises a kit, chip or strip.

3. The use according to claim 1, wherein the CA125, DSC2 and IGKC are expressed at elevated levels in a patient sample from endometriosis ovaries compared to a control sample; the SELL and LILRA3 are expressed at reduced levels in a sample from a patient with ovarian endometriosis; the control sample is an ovarian other benign tumor sample.

4. The use of claim 3, wherein the reagent comprises a mass spectrometric identification reagent, an antibody or antigen-binding fragment thereof, a primer or a probe.

Images