CN118259007A - High-sensitivity detection kit for serum tumor trypsinogen-2 and application of high-sensitivity detection kit in diagnosis of cholangiocarcinoma - Google Patents

High-sensitivity detection kit for serum tumor trypsinogen-2 and application of high-sensitivity detection kit in diagnosis of cholangiocarcinoma Download PDF

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CN118259007A
CN118259007A CN202410398601.6A CN202410398601A CN118259007A CN 118259007 A CN118259007 A CN 118259007A CN 202410398601 A CN202410398601 A CN 202410398601A CN 118259007 A CN118259007 A CN 118259007A
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antibody
tat
trypsinogen
serum
serum tumor
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秦源
黄飚
王毅刚
李佳钰
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Zhejiang University Of Science And Technology Shaoxing Biomedical Research Institute Co ltd
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Abstract

The invention relates to the field of biotechnology, in particular to a high-sensitivity detection kit of serum tumor trypsinogen-2 and application thereof in bile duct cancer diagnosis. Compared with other traditional detection methods, the invention has the characteristics of high sensitivity, simple operation and better detection rate of bile duct cancer.

Description

High-sensitivity detection kit for serum tumor trypsinogen-2 and application of high-sensitivity detection kit in diagnosis of cholangiocarcinoma
Technical Field
The invention relates to the technical field of biology, in particular to a high-sensitivity detection kit of serum tumor trypsinogen-2 and application thereof in diagnosis of bile duct cancer.
Background
Bile duct cancer is a relatively common malignant tumor of the digestive system, mainly originates from cancer of liver epithelial cells or bile duct epithelial cells, and in recent years, the incidence of bile duct cancer tends to rise year by year. At present, the specific pathogenesis of bile duct cancer is not completely known, and gall stones, biliary tract-related chronic inflammation, biliary tract-related parasitic diseases and the like all increase the risk of malignant gall bladder canceration.
Early bile duct cancer lacks specific clinical symptoms, related patients often go to a hospital to visit after obstructive jaundice appears, at the moment, the patients are in middle and late stages of the cancer, the optimal treatment stage is missed, and the survival rate of the patients is low and the survival time is short. Jaundice, abdominal pain, skin itching are the major clinical manifestations of cholangiocarcinoma, with jaundice being the most important sign of the patient. Clinically, patients with yellow skin and sclera staining account for over 90% of the total cholangiocarcinoma patients. In addition, skin itch, nausea and vomiting are common manifestations of the disease, and malignant fluid and other conditions may occur in advanced cholangiocarcinoma.
At present, early diagnosis of bile duct cancer is one of the difficulties in the medical field. The clinical symptoms are hidden, the early diagnosis is difficult, the malignancy degree is high, the prognosis is poor, and the survival rate is low. Imaging examination, such as B-ultrasound, CT, nuclear magnetism, etc., cannot obtain satisfactory clinical information, and other invasive examination has high cost and poor feasibility, and is not suitable for large-scale popularization. Abnormal gene expression and substance metabolism exist in tumor cells and are continuously changed in the process of occurrence and evolution, wherein a part of protein which is expressed abnormally is secreted into tumor microenvironment and further enters blood circulation. Thus, serum-based markers are critical for early diagnosis of tumors. Because blood samples are readily available and less invasive to the patient, serological examinations are more readily accepted by a wide range of patients.
At present, the clinically common tumor markers include CA19-9, CA242, CA125, CEA and the like, and the diagnostic value of CA19-9 in malignant bile duct cancer is still controversial. CA242 is a broad-spectrum tumor marker, and has higher sensitivity but low specificity in diagnosing bile duct cancer. The concentration of CA125 in the serum of 24-47% of patients with bile duct cancer is increased, but in one domestic study, the sensitivity of CA125 is only 25% by measuring and comparing the concentration of a series of markers in the serum of patients with bile duct cancer and bile duct calculus, and the diagnosis value of CA125 is not high. CEA has a low sensitivity of only 13.24% in the diagnosis of cholangiocarcinoma, and is therefore commonly used for combined detection with other targets. Tumor markers specific for bile duct cancer screening or diagnosis have not been found.
Despite the dramatic increase in the discovery of molecular biomarkers in recent years, clinical transformation of these biomarkers remains a challenge. Therefore, the identification of novel serum markers with high sensitivity and specificity, which can be detected in combination with other markers, has important significance for diagnosis and treatment of bile duct cancer, especially early bile duct cancer, and recently finnish scientists report that trypsinogen 2 (TAT-2) is highly expressed in bile duct cancer.
The highly expressed trypsinogen-2 in tumor cells is called as tumor-associated trypsinogen-2 (TAT-2), and researches show that TAT-2 has close relation with various tumors, but the research on the relation between the serum TAT-2 level and cholangiocarcinoma is very few, and whether the TAT-2 can be used as a serum marker for diagnosing cholangiocarcinoma has not been systematically studied.
Trypsinogen is a precursor of trypsin, has a molecular weight of about 25KD, contains two main trypsinogen isozymes, namely trypsinogen-1 (cationic) and trypsinogen-2 (anionic) in pancreatic juice secreted by human pancreas, and is found to still contain trypsinogen-2 in serum samples of patients with all pancreas resections by analysis, which indicates that the expression of trypsinogen-2 is not limited to pancreas. As a result of recent studies, trypsinogen-2 has been found to be mainly distributed in tissues or organs such as skin, esophagus, stomach, small intestine, lung, kidney, liver, bile duct and prostate. Furthermore, the scholars find that the trypsinogen-2 has higher level in tumor cells of cancer patients such as gastric cancer, lung cancer, ovarian cancer and the like, so the trypsinogen-2 highly expressed in the tumor cells is called tumor-related pancreatic eggs
Bai Meiyuan-2 (TAT-2) and studies on TAT-2 were initiated. At present, TAT-2 has been found to cause hydrolysis of peri-tumor matrix proteins to promote tumor invasion. In addition, TAT-2 can also be used as a growth stimulation factor, and can be combined with a receptor on a cell so as to activate the receptor and promote cell proliferation.
Koivunen et al detected levels of TAT-2 in ovarian and colon cancer cells by immunohistochemical staining, found that TAT-2 increased the expression of proteolytic enzymes in malignant cells, indicating that TAT-2 was closely related to ovarian and colon malignancy. Hedstr6m et al measured the concentrations of TAT-2 and trypsin-2-. Alpha.1-antitrypsin (trypsin-2-AAT) in 45 cases of malignant and 61 cases of benign digestive tract disease patients, and found that the levels of TAT-2 and trypsin-2-AAT increased 46% and 42%, respectively, in malignant patients, more significantly than in benign tumor patients (p < 0.05). They have later also measured the concentrations of TAT-1, -2 and tumor-associated trypsin inhibitor (TATI) in bile in 23 patients with benign biliary tract disease, 2 patients with biliary tract cancer and 15 patients with pancreatic cancer, and found that high levels of TAT-1, -2 and TATI were present in the bile in most patients. And under normal conditions, only a small amount of TAT-2 is expressed in human blood, and when certain tissues are destroyed by malignant tumors, a large amount of TAT-2 enters the blood, so that the TAT-2 content in the blood is obviously increased. Therefore, detection of TAT-2 concentration in serum is likely to be helpful for differential diagnosis of cholangiocarcinoma, which first requires establishment of a highly sensitive TAT-2 detection method.
Although TAT-2 detection has been reported, there is no detection reagent available in the clinic, and enzyme-linked immunosorbent assay (ELISA) is the most commonly used method in published studies. The performance characteristics of this assay are suitable for clinical testing using serum or EDTA plasma samples. However, the conventional ELISA method has some defects such as poor sensitivity; enzyme-labeled compounds are unstable; enzymes are easily deactivated; the OD value of the enzyme reaction product only has a linear relation with the enzyme activity in a low concentration range, and when a high concentration of an analyte exists in a serum sample, the Hook phenomenon is serious, and the detection accuracy is reduced. Therefore, there is a need for new immunoassay techniques for highly sensitive, quantitative detection of TAT-2. In recent years, an immunoassay method is vigorously developed, wherein a time-resolved immunofluorescence analysis technology (time-resolved fluroimmunoassay, TRFIA) adopts a dissociation enhancement technology to amplify a fluorescence signal by 100 ten thousand times, and compared with the existing CLTA and ELISA detection technology, the TRFIA technology has higher sensitivity and wider measurement range, and has the advantages of stability, simplicity and convenience in operation, easiness in automation, small influence on the activity of a marked object, multiple-site marking and the like, and is very suitable for highly sensitive detection of TAT-2.
Disclosure of Invention
In order to solve the problems, the invention provides a high-sensitivity detection kit for serum tumor trypsinogen-2 and application thereof in diagnosis of bile duct cancer, and the kit has the advantages of high sensitivity, simplicity in operation and better detection rate for bile duct cancer.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
A high-sensitivity detection kit for serum tumor trypsinogen-2 adopts rare earth ion Eu to mark TAT-2 antibody, captures antigen through TAT-2 antibody on a stationary phase coated plate, combines with europium label antibody to form solid phase antibody-antigen-europium label antibody sandwich immune complex, amplifies fluorescent signal 100 ten thousand times through enhancement liquid, and detects by a time resolution immunofluorescence analyzer to obtain fluorescent intensity in a standard substance and a sample pore plate, thereby calculating and obtaining the content of TAT-2 in a corresponding sample.
Further, the solid phase antibody-antigen-europium label antibody sandwich immune complex is prepared by the following steps:
Preparation of solid phase antibody: diluting recombinant TAT-2 antibody with 100mmol/L NaCO 3-NaHCO3 buffer solution with pH of 9.6 to obtain coating solution with concentration of 1-10 μg/mL, adding 100 μl of coating solution into each well of 96 microwell plate, standing for 2h, removing the sealing solution, vacuum pumping, sealing, and freeze preserving at-20deg.C;
Preparation of Eu-TAT-2 antibody: adding 300 mug of TAT-2 antibody into Ultracel-50K ultrafiltration tube, converting buffer storage condition with marking buffer, mixing with 100 mug of Eu 3+ -DTTA chelate, shaking culturing overnight at 28 ℃, performing Sephadex-G50 column chromatography balanced with eluent containing 0.2% BSA, detecting fluorescence value with full-automatic time-resolved fluorescence immunoassay analyzer, collecting protein peak, and sub-packaging purified antibody and storing at-20deg.C.
Further, the blocking solution contains 5g/L BSA, 8g/L NaCl, 0.2g/L KH 2PO4、2.8g/L Na2HPO4·12H2 O and 1g/L trehalose.
Further, the labeling buffer is Tris-HCl solution containing 1% trehalose and having a concentration of 0.05 mol/L.
Further, the concentration of the standard substance is based on the detection results of the existing serum samples of patients and normal persons, and the minimum value and the maximum value of the standard point are set, so that the concentration values of the detected samples are contained in the range of the set standard point.
The high-sensitivity detection kit of serum tumor trypsinogen-2 can be used for diagnosing bile duct cancer, and comprises the following steps:
Adding 100 mu L of standard substance or serum sample into each hole in a TAT-2 antibody solid-phase coating plate for reaction, adding 200 mu L of reaction buffer solution at the same time, and incubating for 1h at room temperature; washing the plate for 2 times, adding 50 mu L of Eu-TAT-2 antibody diluted by an analysis buffer solution into each hole, carrying out oscillation reaction for 30min at 37 ℃, and washing the plate for 6 times; finally, adding 100 mu L of enhancement solution into each hole, oscillating for 2-5min, and detecting by a time-resolved immunofluorescence analyzer to obtain the fluorescence intensity of the standard substance and the sample pore plate, thereby calculating and obtaining the TAT-2 content in the corresponding sample.
The invention provides a new detection method for bile duct cancer diagnosis, and has the following beneficial effects compared with other traditional detection methods:
1) The detection method has the characteristics of high sensitivity, simplicity in operation and better detection rate for bile duct cancer.
2) The effective period of the reagent reaches more than 1 year by adding the stabilizing agent, so that the intra-batch and inter-batch differences are effectively reduced, and the result of the curative effect evaluation is more comparable.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a working principle diagram of a high-sensitivity detection kit for serum tumor trypsinogen-2 according to an embodiment of the invention.
FIG. 2 is a schematic diagram showing the specificity of an embodiment of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
The invention relates to a high-sensitivity detection kit for serum tumor trypsinogen-2, which adopts rare earth ion Eu to mark TAT-2 antibody, captures antigen through TAT-2 antibody on a stationary phase coated plate, combines with europium label antibody to form solid phase antibody-antigen-europium label antibody sandwich immune complex, amplifies fluorescent signal 100 ten thousand times through enhancement solution, and detects by a time resolution immunofluorescence analyzer to obtain fluorescent intensity in a standard substance and a sample pore plate, thereby calculating and obtaining the content of TAT-2 in a corresponding sample, and the principle is shown in figure 1.
Specific:
Preparation of solid phase antibody: diluting recombinant TAT-2 antibody with 100mmol/L NaCO 3-NaHCO3 buffer solution with pH of 9.6 to obtain coating solution with concentration of 5 μg/mL, adding 100 μl of coating solution into each well of 96 microwell plate, standing overnight at 4deg.C, discarding coating solution, washing for 1 time, adding 200 μl of sealing solution (containing 5g/L BSA, 8g/L NaCl, 0.2g/L KH 2PO4、2.8g/L Na2HPO4·12H2 O and lg/L trehalose) into each well, standing for 2h, discarding sealing solution, vacuum pumping, sealing, and freeze preserving at-20deg.C;
Preparation of Eu-TAT-2 antibody: 300 μg of TAT-2 antibody was added to Ultracel-50K ultrafilter tube, washed repeatedly with labelling buffer (Tris-HCl solution containing 1% trehalose at 0.05 mol/L) for 6 times, mixed with 100 μg of Eu 3 + -DTTA chelate complex, shake-cultured overnight at 28deg.C, sephadex-G50 column chromatography equilibrated with eluent containing 0.2% BSA, the effluent (1 mL/tube) was collected, the fluorescence was detected tube by tube using AutoDelfia 1235 full-automatic time resolved fluorescence immunoassay system, and protein peaks were collected, and the purified antibody was sub-packaged and stored at-20deg.C.
Selection of standard concentration: the minimum value and the maximum value of standard points are set based on the detection results of the existing serum samples of patients and normal persons, so that the measured concentration values of the samples are contained in the range of the set standard points. Specifically, based on the detection results of existing patient and normal human serum samples loaded in the data mining module, the minimum value and the maximum value of the corresponding standard points are obtained, so that the measured sample concentration values are contained in the range of the set standard points.
The high-sensitivity detection kit of serum tumor trypsinogen-2 can be used for diagnosing bile duct cancer, and comprises the following steps:
Adding 100 mu L of standard substance or serum sample into each hole in a TAT-2 antibody solid-phase coating plate for reaction, adding 200 mu L of reaction buffer solution at the same time, and incubating for 1h at room temperature; washing the plate for 2 times, adding 50 mu L of Eu-TAT-2 antibody diluted by an analysis buffer solution into each hole, carrying out oscillation reaction for 30min at 37 ℃, and washing the plate for 6 times; finally, adding 100 mu L of Eu 3+ fluorescence enhancement solution (PERKIN ELMER company) into each hole, oscillating for 2-5min, detecting by a AutoDelfia 1235 full-automatic time-resolved fluorescence immunoassay system, obtaining the fluorescence intensities in the standard substance and the sample pore plate, calculating to obtain the content of TAT-2 in the corresponding sample, and evaluating the content of TAT-2 based on the minimum value and the maximum value of the set standard point.
Clinical data:
9 of 14 clinical cholangiocarcinoma patients have obviously increased TAT-2, and compared with normal patients, TAT-2 has higher positive rate, reaching 64.29 percent. While other tumor markers were negative, TAT-2 was detected with 64.3% sensitivity and 100% specificity by ROC curve analysis (FIG. 2).
TABLE 1 comparison of serum TAT-2 levels in healthy and cholangiocarcinoma patients
Note that: m: a median; QL-QU: quartile spacing.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims (7)

1. A high-sensitivity detection kit for serum tumor trypsinogen-2 is characterized in that: the kit adopts rare earth ion Eu to mark TAT-2 antibody, captures antigen through TAT-2 antibody on a stationary phase coated plate, combines with europium label antibody to form solid phase antibody-antigen-europium label antibody sandwich immune complex, amplifies fluorescence signal 100 ten thousand times through enhancement liquid, and detects by a time resolution immunofluorescence analyzer to obtain fluorescence intensity in a standard substance and a sample pore plate, thereby calculating and obtaining the content of TAT-2 in a corresponding sample.
2. A high sensitivity test kit for serum tumor trypsinogen-2 according to claim 1, wherein: the solid phase antibody-antigen-europium label antibody sandwich immune complex is prepared by the following steps:
Preparation of solid phase antibody: diluting recombinant TAT-2 antibody with 100mmol/L NaCO 3-NaHCO3 buffer solution with pH of 9.6 to obtain coating solution with concentration of 1-10 μg/mL, adding 100 μl of coating solution into each well of 96 microwell plate, standing for 2h, removing the sealing solution, vacuum pumping, sealing, and freeze preserving at-20deg.C;
Preparation of Eu-TAT-2 antibody: adding 300 mug of TAT-2 antibody into Ultracel-50K ultrafiltration tube, converting buffer storage condition with marking buffer, mixing with 100 mug of Eu 3+ -DTTA chelate, shaking culturing overnight at 28 ℃, performing Sephadex-G50 column chromatography balanced with eluent containing 0.2% BSA, detecting fluorescence value with full-automatic time-resolved fluorescence immunoassay analyzer, collecting protein peak, and sub-packaging purified antibody and storing at-20deg.C.
3. A high sensitivity test kit for serum tumor trypsinogen-2 according to claim 2, wherein: the blocking solution contains 5g/L BSA, 8g/L NaCl, 0.2g/L KH 2PO4、2.8g/L Na2HPO4·12H2 O and 1g/L trehalose.
4. A high sensitivity test kit for serum tumor trypsinogen-2 according to claim 2, wherein: the labeling buffer solution is Tris-HCl solution containing 1% trehalose and having a concentration of 0.05 mol/L.
5. A high sensitivity test kit for serum tumor trypsinogen-2 according to claim 1, wherein: the concentration of the standard substance is based on the detection results of the existing serum samples of patients and normal persons, and the minimum value and the maximum value of the standard point are set, so that the concentration values of the measured samples are contained in the range of the set standard point.
6. Use of a highly sensitive detection kit for serum tumor trypsinogen-2 according to claims 1-5 for diagnosis of cholangiocarcinoma.
7. The use of a highly sensitive test kit for serum tumor trypsinogen-2 according to claim 6, wherein: the method comprises the following steps:
Adding 100 mu L of standard substance or serum sample into each hole in a TAT-2 antibody solid-phase coating plate for reaction, adding 200 mu L of reaction buffer solution at the same time, and incubating for 1h at room temperature; washing the plate for 2 times, adding 50 mu L of Eu-TAT-2 antibody diluted by an analysis buffer solution into each hole, carrying out oscillation reaction for 30min at 37 ℃, and washing the plate for 6 times; finally, adding 100 mu L of enhancement solution into each hole, oscillating for 2-5min, and detecting by a time-resolved immunofluorescence analyzer to obtain the fluorescence intensity of the standard substance and the sample pore plate, thereby calculating and obtaining the TAT-2 content in the corresponding sample.
CN202410398601.6A 2024-04-02 2024-04-02 High-sensitivity detection kit for serum tumor trypsinogen-2 and application of high-sensitivity detection kit in diagnosis of cholangiocarcinoma Pending CN118259007A (en)

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