WO2017201748A1 - Procédé de détection d'endométriose et son application - Google Patents

Procédé de détection d'endométriose et son application Download PDF

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WO2017201748A1
WO2017201748A1 PCT/CN2016/083719 CN2016083719W WO2017201748A1 WO 2017201748 A1 WO2017201748 A1 WO 2017201748A1 CN 2016083719 W CN2016083719 W CN 2016083719W WO 2017201748 A1 WO2017201748 A1 WO 2017201748A1
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sample
endometriosis
content
superoxide dismutase
antigen
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曾启瑞
陈郁雯
陈惠文
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the invention relates to a detection method and a detection kit for endometriosis, in particular to an intrauterine using a superoxide dismutase 1 (SOD1) or a CD34 antigen (CD34) as a biochemical marker.
  • SOD1 superoxide dismutase 1
  • CD34 CD34 antigen
  • Endometriosis is a gynaecological disease most commonly occurring in women of reproductive age. Ten to 15% of women worldwide suffer from endometriosis and 35 to 50% of menstrual pain. Infertile women suffer for it. Endometriosis, as its name suggests, is the growth of the endometrial glands (Gland) and interstitial cells (Stroma cells) that should have grown in the uterus outside the uterus, and with the normal endometrium. It has the same physiological type.
  • the human uterine wall can be divided into three layers, from the inside to the outside: Endometrium, Myometrium and Serous layer.
  • Ectopic endometrial cells are also affected by changes in physiological hormones, with periodic changes such as Proliferation, Degradation, Bleeding, and the like. If the lesion of the endometriosis occurs in the myometrium, it is called Adenomyosis; if the lesion of the endometriosis occurs in a location other than the uterus, For example, in the ovary or pelvic cavity, it is called endometriosis.
  • the lesion of endometriosis may occur on the surface of the ovary to form implants, invade the ovary to form a congested cyst called chocolate cyst. -filled cyst), which occurs in the Pelvic cavity and is called Pelvic endometriosis, or occurs elsewhere in the uterus.
  • ABR American Society of Reproductive Medicine
  • stage1 minimal endometriosis
  • stage II mild endometriosis
  • stage III moderate endometriosis
  • severe endometriosis severe; stage IV.
  • a second object of the present invention is to provide a method for detecting mild or moderate endometriosis.
  • Another object of the present invention is to provide a method for detecting endometriosis with high sensitivity and high specificity.
  • a further object of the present invention is to provide a method for detecting whether an endometriosis therapy has a therapeutic effect on a patient with endometriosis.
  • the invention provides a method for detecting whether an individual has endometriosis, comprising the following steps:
  • the first reference content is greater than or equal to 36 ng/mL.
  • the individual is a human.
  • the first reference content is 40 ng/mL.
  • the first reference content is 58 ng/mL.
  • the first reference content is 86.45 ng/mL.
  • the sensitivity of the detection method is greater than 65%, and the specificity is 100%.
  • the positive predictive value (PPV) of the detection method is 100%, and the negative predictive value (NPV) is greater than 85%.
  • an Enzyme immunoassay (ELISA), a multiplex ELISA Array, a Western Blot, and a Dot blot hybridization are used.
  • ELISA Enzyme immunoassay
  • a multiplex ELISA Array a Western Blot
  • a Dot blot hybridization are used.
  • protein microarray or protein chip enzyme activity assay, flow cytometry, immunohistochemistry (IHC), immunofluorescence (IF) , immunocytochemical chemistry (ICC), liquid chromatography tandem mass spectrometry (LC/MS/MS), mass spectrometry (MS), high performance liquid chromatography (HPLC) ), or an ultra-micro spectrophotometer (NanoDrop) to detect the content of superoxide dismutase 1 in the sample.
  • IHC immunohistochemistry
  • IF immunofluorescence
  • ICC immunocytochemical chemistry
  • LC/MS/MS liquid chromatography tandem mass spectrometry
  • MS mass spectrometry
  • HPLC
  • the invention provides a method for detecting whether an individual has endometriosis, comprising the following steps:
  • sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof;
  • the present invention provides a detection method for detecting whether an endometriosis treatment for endometriosis has a therapeutic effect, comprising the following steps:
  • the content of superoxide dismutase 1 in the second sample is smaller than the content of superoxide dismutase 1 in the first sample.
  • the second reference content is less than or equal to 132 ng/mL.
  • the second reference content is less than or equal to 78 ng/mL.
  • an enzyme immunoassay (ELISA), a multiplex ELISA Array, a western blot, a dot blot test are used.
  • ELISA enzyme immunoassay
  • a multiplex ELISA Array a western blot
  • a dot blot test are used.
  • IHC immunostaining
  • IF immunofluorescence
  • ICC immunocytochemistry
  • LC/ MS/MS liquid chromatography tandem mass spectrometry
  • MS mass spectrometry
  • HPLC high performance liquid chromatography
  • NanoDrop ultra-micro spectrophotometer
  • the endometriosis therapy is at least one or a combination of a method of treating with a GnRH agonist and other endometriosis therapies.
  • the invention provides a detection method for detecting whether a patient's endometriosis relapses, comprising the following steps:
  • the content of superoxide dismutase 1 in the fourth sample is larger than the content of superoxide dismutase 1 in the third sample.
  • the third reference content is greater than or equal to 78 ng/mL.
  • the third reference content is greater than or equal to 132 ng/mL.
  • the sensitivity of the detection method is greater than 50%, and the specificity is 90%.
  • the positive predictive value (PPV) of the detection method is greater than 70%, and the negative predictive value (NPV) is greater than 75%.
  • an enzyme immunoassay (ELISA), a multiplex ELISA Array, a western blot, a dot blot test are used.
  • ELISA enzyme immunoassay
  • a multiplex ELISA Array a western blot
  • a dot blot test are used.
  • Dot blot hybridization protein microarray or protein chip, enzyme activity assay, flow cytometry, immunostaining (IHC), immunofluorescence staining Immunofluorescence, IF), immunocytochemistry (ICC), liquid chromatography tandem mass spectrometry (LC/MS/MS), mass spectrometry (MS), high performance liquid chromatography (high performance liquid chromatography)
  • the liquid chromatography (HPLC) and ultra-micro spectrophotometer are (NanoDrop) to detect the content of superoxide dismutase 1 in the sample.
  • the invention provides a method for detecting whether an individual has endometriosis, comprising the following steps:
  • sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof;
  • CD34 antigen Hematopoietic progenitor cell antigen CD34; CD34antigen
  • the CD34 antigen reference content is greater than or equal to 0.56 ng/mL.
  • the individual is a human.
  • the CD34 antigen reference content is 0.60 ng/mL.
  • the CD34 antigen reference content is 1.23 ng/mL.
  • the CD34 antigen reference content is 0.8023 ng/mL.
  • the sensitivity of the detection method is greater than 70% and the specificity is 75%.
  • the positive predictive value (PPV) of the detection method is greater than 85%, and the negative predictive value (NPV) is greater than 45%.
  • an enzyme immunoassay (ELISA), a multiplex ELISA Array, a western blot, and a dot blot hybridization are used.
  • ELISA enzyme immunoassay
  • protein microarray Or protein chip enzyme activity assay
  • flow cytometry immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemical staining (immunocytochemistry) , ICC), liquid chromatography tandem mass spectrometer (LC/MS/MS), mass spectrometry (MS), high performance liquid chromatography (HPLC), or ultra-micro spectrophotometer (NanoDrop) detects the amount of CD34 antigen in the sample.
  • IHC immunohistochemistry
  • IF immunofluorescence
  • IF immunocytochemical staining
  • ICC liquid chromatography tandem mass spectrometer
  • MS mass spectrometry
  • HPLC high performance liquid chromatography
  • NanoDrop ultra-micro spectrophotometer
  • the invention provides a detection kit for detecting whether an individual has endometriosis, comprising:
  • a sample input position for the user to input a sample to the test kit wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample of the individual or Combination
  • a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis
  • the signal reading position indicates the first signal representative of endometriosis, when superoxide in the sample.
  • the signal reading position indicates that the second signal is not suffering from endometriosis
  • the baseline content is greater than or equal to 36 ng/mL.
  • the reference content is 40 ng/mL.
  • the reference content is 58 ng/mL.
  • the reference content is 86.45 ng/mL.
  • the detection kit is a biochip, a test strip, a test stick, a test disc, a biochemical analyzer, a test kit, an identification kit, an analysis kit, and an in vitro diagnostic. Medical equipment, a test reagent, an enzyme immunoassay kit (ELISA kit), a clinical biochemical test platform, a protein array, or other test kit.
  • ELISA kit enzyme immunoassay kit
  • the vector further comprises at least one of an antibody against a CA125 antigen and an antibody against a CD34 antigen, and the carrier analyzes the content of superoxide dismutase 1 in the sample.
  • the content of CA125 antigen or CD34 antigen in the sample is simultaneously analyzed.
  • the invention provides a detection kit for detecting whether an individual has endometriosis, comprising:
  • a sample input position for the user to input a sample to the test kit wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample of the individual or Combination
  • a vector on which an antibody against more than one CD34 antigen is covalently bound, physically adsorbed, or otherwise, and the vector is used to obtain the sample from the input position of the sample and analyze the CD34 antigen in the sample.
  • a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis
  • the signal reading position indicates the first signal representative of endometriosis, when the content of the CD34 antigen in the sample is less than or When the reference content is equal to the reference content, the signal reading position indicates the second signal representing the absence of endometriosis;
  • the reference content is greater than or equal to 0.56 ng/mL.
  • the reference content is 1.23 ng/mL.
  • the reference content is 0.8023 ng/mL.
  • the detection kit is a biochip, a test strip, a test stick, a test disc, a biochemical analyzer, a test kit, an identification kit, an analysis kit, and an in vitro diagnostic. Medical equipment, a test reagent, an enzyme immunoassay kit (ELISA kit), a clinical biochemical test platform, a protein array, or other test kit.
  • ELISA kit enzyme immunoassay kit
  • the present invention provides the use of an antibody against superoxide dismutase 1 as a detection kit for preparing endometriosis.
  • the present invention provides a use of an antibody against CD34 antigen, which is a kit for detecting endometriosis.
  • Figure 1 cDNA microarray colorimetric (cDNA) of messenger ribonucleic acid (mRNA) expression in superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 in endometrial tissue Microarray).
  • cDNA cDNA microarray colorimetric
  • FIG. 2A Protein electrophoresis analysis of the amount of superoxide dismutase 1 present in the lesion tissue.
  • Figure 2B Histogram of the expression of superoxide dismutase 1 (protein) in the lesion tissue.
  • FIG. 3A Protein electrophoresis analysis of CD34 antigen expression in lesion tissues.
  • Figure 3B Histogram of the amount of CD34 antigen (protein) expressed in the lesion tissue.
  • FIG. 4A Protein electrophoresis analysis of glutathione S transferase M4 expression in lesion tissues.
  • Figure 4B Histogram of glutathione S transferase M4 (protein) expression in lesion tissue.
  • Figure 5 Data plot of superoxide dismutase 1 (protein) content in serum of endometriosis, GnRHa treated, and control individuals.
  • Figure 6 Data plot of serum CD34 antigen (protein) levels in endometriosis, GnRHa treated, and control individuals.
  • Figure 7 Data plot of glutathione S transferase M4 (protein) content in serum of endometriosis, GnRHa treated, and control individuals.
  • Figure 8 Receiver operating characteristic curve obtained by classifying serum samples of patients with endometriosis and control group by using superoxide dismutase 1 in serum as a biochemical index.
  • Figure 9 Receiver operating characteristic curve obtained by classifying serum samples of patients with endometriosis and GnRHa treatment groups by using superoxide dismutase 1 in serum as a biochemical index.
  • Figure 10 Receiver operating characteristic curve obtained by classifying serum samples of endometriosis patients and control subjects with CD34 antigen in serum as a biochemical index.
  • Figure 11 Receiver operating characteristic curve obtained by classifying serum samples of patients with endometriosis and GnRHa treatment groups using serum glutathione S transferase M4 as a biochemical index.
  • a refers to one or more than one (ie, “at least one") of the subject matter described herein.
  • one patient with endometriosis means one patient with endometriosis or more than one patient with endometriosis.
  • “Individual” refers to a vertebrate suffering from endometriosis or a vertebrate that is considered to require treatment for endometriosis.
  • the individual includes a warm-blooded animal, such as a mammal, such as a primate, particularly a human.
  • Non-human primates are also the individuals referred to here.
  • Individuals include domesticated animals such as cats, dogs, etc., and also include livestock (livestock (eg, cattle, horses, pigs, sheep, goats) and laboratory animals (eg, mice, rabbits, rats, Gerbil, guinea pig, etc.). Thus, both veterinary use and medical preparations are included in the scope of this expectation.
  • Example 1 Acquisition of clinical specimens (clinical Specimen)
  • TMU-JIRB Taipei Medical University-Joint Institutional Review Board
  • Endometriosis group a total of 50 patients with moderate endometriosis (stage III) or severe endometriosis (severe; stage IV), and these patients did not undergo a gonadotropin-releasing hormone inhibitor (GnRH) Agonist; GnRHa) treatment.
  • GnRH gonadotropin-releasing hormone inhibitor
  • a tissue sample is taken from the lesion location of the patient and blood is drawn to obtain a serum sample.
  • the tissue specimen obtained from the location of the lesion in the endometriosis group is called the Ectopic endometrium of the endometriosis patient.
  • GnRHa treatment group a total of 50 patients with moderate endometriosis (stage III) or severe endometriosis (severe; stage IV) who had at least a gonadotropin-releasing hormone inhibitor before being administered surgery (GnRH agonist; GnRHa) treatment for one month.
  • GnRH agonist gonadotropin-releasing hormone inhibitor before being administered surgery
  • a tissue sample is taken from the lesion location of the patient and blood is drawn to obtain a serum sample.
  • the tissue specimen obtained from the lesion location of the GnRHa treatment group was called the Ectopic endometrium of the GnRHa treatment group.
  • Non-endometriosis group (hereinafter referred to as control group): A total of 50 individuals who have not suffered from endometriosis but have other benign diseases, such as uterine myoma. Obtained from the uterus of these individuals during laparoscopic surgery The endometrial tissue is examined and its blood is drawn to obtain a serum sample. Among them, the endometrial tissue sample obtained from the uterus of the control group is called the Eutopic endometrium of the control group.
  • the gene expression in the tissue of the lesion location of the endometriosis group (hereinafter referred to as the lesion tissue) and the endometrial tissue of the control group (hereinafter referred to as the control tissue) were analyzed.
  • the RNA purification kit the product name is RNA II Kit; the label is Macherey-Nagel, Germany
  • the reverse transcription of the extracted ribonucleic acid into complementary DNA (cDNA) and then analysis of the human endometrial tissue by cDNA microarray analysis (cDNA microarray) mRNA expression levels of superoxide dismutase 1 (SOD1), CD34 antigen (CD34), and human glutathione S transferase M4 (GSTM4).
  • SOD1 superoxide dismutase 1
  • CD34 CD34 antigen
  • Figure 1 is a cDNA microarray colorimetric map of the expression of messenger ribonucleic acid (mRNA) of superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 in endometrial tissue.
  • mRNA messenger ribonucleic acid
  • the left column diagram shows the orthodontic intima of the control group
  • the right column shows the ectopic endometrium of the patients in the endometriosis group.
  • Fig. 1 the mRNA levels of superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 in the orthodontic membrane of the control group, endometriosis patients In the ectopic endometrium, superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 The mRNA expression levels were relatively high.
  • the mRNA expression of superoxide dismutase 1 was 4.1 times that in the orthotopic intima; the ectopic endometrium of patients with endometriosis In the ectopic endometrium of the endometriosis group, the mRNA expression of glutathione S transferase M4 was positive intima. 3.5 times in the middle.
  • the inventors Based on the difference in the expression levels of superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 between the endometriosis group and the control group, the inventors selected the above three genes as candidate genes, and Conduct a series of experiments to assess whether the proteins expressed by these three genes can be used to detect whether an individual has endometriosis and to detect whether an endometriosis therapy has an endometriosis patient. Efficacy, an indicator for detecting whether a patient with endometriosis relapses (also referred to as a biochemical indicator in the present invention).
  • GnRHa GnRH agonist
  • the present invention utilizes a known therapeutic effect of a GnRH agonist to evaluate the amount of expression of the above three proteins in the lesion tissue after atrophy and improvement of the lesion tissue in patients with endometriosis Whether the content of the patient's serum will be linked to the atrophy and improvement of the lesion tissue; that is, whether the amount of the above three proteins in the lesion tissue and the serum content of the patient will be evaluated. Because of the atrophy and improvement of endometriotic lesions, it is affected.
  • Example 3-1 Effect of gonadotropin-releasing hormone inhibitor on protein expression in lesion tissues
  • Western blotting method was used to analyze the expression of superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 in the lesion tissues of GnRHa-treated and endometriotic patients.
  • the antibodies used include murine anti-human SOD1 monoclonal antibody (purchased from abcam), murine anti-human CD34 monoclonal antibody (monoclonal mouse) Anti-human CD34; purchased from abcam), and mouse anti-human glutathione S transferase M4 monoclonal antibody (monoclonal mouse anti human GSTM4 antibody; purchased from abnova).
  • 2A is a protein electrophoresis analysis of the expression of superoxide dismutase 1 in the lesion tissue, wherein columns 1 to 4 are lesion tissues of patients randomly selected from the endometriosis group (ie, no sex hormone release) The lesion tissue of the patient treated with the inhibitor; the data obtained by the analysis of CCGnRH(-) was analyzed, and the 5th to 8th columns were randomly selected from the GnRHa treatment group for the lesion tissue of the four patients (ie, The lesion tissue of a patient treated with a gonadotropin-releasing hormone inhibitor; coded as CCGnRH(+)) was obtained for analysis.
  • Figure 2B is a histogram of the amount of superoxide dismutase 1 (SOD1 protein) expressed in the lesion tissue.
  • the horizontal axis of Fig. 2B is the endometriosis group and the GnRHa treatment group from left to right; the vertical axis of Fig. 2B is the performance of SOD1.
  • the vertical axis of Fig. 2B is SOD1 and ⁇ -actin. The ratio.
  • Figure 3A is a diagram of protein electrophoresis analysis of the expression of CD34 antigen in lesion tissues, wherein the left column is a random selection of lesion tissue from a patient in the endometriosis group (ie, treatment with no gonadotropin releasing hormone inhibitor).
  • the lesion tissue of the patient the data obtained by analysis of the code CCGnRH (-) or Chocolate cyst cyst GnRH (-)
  • the right column is the random selection of the lesion tissue of a patient from the GnRHa treatment group (ie, The lesion tissue of a patient treated with a gonadotropin-releasing hormone inhibitor; coded as CCGnRH(+) or Chocolate cyst cyst GnRH(+)).
  • Figure 3B is a histogram of the amount of CD34 antigen (protein) expressed in the lesion tissue.
  • the horizontal axis of Fig. 3B is the endometriosis group and the GnRHa treatment group from left to right; the vertical axis of Fig. 3B is the expression of CD34 antigen.
  • the vertical axis of Fig. 3B is CD34 and ⁇ - The ratio of actin.
  • Fig. 4A is a diagram showing the protein electrophoresis analysis of the expression amount of glutathione S transferase M4 (GSTM4) in the lesion tissue, wherein columns 1 to 4 randomly select the lesion tissues of four patients from the endometriosis group ( That is, the lesion tissue of patients who have not been treated with a gonadotropin-releasing hormone inhibitor; the data obtained by analysis of the code CCGnRH(-) or Chocolate cyst cyst GnRH(-), and the columns 5-8 are treated with GnRHa.
  • GSTM4 glutathione S transferase M4
  • Figure 4B is a histogram of the amount of glutathione S transferase M4 (GSTM4 protein) expressed in the lesion tissue.
  • the horizontal axis of Fig. 4B is the endometriosis group and the GnRHa treatment group from left to right.
  • the vertical axis of Fig. 4B is the expression of GSTM4.
  • the vertical axis of Fig. 4B is the ratio of GSTM4 to GAPDH. .
  • the amount of superoxide dismutase 1 in the lesion specimen compared to the endometriosis group (treated with no gonadotropin releasing hormone inhibitor), GnRHa treatment group (gonadotropin releasing hormone) Inhibitor treatment) did not increase or decrease significantly. That is, the amount of superoxide dismutase 1 present in the lesion tissue is not affected by the treatment of the gonadotropin releasing hormone inhibitor.
  • the GnRHa-treated group (treated with a gonadotropin-releasing hormone inhibitor) compared to the endometriosis group (without treatment with a non-gonadotropin-releasing inhibitor) for the amount of CD34 antigen in the lesion.
  • the GnRHa treatment group (transgonadotropic hormone) was compared with the endometriosis group (without treatment with a non-gonadotropin-releasing inhibitor) for the amount of glutathione S-transferase M4.
  • Example 3-2 Effect of gonadotropin-releasing hormone inhibitor on serum protein content in patients with endometriosis
  • the levels of superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 in serum of endometriosis group, GnRHa treatment group, and control group were analyzed by immunoenzymatic assay (ELISA).
  • the commercial kit used in this example includes the human superoxide dismutase 1 immunoenzyme assay kit (Human SOD1 ELISA kit; ebiosience Cat#: CSB-EL025164HU), human CD34 antigen immunoenzyme assay kit (Human CD34 ELISA kit).
  • Figure 5 is a graph showing the levels of superoxide dismutase 1 in the serum of the endometriosis group, the GnRHa treatment group, and the control group, with the endometriosis group from left to right (ENDO-GnRH ( -)), GnRHa treatment group (ENDO-GnRH (+)), and control group (Non-ENDO).
  • Figure 6 is a data plot of serum CD34 antigen content in the endometriosis group, GnRHa treatment group, and control group, with the endometriosis group (ENDO-GnRH(-)) from left to right.
  • Figure 7 is a data plot of glutathione S-transferase M4 content in the serum of the endometriosis group, the GnRHa treatment group, and the control group, with the endometriosis group from left to right (ENDO- GnRH(-)), GnRHa treatment group (ENDO-GnRH(+)), and control group (Non-ENDO).
  • the GnRHa-treated group (treated with a gonadotropin-releasing hormone inhibitor) was compared with the serum of superoxide dismutase 1 in patients with endometriosis (not treated with gonadotropin-releasing hormone inhibitors).
  • the content of superoxide dismutase 1 in serum decreased significantly, which resulted in the content of superoxide dismutase 1 in the serum of GnRHa treatment group between control group and endometriosis group, and between the two groups.
  • the present embodiment divides the individual into non-endometriosis patients, endometriosis patients whose treatment is reduced in severity, and severe endometriosis according to the severity of endometriosis.
  • the levels of superoxide dismutase 1 in the serum of the above three groups of individuals were found to increase in three steps.
  • the level of superoxide dismutase 1 in serum may be used as the following indicators:
  • the antigen content in the serum of patients with endometriosis was significantly higher than that of the serum of the control group (p ⁇ 0.05). . Accordingly, the inventors believe that the CD34 content in serum may be used as an indicator to detect whether an individual has endometriosis, but it is still necessary to evaluate its potential as an indicator by the Receiver operating characteristic curve (ROC). Sex.
  • ROC Receiver operating characteristic curve
  • the serum glutathione S-transferase M4 content compared with the endometriosis group (treated with non-gonadotropin-releasing hormone inhibitor), the serum glutathione S-transferase M4 content, GnRHa treatment group (transgonadotropin-releasing hormone inhibitor treatment)
  • the content of glutathione S transferase M4 in the serum of patients increased significantly. Accordingly, the inventors believe that the serum glutathione S transferase M4 content may be used as a test for endometriosis therapy for a patient with endometriosis, and to detect a patient An indicator of whether or not endometriosis recurs.
  • Example 3-2 The data of each group in Example 3-2 was analyzed by the statistical software GraphPad Prism 6.01 (GraphPad Software, Inc) and MedCalc 15.2 (MedCalc Software bvba). Receiver operating characteristic curve (ROC).
  • biochemical indicator concentrations as classification thresholds (or cut-off values) .
  • the biochemical index content in the serum sample is higher than the threshold value, the serum sample is classified as positive; when the biochemical index content in the serum sample is lower than the threshold value, the serum sample is classified as negative.
  • the specimen of a patient with endometriosis is classified as positive, it is classified as a true positive classification result. If the specimen of an endometriotic patient is classified as negative, it is defined as a false negative classification result.
  • a specimen other than an endometriotic individual is classified as positive, it is defined as a false positive classification result. If the specimen of a non-endometriosis individual is classified as negative, it is defined as a true negative classification result.
  • the ratio of true positive classification results (TPR) and the proportion of false positive classification results (FPR) were counted.
  • the ratio of the false positive classification result (FPR) is taken as the abscissa, and the ratio of the true positive classification result (TPR) is taken as the ordinate, and one coordinate point in the receiver operating characteristic curve is obtained.
  • TPRn true positive classification results
  • FPRn false positive classification result
  • the coordinate points such as (FPR1, TPR1), (FPR2, TPR2), (FPR3, TPR3), ... (FPRn, TPRn) are sequentially drawn on the coordinate axes, and the receiver operation characteristic graph can be drawn.
  • AUC area under the receiver operating curve
  • the AUC value is 1
  • the correct rate when detecting by the threshold is 100%.
  • the AUC value is 0, it means that the correct rate of detection is 0%.
  • the detection method generally adopted by the market, the AUC is greater than 0.5.
  • FIG. 8 is a receiver operating characteristic curve obtained by classifying serum samples of patients with endometriosis and control groups by using superoxide dismutase 1 in serum as a biochemical index.
  • the content of superoxide dismutase 1 in serum was used as the best cut-off value for detecting endometriosis was 86.45229 ng/mL. That is, when the content of superoxide dismutase 1 in the individual serum sample exceeds 86.45229 ng/mL, the diagnosis is endometriosis, and if it is less than or equal to 86.45229 ng/mL, the diagnosis is that the uterus is not affected.
  • Membrane ectopic disease is used as the best cut-off value for detecting endometriosis.
  • this threshold to detect whether or not suffering from endometriosis, the sensitivity is 66.67%, the specificity is 100%, the positive predictive value (PPV) is 100%, and the negative predictive value (negative predictive value; NPV) is 86.302533%. That is, the use of this threshold to detect whether or not suffering from endometriosis has the advantages of high sensitivity, high specificity, high positive predictive value, and high negative predictive value.
  • the suboptimal threshold for the detection of whether or not suffering from endometriosis was 58.31347 ng/mL using the content of superoxide dismutase 1 in serum. That is, when the content of superoxide dismutase 1 in the individual serum sample exceeds 58.31347 ng/mL, the diagnosis is endometriosis, and if it is less than or equal to 58.31347 ng/mL, the diagnosis is that the uterus is not affected.
  • Membrane ectopic disease is 58.31347 ng/mL using the content of superoxide dismutase 1 in serum. That is, when the content of superoxide dismutase 1 in the individual serum sample exceeds 58.31347 ng/mL, the diagnosis is endometriosis, and if it is less than or equal to 58.31347 ng/mL, the diagnosis is that the uterus is not affected.
  • this threshold to detect the presence or absence of endometriosis, the sensitivity is 85.71%, specificity was 80%, positive predictive value (PPV) was 67.11303%, and negative predictive value (NPV) was 92.160831%. That is, the use of this threshold to detect whether or not suffering from endometriosis also has the advantages of high sensitivity, high specificity, high positive predictive value, and high negative predictive value.
  • the reference concentration is greater than or equal to 36 ng/mL when the amount of superoxide dismutase 1 in the serum is used as a test for endometriosis.
  • the baseline concentration is 40 ng/mL.
  • the baseline concentration is 58 ng/mL.
  • the baseline concentration is 86.45 ng/mL.
  • the baseline concentration is between 36 and 159.5334 ng/mL.
  • FIG. 9 is a receiver operating characteristic curve obtained by classifying serum samples of endometriosis patients and GnRHa treatment groups by using superoxide dismutase 1 in serum as a biochemical index.
  • Figure 9 is suitable as a basis for evaluating whether "the use of serum superoxide dismutase 1 as an indicator of recurrence of endometriosis" is appropriate.
  • the area under the receiver operating characteristic curve (AUC) is 0.695, and the p value is ⁇ 0.05, the analysis result showing an area under the receiver operating characteristic curve of 0.695 is sufficient. Based on the analysis of AUC greater than 0.5, the serum superoxide dismutase 1 content is indeed suitable for detecting whether the patient has recurrent endometriosis.
  • the optimal threshold for detecting the recurrence of endometriosis in patients by using superoxide dismutase 1 in serum is 132 ng/mL. That is, after treatment, the content of superoxide dismutase 1 in the patient's serum sample increases again, and when it exceeds 132 ng/mL, it is diagnosed as recurrence of endometriosis, if it is less than or equal to 132 ng/mL, Diagnostics Endometriosis did not recur.
  • this threshold to detect recurrence of endometriosis, the sensitivity is 52.38%, the specificity is 90%, and the positive predictive value (PPV) is 71.38188%, and the negative predictive value. (negative predictive value; NPV) was 79.874879%. That is, the use of this threshold to detect recurrence of endometriosis has the advantages of high sensitivity, high specificity, high positive predictive value, and high negative predictive value.
  • the suboptimal threshold for detecting whether or not the endometriosis of the patient is relapsed by using the superoxide dismutase 1 content in the serum is 78.06242 ng/mL. That is, after treatment, the content of superoxide dismutase 1 in the patient's serum sample increases again, and when it exceeds 78.06242 ng/mL, it is diagnosed as recurrence of endometriosis, if it is less than or equal to 78.06242 ng/mL. At the time, the diagnosis was that endometriosis did not recur.
  • FIG. 10 is a receiver operating characteristic curve obtained by classifying serum samples of patients with endometriosis and control groups by using CD34 antigen in serum as a biochemical index.
  • the area under the receiver operating characteristic curve (AUC) is 0.7286, and the p value is ⁇ 0.05.
  • the results of the analysis showing an area under this receiver operating characteristic curve of 0.7286 are sufficient to believe.
  • the CD34 content in serum is indeed suitable as an indicator for detecting whether an individual has endometriosis.
  • the optimal threshold value for using CD34 in serum as a test for endometriosis is 0.802379 ng/mL. That is, CD34 in individual serum samples When the antigen content exceeds 0.802379 ng/mL, it is diagnosed as suffering from endometriosis. If it is less than or equal to 0.802379 ng/mL, it is diagnosed as having no endometriosis.
  • the sensitivity is 71.43%
  • the specificity is 75%
  • the positive predictive value (PPV) is 87.41282%
  • the negative predictive value negative predictive value
  • NPV negative predictive value
  • the suboptimal threshold for the detection of endometriosis using serum CD34 antigen content was 1.232634 ng/mL. That is, when the content of CD34 antigen in the individual serum sample exceeds 1.232634 ng/mL, it is diagnosed as suffering from endometriosis, and if it is less than or equal to 1.232634 ng/mL, it is diagnosed as having no endometriosis. .
  • the sensitivity is 42.86%
  • the specificity is 83.33%
  • the positive predictive value (PPV) is 88.23392%
  • the negative predictive value negative predictive value
  • NPV negative predictive value
  • the reference concentration is greater than or equal to 0.56 ng/mL when the CD34 antigen content in the serum is used as a test for endometriosis.
  • the baseline concentration is 0.60 ng/mL.
  • the baseline concentration is 1.23 ng/mL.
  • the baseline concentration is 0.8023 ng/mL.
  • the reference concentration is between 0.3666374 and 2.051715 ng/mL.
  • FIG. 11 is a receiver operating characteristic curve obtained by classifying serum samples of endometriosis patients and GnRHa treatment groups by using glutathione S transferase M4 in serum as a biochemical index. .
  • Figure 11 is suitable as a basis for evaluating whether "the use of serum glutathione S-transferase M4 as an indicator of recurrence of endometriosis" is appropriate.
  • Example 3 of the present invention From the results of Example 3 of the present invention, it was found that the content of superoxide dismutase 1 in the serum of patients with endometriosis was significantly increased.
  • the receiver operating characteristic curve of Example 4 further indicates that superoxide dismutase 1 in serum can be used as a basis for detecting whether an individual has endometriosis, and has high sensitivity, high specificity, and high positive predictive value. And the advantages of high negative predictive values. Therefore, the content of superoxide dismutase 1 in serum is a good indicator for detecting whether an individual has endometriosis. The detection effect is better than the currently used CA-125 detection method.
  • the present invention divides an individual into a non-endometriosis patient, an endometriosis patient whose treatment is reduced in severity, and a severe endometriosis depending on the severity of endometriosis. Suffering, it was found that the content of superoxide dismutase 1 in the serum showed three steps in sequence according to the above three severity levels. Further, after analyzing the receiver operating characteristic curves of FIG. 8 and FIG.
  • the content of superoxide dismutase 1 in serum can be used as at least three indicators:
  • the moderate endometriosis patient refers to a patient with endometriosis whose degree of endometriosis is less than or equal to moderate endometrial dysplasia.
  • the patient with moderate endometriosis refers to a mild endometriosis endometriosis patient or a mild endometriosis (mild) endometriosis Patients with symptoms.
  • the endometriosis therapy uses a GnRH agonist At least one or combination of methods of treatment and other endometriosis therapies.
  • the detection method is: obtaining the serum sample of the patient (ie, the first serum sample) at the first time before the patient is administered the endometriosis therapy; detecting the super in the first serum sample The content of the oxide dismutase 1; waiting for the second time after the patient is administered the endometriosis therapy, the serum sample of the patient (ie, the second serum sample) is obtained; And detecting the superoxide dismutase 1 content in the second serum sample.
  • the endometriosis therapy is diagnosed as having a therapeutic effect on a patient with endometriosis; wherein the second baseline concentration is less than or equal to 132 ng/mL.
  • the second reference concentration is less than or equal to 78 ng/mL.
  • the content of superoxide dismutase 1 in serum can be used as an indicator of whether endometriosis therapy has an effect on a patient with endometriosis
  • the present invention it is also received by the recipient.
  • the operating characteristic curve analysis, and the analysis results are the same as the values of the superoxide dismutase 1 column in Table 2, the only difference is that the greater than (>) sign in the cut-off should be changed to less than ( ⁇ ) symbol. . That is, preferably, after treatment, when the content of superoxide dismutase 1 in the serum sample of the patient is less than 78.06242 ng/mL, the endometriosis therapy is diagnosed as endometriosis. Suffering from curative effect.
  • the endometriosis therapy is diagnosed as having curative effect on the patient with endometriosis.
  • the detection method is: obtaining a serum sample (ie, a third serum sample) of the patient at one time point (ie, the third time point) after the patient is administered the endometriosis treatment; Detecting the content of superoxide dismutase 1 in the third serum sample; at another time point after the plurality of days (ie, the fourth time point), obtaining the serum sample of the patient (ie, the fourth serum sample); The content of superoxide dismutase 1 in the fourth serum sample was detected.
  • a serum sample ie, a third serum sample
  • the detection method is: obtaining a serum sample (ie, a third serum sample) of the patient at one time point (ie, the third time point) after the patient is administered the endometriosis treatment; Detecting the content of superoxide dismutase 1 in the third serum sample; at another time point after the plurality of days (ie, the fourth time point), obtaining the serum sample of the patient (ie, the fourth serum sample); The
  • the patient is diagnosed with recurrence of endometriosis; wherein the third baseline concentration is greater than or equal to 78 ng/mL.
  • the third baseline concentration is greater than or equal to 132 ng/mL.
  • the plurality of days is, for example, one week, one month, one year, two years, three years, five years, or He is a few days old.
  • Example 4 From the results of Example 3 of the present invention, it was found that the serum CD34 antigen content in patients with endometriosis was significantly increased.
  • the receiver operating characteristic curve of Example 4 further indicates that the CD34 antigen in serum can be used as a basis for detecting whether an individual has endometriosis, and has high sensitivity, high specificity, high positive predictive value, and high negative.
  • the present invention provides a detection kit for detecting whether an individual has endometriosis, comprising:
  • a sample input position for the user to input a sample to the test kit wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample of the individual or Combination
  • a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis
  • the signal reading position indicates the first signal representing endometriosis, when the sample is in the sample
  • the signal reading position indicates that the second signal is not suffering from endometriosis
  • the reference concentration is greater than or equal to 36 ng/mL.
  • the reference concentration is 40 ng/mL.
  • the reference concentration is 58 ng/mL.
  • the reference concentration is 86.45 ng/mL.
  • the detection kit is a biochip, a test strip, a test stick, a test disc, a biochemical analyzer, a screening kit, a test kit, an authentication kit, and an analysis sleeve.
  • a biochip a biochip, a test strip, a test stick, a test disc, a biochemical analyzer, a screening kit, a test kit, an authentication kit, and an analysis sleeve.
  • an in vitro diagnostic medical device a model determination platform, a reagent test paper, a test reagent, an enzyme immunoassay reagent, an antiserum screening platform, a specific identification platform, a clinical biochemical test platform, a rapid test reagent platform , a protein array, or other test kit.
  • the vector further comprises at least one of an antibody of CA-125 antigen and an antibody of CD34 antigen, and the vector analyzes the content of superoxide dismutase 1 in the sample simultaneously.
  • the content of CA-125 antigen or CD34 antigen in the sample is particularly preferred.
  • the invention further provides another detection kit for detecting whether an individual has endometriosis, comprising:
  • a sample input position for the user to input a sample to the test kit wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample of the individual or Combination
  • a vector on which an antibody against more than one CD34 antigen is covalently bound, physically adsorbed, or otherwise, and the vector is used to obtain the sample from the input position of the sample and analyze the CD34 antigen in the sample.
  • a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis
  • the signal reading position indicates the first signal representing endometriosis, and the content of CD34 antigen in the sample When the reference concentration is less than the reference concentration, the signal reading position indicates that the second signal is not suffering from endometriosis;
  • the reference concentration is greater than or equal to 0.56 ng/mL.
  • the reference concentration is 1.23 ng/mL.
  • the reference concentration is 0.8023 ng/mL.
  • the detection kit is a biochip, a test strip, a test stick, a test disc, a biochemical analyzer, a screening kit, a test kit, an authentication kit, and an analysis sleeve.
  • a biochip a biochip, a test strip, a test stick, a test disc, a biochemical analyzer, a screening kit, a test kit, an authentication kit, and an analysis sleeve.
  • an in vitro diagnostic medical device a model determination platform, a reagent test paper, a test reagent, an enzyme immunoassay reagent, an antiserum screening platform, a specific identification platform, a clinical biochemical test platform, a rapid test reagent platform , a protein array, or other test kit.
  • the invention further provides the use of an antibody against superoxide dismutase 1 as a detection kit for preparing endometriosis.
  • the invention further provides the use of an antibody against CD34 antigen, which is a detection kit for preparing endometriosis.

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Abstract

L'invention concerne un procédé de détection de l'endométriose. Le procédé comprend : la détection et l'acquisition d'une mesure des taux de superoxyde dismutase 1 sérique ou d'antigène CD34 humain, et la comparaison de la mesure avec une concentration standard prédéfinie de la superoxyde dismutase sérique ou de l'antigène CD34 humain. L'invention fournit un étalon supplémentaire servant au diagnostic de l'endométriose.
PCT/CN2016/083719 2016-05-27 2016-05-27 Procédé de détection d'endométriose et son application Ceased WO2017201748A1 (fr)

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Cited By (1)

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