WO2019033866A1 - 肿瘤血液标志物及其应用 - Google Patents

肿瘤血液标志物及其应用 Download PDF

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WO2019033866A1
WO2019033866A1 PCT/CN2018/094401 CN2018094401W WO2019033866A1 WO 2019033866 A1 WO2019033866 A1 WO 2019033866A1 CN 2018094401 W CN2018094401 W CN 2018094401W WO 2019033866 A1 WO2019033866 A1 WO 2019033866A1
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gapdh
tumor
cancer
detecting
blood sample
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French (fr)
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王泽宋
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山东泽济生物科技有限公司
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Publication of WO2019033866A1 publication Critical patent/WO2019033866A1/zh

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90203—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the invention relates to a tumor blood marker and an application thereof, in particular to the diagnosis and prognosis of various tumors such as liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer and colorectal cancer by detecting the content of GAPDH in human blood. Evaluation and efficacy monitoring techniques.
  • the marker GAPDH of the present invention can be used for, but not limited to, tumor screening, risk assessment of tumors in a subject, differentiation of stages of tumor progression, identification of therapeutic effects of tumors, and risk analysis of tumor progression.
  • Malignant tumors are common and frequently-occurring diseases in the world and are one of the major diseases that endanger human health.
  • China there were about 4.292 million new cases of cancer in 2015, an average of 12,000 cases per day.
  • lung and bronchial cancer, gastric cancer, liver cancer, esophageal cancer, and colorectal cancer account for three-quarters of all cancer deaths (CA Cancer J Clin. 2016; 66: 115-132).
  • the incidence of malignant tumors in China has been continuously high in recent years and has become the leading cause of death among domestic residents.
  • Glyceraldehyde 3-phosphate dehydrogenase is an important enzyme in the glycolysis process with a molecular weight of 37kDa, catalyzing the glyceraldehyde 3-phosphate to 1,3-diphosphate. D-glycerate 1,3-bisphosphate.
  • GAPDH is also involved in many non-metabolic regulatory processes, including transcriptional activation (Oncogene. 2007; 26(18): 2606–20).
  • GAPDH GAPDH plays an important role in the glycolysis process, and its anti-apoptotic function is also important for the proliferation and protection of tumor cells.
  • GAPDH can protect telomere shortening caused by the action of chemotherapy drugs.
  • the conditions such as oxidative stress destroy the function of GAPDH, the cells will age or die (Clinical and Experimental Pharmacology & Physiology.
  • the inventors of the present study found that the expression level of GAPDH in blood (serum and/or plasma) can be used for the diagnosis and staging of various tumors such as liver cancer, lung cancer, breast cancer, gastric cancer and colorectal cancer, as well as the monitoring of tumor patients. And efficacy evaluation.
  • the present invention provides the use of GAPDH as a tumor blood marker.
  • the invention provides reagents for detecting GAPDH in a blood sample for preparing for tumor screening, risk assessment of a tumor in a subject, differentiation of tumor progression stages, identification of tumor treatment efficacy, and/or risk analysis of tumor progression.
  • the application of the test composition is reagents for detecting GAPDH in a blood sample for preparing for tumor screening, risk assessment of a tumor in a subject, differentiation of tumor progression stages, identification of tumor treatment efficacy, and/or risk analysis of tumor progression.
  • the blood sample is a serum or plasma sample.
  • the tumor comprises liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma or thyroid tumor.
  • the reagent for detecting GAPDH in a blood sample comprises an agent for detecting a concentration of GAPDH in a blood sample by the following method: a protein immunoblotting method, an enzyme-linked immunosorbent sandwich method.
  • the reagent for detecting GAPDH in a blood sample comprises: an antibody that specifically binds to GAPDH or a polypeptide fragment thereof.
  • the GAPDH has the amino acid sequence set forth in SEQ ID No. 1, and the polypeptide fragment of the GAPDH comprises the amino acid sequence 40-160 of the amino acid sequence of the GAPDH ( That is, a polypeptide fragment consisting of amino acids 40-160 of SEQ ID No. 1 or a polypeptide fragment consisting of amino acids 180-335 (i.e., amino acids 180-335 of SEQ ID No. 1).
  • GAPDH is a combined diagnosis with at least one other tumor marker.
  • performing tumor screening, risk assessment of tumors in a subject, differentiation of tumor progression stages, identification of therapeutic effects of tumor treatment, and/or risk analysis of tumor progression including detection of GAPDH in blood samples and at least one other tumor a marker, the at least one other tumor marker including but not limited to: AFP, CEA, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, AFU, SF, POA, TSGF.
  • the present invention provides a kit for use in tumor screening, risk assessment of a tumor in a subject, differentiation of a stage of tumor progression, identification of a therapeutic effect of a tumor, and/or risk analysis of tumor progression. It includes: a reagent for detecting GAPDH in a blood sample.
  • the reagent for detecting GAPDH in a blood sample includes an agent for detecting a concentration of GAPDH in a blood sample by the following method: protein immunoblotting method, enzyme-linked immunosorbent method, luminescence Immunoassay or colloidal gold method.
  • the reagent for detecting GAPDH in a blood sample comprises: an antibody that specifically binds to GAPDH or a polypeptide fragment thereof; preferably, the polypeptide fragment of the GAPDH includes GAPDH A polypeptide fragment consisting of amino acids 40-160 of the N-terminus of the amino acid sequence, or a polypeptide fragment consisting of amino acids 180-335.
  • the above kit of the present invention further comprises: a 96-well enzyme label, a standard dilution, a sample dilution, a wash concentrate, a color developing solution, and a stop solution.
  • the kit of the present invention comprises: a human GAPDH protein (humanized protein as a standard protein in a kit), a monoclonal antibody against human GAPDH protein, and a related reagent (96) Pore enzyme plate, standard dilution, sample dilution, wash concentrate, color solution and stop solution).
  • the method further includes a specification for labeling the detection parameter and the related judgment reference value, for example, the concentration of the standard product, the dilution factor of the sample, the monitoring range of the standard curve, the accuracy, the minimum detection limit, and the components in the reaction system.
  • the amount, the reaction temperature, the time, and the like, the judgment reference values are, for example, reference values for different tumors and the like.
  • the invention also provides a method of determining whether a subject has or is at risk of developing a cancer, the method comprising:
  • the reference value for determining liver cancer is 5.95 ⁇ g/ml (may also be rounded to 6 ⁇ g/ml), that is, the concentration of GAPDH in the blood sample of the subject is in the range of 0-6 ⁇ g/ml.
  • the time is considered to be a normal value, and if the concentration of GAPDH in the blood sample of the subject is higher than or equal to 6 ⁇ g/ml, the subject is judged to have a tumor or is at risk of developing cancer.
  • the increase or decrease of 25% is regarded as having the same judgment meaning, that is, the determination reference value for the liver cancer is 6 ⁇ g/ml, when the detection result is greater than or When it is equal to 4.5 ⁇ g/ml, the subject may also be judged to have a tumor or have a cancer risk.
  • the invention also provides a classification for distinguishing stages of tumor progression, the method comprising:
  • the present invention also provides a method for monitoring a condition of a tumor patient, the method comprising:
  • the reference value may be selected from 10-50%. In a specific embodiment of the invention, the reference value is 30%. Moreover, the reference value of ⁇ 25% in the present invention has the same judgment meaning, that is, when the reference value is 30% and the concentration of GAPDH in the blood sample of the tumor patient is decreased by 22.5%, it can be judged that the disease progresses well.
  • the “period” may be set according to the patient's condition, and "a treatment” includes daily treatment, one-cycle treatment, and/or multi-cycle treatment.
  • the present invention also provides a method for evaluating a therapeutic effect on a tumor patient, the method comprising:
  • the concentration of GAPDH in the serum or plasma sample before and after treatment of the subject such as the concentration of GAPDH in the sample after treatment is lower than before treatment, and the percentage of decrease reaches or exceeds the set reference value.
  • the treatment method or the drug is beneficial for the relief of the disease; if the concentration of the sample GAPDH is increased after treatment, there is no significant difference, or the change range does not reach or exceed the set reference value, the therapeutic method or the drug is judged to be inferior.
  • the reference value may be selected from 10-50%.
  • the concentration of GAPDH in the serum of a lung cancer patient before treatment is 22.63 ⁇ g/ml, and the concentration after treatment is decreased to 20.41 ⁇ g/ml, 9.81%, and 10% after rounding;
  • the concentration of GAPDH in the serum of a lung cancer patient before treatment is 46.51 ⁇ g/ml, and after treatment, the concentration is decreased to 24.20 ⁇ g/ml, which is decreased by 47.97%, and is 50% after rounding.
  • the reference value is 30%.
  • the reference value ⁇ 25% has the same judgment meaning, that is, when the reference value is 30%, and the concentration of GAPDH in the blood sample of the tumor patient is decreased by 22.5%, the therapeutic effect is also determined to be good.
  • the “period” may be set according to the patient's condition, and "a treatment” includes daily treatment, one-cycle treatment, and/or multi-cycle treatment.
  • the invention also provides a method of determining whether a subject has or is at risk of developing cancer by a joint diagnosis, the method comprising:
  • the subject was identified as a tumor patient or was at risk of developing a tumor.
  • the concentration reference value of the GAPDH is 5.57-11.75 ⁇ g/ml, and the reference value ⁇ 25% has the same judgment significance.
  • the invention also provides a method of classifying a stage of tumor progression by joint diagnosis, the method comprising:
  • the present invention also provides a method for monitoring a tumor patient by joint diagnosis, the method comprising:
  • the concentration of GAPDH and at least one other tumor marker in the serum or plasma sample at the beginning and end of the subject for a period of time, such as when the concentration of the sample GAPDH and at least one other tumor marker is lower than the beginning, and If the percentage reduction reaches or exceeds the set reference value, the condition is relieved; if the concentration of the sample GAPDH and at least one other tumor marker is increased at the end, there is no significant difference, or the change range does not reach or exceed the set value.
  • the reference value determines that the condition has not been alleviated.
  • the present invention also provides a method for evaluating a therapeutic effect of a tumor patient by joint diagnosis, the method comprising:
  • the treatment method or drug is beneficial for the disease to be relieved; for example, the concentration of the sample GAPDH and at least one other tumor marker after treatment is increased and there is no significant difference before the treatment. Or the magnitude of the change does not reach or exceed the set reference value, then the treatment method or the drug is judged to be inferior.
  • the tumor includes, but is not limited to, liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma or thyroid tumor.
  • the at least one other tumor marker includes, but is not limited to, AFP, CEA, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, AFU, SF, POA, TSGF.
  • tumor means, but is not limited to, a malignant tumor, and is suitable for use in an early stage of benign tumor or tumorigenesis.
  • the present invention by detecting the content of GAPDH in a human blood sample, it is possible to perform diagnosis, prognosis evaluation and therapeutic effect monitoring on liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer and the like, and can be used for, but not limited to, tumors. Screening, risk assessment of tumors in subjects, differentiation of tumor progression stages, identification of tumor treatment efficacy, and risk analysis of tumor progression.
  • FIG 1 shows the results of protein immunoblotting of GAPDH in plasma of patients with different tumors.
  • Figure 2 is a ROC curve of a liver cancer patient versus a healthy person.
  • Figure 3 is a ROC curve of a lung cancer patient versus a healthy person.
  • Figure 4 is a ROC curve of a gastric cancer patient versus a healthy person.
  • Figure 5 is a ROC curve of a colorectal cancer patient versus a healthy person.
  • Figure 6 is a ROC curve of a pancreatic cancer patient versus a healthy person.
  • Figure 7 is a summary of the results of serum GAPDH levels in a variety of tumor patients.
  • Figure 8 shows the secondary structure, hydrophilicity, flexibility, antigenic index and surface likelihood of the GAPDH protein.
  • tumor as used in the present invention means, but is not limited to, a malignant tumor, and the tumor marker and the detection kit thereof according to the present invention are equally applicable to an early stage of benign tumor or tumorigenesis.
  • blood sample refers to a sample obtained from the blood of a subject, and specifically includes serum and/or plasma samples.
  • the term "healthy person” as used in the present invention refers to a population that has not been diagnosed as a non-tumor patient by biochemical, imaging or pathological methods.
  • sensitivity refers to a case in which a tumor is detected by a pathological method, and the result of the kit detection is also a positive probability.
  • the “specificity” as used in the present invention means the probability that the test result of the kit in a healthy population is also negative.
  • the plasma of healthy people and different types of tumor patients was collected, and the concentration of GAPDH in plasma was detected by Western blotting, which proved that GAPDH can be used as a tumor marker.
  • the blood of healthy people and pathologically diagnosed patients with gastric cancer, lung cancer, and liver cancer were collected and collected in blood-filled anticoagulation tubes, which were inverted 8-10 times.
  • the plasma was separated by centrifugation (800-1000 rpm, 10 minutes), and plasma was dispensed into an EP tube (50 ⁇ L each) and stored rapidly at -20 ° C for cryopreservation.
  • Each plasma sample was diluted 20-fold, and then 5 x loading buffer was added and heated at 100 ° C for 10 minutes.
  • the GAPDH in the treated samples was detected by protein immunoblotting.
  • GAPDH was detected in plasma samples from healthy people and different tumor patients by protein immunoblotting. GAPDH levels in plasma of patients with gastric cancer, lung cancer and liver cancer were significantly higher than those in healthy people, indicating that GAPDH in blood can be used. Tumor markers distinguish between healthy people and cancer patients.
  • the blood of healthy people and liver cancer patients were collected.
  • the concentration of GAPDH in serum was determined by Enzyme linked immunosorbent assay (Elisa), and the effectiveness of GAPDH in the diagnosis of liver cancer was evaluated.
  • Healthy person A person who has not been diagnosed by biochemical, imaging or pathological methods and is considered to be a non-tumor patient.
  • Liver cancer patients patients diagnosed with liver cancer by pathological diagnosis, including different types and different stages of liver cancer.
  • the blood of healthy people and liver cancer patients were collected separately.
  • the blood was collected and allowed to stand at room temperature for 20 minutes.
  • the serum was separated by centrifugation (800-1000 rpm, 10 minutes), and the serum was dispensed into an EP tube (50 ⁇ L each). Store at -20 ° C for free.
  • the time of collection of samples of liver cancer patients receiving drug treatment was as follows: serum samples were collected 1 time before the start of treatment, and samples were collected once after the end of the treatment period.
  • the concentration of GAPDH in the collected serum samples was tested by the Elisa method.
  • the concentration of GAPDH in the clinical samples was counted, and the true positive (sensitivity) and false positive (1-specificity) were plotted as the receiver operating characteristic curve (ROC curve) as the ordinate and abscissa, respectively, and calculated.
  • ROC curve receiver operating characteristic curve
  • the area under the ROC curve and 95% CI yielded a correlation between GAPDH and tumor to evaluate the value of adjuvant diagnosis.
  • the serum of healthy people and lung cancer patients were collected, and the concentration of GAPDH in serum was detected by Elisa method, and the effectiveness of GAPDH in the diagnosis of lung cancer was evaluated.
  • some patients with lung cancer were followed up, blood samples were collected before and after treatment, and the changes of serum GAPDH concentration were measured to evaluate the effectiveness of GAPDH in disease monitoring and efficacy evaluation.
  • Healthy person A person who has not been diagnosed by biochemical, imaging or pathological methods and is considered to be a non-tumor patient.
  • Patients with lung cancer Patients diagnosed with lung cancer by pathological diagnosis, including different types and different stages of lung cancer.
  • the blood of healthy people and lung cancer patients were collected separately.
  • the blood was collected and allowed to stand at room temperature for 20 minutes.
  • the serum was centrifuged (800-1000 rpm, 10 minutes), and the serum was dispensed into an EP tube (50 ⁇ L each). Store at -20 ° C for free.
  • the concentration of GAPDH in the collected serum samples was tested by the Elisa method.
  • the concentration of GAPDH in the clinical samples was counted, and the true positive (sensitivity) and false positive (1-specificity) were plotted as ROC curves on the ordinate and abscissa respectively, and the area under the ROC curve and 95% CI were calculated to obtain GAPDH and tumor.
  • the blood of healthy people and gastric cancer patients were collected.
  • the Elisa method was used to detect the concentration of GAPDH in serum, and the effectiveness of GAPDH in the diagnosis of gastric cancer was evaluated.
  • some patients with gastric cancer were followed up, blood samples were collected before and after treatment, and the changes of serum GAPDH concentration were measured to evaluate the effectiveness of GAPDH in disease monitoring and efficacy evaluation.
  • Healthy person A person who has not been diagnosed by biochemical, imaging or pathological methods and is considered to be a non-tumor patient.
  • Patients with gastric cancer patients diagnosed with gastric cancer by pathological diagnosis, including different types and different stages of gastric cancer.
  • the blood of healthy people and gastric cancer patients were collected separately.
  • the blood was collected and allowed to stand at room temperature for 20 minutes.
  • the serum was separated by centrifugation (800-1000 rpm, 10 minutes), and the serum was dispensed into an EP tube (50 ⁇ L each). Store at -20 ° C for free.
  • the time of collection of samples of gastric cancer patients receiving drug treatment was as follows: serum samples were collected once before the start of treatment, and samples were collected once after the end of the treatment period.
  • the concentration of GAPDH in the collected serum samples was tested by the Elisa method.
  • the concentration of GAPDH in the clinical samples was counted, and the true positive (sensitivity) and false positive (1-specificity) were plotted as ROC curves on the ordinate and abscissa respectively, and the area under the ROC curve and 95% CI were calculated to obtain GAPDH and cancer.
  • the serum of healthy people and colorectal cancer patients were collected.
  • the concentration of GAPDH in serum was measured by Elisa method, and the effectiveness of GAPDH in the diagnosis of colorectal cancer was evaluated.
  • Healthy person A person who has not been diagnosed by biochemical, imaging or pathological methods and is considered to be a non-tumor patient.
  • Patients with colorectal cancer patients diagnosed with colorectal cancer by pathological diagnosis, including different types and different stages of colorectal cancer.
  • the blood of healthy people and colorectal cancer patients were collected separately.
  • the blood was collected and allowed to stand at room temperature for 20 minutes.
  • the serum was separated by centrifugation (800-1000 rpm, 10 minutes), and the serum was dispensed into an EP tube (50 ⁇ L each). Store at -20 ° C for cryopreservation.
  • the concentration of GAPDH in the collected serum samples was tested by the Elisa method.
  • the concentration of GAPDH in the clinical samples was counted, and the true positive (sensitivity) and false positive (1-specificity) were plotted as ROC curves on the ordinate and abscissa respectively, and the area under the ROC curve and 95% CI were calculated to obtain GAPDH and tumor.
  • the blood of healthy people and patients with pancreatic cancer were collected, and the concentration of GAPDH in serum was measured by Elisa method to evaluate the effectiveness of GAPDH in the diagnosis of pancreatic cancer.
  • some patients with pancreatic cancer were followed up, blood samples were collected before and after treatment, and the changes of serum GAPDH concentration were measured to evaluate the effectiveness of GAPDH in disease monitoring and efficacy evaluation.
  • Healthy person A person who has not been diagnosed by biochemical, imaging or pathological methods and is considered to be a non-tumor patient.
  • Patients with pancreatic cancer patients diagnosed with pancreatic cancer by pathological diagnosis, including different types and different stages of pancreatic cancer.
  • the blood of healthy people and patients with pancreatic cancer were collected separately.
  • the blood was collected and allowed to stand at room temperature for 20 minutes.
  • the serum was separated by centrifugation (800-1000 rpm, 10 minutes), and the serum was dispensed into an EP tube (50 ⁇ L each). Store frozen at -20 °C.
  • the time of collection of samples of patients with pancreatic cancer who received medical treatment was: serum samples were collected 1 time before the start of treatment, and samples were collected once after the end of the treatment period.
  • the concentration of GAPDH in the collected serum samples was tested by the Elisa method.
  • the concentration of GAPDH in the clinical samples was counted, and the true positive (sensitivity) and false positive (1-specificity) were plotted as ROC curves on the ordinate and abscissa respectively, and the area under the ROC curve and 95% CI were calculated to obtain GAPDH and tumor.
  • the blood of healthy people and pathologically diagnosed patients with liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma, thyroid tumor were collected.
  • the blood was collected and allowed to stand at room temperature for 20 minutes, and centrifuged. Serum (800-1000 rpm, 10 minutes), serum was dispensed into EP tubes (50 ⁇ L per serving) and quickly stored frozen at -20 °C.
  • the concentration of GAPDH in the collected serum samples was tested by the Elisa method.
  • the levels of GAPDH in the serum of patients with liver cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, lymphoma, and thyroid tumor are significantly higher than those in healthy people, indicating that GAPDH in the blood can be It is used as a marker for a variety of tumors to distinguish between healthy and cancer patients.
  • the amino acid sequence of GAPDH was first obtained from the NCBI database, and the secondary structure and antigen domain of GAPDH were predicted using DNASTAR Protean software.
  • the software used the Gamier-Robson and Chou-Fasman algorithms to predict the secondary structure of proteins ( ⁇ -helix, ⁇ -sheet, corner and random coil); the Kyte-Doolittle algorithm was used to predict the hydrophilicity of the protein;
  • the flexibility of the protein was predicted by the Karplus-Schultz algorithm; the antigenic index of the protein was predicted using the Jameson-Wolf algorithm; the surface potential of the protein was predicted using the Plot-Emini algorithm.
  • this example is directed to positions 40-160 and 180-335 of SEQ ID No. 1, respectively.
  • a paired antibody is prepared from a peptide consisting of an amino acid sequence.
  • Antiserum was obtained by immunizing BALB/c mice with antigen and detecting the antigen. After successful detection, B cells were isolated and fused with myeloma cells to prepare monoclonal hybridoma cells.
  • This embodiment provides a kit for detecting GAPDH in a blood sample.
  • the kit comprises a GAPDH standard protein, a monoclonal antibody against GAPDH (including primary and secondary antibodies), a 96-well microplate, a standard dilution, a sample dilution, a wash concentrate, a color developing solution, and a stop solution. Other experimental aids.
  • a method for detecting GAPDH in a blood sample of an individual to be tested using the kit is as follows:
  • the standard curve detected by this kit ranges from 0 to 10 ⁇ g/ml, and the minimum detection limit is 0.10 ⁇ g/ml.
  • a kit prepared according to the above Example 8 or a monoclonal antibody against GAPDH, respectively, was used to provide different kits for detecting GAPDH in blood samples.
  • the sensitivity of these kits for different cancers is shown in the table below.

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Abstract

本发明提供了一种肿瘤血液标志物及其应用，具体地说，本发明提供了检测血液样本中GAPDH的试剂在制备用于肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定和/或肿瘤进展的风险分析的检测用组合物中的应用。本发明还提供了用于检测血液样本中GAPDH浓度的试剂盒和检测方法。

Description

肿瘤血液标志物及其应用 技术领域
本发明是关于一种肿瘤血液标志物及其应用，具体是关于通过检测人血液中的GAPDH的含量对肝癌、肺癌、乳腺癌、胃癌、食道癌、结直肠癌等多种肿瘤进行诊断、预后评价和疗效监测的技术。本发明的标志物GAPDH可用于包括但不限于肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定以及肿瘤进展的风险分析。
背景技术
恶性肿瘤是全球性的常见病及多发病，是危害人类健康的重大疾病之一。我国2015年约有429.2万例新增癌症病例，平均每天新增1.2万例；同时约有281.4万人死于癌症，平均每天死亡例数达7500例。其中肺和支气管癌、胃癌、肝癌、食道癌和结直肠癌占所有癌症死亡情况的四分之三(CA Cancer J Clin.2016；66:115-132)。我国恶性肿瘤发病率近年来呈持续高发趋势，已成为国内居民的首要死因。
甘油醛-3-磷酸脱氢酶(Glyceraldehyde 3-phosphate dehydrogenase，GAPDH)是糖酵解过程中重要的酶，分子量37kDa，催化3-磷酸甘油醛(glyceraldehyde 3-phosphate)到1,3-二磷酸甘油酸的反应(D-glycerate 1,3-bisphosphate)。除此众所周知的代谢调控功能外，近几年研究证明GAPDH还参与了许多非代谢调控过程，包括转录激活(Oncogene.2007；26(18):2606–20)等。
有研究报道在黑色素瘤(Anticancer Research.2013；35(1):439–44)和非小细胞肺癌组织中(PLOS ONE.2013；8(4):e61262)GAPDH的mRNA水平显著上升，且表达水平与肿瘤恶性程度正相关。这是因为GAPDH在糖酵解过程中起到了重要作用，其抗凋亡功能对肿瘤细胞的增殖和保护也十分重要，例如GAPDH可以保护因化疗药物的作用导致的端粒缩短。但如果氧化压力等条件破坏了GAPDH的功能，细胞就会老化或者死亡(Clinical and Experimental Pharmacology&Physiology.2012；39(8):674–9)，GAPDH的缺失同样会导致肿瘤细胞老化(Biochemical and Biophysical Research Communications.2011；411(2):409–15)。曾有文章报道肿瘤中GAPDH的转录水平升高，例如利用荧光定量PCR方法检测乳腺癌患者血清中的游离DNA，结果 发现84.5％的乳腺癌患者DNA检测为阳性，I～II期乳腺癌患者阳性率为84％(肿瘤2011；31(12)：1099-1102)。目前，相关研究主要集中在几个瘤种中GAPDH基因水平，特别是mRNA的表达量的变化与肿瘤的关系。
经查，未见关于肿瘤患者血清/血浆中GAPDH的含量与肿瘤的相关性的报道，其与肿瘤发生发展的关系更是有待进一步研究。
发明内容
本案发明人通过研究发现，血液(血清和/或血浆)中GAPDH的表达水平可用于肝癌、肺癌、乳腺癌、胃癌、结直肠癌等多种肿瘤的诊断和分期分型以及肿瘤患者的病情监测和疗效评价。
从而，本发明提供了GAPDH作为肿瘤血液标志物的应用。
一方面，本发明提供了检测血液样本中GAPDH的试剂在制备用于肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定和/或肿瘤进展的风险分析的检测用组合物中的应用。
根据本发明的具体实施方案，本发明的上述应用中，所述血液样本为血清或血浆样本。
根据本发明的具体实施方案，本发明的上述应用中，所述肿瘤包括肝癌、肺癌、乳腺癌、胃癌、食道癌、结直肠癌、胰腺癌、宫颈癌、淋巴瘤或甲状腺瘤。
根据本发明的具体实施方案，本发明的上述应用中，所述检测血液样本中GAPDH的试剂包括采用以下方法检测血液样本中GAPDH浓度的试剂：蛋白免疫印记方法、酶联免疫夹心法。
根据本发明的具体实施方案，本发明的上述应用中，所述检测血液样本中GAPDH的试剂包括：与GAPDH或其多肽片段特异性结合的抗体。
根据本发明的具体实施方案，本发明的上述应用中，所述GAPDH具有如SEQ ID No.1所示的氨基酸序列，所述GAPDH的多肽片段包括GAPDH氨基酸序列N端第40-160位氨基酸(即SEQ ID No.1所示第40-160位氨基酸)组成的多肽片段、或第180-335位氨基酸(即SEQ ID No.1所示第180-335位氨基酸)组成的多肽片段。
根据本发明的具体实施方案，本发明的上述应用中，GAPDH是与至少一种其它肿瘤标志物进行联合诊断。具体地，进行肿瘤筛查、受试者发生肿瘤的风险评估、肿 瘤进展阶段的区分、肿瘤治疗疗效的鉴定和/或肿瘤进展的风险分析时，包括检测血液样本中GAPDH以及至少一种其它肿瘤标志物，所述至少一种其他肿瘤标志物包括但不局限于：AFP、CEA、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、AFU、SF、POA、TSGF。
另一方面，本发明还提供了一种用于肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定和/或肿瘤进展的风险分析的检测用试剂盒，其包括：检测血液样本中GAPDH的试剂。
根据本发明的具体实施方案，本发明的上述试剂盒中，所述检测血液样本中GAPDH的试剂包括采用以下方法检测血液样本中GAPDH浓度的试剂：蛋白免疫印记方法、酶联免疫夹心法、发光免疫分析法或胶体金法。
根据本发明的具体实施方案，本发明的上述试剂盒中，所述检测血液样本中GAPDH的试剂包括：与GAPDH或其多肽片段特异性结合的抗体；优选地，所述GAPDH的多肽片段包括GAPDH氨基酸序列N端第40-160位氨基酸组成的多肽片段、或第180-335位氨基酸组成的多肽片段。
根据本发明的具体实施方案，本发明的上述试剂盒还包括：96孔酶标版，标准品稀释液，样品稀释液，洗涤浓缩液，显色液和终止液。
在本发明一具体实施方案中，本发明的试剂盒包括：人源GAPDH蛋白(人源化的该蛋白在试剂盒中作为标准蛋白)、抗人源GAPDH蛋白的单克隆抗体、相关试剂(96孔酶标版，标准品稀释液，样品稀释液，洗涤浓缩液，显色液和终止液)。优选还进一步包括标注检测参数及相关判断参考值的说明书等，所述的检测参数例如：标准品的浓度，样品稀释倍数，标准曲线的监测范围、精度、最低检测限度，反应体系中各组分的量，反应温度和时间等，所述的判断参考值例如针对不同肿瘤的参考值等。
另一方面，本发明还提供了一种确定受试者是否患有肿瘤或处于患癌风险的方法，该方法包括：
a.获得受试者的血液样本；
b.测定受试者血清或血浆样本中GAPDH的浓度；
c.将受试者血清或血浆样本中GAPDH的浓度与参考浓度进行比较，样本中GAPDH的浓度大于或等于参考浓度时，该受试者被判定为肿瘤患者或具有罹患肿瘤的风险。
在本发明的一具体实施方式中，罹患肝癌的判定参考值是5.95μg/ml(也可四舍五入为6μg/ml)，即受试者血液样品中GAPDH的浓度在0-6μg/ml的范围内时被认为是正常值，如果受试者血液样品中GAPDH的浓度高于或等于6μg/ml则该受试者被判定患有肿瘤或有患癌风险。
在本发明中，受试者的检测结果在排除检测方法引起的误差后，上升或下降25％被视为具有同等判定意义，即针对肝癌的判定参考值是6μg/ml，当检测结果大于或等于4.5μg/ml时，也可判定该受试者患有肿瘤或有患癌风险。
另一方面，本发明还提供了一种区分肿瘤进展阶段的分类，该方法包括：
a.获得受试者的血液样本；
b.测定受试者血清或血浆样本中GAPDH的浓度；
c.将受试者血清或血浆样本中GAPDH的浓度与一系列参考浓度进行比较，判定肿瘤的进展阶段。
另一方面，本发明还提供了一种对肿瘤患者进行病情监测的方法，该方法包括：
获得受试者一段时间开始和结束时的血液样本；
测定受试者血清或血浆样本中GAPDH的浓度；
将受试者一段时间开始和结束时血清或血浆样本中GAPDH的浓度进行比较，如结束时样本GAPDH的浓度低于开始时，且降低百分比达到或超过设定的参考值，则判定病情缓解；如结束时样本GAPDH的浓度与开始时比较增多、无明显差异或变化幅度未达到或超过设定的参考值，则判定病情未缓解。
其中所述参考值可选自10-50％。在本发明的一具体实施方案中，该参考值为30％。并且，本发明中所述参考值±25％具有同等判定意义，即当所述参考值为30％、肿瘤患者血液样本中GAPDH的浓度降低22.5％时也可判定为病情进展较好。其中所述“一段时间”可根据病人病情发展而设定，“一段治疗”包括每天的治疗、一个周期的治疗和/或多周期的治疗。
另一方面，本发明还提供了一种对肿瘤患者进行疗效评价的方法，该方法包括：
a.获得受试者接受某次治疗的治疗前和治疗后血液样本；
b.测定受试者血清或血浆样本中GAPDH的浓度；
c.将受试者某次治疗的治疗前和治疗后血清或血浆样本中GAPDH的浓度进行比较，如治疗后样本GAPDH的浓度低于治疗前，且降低百分比达到或超过设定的参 考值，则治疗方法或药物有益于病情缓解；如治疗后样本GAPDH的浓度与治疗前比较增多、无明显差异或变化幅度未达到或超过设定的参考值，则判定治疗方法或药物的疗效较差。
其中所述参考值可选自10-50％。在本发明的一个具体实施例中，一位肺癌患者治疗前血清中GAPDH浓度为22.63μg/ml，经过治疗后浓度降低为20.41μg/ml，降低9.81％，四舍五入后为10％；在本发明的另一个具体实施例中，一位肺癌患者治疗前血清中GAPDH浓度为46.51μg/ml，经过治疗后浓度降低为24.20μg/ml，降低47.97％，四舍五入后为50％。在本发明的一具体实施方案中，该参考值为30％。并且，所述参考值±25％具有同等判定意义，即当所述参考值为30％、肿瘤患者血液样本中GAPDH的浓度降低22.5％时也可判定为疗效较好。其中所述“一段时间”可根据病人病情发展而设定，“一段治疗”包括每天的治疗、一个周期的治疗和/或多周期的治疗。
另一方面，本发明还提供了一种通过联合诊断确定受试者是否患有肿瘤或处于患癌风险中的方法，该方法包括：
a.获得受试者的血液样本；
b.测定受试者血清或血浆样本中GAPDH的浓度以及至少一种其它肿瘤标志物的浓度；
c.将受试者血清或血浆样本中GAPDH的浓度以及至少一种其它肿瘤标志物的浓度与设定的参考值进行比较，样本中GAPDH以及至少一种其它肿瘤标志物的浓度大于或等于参考浓度时，该受试者被鉴定为肿瘤患者或具有罹患肿瘤的风险。
其中所述GAPDH的浓度参考值为5.57-11.75μg/ml，且参考值±25％具有同等判定意义。
另一方面，本发明还提供了一种通过联合诊断对肿瘤进展阶段进行分类的方法，该方法包括：
a.获得受试者的血液样本；
b.测定受试者血清或血浆样本中GAPDH以及至少一种其它肿瘤标志物的浓度；
c.将受试者血清或血浆样本中GAPDH以及至少一种其它肿瘤标志物的浓度与一系列参考浓度进行比较，判定肿瘤的进展阶段。
另一方面，本发明还提供了一种通过联合诊断对肿瘤患者进行病情监测的方法，该方法包括：
获得受试者一段时间开始和结束时的血液样本；
测定受试者血清或血浆样本中GAPDH以及至少一种其它肿瘤标志物的浓度；
将受试者一段时间开始和结束时血清或血浆样本中GAPDH以及至少一种其它肿瘤标志物的浓度进行比较，如结束时样本GAPDH以及至少一种其它肿瘤标志物的浓度低于开始时，且降低百分比达到或超过设定的参考值，则判定病情缓解；如结束时样本GAPDH以及至少一种其它肿瘤标志物的浓度与开始时比较增多、无明显差异或变化幅度未达到或超过设定的参考值，则判定病情未缓解。
另一方面，本发明还提供了一种通过联合诊断对肿瘤患者进行疗效评价的方法，该方法包括：
a.获得受试者接受某次治疗的治疗前和治疗后血液样本；
b.测定受试者血清或血浆样本中GAPDH以及至少一种其它肿瘤标志物的浓度；
c.将受试者某次治疗的治疗前和治疗后血清或血浆样本中GAPDH以及至少一种其它肿瘤标志物的浓度进行比较，如治疗后样本GAPDH以及至少一种其它肿瘤标志物的浓度低于治疗前，且降低百分比达到或超过设定的参考值，则治疗方法或药物有益于病情缓解；如治疗后样本GAPDH以及至少一种其它肿瘤标志物的浓度与治疗前比较增多、无明显差异或变化幅度未达到或超过设定的参考值，则判定治疗方法或药物的疗效较差。
本发明的上述方法中，所述肿瘤包括但不局限于肝癌、肺癌、乳腺癌、胃癌、食道癌、结直肠癌、胰腺癌、宫颈癌、淋巴瘤或甲状腺瘤。
本发明的上述方法中，所述至少一种其他肿瘤标志物包括但不局限于：AFP、CEA、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、AFU、SF、POA、TSGF。
本发明所涉及的“肿瘤”表示但不限于恶性肿瘤，同时适用于良性肿瘤或肿瘤发生的早期阶段。
本发明中，通过检测人血液样本中的GAPDH的含量，能对肝癌、肺癌、乳腺癌、胃癌、食道癌、结直肠癌等肿瘤进行诊断、预后评价和疗效监测，可用于包括但不限于肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定以及肿瘤进展的风险分析。
附图说明
图1是不同肿瘤患者血浆中GAPDH的蛋白免疫印记结果。
图2是肝癌患者相对于健康人的ROC曲线。
图3是肺癌患者相对于健康人的ROC曲线。
图4是胃癌患者相对于健康人的ROC曲线。
图5是结直肠癌患者相对于健康人的ROC曲线。
图6是胰腺癌患者相对于健康人的ROC曲线。
图7是多种肿瘤患者血清GAPDH含量检测结果汇总。
图8显示GAPDH蛋白的二级结构、亲水性、柔韧性、抗原指数和表面可能性。
具体实施方式
下面结合具体实施例进一步阐明本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
本发明中所述“肿瘤”表示但不限于恶性肿瘤，本发明所涉及的肿瘤标志物及其检测试剂盒同样适用于良性肿瘤或肿瘤发生的早期阶段。
本发明中所述“血液样本”是指从受试者的血液获得的样本，具体包括血清和/或血浆样本。
本发明中所述“健康人”是指未经生化、影像或病理学方法确诊，暂被认为非肿瘤患者的人群。
本发明中所述“灵敏度”是指用病理学方法检测为肿瘤的病例，其试剂盒检测结果同样是阳性的概率。
本发明中所述“特异度”是指健康人群的试剂盒检测结果同样是阴性的概率。
实施例1
收集健康人与不同类型肿瘤患者的血浆，采用蛋白免疫印记的方法(Western blot)检测血浆中GAPDH的浓度，证明GAPDH可用作肿瘤标志物。
具体实验方法如下：
1.样本收集
分别收集健康人和病理学诊断为胃癌、肺癌、肝癌的患者的血液，收集血液装抗 凝管中，颠倒8-10次。离心分离血浆(800-1000转/分钟，10分钟)，将血浆分装至EP管中(每份50μL)，迅速放置于-20℃冷冻保存。
2.样本处理
每例血浆样本稀释20倍后加入5×上样缓冲液，100℃加热10分钟。
3.样本检测
采用蛋白免疫印记方法对处理后样本中的GAPDH进行检测。
4.结果分析
如图1所示，采用蛋白免疫印记方法对健康人和不同肿瘤患者血浆样本中的GAPDH进行检测，胃癌、肺癌、肝癌患者血浆中GAPDH含量明显高于健康人，说明血液中GAPDH可被用作肿瘤标志物，区分健康人与肿瘤患者。
实施例2
收集健康人与肝癌患者的血液，采用酶联免疫夹心法(Enzyme linked immunosorbent assay，Elisa)检测血清中GAPDH的浓度，对GAPDH在肝癌诊断中的有效性进行评价。
具体实验方法如下：
1.样本选择
健康人：未经生化、影像或病理学方法确诊，暂被认为非肿瘤患者的人群。
肝癌患者：经病理学诊断确诊为肝癌的患者，包括肝癌的不同类型和不同分期。
2.样本收集
分别收集健康人和肝癌患者的血液，收集血液室温静置20分钟，离心分离血清(800-1000转/分钟，10分钟)，将血清分装至EP管中(每份50μL)，迅速放置于-20℃冷冻保存。
对接受药物治疗的肝癌患者样本的收集时间点为：治疗开始前收集血清样本1次，治疗周期结束后收集样本1次。
3.样本检测
采用Elisa方法对收集的血清样本中GAPDH的浓度进行检测。
4.数据分析
统计临床样本中GAPDH的浓度值，将真阳性(灵敏度)和假阳性(1－特异度)分别作为纵坐标和横坐标绘制受试者工作特征曲线(Receiver Operating Characteristic curve，ROC曲线)，并计算ROC曲线下面积和95％CI得出GAPDH与肿瘤的相关性，以评价辅助诊断的价值。
5.研究结果
(1)样本构成
样本构成 例数
健康人 67
肝癌患者 52
总数 119
(2)ROC曲线及评价标准(图2)
变量 数值
临界值 5.95(μg/ml)
灵敏度 84.62％
特异度 74.63％
曲线下面积 0.8407
(3)药物治疗前后疗效评价
序号 治疗前(μg/ml) 治疗后(μg/ml)
1 17.16 4.20
2 15.86 3.60
3 12.63 2.60
实施例3
一方面收集健康人与肺癌患者的血清，采用Elisa方法检测血清中GAPDH的浓度，对GAPDH在肺癌诊断中的有效性进行评价。另一方面对部分肺癌患者进行跟踪，收集患者在治疗前和治疗后的血样，检测其血清中GAPDH浓度的变化，对GAPDH在病情监测和疗效评价方面的有效性进行评价。
具体实验方法如下：
1.样本选择
健康人：未经生化、影像或病理学方法确诊，暂被认为非肿瘤患者的人群。
肺癌患者：经病理学诊断确诊为肺癌的患者，包括肺癌的不同类型和不同分期。
2.样本收集
分别收集健康人和肺癌患者的血液，收集血液室温静置20分钟，离心分离血清(800-1000转/分钟，10分钟)，将血清分装至EP管中(每份50μL)，迅速放置于-20℃冷冻保存。
3.样本检测
采用Elisa方法对收集的血清样本中GAPDH的浓度进行检测。
4.数据分析
统计临床样本中GAPDH的浓度值，将真阳性(灵敏度)和假阳性(1－特异度)分别作为纵坐标和横坐标绘制ROC曲线，并计算ROC曲线下面积和95％CI得出GAPDH与肿瘤的相关性，以评价辅助诊断的价值。
5.研究结果
(1)样本构成
样本构成 例数
健康人 67
肺癌患者 56
总数 123
(2)ROC曲线及评价标准(图3)
变量 数值
临界值 10.58(μg/ml)
灵敏度 80.36％
特异度 88.06％
曲线下面积 0.9043
(3)药物治疗前后疗效评价
序号 治疗前(μg/ml) 治疗后(μg/ml)
1 46.51 24.20
2 23.84 8.60
3 12.63 6.60
4 22.63 20.41
实施例4
一方面收集健康人与胃癌患者的血液，采用Elisa方法检测血清中GAPDH的浓度，对GAPDH在胃癌诊断中的有效性进行评价。另一方面对部分胃癌患者进行跟踪，收集患者在治疗前和治疗后的血样，检测其血清中GAPDH浓度的变化，对GAPDH在病情监测和疗效评价方面的有效性进行评价。
具体实验方法如下：
1.样本选择
健康人：未经生化、影像或病理学方法确诊，暂被认为非肿瘤患者的人群。
胃癌患者：经病理学诊断确诊为胃癌的患者，包括胃癌的不同类型和不同分期。
2.样本收集
分别收集健康人和胃癌患者的血液，收集血液室温静置20分钟，离心分离血清(800-1000转/分钟，10分钟)，将血清分装至EP管中(每份50μL)，迅速放置于-20℃冷冻保存。
对接受药物治疗的胃癌患者样本的收集时间点为：治疗开始前收集血清样本1次，治疗周期结束后收集样本1次。
3.样本检测
采用Elisa方法对收集的血清样本中GAPDH的浓度进行检测。
4.数据分析
统计临床样本中GAPDH的浓度值，将真阳性(灵敏度)和假阳性(1－特异度)分别作为纵坐标和横坐标绘制ROC曲线，并计算ROC曲线下面积和95％CI得出GAPDH与癌症的相关性，以评价辅助诊断的价值。
5.研究结果
(1)样本构成
样本构成 例数
健康人 67
胃癌患者 48
总数 115
(2)ROC曲线及评价标准(图4)
变量 数值
临界值 5.57(μg/ml)
灵敏度 77.08％
特异度 70.15％
曲线下面积 0.7976
(3)药物治疗前后疗效评价
序号 治疗前(μg/ml) 治疗后(μg/ml)
1 55.81 27.34
2 45.62 22.65
3 19.55 5.30
实施例5
收集健康人与结直肠癌患者的血清，采用Elisa方法检测血清中GAPDH的浓度，对GAPDH在结直肠癌诊断中的有效性进行评价。
具体实验方法如下：
1.样本选择
健康人：未经生化、影像或病理学方法确诊，暂被认为非肿瘤患者的人群。
结直肠癌患者：经病理学诊断确诊为结直肠癌的患者，包括结直肠癌的不同类型和不同分期。
2.样本收集
分别收集健康人和结直肠癌患者的血液，收集血液室温静置20分钟，离心分离血清(800-1000转/分钟，10分钟)，将血清分装至EP管中(每份50μL)，迅速放置于-20℃冷冻保存。
3.样本检测
采用Elisa方法对收集的血清样本中GAPDH的浓度进行检测。
4.数据分析
统计临床样本中GAPDH的浓度值，将真阳性(灵敏度)和假阳性(1－特异度)分别作为纵坐标和横坐标绘制ROC曲线，并计算ROC曲线下面积和95％CI得出GAPDH与肿瘤的相关性，以评价辅助诊断的价值。
5.研究结果
(1)样本构成
样本构成 例数
健康人 67
结直肠癌患者 30
总数 97
(2)ROC曲线及评价标准(图5)
变量 数值
临界值 11.75(μg/ml)
灵敏度 73.33％
特异度 89.55％
曲线下面积 0.8474
实施例6
一方面收集健康人与胰腺癌患者的血液，采用Elisa方法检测血清中GAPDH的浓度，对GAPDH在胰腺癌诊断中的有效性进行评价。另一方面对部分胰腺癌患者进行跟踪，收集患者在治疗前和治疗后的血样，检测其血清中GAPDH浓度的变化，对GAPDH在病情监测和疗效评价方面的有效性进行评价。
具体实验方法如下：
1.样本选择
健康人：未经生化、影像或病理学方法确诊，暂被认为非肿瘤患者的人群。
胰腺癌患者：经病理学诊断确诊为胰腺癌的患者，包括胰腺癌的不同类型和不同分期。
2.样本收集
分别收集健康人和胰腺癌患者的血液，收集血液室温静置20分钟，离心分离血清(800-1000转/分钟，10分钟)，将血清分装至EP管中(每份50μL)，迅速放置于-20℃冷冻保存。
对接受药物治疗的胰腺癌患者样本的收集时间点为：治疗开始前收集血清样本1次，治疗周期结束后收集样本1次。
3.样本检测
采用Elisa方法对收集的血清样本中GAPDH的浓度进行检测。
4.数据分析
统计临床样本中GAPDH的浓度值，将真阳性(灵敏度)和假阳性(1－特异度)分别作为纵坐标和横坐标绘制ROC曲线，并计算ROC曲线下面积和95％CI得出GAPDH与肿瘤的相关性，以评价辅助诊断的价值。
5.研究结果
(1)样本构成
样本构成 例数
健康人 67
胰腺癌患者 24
总数 91
(2)ROC曲线及评价标准(图6)
变量 数值
临界值 5.94(μg/ml)
灵敏度 79.17％
特异度 74.63％
曲线下面积 0.8259
实施例7
收集健康人与不同类型肿瘤患者的血液，采用Elisa方法检测血清中GAPDH的浓度，证明GAPDH可用作多种肿瘤的标志物。
具体实验方法如下：
1.样本收集
分别收集健康人和病理学诊断肝癌、肺癌、乳腺癌、胃癌、食道癌、结直肠癌、胰腺癌、宫颈癌、淋巴瘤、甲状腺瘤的患者的血液，收集血液室温静置20分钟，离心分离血清(800-1000转/分钟，10分钟)，将血清分装至EP管中(每份50μL)，迅速放置于-20℃冷冻保存。
2.样本检测
采用Elisa方法对收集的血清样本中GAPDH的浓度进行检测。
3.结果分析
如图7所示，肝癌、肺癌、胃癌、结直肠癌、胰腺癌、乳腺癌、食道癌、宫颈癌、淋巴瘤、甲状腺瘤患者血清中GAPDH含量明显高于健康人，说明血液中GAPDH可被用作多种肿瘤的标志物，区分健康人与肿瘤患者。
本实施例中所测得的不同肿瘤患者血清中GAPDH的浓度参见下表。
Figure PCTCN2018094401-appb-000001
实施例8
本实施例中，首先从NCBI数据库中获得GAPDH的氨基酸序列，再利用DNASTAR Protean软件对GAPDH的二级结构和抗原域进行预测。该软件使用Gamier-Robson和Chou-Fasman算法对蛋白质的二级结构(α螺旋、β折叠、转角和无规则卷曲)进行了预测；使用Kyte-Doolittle算法对蛋白的亲水性进行了预测；使用Karplus-Schultz算法对蛋白的柔韧性进行了预测；使用Jameson-Wolf算法对蛋白的抗原指数进行了预测；使用Plot-Emini算法对蛋白的表面可能性进行了预测。综合以上二级结构、亲水性、柔韧性、抗原指数和表面可能性的预测结果(图8)，本实施例分别针对SEQ ID No.1所示第40-160位和第180-335位氨基酸序列组成的肽段制备配对抗体。
抗体具体制备过程可参照现有技术进行，简述如下：
1制备抗原。分别针对SEQ ID No.1所示第40-160位和第180-335位氨基酸序列进行基因合成，构建载体并利用原核表达系统表达抗原。
2获得单克隆杂交瘤细胞。使用抗原免疫BALB/c小鼠获得抗血清并对抗原进行检测，检测成功后分离B细胞并与骨髓瘤细胞融合制备单克隆杂交瘤细胞。
3获得配对抗体。对不同杂交瘤细胞产生的不同单克隆抗体进行两两配对，并用原核表达的全长蛋白作为抗原进行检测，获得至少一对满足检测要求的配对抗体。
实施例9
本实施例提供了一种检测血液样本中GAPDH的试剂盒。
所述试剂盒包含GAPDH标准蛋白，抗GAPDH的单克隆抗体(包括一抗和二抗)，96孔酶标版，标准品稀释液，样本稀释液，洗涤浓缩液，显色液和终止液以及其它实验辅助材料。
应用所述试剂盒检测待测个体血液样本中GAPDH的方法简述如下：
1.将50μl稀释好的标准品或样本加入包被好的96孔酶标板，37℃放置30-60min。
2.弃去孔内液体，每孔加300μl洗涤液洗涤三次。
3.每孔加入50μl二抗溶液，37℃放置30-60min。
4.弃去孔内液体，每孔加300μl洗涤液洗涤三次。
5.每孔加入100μl显色剂，37℃避光放置10-15min。
6.每孔加入50μl终止液，450nm波长处读取吸光值。
本试剂盒检测的标准曲线范围为0-10μg/ml，最低检测限度为0.10μg/ml。
本实施例中，分别采用按照上述实施例8制备的抗体、或者商购抗GAPDH的单克隆抗体，提供了不同的检测血液样本中GAPDH的试剂盒。这些试剂盒对不同癌症的检测灵敏度结果参见下表。
Figure PCTCN2018094401-appb-000002
上述结果表明，本发明的检测血液样本中GAPDH的试剂盒具有良好的检测灵敏度。

Claims (12)

  1. 检测血液样本中GAPDH的试剂在制备用于肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定和/或肿瘤进展的风险分析的检测用组合物中的应用。
  2. 根据权利要求1所述的应用，其中，所述血液样本为血清或血浆样本。
  3. 根据权利要求1所述的应用，其中，所述肿瘤包括肝癌、肺癌、乳腺癌、胃癌、食道癌、结直肠癌、胰腺癌、宫颈癌、淋巴瘤或甲状腺瘤。
  4. 根据权利要求1所述的应用，其中，所述检测血液样本中GAPDH的试剂包括采用以下方法检测血液样本中GAPDH浓度的试剂：蛋白免疫印迹法、酶联免疫法、发光免疫分析法或胶体金法。
  5. 根据权利要求1所述的应用，其中，所述检测血液样本中GAPDH的试剂包括：与GAPDH或其多肽片段特异性结合的抗体。
  6. 根据权利要求5所述的应用，其中，所述GAPDH的多肽片段包括GAPDH氨基酸序列N端第40-160位氨基酸组成的多肽片段、或第180-335位氨基酸组成的多肽片段。
  7. 根据权利要求1～6任一项所述的应用，其中，进行肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定和/或肿瘤进展的风险分析时，包括检测血液样本中GAPDH以及至少一种其它肿瘤标志物，所述至少一种其他肿瘤标志物包括但不局限于：AFP、CEA、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、AFU、SF、POA、TSGF。
  8. 一种用于肿瘤筛查、受试者发生肿瘤的风险评估、肿瘤进展阶段的区分、肿瘤治疗疗效的鉴定和/或肿瘤进展的风险分析的检测用试剂盒，其包括：
    检测血液样本中GAPDH的试剂。
  9. 根据权利要求8所述的试剂盒，其中，所述检测血液样本中GAPDH的试剂包括采用以下方法检测血液样本中GAPDH浓度的试剂：蛋白免疫印记方法、酶联免疫夹心法、发光免疫分析法或胶体金法。
  10. 根据权利要求9所述的试剂盒，其中，所述检测血液样本中GAPDH的试剂包括：与GAPDH或其多肽片段特异性结合的抗体。
  11. 根据权利要求10所述的试剂盒，其中，所述GAPDH的多肽片段包括GAPDH 氨基酸序列N端第40-160位氨基酸组成的多肽片段、或第180-335位氨基酸组成的多肽片段。
  12. 根据权利要求8所述的试剂盒，该试剂盒还可以进一步包括：
    96孔酶标版，标准品稀释液，样品稀释液，洗涤浓缩液，显色液和终止液。
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