TUMOR BLOOD MARKER, USE THEREOF, AND KIT COMPRISING THE SAME

Abstract

The present invention provides a tumor blood marker and a use thereof. Specifically, the present invention provides a use of a reagent, which is used to detect GAPDH in a blood sample, in a preparation of a detecting composition for tumor screening, risk evaluation of tumor development in subjects, distinction of tumor progression stages, identification of therapeutic efficacy of tumor and/or risk analysis of tumor progression. The present invention also provides a kit and a method for detecting GAPDH concentrations in blood samples.

Claims

1. A use of a reagent, the reagent is used to detect GAPDH in a blood sample, wherein the use of the reagent in a preparation of a detecting composition is for a tumor screening, a risk evaluation of tumor development in a subject, a distinction of tumor progression stages, an identification of therapeutic efficacy of tumor and/or a risk analysis of tumor progression.

2. The use according to claim 1, wherein the blood sample is a serum or plasma sample.

3. The use according to claim 1, wherein the tumor includes a liver cancer, a lung cancer, a breast cancer, a gastric cancer, an esophageal cancer, a colorectal cancer, a pancreatic cancer, a cervical cancer, a lymphoma, or a thyroid tumor.

4. The use according to claim 1, wherein the reagent used to detect GAPDH in the blood sample includes a reagent used to detect GAPDH in a blood sample in a Western blotting, an enzyme-linked immunosorbent assay, a luminescent immunoassay, or a colloidal gold assay.

5. The use according to claim 1, wherein the reagent used to detect GAPDH in the blood sample includes a pair of antibodies specifically binding to GAPDH or a polypeptide fragment thereof.

6. The use according to claim 5, wherein the polypeptide fragment of GAPDH comprises a polypeptide fragment consisting of N-terminal amino acids 40-160 of the GAPDH amino acid sequence, or a polypeptide fragment consisting of N-terminal amino acids 180-335 of the GAPDH amino acid sequence.

7. The use according to claim 1, wherein the tumor screening, the risk evaluation of tumor development in the subject, the distinction of tumor progression stages, the identification of therapeutic efficacy of tumor and/or the risk analysis of tumor progression each comprises a detection of GAPDH and at least one additional tumor marker in the blood sample, and the at least one additional tumor marker comprises, but not limited to, AFP, CEA, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, AFU, SF, POA, and TSGF.

8. An assay kit for a tumor screening, a risk evaluation of tumor development in subjects, a distinction of tumor progression stages, an identification of therapeutic efficacy of tumor and/or a risk analysis of tumor progression, comprising: a reagent used to detect GAPDH in a blood sample.

9. The kit according to claim 8, wherein the reagent used to detect GAPDH in the blood sample comprises a reagent used to detect GAPDH in a blood sample in a Western blotting, an enzyme-linked immunosorbent assay, a luminescent immunoassay or a colloidal gold assay.

10. The kit according to claim 9, wherein the reagent used to detect GAPDH in the blood sample includes a pair of antibodies specifically binding to GAPDH or a polypeptide fragment thereof.

11. The kit according to claim 10, wherein the polypeptide fragment of GAPDH comprises a polypeptide fragment consisting of N-terminal amino acids 40-160 of the GAPDH amino acid sequence, or a polypeptide fragment consisting of N-terminal amino acids 180-335 of the GAPDH amino acid sequence.

12. The kit according to claim 8, the kit further comprises: a 96-well ELISA Plate, a standard dilution solution, a sample dilution solution, a concentrated solution for washing, a color-developing solution and a stopping solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 is a Western blotting result of GAPDH in plasma from patients with different tumors.

[0061] FIG. 2 is a ROC curve of liver cancer patients relative to healthy subjects.

[0062] FIG. 3 is a ROC curve of lung cancer patients relative to healthy subjects.

[0063] FIG. 4 is a ROC curve of gastric cancer patients relative to healthy subjects.

[0064] FIG. 5 is a ROC curve of colorectal cancer patients relative to healthy subjects.

[0065] FIG. 6 is a ROC curve of pancreatic cancer patients relative to healthy subjects.

[0066] FIG. 7 is a summary of the detection results of serum GAPDH contents in patients with a variety of tumor.

[0067] FIG. 8 shows the secondary structure, hydrophilicity, flexibility, antigenic index and surface probability of a GAPDH protein.

DETAILED DESCRIPTION

[0068] The present invention will be further illustrated in connection with the following specific embodiments. It should be understood that these examples are only to explain the present invention, but not intended to limit the scope of the invention. The experimental methods in the following examples which are not specified with the specific conditions were usually carried out in accordance with conventional conditions, or according to the conditions recommended by the manufacturer.

[0069] The term 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 a benign tumor or an early stage of tumorigenesis.

[0070] The term blood sample as used in the present invention refers to a sample obtained from the blood of a subject, specifically including serum and/or plasma samples.

[0071] The term healthy subjects as used in the present invention refers to a population that is considered at the moment as a non-tumor patient, because he or she has not been subjected to any diagnosis by biochemical, imaging or pathological methods.

[0072] The term sensitivity as used in the present invention refers to a probability, for a case in which a tumor is detected by a pathological method, to also have a positive detection result by the kit.

[0073] The specificity as used in the present invention refers to a probability for a healthy subject to also have a negative detection result by the kit.

Example 1

[0074] The plasma was collected from healthy subjects and patients having different types of tumors. Western blotting was used to detect the concentration of GAPDH in the plasma, which proved that GAPDH can be used as a tumor marker.

[0075] The specific experimental process is as follows:

[0076] 1. Sample Collection

[0077] Blood was collected from each of healthy subjects and patients pathologically diagnosed as having gastric cancer, lung cancer, or liver cancer, into anticoagulation tubes, and the tubes were inverted 8-10 times. The plasma was separated by centrifugation at 800-1000 rpm for 10 minutes, and the separated plasma was dispensed into EP tubes (50 L each part), and quickly stored at 20 C. for cryopreservation.

[0078] 2. Sample Processing

[0079] Each sample of plasma was diluted 20 times and then added with 5 loading buffer and heated at 100 C. for 10 minutes.

[0080] 3. Sample Detection

[0081] The treated samples were detected for GAPDH by Western blotting.

[0082] 4. Result Analysis

[0083] As shown in FIG. 1, GAPDH was detected by Western blotting in plasma samples from healthy subjects and the patients with different tumors. GAPDH levels in plasma of patients with gastric cancer, lung cancer or liver cancer were significantly higher than that in healthy subjects, indicating that GAPDH in blood can be used as a tumor marker distinguishing healthy subjects from cancer patients.

Example 2

[0084] The blood of healthy subjects and liver cancer patients was collected. The concentration of GAPDH in serum was detected by Enzyme linked immunosorbent assay (ELISA), and the effectiveness of GAPDH in the diagnosis of liver cancer was evaluated.

[0085] The specific experimental process is as follows:

[0086] 1. Sample Selection

[0087] Healthy subjects: a population who has not been subjected to any diagnosis by biochemical, imaging or pathological methods and is considered to be a non-tumor patient.

[0088] Liver cancer patients: the patients diagnosed as having liver cancer by pathological diagnosis, including different types and different stages of liver cancer.

[0089] 2. Sample Collection

[0090] Blood was collected from each of healthy subjects and liver cancer patients, and allowed to stand at room temperature for 20 minutes. The serum was separated by centrifugation at 800-1000 rpm for 10 minutes, and the separated serum was dispensed into EP tubes (50 L each part), and quickly stored at 20 C. for cryopreservation.

[0091] The samples (serum samples) of liver cancer patients receiving drug treatments were collected once before the start of treatment, and collected once after the end of the treatment period.

[0092] 3. Sample Detection

[0093] The collected serum samples were detected by ELISA for concentration of GAPDH therein.

[0094] 4. Data Analysis

[0095] The concentration of GAPDH in the clinical samples was counted, and a Receiver Operating Characteristic curve (ROC curve) was plotted by using the true positive and false positive (1-specificity) as the ordinate and the abscissa, respectively. The area under ROC curve and 95% CI were calculated, to determine the correlation between GAPDH and tumor, so as to evaluate the value of the auxiliary diagnosis.

[0096] 5. Research Result

[0097] (1) Subjects category

TABLE-US-00001 Subjects category Case number Healthy subjects 67 Liver cancer patients 52 Total 119

[0098] (2) ROC curve and evaluation criteria (FIG. 2)

TABLE-US-00002 Variable Numerical value Threshold 5.95(g/ml) Sensitivity 84.62% Specificity 74.63% Area under the curve 0.8407

[0099] (3) Evaluation of treating efficacy for the medicine therapy

TABLE-US-00003 No. Before treatment (g/ml) After treatment (g/ml) 1 17.16 4.20 2 15.86 3.60 3 12.63 2.60

Example 3

[0100] On the one hand, the serums were collected from healthy subjects and lung cancer patients, and the concentration of GAPDH in serum was detected by ELISA procedure such that the effectiveness of GAPDH in the diagnosis of lung cancer was evaluated. On the other hand, some patients with lung cancer were followed up, from which patients the blood samples were collected before and after treatment. The blood samples were tested for changes in serum GAPDH concentration, such that the effectiveness of GAPDH in condition monitoring and therapy efficacy evaluation was evaluated.

[0101] The specific experimental process is as follows:

[0102] 1. Sample Selection

[0103] Healthy subjects: a population who has not been subjected to any diagnosis by biochemical, imaging or pathological methods and is considered to be a non-tumor patient.

[0104] Lung cancer patients: the patients diagnosed as having lung cancer by pathological diagnosis, including different types and different stages of lung cancer.

[0105] 2. Sample Collection

[0106] Blood was collected from each of healthy subjects and pathologically diagnosed lung cancer patients, and allowed to stand at room temperature for 20 minutes. The serum was separated by centrifugation at 800-1000 rpm for 10 minutes, and the separated serum was dispensed into EP tubes (50 L each part), and quickly stored at 20 C. for cryopreservation.

[0107] 3. Sample Detection

[0108] The collected serum samples were detected by ELISA for concentration of GAPDH therein.

[0109] 4. Data Analysis

[0110] The concentration of GAPDH in the clinical samples was counted, and a Receiver Operating Characteristic curve (ROC curve) was plotted by using the true positive and false positive (1-specificity) as the ordinate and the abscissa, respectively. The area under ROC curve and 95% CI were calculated, to determine the correlation between GAPDH and tumor, so as to evaluate the value of the auxiliary diagnosis.

[0111] 5. Research result

[0112] (1) Subjects Category

TABLE-US-00004 Subjects category Case number Healthy subjects 67 Lung cancer patients 56 Total 123

[0113] (2) ROC Curve and Evaluation Criteria (FIG. 3)

TABLE-US-00005 Variable Numerical value Threshold 10.58(g/ml) Sensitivity 80.36% Specificity 88.06% Area under the curve 0.9043

[0114] (3) Evaluation of Treating Efficacy for the Medicine Therapy

TABLE-US-00006 No. Before treatment (g/ml) After treatment (g/ml) 1 46.51 24.20 2 23.84 8.60 3 12.63 6.60 4 22.63 20.41

Example 4

[0115] On the one hand, the serums were collected from healthy subjects and gastric cancer patients, and the concentration of GAPDH in serum was detected by ELISA procedure such that the effectiveness of GAPDH in the diagnosis of gastric cancer was evaluated. On the other hand, some patients with gastric cancer were followed up, from which patients the blood samples were collected before and after treatment. The blood samples were tested for changes in serum GAPDH concentration, such that the effectiveness of GAPDH in condition monitoring and therapy efficacy evaluation was evaluated.

[0116] The specific experimental process is as follows:

[0117] 1. Sample Selection

[0118] Healthy subjects: a population who has not been subjected to any diagnosis by biochemical, imaging or pathological methods and is considered to be a non-tumor patient. Gastric cancer patients: the patients diagnosed as having gastric cancer by pathological diagnosis, including different types and different stages of gastric cancer.

[0119] 2. Sample Collection

[0120] Blood was collected from each of healthy subjects and pathologically diagnosed gastric cancer patients, and allowed to stand at room temperature for 20 minutes. The serum was separated by centrifugation at 800-1000 rpm for 10 minutes, and the separated serum was dispensed into EP tube (50 L each part), and quickly stored at 20 C. for cryopreservation.

[0121] The samples (serum samples) of gastric cancer patients receiving drug treatment were collected once before the start of treatment, and collected once after the end of the treatment period.

[0122] 3. Sample Detection

[0123] The collected serum samples were detected by ELISA for concentration of GAPDH therein.

[0124] 4. Data Analysis

[0125] The concentration of GAPDH in the clinical samples was counted, and a Receiver Operating Characteristic curve (ROC curve) was plotted by using the true positive and false positive (1-specificity) as the ordinate and the abscissa, respectively. The area under ROC curve and 95% CI were calculated, to determine the correlation between GAPDH and cancer, so as to evaluate the value of the auxiliary diagnosis.

[0126] 5. Research result

[0127] (1) Subjects Category

TABLE-US-00007 Subjects category Case number Healthy subjects 67 Gastric cancer patients 48 Total 115

[0128] (2) ROC Curve and Evaluation Criteria (FIG. 4)

TABLE-US-00008 Variable Numerical value Threshold 5.57(g/ml) Sensitivity 77.08% Specificity 70.15% Area under the curve 0.7976

[0129] (3) Evaluation of Treating Efficacy for the Medicine Therapy

TABLE-US-00009 No. Before treatment(g/ml) After treatment(g/ml) 1 55.81 27.34 2 45.62 22.65 3 19.55 5.30

Example 5

[0130] The blood of healthy subjects and colorectal cancer patients were collected. The concentration of GAPDH in serum was detected by Enzyme linked immunosorbent assay (ELISA), and the effectiveness of GAPDH in the diagnosis of colorectal cancer was evaluated.

[0131] The specific experimental process is as follows:

[0132] 1. Sample Selection

[0133] Healthy subjects: a population who has not been subjected to any diagnosis by biochemical, imaging or pathological methods and is considered to be a non-tumor patient. Colorectal cancer patients: the patients diagnosed as having colorectal cancer by pathological diagnosis, including different types and different stages of colorectal cancer.

[0134] 2. Sample Collection

[0135] Blood was collected from each of healthy subjects and pathologically diagnosed colorectal cancer patients, and allowed to stand at room temperature for 20 minutes. The serum was separated by centrifugation at 800-1000 rpm for 10 minutes, and the separated serum was dispensed into EP tube (50 L each part), and quickly stored at 20 C. for cryopreservation.

[0136] 3. Sample Detection

[0137] The collected serum samples were detected by ELISA for concentration of GAPDH therein.

[0138] 4. Data Analysis

[0139] The concentration of GAPDH in the clinical samples was counted, and a Receiver Operating Characteristic curve (ROC curve) was plotted by using the true positive and false positive (1-specificity) as the ordinate and the abscissa, respectively. The area under ROC curve and 95% CI were calculated, to determine the correlation between GAPDH and tumor, so as to evaluate the value of the auxiliary diagnosis.

[0140] 5. Research Result

[0141] (1) Subjects Category

TABLE-US-00010 Subjects category Case number Healthy subjects 67 Colorectal cancer patients 30 Total 97

[0142] (2) ROC Curve and Evaluation Criteria (FIG. 5)

TABLE-US-00011 Variable Numerical value Threshold 11.75(g/ml) Sensitivity 73.33% Specificity 89.55% Area under the curve 0.8474

Example 6

[0143] On the one hand, the serums were collected from healthy subjects and pancreatic cancer patients, and the concentration of GAPDH in serum was detected by ELISA procedure such that the effectiveness of GAPDH in the diagnosis of pancreatic cancer was evaluated. On the other hand, some patients with pancreatic cancer were followed up, from which patients the blood samples were collected before and after treatment. The blood samples were tested for changes in serum GAPDH concentration, such that the effectiveness of GAPDH in condition monitoring and therapy efficacy evaluation was evaluated.

[0144] The specific experimental process is as follows:

[0145] 1. Sample Selection

[0146] Healthy subjects: a population who has not been subjected to any diagnosis by biochemical, imaging or pathological methods and is considered to be a non-tumor patient. Pancreatic cancer patients: the patients diagnosed as having pancreatic cancer by pathological diagnosis, including different types and different stages of pancreatic cancer.

[0147] 2. Sample Collection

[0148] Blood was collected from each of healthy subjects and pathologically diagnosed pancreatic cancer patients, and allowed to stand at room temperature for 20 minutes. The serum was separated by centrifugation at 800-1000 rpm for 10 minutes, and the separated serum was dispensed into EP tube (50 L each part), and quickly stored at 20 C. for cryopreservation.

[0149] The samples (serum samples) of pancreatic cancer patients receiving drug treatment were collected once before the start of treatment, and collected once after the end of the treatment period.

[0150] 3. Sample Detection

[0151] The collected serum samples were detected by ELISA for concentration of GAPDH therein.

[0152] 4. Data Analysis

[0153] The concentration of GAPDH in the clinical samples was counted, and a Receiver Operating Characteristic curve (ROC curve) was plotted by using the true positive and false positive (1-specificity) as the ordinate and the abscissa, respectively. The area under ROC curve and 95% CI were calculated, to determine the correlation between GAPDH and tumor, so as to evaluate the value of the auxiliary diagnosis.

[0154] 5. Research Result

[0155] (1) Subjects Category

TABLE-US-00012 Subjects category Case number Healthy subjects 67 Pancreatic cancer patients 24 Total 91

[0156] (2) ROC Curve and Evaluation Criteria (FIG. 6)

TABLE-US-00013 Variable Numerical value Threshold 5.94(g/ml) Sensitivity 79.17% Specificity 74.63% Area under the curve 0.8259

Example 7

[0157] The blood of healthy subjects and patients with different types of tumors was collected. The concentration of GAPDH in serum was detected by Enzyme linked immunosorbent assay (ELISA), in order to prove that GAPDH can be used as a marker for various tumors.

[0158] The specific experimental process is as follows:

[0159] 1. Sample Collection

[0160] Blood was collected from each of healthy subjects and patients pathologically diagnosed with liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma, or thyroid tumor, and allowed to stand at room temperature for 20 minutes. The serum was separated by centrifugation at 800-1000 rpm for 10 minutes, and the separated serum was dispensed into EP tube (50 L each part), and quickly stored at 20 C. for cryopreservation.

[0161] 2. Sample Detection

[0162] The collected serum samples were detected by ELISA for concentration of GAPDH therein.

[0163] 3. Result Analysis

[0164] As shown in FIG. 7, 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, or thyroid tumor are significantly higher than that of the healthy subjects, indicating that GAPDH in the blood can be used as a marker for a variety of tumors, distinguishing between healthy subjects and tumor patients.

[0165] The concentrations of GAPDH in patients with different tumors measured in this Examples are shown in table below.

TABLE-US-00014 Average concentration Category Case number (g/ml) Healthy subjects 67 3.66 Liver cancer patients 52 20.22 Lung cancer patients 56 41.74 Gastric cancer patients 48 29.16 Colorectal cancer patients 30 20.43 Pancreatic cancer patients 24 30.05 Breast cancer patients 9 18.70 Esophageal cancer patients 9 22.57 Cervical cancer patients 7 24.10 Lymphoma patients 6 15.75 Thyroid tumor patients 5 17.88

Example 8

[0166] In this example, the amino acid sequence of GAPDH was first obtained from the NCBI database, and then the secondary structure and antigen domain of GAPDH were predicted using DNASTAR Protean software. The software employs the Gamier-Robson and Chou-Fasman algorithms for prediction of the secondary structure of the protein (-helix, -sheet, turn and random coil); the Kyte-Doolittle algorithm for prediction of the hydrophilicity of the protein; the Karplus-Schultz algorithm for prediction of the flexibility of the protein; the Jameson-Wolf algorithm for prediction of the antigenic index of the protein; the Plot-Emini algorithm for prediction of the surface probability of the protein. The results of the above predictions for secondary structure, hydrophilicity, flexibility, antigenic index and surface probability were considered in combination (FIG. 8), the paired-antibodies were prepared against a peptide consisting of amino acids 40-160 and a peptide consisting of amino acids 180-335 of SEQ ID No. 1, respectively.

[0167] The specific preparation process of the antibody can be carried out by referring to the prior art, and is briefly described as follows:

[0168] 1. Preparation of Antigen.

[0169] Genes were synthesized for amino acid sequences 40-160 and 180-335 of SEQ ID No. 1, respectively, the vectors thereof were constructed, and the antigens were expressed in prokaryotic expression system.

[0170] 2. Obtaining Monoclonal Hybridoma Cells.

[0171] Anti-serum was obtained by immunizing BALB/c mice with the antigen and used to detect the antigen. After successful detection, B cells were isolated and fused with myeloma cells to prepare monoclonal hybridoma cells.

[0172] 3. Obtaining Paired Antibodies.

[0173] The different monoclonal antibodies produced by different hybridoma cells were paired pair wisely, and tested by using prokaryotic expressed full-length protein as antigen, and it was obtained at least one pair of antibodies that meet the detection requirements.

Example 9

[0174] This Example provides an assay kit for detecting GAPDH in a blood sample. The kit comprises a GAPDH standard protein, a pair of monoclonal antibodies against GAPDH (including primary and secondary antibodies), a 96-well ELISA Plate, a standard dilution solution, a sample dilution solution, a concentrated solution for washing, a color-developing solution and a stopping solution, as well as other experimental aiding materials.

[0175] GAPDH in a blood sample of an individual to be tested can be detected using the kit by a method as follows:

[0176] 1. Adding 50 l of the diluted standard or sample to the coated 96-well ELISA Plate, and allowing the plate to be still at 37 C. for 30-60 min.

[0177] 2. Discarding the liquid in the well, and washing three times with 300 l of washing solution per well.

[0178] 3. Adding 50 l of secondary antibody solution to each well, and allowing the plate to be still at 37 C. for 30-60 min.

[0179] 4. Discarding the liquid in the well, and washing three times with 300 l of washing solution per well.

[0180] 5. Adding 100 l of color-developing solution to each well and allowing the plate to be still at 37 C. for 10-15 min.

[0181] 6. Adding 50 l of stopping solution to each well, and reading the absorbance at 450 nm.

[0182] The standard curve range detected by the kit is 0-10 g/ml, and the minimum detection limit is 0.10 g/ml.

[0183] In this example, by employing antibodies prepared according to Example 8, or commercial monoclonal antibodies against GAPDH, different kits are provided to detect GAPDH in a blood sample. The detection sensitivities of these kits used for different cancers are shown in the following table.

TABLE-US-00015 Detection sensitivity of Detection sensitivity of kits containing commercial kits containing antibodies Cancers antibodies prepared in Example 8 Liver cancer 60.36% 84.62% Lung cancer 58.69% 80.36% Gastric 55.42% 77.08% cancer Colorectal 64.57% 73.33% cancer Pancreatic 60.06% 79.17% cancer

[0184] The above results indicate that the kits used to detect GAPDH in a blood sample of the present invention have a good detection sensitivity.