WO2017201748A1 - Method for detecting endometriosis and application thereof - Google Patents

Abstract

A method for detecting endometriosis. The method comprises: detecting and acquiring a measurement of the levels of serum superoxide dismutase 1 or human CD34 antigen, and comparing the measurement with a preset standard concentration of the serum superoxide dismutase or human CD34 antigen. The invention provides a supplementary standard for the diagnosis of endometriosis.

Description

Method for detecting endometriosis and its application [Technical Field]

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. Membrane ectopic detection method and detection kit.

【current technology】

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.

Among them, 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.

The American Society of Reproductive Medicine (ASRM) uses the severity of the disease to define endometriosis as a fourth grade, namely minimal endometriosis (stage1), mild endometriosis (mild). ;stageII), moderate endometriosis (moderate; stage III), And severe endometriosis (severe; stage IV).

In 1986, Barbieri first proposed that the cancer antigen 125 (CA-125) in patients with endometriosis would increase, and the subsequent application was to detect biochemical indicators of endometriosis. However, the sensitivity of CA-125 antigen for detecting endometriosis was only 18.6%, the specificity was only 19.10%, and the positive predictive value (PPV) was only 19.55%, negative. The negative predictive value (NPV) was 80.40%, and the CA-125 antigen in the serum of patients with early endometriosis was hardly elevated. Therefore, the clinical content of CA-125 antigen in serum can only be used as a biochemical indicator for the detection of advanced endometriosis (Stage III-IV). Mild or moderate endometriosis (stage I-II) can only be detected by invasiveness laparoscopy, lack of detection of mild or moderate endometrial abnormalities by serum samples. A convenient way of bits.

Therefore, it is necessary to find a biochemical indicator that can effectively detect early, mild, or moderate endometriosis. In addition, it is necessary to establish a method for detecting endometriosis using a serum sample, which has high sensitivity, high specificity, high positive predictive value, and high negative predictive value.

[Summary of the Invention]

It is an object of the present invention to provide a method for detecting early detection of early endometriosis.

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.

It is still another object of the present invention to provide a method for detecting endometriosis having a high positive predictive value and a high negative predictive value.

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.

It is still another object of the present invention to provide a method for detecting whether a patient's endometriosis is recurring.

It is a further object of the present invention to provide a test kit for detecting whether an individual is suffering from endometriosis.

The invention provides a method for detecting whether an individual has endometriosis, comprising the following steps:

(a1) obtaining a sample of the individual (Specimen), wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof;

(b1) detecting the content of superoxide dismutase 1 (SOD 1) in the sample;

(c1) when the content of superoxide dismutase 1 in the sample is greater than a first reference content, the individual is diagnosed with endometriosis;

Wherein, the first reference content is greater than or equal to 36 ng/mL.

According to the above invention, the individual is a human.

According to the above invention, in the step (c1), the first reference content is 40 ng/mL.

According to the above invention, in the step (c1), the first reference content is 58 ng/mL.

According to the above invention, in the step (c1), the first reference content is 86.45 ng/mL.

According to the above invention, the sensitivity of the detection method is greater than 65%, and the specificity is 100%.

According to the above invention, the positive predictive value (PPV) of the detection method is 100%, and the negative predictive value (NPV) is greater than 85%.

According to the above invention, in the step (b1), an Enzyme immunoassay (ELISA), a multiplex ELISA Array, a Western Blot, and 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.

The invention provides a method for detecting whether an individual has endometriosis, comprising the following steps:

(a2) obtaining a sample of the individual, wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof;

(b2) detecting the content of superoxide dismutase 1 (SOD 1) in the sample;

(c2) when the superoxide dismutase 1 content in the sample is greater than 86.45 ng/mL and less than or equal to 132 ng/mL, the individual is diagnosed with moderate endometriosis; The superoxide dismutase 1 content greater than 132 ng/mL was diagnosed as having severe endometriosis in the individual.

The present invention provides a detection method for detecting whether an endometriosis treatment for endometriosis has a therapeutic effect, comprising the following steps:

(a3) obtaining a first sample of the patient with endometriosis at a first time before the treatment, wherein the first sample is a serum sample, a plasma sample, and a blood test At least one or a combination of the bodies;

(b3) detecting the content of superoxide dismutase 1 (SOD 1) in the first sample;

(c3) waiting until the patient with endometriosis completes the course of endometriosis therapy;

(d3) obtaining a second sample of the endometriosis patient at a second time point, wherein the second sample is at least one of a serum sample, a plasma sample, and a blood sample One or a combination thereof;

(e3) detecting the content of superoxide dismutase 1 in the second sample;

When the content of superoxide dismutase 1 in the second sample is less than a second reference content, it is diagnosed that the endometriosis therapy is effective for the patient with endometriosis.

According to the above invention, preferably, the content of superoxide dismutase 1 in the second sample is smaller than the content of superoxide dismutase 1 in the first sample.

According to the above invention, in the step (e3), the second reference content is less than or equal to 132 ng/mL.

According to the above invention, in the step (e3), the second reference content is less than or equal to 78 ng/mL.

According to the above invention, in the step (b3) or (e3), an enzyme immunoassay (ELISA), 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 Flow cytometry, immunostaining (IHC), immunofluorescence (IF), immunocytochemistry (ICC), liquid chromatography tandem mass spectrometry (LC/ MS/MS), mass spectrometry (MS), high performance liquid chromatography (HPLC), or ultra-micro spectrophotometer (NanoDrop) detection of superoxide dismutase 1 in the sample The content.

According to the above invention, 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:

(a4) Waiting until the endometriosis treatment of the endometriosis patient completes the treatment of the endometriosis therapy, at the third time point, obtaining the third sample of the patient, wherein the third sample Is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof;

(b4) detecting the content of superoxide dismutase 1 (SOD 1) in the third sample;

(c4) after a number of days;

(d4) obtaining a fourth sample of the patient at a fourth time point, wherein the fourth sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof; as well as

(e4) detecting the content of superoxide dismutase 1 in the fourth sample;

When the content of superoxide dismutase 1 in the fourth sample is greater than a third reference content, it is diagnosed that the patient has recurrence of endometriosis.

According to the above invention, preferably, the content of superoxide dismutase 1 in the fourth sample is larger than the content of superoxide dismutase 1 in the third sample.

According to the above invention, in the step (e4), the third reference content is greater than or equal to 78 ng/mL.

According to the above invention, in the step (e4), the third reference content is greater than or equal to 132 ng/mL.

According to the above invention, the sensitivity of the detection method is greater than 50%, and the specificity is 90%.

According to the above invention, the positive predictive value (PPV) of the detection method is greater than 70%, and the negative predictive value (NPV) is greater than 75%.

According to the above invention, in the step (b4) or (e4), an enzyme immunoassay (ELISA), 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:

(A) obtaining a sample of the individual, wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof;

(B) detecting the content of a CD34 antigen (Hematopoietic progenitor cell antigen CD34; CD34antigen) in the sample;

(C) when the CD34 antigen content in the sample is greater than a CD34 antigen reference content, the individual is diagnosed with endometriosis;

Wherein, the CD34 antigen reference content is greater than or equal to 0.56 ng/mL.

According to the above invention, the individual is a human.

According to the above invention, in the step (C), the CD34 antigen reference content is 0.60 ng/mL.

According to the above invention, in the step (C), the CD34 antigen reference content is 1.23 ng/mL.

According to the above invention, in the step (C), the CD34 antigen reference content is 0.8023 ng/mL.

According to the above invention, the sensitivity of the detection method is greater than 70% and the specificity is 75%.

According to the above invention, the positive predictive value (PPV) of the detection method is greater than 85%, and the negative predictive value (NPV) is greater than 45%.

According to the above invention, in the step (B), an enzyme immunoassay (ELISA), a multiplex ELISA Array, a western blot, and a dot blot hybridization are used. ), 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.

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 more than one antibody of superoxide dismutase 1 is carried by covalent bonding, physical adsorption, or other means, and the vector is used to obtain the sample from the input position of the sample and analyze the sample The content of superoxide dismutase 1 in the medium;

a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis;

Wherein, when the content of superoxide dismutase 1 in the sample is greater than a reference content, the signal reading position indicates the first signal representative of endometriosis, when superoxide in the sample When the content of the dismutase 1 is less than or equal to the reference content, the signal reading position indicates that the second signal is not suffering from endometriosis;

Moreover, the baseline content is greater than or equal to 36 ng/mL.

According to the above invention, the reference content is 40 ng/mL.

According to the above invention, the reference content is 58 ng/mL.

According to the above invention, the reference content is 86.45 ng/mL.

According to the above invention, 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.

According to the above invention, 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. Content;

a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis;

Wherein, when the content of the CD34 antigen in the sample is greater than a reference content, 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;

Moreover, the reference content is greater than or equal to 0.56 ng/mL.

According to the above invention, the reference content is 1.23 ng/mL.

According to the above invention, the reference content is 0.8023 ng/mL.

According to the above invention, 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.

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.

[Description of the Drawings]

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).

Figure 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.

Figure 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.

Figure 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.

[Embodiment]

In this document, "a" refers to one or more than one (ie, "at least one") of the subject matter described herein. For example, "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.

All numerical values herein should be understood as modified by "approximately".

The following specific examples of the invention are merely illustrative of the invention and are not intended to limit the scope of the invention in any way.

Example 1: Acquisition of clinical specimens (clinical Specimen)

Under the supervision of the Taipei Medical University-Joint Institutional Review Board (TMU-JIRB), the clinical specimens required for the trial were obtained from 2013 to 2015. Among them, the trial is divided into three groups, including:

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. During laparoscopic surgery, a tissue sample is taken from the lesion location of the patient and blood is drawn to obtain a serum sample. Among them, 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. During laparoscopic surgery, a tissue sample is taken from the lesion location of the patient and blood is drawn to obtain a serum sample. Among them, 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.

Among them, according to ASRM classification criteria for endometriosis, moderate endometriosis (moderate; stage III) and severe endometriosis (severe; stage IV) are characterized by the formation of chocolate cysts and more serious dip Adhesion situation. In this study, patients in the GnRHa-treated group and the endometriosis group were moderately intimal (stage III) or severe endometriosis, and were treated with GnRHa and endometriosis. The tissue examination system obtained from the location of the lesion in the group of patients was taken from the tissue in the chocolate cyst.

Example 2: Analysis of gene expression in lesion tissues

RNA II Kit；厂牌为Macherey-Nagel，德国)抽取上述组织中的核醣核酸(RNA)，经反转录套组(reverse transcription kits；产品名称为

ⅢReverse Transcriptase；厂牌为Invitrogen，美国)将抽取所得的核醣核酸反转录为互补脱氧核醣核酸(complementary DNA,cDNA)后，再利用cDNA微数组分析法(cDNA microarray)分析上述人类子宫内膜组织中之超氧化物歧化酶1(SOD1)、CD34抗原(CD34)、以及人类谷胱甘肽S转移酶M4(GSTM4)之mRNA表现量。其中，所述cDNA微数组分析法系利用商业上可获得之人类基因表现微数组芯片(human gene expression microarray)进行分析，例如自威健股份有限公司(台湾)购买所述人类基因表现微数组芯片。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. First, use the RNA purification kit; the product name is

RNA II Kit; the label is Macherey-Nagel, Germany) extracts the ribonucleic acid (RNA) from the above tissues, and the reverse transcription kits (product name is

IIIReverse Transcriptase; The label is Invitrogen, USA) 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). Wherein, the cDNA microarray analysis method is analyzed by using a commercially available human gene expression microarray, for example, the human gene expression microarray chip is purchased from Weijian Co., Ltd. (Taiwan). .

Referring to Figure 1, 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. In Fig. 1, the left column diagram shows the orthodontic intima of the control group, and the right column shows the ectopic endometrium of the patients in the endometriosis group.

As can be seen from 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. Among them, in the ectopic endometrium of patients with endometriosis, 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.

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).

Example 3: Effect of GnRH agonist therapy on the expression of candidate genes

GnRH agonist (GnRHa) is an important drug used clinically to suppress endometriosis and relieve pain caused by endometriosis. It is known that gonadotropin-releasing hormone inhibitors can attenuate and reduce the lesion tissue of endometriosis patients with moderate endometriosis (stage III) or severe endometriosis (severe; stage IV). Achieve good therapeutic results. Therefore, 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).

Please refer to Figures 2A-4B. 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. In detail, the vertical axis of Fig. 2B is SOD1 and β-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. In detail, the vertical axis of Fig. 3B is CD34 and β- 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. The data of the lesions of four patients (ie, the lesions of patients treated with gonadotropin-releasing hormone inhibitors; codenamed CCGnRH(+) or Chocolate cyst cyst GnRH(+)) were randomly selected from the group. . 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. In detail, the vertical axis of Fig. 4B is the ratio of GSTM4 to GAPDH. .

2A and 2B, 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) The amount of superoxide dismutase 1 in the lesion sample 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.

As can be seen from Figures 3A and 3B, 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 CD34 antigen expression of the lesions was less and there was a significant difference between the two (p=0.036). That is, the amount of CD34 antigen expression in the lesion tissue was significantly reduced by gonadotropin-releasing factor inhibitor therapy.

4A and 4B, 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. Glutathione S-transferase M4 was more abundant in the lesions treated with the release factor inhibitor, and there was a significant difference between the two (p=0.041). That is, the amount of glutathione S transferase M4 in the lesion tissue was significantly increased by the gonadotropin releasing hormone inhibitor therapy.

According to the experience of the inventors, changes in the expression levels of various proteins in tissues are not necessarily absolutely related to changes in the expression levels of various proteins in serum. Therefore, the inventors simultaneously evaluated whether the content of the above three proteins in the patient's serum was affected by atrophy and improvement of endometriotic lesion tissue.

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). Cusabio Cat#: CSB-EL002837HU), and the Human Glutathione S Transferase M4 Immunozyme Assay Kit (Human GSTM4 ELISA kit; cusbio Cat#: ELH-NGFR-001), But not limited to this. The contents of superoxide dismutase 1, CD34 antigen, and glutathione S transferase M4 in the serum samples were determined according to the instructions of each kit.

See Figures 5-7. 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. , GnRHa treatment group (ENDO-GnRH (+)), and control group (Non-ENDO). 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).

As can be seen from Fig. 5, superoxide dismutase 1 in the serum of patients with endometriosis (not treated with gonadotropin releasing hormone inhibitor) was compared with the level of superoxide dismutase 1 in the serum of the control group. The content was significantly higher (p<0.01). Accordingly, the inventors believe that the level of superoxide dismutase 1 in serum may be used as an indicator to detect whether an individual has endometriosis, but still needs to be evaluated by Receiver operating characteristic curve (ROC). Its possibility as an indicator.

In addition, 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. There are significant differences. That is, 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. In patients, the levels of superoxide dismutase 1 in the serum of the above three groups of individuals were found to increase in three steps.

Accordingly, the inventors believe that the level of superoxide dismutase 1 in serum may be used as the following indicators:

1. Distinguish between non-endometriosis individuals, patients with moderate endometriosis, and severe uterus Indicators of patients with endometriosis;

2. To test whether an endometriosis therapy has an effect on a patient with endometriosis;

3. An indicator for detecting whether a patient's endometriosis relapses.

Although the inventors initially thought that the content of superoxide dismutase 1 in serum may be used as the above index, it is still necessary to evaluate the possibility of being used as an index by the receiver operating characteristic curve (ROC).

As can be seen from Fig. 6, the antigen content in the serum of patients with endometriosis (not treated with gonadotropin-releasing hormone inhibitor) 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.

There was no significant difference in serum CD34 levels between the endometriosis group (treated with no gonadotropin-releasing hormone inhibitor) and the GnRHa-treated group (treated with gonadotropin-releasing hormone inhibitor).

As can be seen from Fig. 7, there was no significant difference in the serum glutathione S transferase M4 content between the control group and the endometriosis group (no treatment with the gonadotropin releasing hormone inhibitor).

However, 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.

Although the inventors initially thought that the content of glutathione S transferase M4 in serum may be used as the above index, it is still necessary to evaluate the possibility of being used as an index by the Receiver operating characteristic curve (ROC).

Example 4: Receiver operating characteristic curve (ROC) analysis

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).

The following is an example of the detection of endometriosis in this case. The principle of drawing the receiver's operating characteristic curve is briefly explained. If there are any deficiencies, please refer to the statistics textbook.

Using superoxide dismutase 1, CD34 antigen, or glutathione S transferase M4 as biochemical indicators, sequentially set different biochemical indicator concentrations as classification thresholds (or cut-off values) . When 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.

Among them, if 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.

If 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.

After classifying all the serum samples by a single threshold, 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. For example, after the nth threshold is used as the classification basis, all the serum samples are classified one by one, and the true positive ratio is TPRn, and the false positive ratio is FPRn, which obtains a coordinate point in the receiver operating characteristic curve ( FPRn, TPRn).

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.

Integrate the receiver operating characteristic curve to obtain the area under the receiver operating curve (AUC), and then evaluate the biochemical index content in the serum sample as the detection method, and obtain the correct detection result. Probability. Because the area is found in the 1x1 square, the AUC must be between 0 and 1.

The closer the AUC value is to one, the higher the correct rate is, and the closer to 0, the lower the correct rate. If the AUC value is 1, it means that there is at least one threshold, and the correct rate when detecting by the threshold is 100%. If 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.

Please refer to FIG. 8. 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.

As can be seen from Fig. 8, when serum superoxide dismutase 1 is used as a classification index for evaluating whether an individual has endometriosis, the area under the receiver operating characteristic curve (AUC) is 0.9048, and the p value is <0.001, the analysis result showing that the area under the receiver operating characteristic curve is 0.9048 is sufficient. Based on the analysis results of AUC greater than 0.5 and close to 1, the serum superoxide dismutase 1 content is indeed suitable as an indicator for detecting whether an individual has endometriosis.

Therefore, we further analyze the preferred threshold and the sensitivity, specificity, positive predictive value (PPV), and Negative predictive value (NPV).

Please refer to Table 1. The results of the analysis of the threshold values of superoxide dismutase 1, CD34 antigen, or glutathione S transferase M4 in serum as a threshold for detecting endometriosis.

As can be seen from Table 1, 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. Using 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.

As can be seen from Table 1, 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. Using 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.

Preferably, the reference concentration (threshold) 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. Preferably, the baseline concentration is 40 ng/mL. Preferably, the baseline concentration is 58 ng/mL. Preferably, the baseline concentration is 86.45 ng/mL. Preferably, the baseline concentration is between 36 and 159.5334 ng/mL.

Please refer to FIG. 9. 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.

The inventors believe that 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.

As can be seen from Fig. 9, when serum superoxide dismutase 1 is used as a classification index for evaluating whether an individual has recurrent endometriosis, 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.

Therefore, we further analyze the preferred threshold and the sensitivity, specificity, positive predictive value (PPV), and Negative predictive value (NPV).

See Table 2 for the analysis of the threshold values of superoxide dismutase 1, CD34 antigen, or glutathione S transferase M4 in serum as a threshold for detecting recurrence of endometriosis.

As can be seen from Table 2, 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. Using 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.

As can be seen from Table 2, 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. Using this threshold to detect recurrence of endometriosis, the sensitivity was 71.43%, the specificity was 70%, and the positive predictive value (PPV) was 53.1355%. Negative predictive value. (negative predictive value; NPV) is 83.727266%. 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.

Please refer to FIG. 10. 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.

As can be seen from Fig. 10, when the CD34 antigen in serum is used as a classification index for evaluating whether an individual has endometriosis, 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. Based on the analysis results of AUC greater than 0.5 and close to 1, the CD34 content in serum is indeed suitable as an indicator for detecting whether an individual has endometriosis.

Therefore, we further analyze the preferred threshold and the sensitivity, specificity, positive predictive value (PPV), and Negative predictive value (NPV).

As can be seen from Table 1, 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. Using this threshold to detect whether or not suffering from endometriosis, the sensitivity is 71.43%, the specificity is 75%, and the positive predictive value (PPV) is 87.41282%, and the negative predictive value (negative predictive value; NPV) is 45.653395%. That is, the use of this threshold to detect whether or not suffering from endometriosis has the advantages of high sensitivity, high specificity, and high positive 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. . Using this threshold to detect whether or not suffering from endometriosis, the sensitivity is 42.86%, the specificity is 83.33%, and the positive predictive value (PPV) is 88.23392%, and the negative predictive value (negative predictive value; NPV) is 33.333538%. That is, the use of this threshold to detect whether or not suffering from endometriosis also has the advantage of high specificity and high positive predictive value.

Preferably, the reference concentration (threshold) 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. Preferably, the baseline concentration is 0.60 ng/mL. Preferably, the baseline concentration is 1.23 ng/mL. Preferably, the baseline concentration is 0.8023 ng/mL. Preferably, the reference concentration is between 0.3666374 and 2.051715 ng/mL.

Please refer to FIG. 11. 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. .

The inventors believe that 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.

As can be seen from Fig. 11, when the serum glutathione S transferase M4 is used as a classification index for evaluating whether the individual has recurrent endometriosis, the area under the receiver operating characteristic curve (AUC) is 0.6418, but The p value was 0.137 (>0.05), indicating that the analysis of the area under the receiver operating characteristic curve of 0.6418 was not sufficient.

This result shows that although Figure 7 shows a significant difference in glutathione S transferase M4 content in serum samples between the endometriosis group and the GnRHa treatment group, it is operated by the recipient. After the characteristic curve analysis, the content of glutathione S transferase M4 in the serum sample was not suitable as a result of the evaluation index. There is no inevitable connection between the statistical difference in the display experiment and the evaluation index.

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.

In addition, 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. 9, it is proved that the content of superoxide dismutase 1 in the serum can be used as a test for which kind of severity the above-mentioned individuals belong to (non-endometriosis patients, The basis for treatment of patients with endometriosis who have reduced severity, or patients with severe endometriosis.

It can be seen that the content of superoxide dismutase 1 in serum can be used as at least three indicators:

1. Differentiate between non-endometriosis individuals, patients with moderate endometriosis, and patients with severe endometriosis. When the content of superoxide dismutase 1 in the serum sample is less than or equal to 86.45 ng/mL, it is diagnosed as a non-endometriosis individual; when the serum sample has a superoxide dismutase 1 content greater than 86.45 ng / When the mL is less than or equal to 132 ng/mL, the patient is diagnosed with moderate endometriosis; when the serum superoxide dismutase 1 content is greater than 132 ng/mL, the diagnosis of severe endometriosis is diagnosed. Patients with symptoms.

Preferably, the moderate endometriosis patient refers to a patient with endometriosis whose degree of endometriosis is less than or equal to moderate endometrial dysplasia. Preferably, the patient with moderate endometriosis refers to a mild endometriosis endometriosis patient or a mild endometriosis (mild) endometriosis Patients with symptoms.

2. To test whether an endometriosis therapy has an effect on a patient with endometriosis. 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. When the content of superoxide dismutase 1 in the second serum sample is less than the content of superoxide dismutase 1 in the first serum sample, and the content of superoxide dismutase 1 in the second serum sample is less than At a second baseline concentration, 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. Preferably, the second reference concentration is less than or equal to 78 ng/mL.

Among them, "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", and in 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. Preferably, after treatment, when the content of superoxide dismutase 1 in the serum sample of the patient is less than 132 ng/mL, the endometriosis therapy is diagnosed as having curative effect on the patient with endometriosis.

3. An indicator for detecting whether a patient's endometriosis relapses. 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. When the content of superoxide dismutase 1 in the fourth serum sample is greater than the content of superoxide dismutase 1 in the third serum sample, and the content of superoxide dismutase 1 in the fourth serum sample is greater than At a third baseline concentration, the patient is diagnosed with recurrence of endometriosis; wherein the third baseline concentration is greater than or equal to 78 ng/mL. Preferably, the third baseline concentration is greater than or equal to 132 ng/mL.

Preferably, 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.

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 advantage of the predicted value. Therefore, the serum CD34 antigen content is a good indicator for detecting whether an individual has endometriosis, and the detection effect is better than the currently used CA-125 detection method.

According to various embodiments of the present invention, 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 vector on which more than one antibody of superoxide dismutase 1 is carried by covalent bonding, physical adsorption, or other means, and the vector is used to obtain the sample from the input position of the sample and analyze the sample The content of superoxide dismutase 1 in the medium;

a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis;

Wherein, when the content of superoxide dismutase 1 in the sample is greater than or equal to a reference concentration, the signal reading position indicates the first signal representing endometriosis, when the sample is in the sample When the content of superoxide dismutase 1 is less than the reference concentration, the signal reading position indicates that the second signal is not suffering from endometriosis;

Moreover, the reference concentration is greater than or equal to 36 ng/mL.

Preferably, the reference concentration is 40 ng/mL. Preferably, the reference concentration is 58 ng/mL. Preferably, the reference concentration is 86.45 ng/mL.

Preferably, 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. Group, 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.

Preferably, 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.

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. Content;

a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis;

Wherein, when the content of the CD34 antigen in the sample is greater than or equal to a reference concentration, 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;

Moreover, the reference concentration is greater than or equal to 0.56 ng/mL.

Preferably, the reference concentration is 1.23 ng/mL. Preferably, the reference concentration is 0.8023 ng/mL.

Preferably, 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. Group, 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.

Although specific aspects of the invention have been described and illustrated, such aspects should be considered merely as The exemplification of the invention is not intended to limit the scope of the invention. All of the publications and patent applications cited in this specification are hereby incorporated by reference in their entirety in their entirety in their entirety in the the the the the the the Although the foregoing invention has been described in some detail, by way of illustration and example embodiments, the embodiments of the invention Certain changes and modifications may be made in accordance with the teachings of the invention.

Table 1. Analysis of the use of superoxide dismutase 1, CD34 antigen, or glutathione S transferase M4 in serum as a threshold for detecting whether or not there is endometriosis

Table 2. Analysis of the use of superoxide dismutase 1, CD34 antigen, or glutathione S transferase M4 in serum as a threshold for detecting the recurrence of endometriosis

Claims (40)

A method of detecting whether an individual has endometriosis comprises the following steps: (a1) obtaining a sample of the individual, wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof; (b1) detecting the content of superoxide dismutase 1 (SOD 1) in the sample; (c1) when the content of superoxide dismutase 1 in the sample is greater than a first reference concentration, the individual is diagnosed with endometriosis; Wherein, the first reference concentration is greater than or equal to 36 ng/mL. The method of claim 1 wherein the individual is a human. The method of claim 1, wherein in step (c1), the first reference concentration is 40 ng/mL. The method of claim 1 wherein in step (c1) the first reference concentration is 58 ng/mL. The method of claim 1, wherein in step (c1), the first reference concentration is 86.45 ng/mL. The method of claim 5, wherein the detection method has a sensitivity greater than 65% and a specificity of 100%. The method of claim 5, wherein the positive predictive value (PPV) of the detection method is 100% and the negative predictive value (NPV) is greater than 85%. The method according to claim 1, wherein in step (b1), an enzyme immunoassay (ELISA), a multiplex ELISA Array, a western blot, and a dot ink are used. Dot blot hybridization, protein microarray or protein chip, enzyme activity assay, flow cytometry, immunostaining (IHC), immunofluorescence staining (Immunofluorescence, IF), immunocytochemical chemistry (ICC), liquid chromatography tandem mass spectrometry (LC/MS/MS), mass spectrometry (MS), high performance liquid chromatography (high) Performance liquid chromatography (HPLC), or ultra-micro spectrophotometer (NanoDrop) was used to detect the content of superoxide dismutase 1 in the sample. A method of detecting whether an individual has endometriosis comprises the following steps: (a2) obtaining a sample of the individual, wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof; (b2) detecting the content of superoxide dismutase 1 (SOD 1) in the sample; (c2) when the superoxide dismutase 1 content in the sample is greater than 86.45 ng/mL and less than or equal to 132 ng/mL, the individual is diagnosed with moderate endometriosis; The superoxide dismutase 1 content is greater than 132 ng/mL, and the individual is diagnosed with severe endometriosis; A test method for detecting whether an endometriosis therapy is effective for an endometriosis patient, comprising the following steps: (a3) obtaining a first sample of the patient with endometriosis at a first time before the treatment, wherein the first sample is a serum sample, a plasma sample, and a blood test At least one or a combination of the bodies; (b3) detecting the content of superoxide dismutase 1 (SOD 1) in the first sample; (c3) waiting until the patient with endometriosis completes the course of endometriosis therapy; (d3) obtaining a second sample of the endometriosis patient at a second time point, wherein the second sample is at least one of a serum sample, a plasma sample, and a blood sample One or a combination thereof; (e3) detecting the content of superoxide dismutase 1 in the second sample; When the content of superoxide dismutase 1 in the second sample is less than a second reference concentration, and the content of superoxide dismutase 1 in the second sample is smaller than the superoxide dismutase in the first sample 1 content, it is diagnosed that the endometriosis therapy has curative effect on patients with endometriosis. The method of claim 10, wherein in step (e3), the second reference concentration is less than or equal to 132 ng/mL. The method of claim 11, wherein in step (e3), the second reference concentration is less than or equal to 78 ng/mL. The method according to claim 10, wherein in step (b3) or (e3), an enzyme immunoassay (ELISA), a multiplex ELISA Array, and a western blot method are used. ), Dot blot hybridization, protein microarray or protein chip, enzyme activity assay Assay), flow cytometry, immunostaining (IHC), immunofluorescence (IF), immunocytochemistry (ICC), liquid chromatography tandem mass spectrometry Instrumentation (LC/MS/MS), mass spectrometry (MS), high performance liquid chromatography (HPLC), or ultra-micro spectrophotometer (NanoDrop) for detection of superoxide dismutase 1 The content in the sample. The method of claim 10, wherein the endometriosis therapy is at least one or a combination of a method of treating with a GnRH agonist and other endometriosis therapies. A test method for detecting whether a patient's endometriosis relapses includes the following steps: (a4) Waiting until the endometriosis treatment of the endometriosis patient completes the treatment of the endometriosis therapy, at the third time point, obtaining the third sample of the patient, wherein the third sample Is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof; (b4) detecting the content of superoxide dismutase 1 (SOD 1) in the third sample; (c4) after a number of days; (d4) obtaining a fourth sample of the patient at a fourth time point, wherein the fourth sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof; as well as (e4) detecting the content of superoxide dismutase 1 in the fourth sample; When the content of superoxide dismutase 1 in the fourth sample is greater than a third reference concentration, and the content of superoxide dismutase 1 in the fourth sample is greater than the superoxide disproportionation in the third sample The enzyme 1 content was diagnosed as a recurrence of endometriosis in the patient. The method according to claim 15, wherein in the step (e4), the third reference concentration is greater than or equal to 78 ng/mL. The method according to claim 16, wherein in the step (e4), the third reference concentration is greater than or equal to 132 ng/mL. The method of claim 17 wherein the detection method has a sensitivity greater than 50% and a specificity of 90%. The method of claim 17, the positive predictive value of the detection method (positive The predictive value (PPV) is greater than 70% and the negative predictive value (NPV) is greater than 75%. The method according to claim 15, wherein in step (b4) or (e4), an enzyme immunoassay (ELISA), a multiplex ELISA Array, and a western blot method are used. ), Dot blot hybridization, protein microarray or protein chip, enzyme activity assay, flow cytometry, immunochromatography (IHC), Immunofluorescence (IF), immunocytochemistry (ICC), liquid chromatography tandem mass spectrometry (LC/MS/MS), mass spectrometry (MS), high performance liquid layer The content of superoxide dismutase 1 in the sample is detected by a high performance liquid chromatography (HPLC) or an ultra-micro spectrophotometer (NanoDrop). A method of detecting whether an individual has endometriosis comprises the following steps: (A) obtaining a sample of the individual, wherein the sample is at least one of a serum sample, a plasma sample, and a blood sample, or a combination thereof; (B) detecting the content of a CD34 antigen (Hematopoietic progenitor cell antigen CD34; CD34antigen) in the sample; (C) when the CD34 antigen content in the sample is greater than a CD34 antigen reference concentration, the individual is diagnosed with endometriosis; Wherein, the CD34 antigen reference concentration is greater than or equal to 0.56 ng/mL. The method of claim 21 wherein the individual is a human. The method of claim 21, wherein the CD34 antigen reference concentration is 0.60 ng/mL in step (C). The method of claim 21, wherein the CD34 antigen reference concentration is 1.23 ng/mL in step (C). The method of claim 21, wherein the CD34 antigen reference concentration is 0.8023 ng/mL in step (C). The method of claim 25, wherein the detection method has a sensitivity greater than 70% and a specificity of 75%. The method of claim 25, the positive predictive value of the detection method (positive The predictive value (PPV) is greater than 85%, and the negative predictive value (NPV) is greater than 45%. The method according to claim 21, wherein in step (B), an enzyme immunoassay (ELISA), a multiplex ELISA Array, a western blot, and a dot ink are used. Dot blot hybridization, protein microarray or protein chip, enzyme activity assay, flow cytometry, immunostaining (IHC), immunofluorescence staining (Immunofluorescence, IF), immunocytochemical chemistry (ICC), liquid chromatography tandem mass spectrometry (LC/MS/MS), mass spectrometry (MS), high performance liquid chromatography (high) Performance liquid chromatography (HPLC), or ultra-micro spectrophotometer (NanoDrop) was used to detect the content of CD34 antigen in the sample. A test 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 more than one antibody of superoxide dismutase 1 is carried by covalent bonding, physical adsorption, or other means, and the vector is used to obtain the sample from the input position of the sample and analyze the sample The content of superoxide dismutase 1 in the medium; a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis; Wherein, when the content of superoxide dismutase 1 in the sample is greater than a reference concentration, the signal reading position indicates the first signal representing endometriosis, when superoxide in the sample When the content of the dismutase 1 is less than or equal to the reference concentration, the signal reading position indicates the second signal indicating that the endometriosis is not affected; Moreover, the reference concentration is greater than or equal to 36 ng/mL. The test kit of claim 29, wherein the reference concentration is 40 ng/mL. The test kit of claim 29, wherein the reference concentration is 58 ng/mL. The test kit of claim 29, wherein the reference concentration is 86.45 ng/mL. A detection kit according to claim 29, wherein 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, an in vitro diagnostic medical device, a test reagent, an enzyme immunoassay kit (ELISA) Kit), a clinical biochemical test platform, a protein array, or other test kit. The test kit according to claim 29, further comprising at least one of an antibody of CA-125 antigen and an antibody against CD34 antigen, and wherein the carrier analyzes the content of superoxide dismutase 1 in the sample In the middle, the content of CA-125 antigen or CD34 antigen in the sample is simultaneously analyzed. A test 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. Content; a signal reading position for displaying a first signal representative of one of endometriosis or a second signal representative of endometriosis; Wherein, when the content of the CD34 antigen in the sample is greater than a reference concentration, 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 concentration is equal to the reference concentration, the signal reading position indicates that the second signal is not suffering from endometriosis; Moreover, the reference concentration is greater than or equal to 0.56 ng/mL. A test kit according to claim 35, wherein the reference concentration is 1.23 ng/mL. The test kit of claim 35, wherein the reference concentration is 0.8023 ng/mL. The test kit according to claim 35, wherein the test kit is a biochip, a test strip, a test stick, a test disc, a biochemical analyzer, a test kit, an identification kit, an analysis A kit, an in vitro diagnostic medical device, a test reagent, an enzyme immunoassay kit (ELISA kit), a clinical biochemical test platform, a protein array, or other test kit. The use of an antibody to superoxide dismutase 1 is a test kit for the preparation of endometriosis. The use of an antibody against CD34 antigen is a test kit for the preparation of endometriosis.

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