CN111579787B - A test strip for early esophageal squamous cell carcinoma screening - Google Patents

Abstract

The invention belongs to the field of medical biotechnology, and discloses a test strip for early esophageal squamous cell carcinoma screening. The test strip includes a binding pad and a chromatography pad, the binding pad is coated with a capture antibody, and the capture antibody is MPST antibody A A mixture of , NME1 antibody A, MPST antibody A, and NME1 antibody A with detectable markers; there are two detection lines and a quality control line on the chromatography pad, and detection antibodies are immobilized on the detection lines. The detection antibodies immobilized on each detection line are MPST antibody B, NME1 antibody B, and antibody A and antibody B of the same antigen recognize different epitopes of the antigen; goat anti-mouse IgG is immobilized on the quality control line. The test strip of the invention can rapidly detect MPST protein and NME1 protein in human serum, realize the combined detection of two tumor markers, and can effectively detect esophageal squamous cell carcinoma, especially early esophageal squamous cell carcinoma according to the detection result, which greatly improves the early stage Detection rate of esophageal squamous cell carcinoma.

Description

A test strip for early esophageal squamous cell carcinoma screening

technical field

The invention belongs to the field of medicine and biotechnology, and in particular relates to a test strip for early esophageal squamous cell carcinoma screening.

Background technique

Esophageal squamous cell carcinoma (ESCC) is a squamous cell carcinoma that occurs in human esophageal epithelial tissue, and is a malignant tumor of the digestive tract, which is extremely common in clinical practice. Esophageal squamous cell carcinoma will not only reduce people's quality of life, endanger people's life and health, but also bring heavy disasters to the patient's family and a heavy burden to the country. Esophageal squamous cell carcinoma is a common malignant tumor of the digestive system in the world, and there are typical high-incidence areas of esophageal squamous cell carcinoma all over the world. The high-incidence areas of esophageal squamous cell carcinoma in China have prominent aggregation characteristics. Over the years, many typical high-incidence areas of esophageal squamous cell carcinoma have been discovered, such as Linxian in Henan, Cixian in Hebei, Feicheng in Shandong, and Yangquan in Shanxi. Esophageal squamous cell carcinoma has high morbidity and mortality in China. In recent years, relevant medical studies have shown that, among malignant tumors, esophageal squamous cell carcinoma ranks fifth in incidence and fourth in mortality, after lung cancer, liver cancer, and gastric cancer.

The main reason for the high mortality rate of esophageal squamous cell carcinoma at present is the lack of patients suitable for early diagnosis of esophageal squamous cell carcinoma and asymptomatic people (some patients with early esophageal squamous cell carcinoma have been confirmed to have atypical hyperplasia or early cancer by endoscopic biopsy, but patients do not There is no discomfort, this population is called esophageal squamous cell carcinoma asymptomatic population) detection of economical, efficient and sensitive screening indicators and methods, which leads to 80% of the first visit patients are middle-advanced patients, despite active surgical treatment, However, the prognosis is still poor, with a 5-year survival rate of only about 10%.

At present, the most common method for early screening of esophageal squamous cell carcinoma is esophageal endoscopy, but due to the trauma, high cost and low efficiency of endoscopic screening (for example, routine high-risk endoscopic screening is described in the above "asymptomatic population", early The detection rate of esophageal squamous cell carcinoma is only about 2%, and more than 90% of "asymptomatic people" are "accompanying examinations"), which limits the promotion and application of endoscopic screening in the early detection of esophageal squamous cell carcinoma in asymptomatic people. Therefore, the discovery of a new efficient, convenient and non-invasive method for early screening of asymptomatic people with esophageal squamous cell carcinoma can not only effectively improve the efficiency of early screening of esophageal squamous cell carcinoma, reduce the difficulty of large-scale screening, and further improve the survival time of esophageal squamous cell carcinoma patients. Improve the prognosis, and can provide new ideas for the prevention and treatment of esophageal squamous cell carcinoma.

Tumor markers are abnormally expressed biologically active substances produced by tumor tissues and cells, and can identify or diagnose tumors according to their biochemical post-immune properties. Although some tumor markers commonly used in clinic, such as CA125 (cancer antigen 125), CA199 (cancer antigen 199), CEA (carcinoembryonic antigen) and SCCA (squamous cell carcinoma antigen), can be used for the diagnosis of esophageal squamous cell carcinoma, Its sensitivity and specificity are not high, especially the diagnostic value of early esophageal squamous cell carcinoma is not high enough. On the basis of the genomics database established by whole genome association analysis, whole genome sequencing, whole genome exome sequencing and other technologies in the previous stage, the research team used protein chip technology to analyze patients with early esophageal squamous cell carcinoma, asymptomatic high-risk groups in high-incidence areas, and high-risk groups. Normal human serum was detected and analyzed, and two tumor serum markers, MPST protein and NME1 protein, which were differentially expressed in esophageal squamous cell carcinoma patients and normal people, were screened out.

3-Mercaptopyruvate sulfurtransferase (3-Mercaptopyruvate sulfurtransferase, MPST or 3-MST) consists of 297 amino acids and mainly exists in the cytoplasm. Usually MPST exists as a monomer or a disulfide-linked homodimer , whose function is to catalyze the transfer of sulfide ions to select thiol compounds (such as cyanide), and participate in cyanide detoxification and homocysteine interpretation. The study showed that neuroblastoma SH-SY5Y cells were used as the research object to observe the role of telangiectasia ataxia mutant gene (ATM) in NaAsO2-induced SH-SY5Y cell cycle changes, and exogenously overexpressed SH-SY5Y cells. MPST protein, observe the effect of MPST overexpression in NaAsO2 on neuronal cell growth and cycle changes, it can be observed that MPST overexpression will affect cell growth cycle, in the latest research in our laboratory, there is evidence that MPST overexpression affects Atypical hyperplasia of esophageal squamous cells, the advanced stage of atypical hyperplasia is esophageal squamous cell carcinoma. This shows that MPST is expected to become the latest powerful indicator for early screening of asymptomatic people with esophageal squamous cell carcinoma.

The non-metastatic cells (NME) gene family, formerly known as nm23 (non-metastaticclone 23), as an NME protein-encoding gene, was originally isolated from mouse melanoma cells and was the first tumor metastasis inhibitory related gene discovered. Gene. In yeast, the nucleoside diphosphate kinase (NDPK) responsible for the transfer of phosphate groups from nucleoside triphosphates to nucleoside diphosphates was described for the first time. Protein sequences encoding putative or experimentally validated NDPK activities were subsequently identified. To date, 10 genes containing partial or complete NDPK domains, namely NME family members, have been identified in humans. The proteins of this family can be divided into two categories according to sequence characteristics and NDPK activity. Type Ⅰ NME proteins (NME1, NME2, NME3, NME4) present a particularly conserved region and active site, and type Ⅱ NME proteins (NME5, NME6, NME7, NME8, NME9) present highly diverse domains. Although the structures of NME1, NME2 and NME3 are similar, there are certain differences in their structures. It is worth noting that studies have shown that low expression of NME1 is associated with poor prognosis, poor survival, and lymph node infiltration in patients. Histopathological indicators of tumors.

MPST protein and NME1 protein are lowly expressed in normal human serum, and highly expressed in the serum of patients with esophageal squamous cell carcinoma. Furthermore, further statistical tests found that the positive rates of MPST protein and NME1 protein in the population of patients with esophageal squamous cell carcinoma were significantly higher than In the normal population, there are significant differences. Therefore, MPST protein and NME1 protein can be used for the diagnosis of esophageal squamous cell carcinoma. In response to this discovery, the research team used MPST protein and NME1 protein as detection indicators to prepare products for early esophageal squamous cell carcinoma detection, which were used for early esophageal squamous cell carcinoma screening (non-invasive, simple, economy), in order to improve the early detection rate of esophageal squamous cell carcinoma, improve the quality of life of patients, and reduce the burden on families and society.

SUMMARY OF THE INVENTION

In view of the problems and deficiencies in the prior art, one of the objectives of the present invention is to provide a marker for early esophageal squamous cell carcinoma screening, and the second objective of the present invention is to provide a marker for early esophageal squamous cell carcinoma Application of the detection reagent for the marker of cancer screening, the third object of the present invention is to provide a test strip for early esophageal squamous cell carcinoma screening.

For realizing the purpose of the invention, the technical scheme adopted in the present invention is as follows:

The present invention first provides a marker for early esophageal squamous cell carcinoma screening, wherein the marker is any one of MPST protein and NME1 protein or a combination of the two.

The present invention also provides an application of the above-mentioned marker detection reagent in preparing a product for early esophageal squamous cell carcinoma screening.

According to the above application, preferably, the detection reagent is an antibody that specifically binds to the marker. More preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

According to the above application, preferably, the product detects the marker in the sample by immunochromatography or enzyme-linked immunosorbent assay.

According to the above application, preferably, the sample is serum.

According to the above application, preferably, the product is a test strip or a kit.

The present invention also provides a test strip for early esophageal squamous cell carcinoma screening, the test strip includes a binding pad and a chromatography pad, the binding pad is coated with a capture antibody, and the capture antibody is MPST antibody A. A mixture of NME1 antibody A, MPST antibody A, and NME1 antibody A with detectable labels; two detection lines and a quality control line are arranged on the chromatography pad, and detection lines are fixed on the detection lines Antibodies, the two detection lines immobilized detection antibodies are MPST antibody B and NME1 antibody B respectively, and the same antigen antibody A and antibody B recognize different epitopes of the antigen; goat anti-mouse IgG is immobilized on the quality control line.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, the marker is colloidal gold particles. More preferably, the diameter of the colloidal gold particles ranges from 25 to 35 nm.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, the MPST antibody A and the NME1 antibody A are MPST monoclonal antibody and NME1 monoclonal antibody, respectively.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, MPST antibody B and NME1 antibody B are MPST polyclonal antibody and NME1 polyclonal antibody, respectively.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, the test strip further comprises a sample pad, a sample suction pad and a bottom plate, the sample pad, the binding pad, the chromatography pad and the sample suction pad One end of the binding pad is pressed under the sample pad, and the other end of the binding pad is pressed above the chromatography pad; one end of the chromatography pad is pressed under the binding pad, and the other end of the chromatography pad Press down on the suction pad.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, the mass ratio of MPST monoclonal antibody to NME1 monoclonal antibody in the capture antibody is 1:1.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, the two detection lines and one quality control line on the chromatography pad are arranged in the following order: One end of the pad is followed by a quality control line C coated with goat anti-mouse IgG, a detection line T2 with an MPST polyclonal antibody immobilized, and a detection line T1 with an NME1 polyclonal antibody immobilized.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, the interval between the detection lines T1, T2 and the quality control line C on the chromatography pad is not less than 5 mm.

According to the above-mentioned test strip for early esophageal squamous cell carcinoma screening, preferably, the material of the sample pad is absorbent glass fiber, the material of the binding pad is glass fiber membrane, and the material of the chromatography pad is Nitrocellulose membrane; the material of the suction pad is absorbent paper or absorbent glass fiber membrane; the bottom plate is PVC bottom plate, cardboard or hard fiberboard.

The present invention also provides a kit comprising the above-mentioned test strip for early esophageal squamous cell carcinoma screening.

According to the above-mentioned kit, preferably, the kit further comprises a pipette, a serum sample cup and a disposable syringe.

The method of using the test strip for early esophageal squamous cell carcinoma screening of the present invention is:

Drop the serum sample to be tested on the sample pad of the test strip or insert the sample pad of the test strip into the serum sample to be tested, observe the color change of the detection line and the quality control line within 5-15 minutes after removing the test strip, and record the results.

Wherein, the serum sample can be diluted with a sample diluent before detection. Wherein, the sample diluent is 0.02M PBS buffer containing 0.9% NaCl, 0.1% Tween-20 and 0.1% BSA.

The method for judging the results of the combined screening test strip for early esophageal squamous cell carcinoma of the present invention is as follows:

Positive result: if at least one of the test lines of the test strip T1 and T2 develops color, and the quality control line C develops color, the test result is judged to be positive.

Negative result: The T1 and T2 test lines on the test strip chromatography pad did not develop color, only the quality control line C developed color, and the test result was judged to be negative.

Invalid result: The quality control line C has no color, and the test result is judged to be invalid and needs to be re-tested.

The detection principle of the early esophageal squamous cell carcinoma combined screening test strip of the present invention is as follows:

When the serum sample to be tested is dripped on the sample pad of the test strip, the serum sample to be tested will flow to the direction of the suction pad under the capillary action of the water-absorbing material of the suction pad, and when it flows onto the binding pad, the binding pad will be wrapped It is dissolved by colloidal gold-labeled MPST monoclonal antibody and colloidal gold-labeled NME1 monoclonal antibody, wherein the colloidal gold-labeled MPST monoclonal antibody is combined with the MPST antigen (MPST protein) that may be contained in the serum sample to form colloidal gold-labeled MPST monoclonal Antibody-MPST antigen complex, colloidal gold-labeled NME1 monoclonal antibody binds to NME1 antigen (NME1 protein) that may be contained in serum samples to form colloidal gold-labeled NME1 monoclonal antibody-NME1 antigen complex; due to capillary effect, two colloidal The gold-labeled capture antibody-antigen complex moves on the chromatography pad, and when it moves to the detection line T1, the colloidal gold-labeled NME1 monoclonal antibody-NME1 antigen complex specifically binds to the NME1 polyclonal antibody to form a solidified immune complex , was trapped on the detection line T1; when moving to the detection line T2, the colloidal gold-labeled MPST monoclonal antibody-MPST antigen complex specifically combined with the MPST polyclonal antibody to form a solidified immune complex, which was trapped in the detection line On T2; the free colloidal gold-labeled capture antibody continues to move forward due to the capillary effect, binds with the goat anti-mouse IgG coated on the quality control line C and is trapped on the quality control line; the excess unbound material is Continue to move to the suction pad under capillary action. Therefore, the MPST and NME1 tumor-associated antigens in the sample to be tested can be qualitatively determined by the color development of the detection lines T1 and T2 and the color development of the quality control line.

Since the expression abundance of tumor-associated antigens in serum is proportional to the expression abundance of their corresponding autoantibodies, the high expression of tumor-associated antigens will also lead to high expression of their corresponding autoantibodies. Therefore, MPST protein and NME1 protein can also be used as detection reagents to detect the autoantibodies corresponding to MPST protein and NME1 protein in serum, and the autoantibodies of the two tumor-associated antigens can be used as the diagnosis and screening indicators of esophageal squamous cell carcinoma. .

Compared with the prior art, the positive beneficial effects obtained by the present invention are:

(1) The present invention uses MPST protein and NME1 protein for early screening and detection of esophageal squamous cell carcinoma for the first time. By detecting the expression levels of MPST protein and NME1 protein in human serum, esophageal squamous cell carcinoma can be effectively detected, especially in the early stage. Esophageal squamous cell carcinoma; moreover, when MPST protein and NME1 protein are used as a combination for screening and detection of early esophageal squamous cell carcinoma, the detection sensitivity is as high as 86.3% (that is, these two proteins are correctly diagnosed in patients with early esophageal squamous cell carcinoma). The rate of diagnosis of early esophageal squamous cell carcinoma was 86.3%), and the specificity reached 83.1% (that is, the rate of non-esophageal squamous cell carcinoma patients diagnosed with these two proteins was 83.1%) Therefore, the marker of the present invention has high sensitivity and specificity, greatly improves the detection rate of early esophageal squamous cell carcinoma, and the detection rate of esophageal squamous cell carcinoma is much higher than the existing clinical endoscopic screening The detection rate of esophageal squamous cell carcinoma can be used for large-scale screening of asymptomatic people in high-risk areas of esophageal squamous cell carcinoma, which is conducive to the early detection of asymptomatic high-risk groups of esophageal squamous cell carcinoma, thereby greatly reducing the mortality rate of esophageal squamous cell carcinoma patients. Squamous cell carcinoma patients and families bring great benefits.

(2) The test strip of the present invention can rapidly detect MPST protein and NME1 protein in human serum, realizing the combined detection of two tumor markers, and the test strip has high sensitivity and specificity. The high detection accuracy of cancer has greatly improved the detection rate of early esophageal squamous cell carcinoma, which is conducive to the early detection of asymptomatic high-risk groups of esophageal squamous cell carcinoma, and also provides important information for long-term tracking of asymptomatic high-risk groups in high-risk areas of esophageal squamous cell carcinoma. The detection method has broad market prospects and social benefits.

(3) The test strip used for early esophageal squamous cell carcinoma screening of the present invention is easy to operate, easy to use, and takes a short time to detect the result. It only needs to insert its test end into the sample liquid to be tested for about 10s, and then within 15min The detection results can be determined, which greatly improves the diagnosis efficiency of early esophageal squamous cell carcinoma.

(4) The test strip used for early esophageal squamous cell carcinoma screening of the present invention does not require other instruments and reagents, and non-professionals can also detect at any time, which can greatly reduce the detection cost and detection cost, and the human operation error is small and stable good sex.

(5) The detection sample of the test strip used for early esophageal squamous cell carcinoma screening of the present invention is serum, which requires less blood, less suffering for the masses, and high acceptance.

Description of drawings

FIG. 1 is a schematic structural diagram of a test strip for early esophageal squamous cell carcinoma screening prepared in Example 1 of the present invention.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments and accompanying drawings. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Tumor markers are abnormally expressed biologically active substances produced by tumor tissues and cells, and can identify or diagnose tumors according to their biochemical post-immune properties. On the basis of the genomics database established by whole genome association analysis, whole genome sequencing, whole genome exome sequencing and other technologies in the previous stage, the research team used protein chip technology to analyze patients with early esophageal squamous cell carcinoma, asymptomatic high-risk groups in high-incidence areas, and high-risk groups. Normal human serum was detected and analyzed, and two tumor serum markers, MPST protein and NME1 protein, which were differentially expressed in esophageal squamous cell carcinoma patients and normal people, were screened out. MPST protein and NME1 protein are lowly expressed in normal human serum, and highly expressed in the serum of patients with esophageal squamous cell carcinoma. Furthermore, further statistical tests found that the positive rates of MPST protein and NME1 protein in the population of patients with esophageal squamous cell carcinoma were significantly higher than In the normal population, there are significant differences. This shows that MPST protein and NME1 protein can be used for the diagnosis of esophageal squamous cell carcinoma. In response to this finding, the research team used MPST protein and NME1 protein as detection indicators to prepare test strips for the detection of early esophageal squamous cell carcinoma, which were used to detect the expression levels of MPST protein and NME1 protein in the sample, so as to realize the early screening of esophageal squamous cell carcinoma. check.

The experimental methods described in the following examples, unless otherwise specified, are conventional techniques in this technical field, or in accordance with the conditions suggested by the manufacturer; the reagents, materials and instruments used are not marked with the manufacturer, all can be purchased through the market. Regular product obtained.

Example 1: Preparation of test strips

1. Experimental materials

The biologically active raw materials used in the present invention are all commercially available products. Among them, MPST monoclonal antibody, NME1 monoclonal antibody, MPST polyclonal antibody, and NME1 polyclonal antibody were purchased from Wuhan Ai Meijie Biotechnology Co., Ltd., the product numbers are: H00004357-M01, H00004830-M01, H00004357-B01, H00004830- A01.

PVC bottom plate, nitrocellulose membrane, glass fiber membrane, absorbent paper, etc. are all existing commercial products.

2. Preparation of binding pads

(1) Preparation of colloidal gold:

The colloidal gold solution with a diameter of 25-35 nm was prepared by trisodium citrate reduction method. Add 300 mL of 0.01% chloroauric acid solution to the cleaned flask, and heat to boiling. Quickly add 4.8 mL of 0.1% trisodium citrate aqueous solution under magnetic stirring, continue to heat and boil for 15 min, until the color gradually stabilizes to a translucent wine red. The flask was cooled at room temperature, restored to its original volume with distilled water, and stored at 4°C.

(2) Preparation of colloidal gold-labeled MPST monoclonal antibody and colloidal gold-labeled NME1 monoclonal antibody:

Adjust the pH of the colloidal gold solution to 8.0 with 0.1M potassium carbonate solution, add MPST monoclonal antibody solution and NME1 monoclonal antibody solution to the colloidal gold solution, mix well, leave at room temperature for 15 minutes, add stabilizer (10% BSA solution) After mixing, let stand for 10min at room temperature, centrifuge at 12000rpm for 30min, discard the supernatant, and resuspend the precipitate with buffer to obtain a mixed solution of colloidal gold particle-labeled MPST monoclonal antibody and colloidal gold particle-labeled NME1 monoclonal antibody (the mixed solution). The mass ratio of MPST monoclonal antibody and NME1 monoclonal antibody in the solution is 1:1), that is, the capture antibody solution; wherein, the buffer is PBS containing 1% BSA and 5% sucrose at 0.01mol/L pH8.0 buffer (pH 8.0).

(3) Preparation of the binding pad:

The glass fiber membrane was selected as the bonding pad material, and the glass fiber membrane was soaked in the pretreatment solution (pretreated as borate buffer containing 2% BSA, 3% sucrose, 0.6M NaCl, 0.2% Tween-20) for 10 minutes. ～15min, then dried at 36～38℃ or vacuum freeze-dried; the pretreated glass fiber membrane was immersed in the above capture antibody solution, fully soaked, taken out, freeze-dried to obtain a binding pad.

3. Preparation of chromatography pads

A nitrocellulose membrane was selected as the chromatography pad material, and the positions of two detection lines T1, T2 and one quality control line C were marked on the nitrocellulose membrane, with an interval of 6 mm between them. Dilute the NME1 polyclonal antibody and MPST polyclonal antibody to 1 mg/mL, and dilute the goat anti-mouse IgG to 2 mg/mL, and use a microtome to make two detection lines T1, T2 and quality control lines on the nitrocellulose membrane respectively. At the position of C, scribe the line at a dosage of 0.1-0.5 μL/mm, and dry it at 37 °C overnight; then immerse the nitrocellulose membrane in 0.01 mol/L PBS buffer (pH 8.0) containing 1% BSA, and then remove the nitric acid The cellulose membrane was washed with PBS buffer and dried to obtain a chromatography pad.

4. Sample pad preparation

The material of the sample pad is glass fiber membrane, soak the glass fiber membrane in the sample pad sealing solution for 30 minutes, and dry at 37°C to obtain the sample pad; wherein the sample pad sealing solution contains 1% BSA, 1.0% to 2.0% sucrose , 0.1%-0.5% Tween-20, 0.1-1.0% PVP polyvinylpyrrolidone in 0.01mol/L PBS buffer (pH=7.4).

5. Test strip assembly

The test strip is composed of a sample pad, a binding pad, a chromatography pad, a sample suction pad and a bottom plate, and the material of the sample suction pad is absorbent filter paper.

The chromatography pad is attached to the middle of the bottom plate, which is the detection area and the quality control area, that is, the detection lines T1, T2 and the quality control line C are set on the chromatography pad to interpret the results; the right end of the chromatography pad (upper section) is a handheld The left end of the chromatography pad is fixed with a binding pad, which is coated with colloidal gold-labeled MPST monoclonal antibody and NME1 monoclonal antibody; the binding pad and the aspirating pad It overlaps with the two ends of the chromatography pad respectively; the end (lower section) of the binding pad away from the chromatography pad is fixed with a sample pad, which is used to contact the sample to be tested. After processing, a test strip for early esophageal squamous cell carcinoma screening was obtained (see Figure 1).

6. How to use the test strip

Drop the serum sample to be tested on the sample pad of the test strip, lay the test strip flat, observe the color change of the detection line and the quality control line within 5-15 minutes, and record the results.

7. Determination of test results of test strips

Positive result: if at least one of the test lines of the test strip T1 and T2 develops color, and the quality control line C develops color, the test result is judged to be positive.

Negative result: The T1 and T2 test lines on the test strip chromatography pad did not develop color, only the quality control line C developed color, and the test result was judged to be negative.

Invalid result: The quality control line C has no color, and the test result is judged to be invalid and needs to be re-tested.

8. Precautions for test strip detection

(1) The sample to be tested is detected at room temperature (about 20°C).

(2) The tested samples are required to be fresh samples. The samples were collected and tested within 1 hour to ensure the reliability of the results.

(3) This test strip is only for in vitro detection and screening, and cannot be used as a confirmation reagent. If a positive result is obtained, further upper gastrointestinal endoscopy must be carried out.

(4) The observation result after 30min is invalid.

(5) The test strip should be kept away from light.

Example 2: Analysis of the diagnostic value of test strips

The test strip prepared in Example 1 of the present invention was used to detect the serum samples of patients with early esophageal squamous cell carcinoma and normal people diagnosed by pathology, and to evaluate and analyze the value of the test strip of the present invention for screening and diagnosis of early esophageal squamous cell carcinoma.

1. Sample source

Serum samples were collected from the First Affiliated Hospital of Zhengzhou University Henan Provincial Key Laboratory of Esophageal Squamous Cell Carcinoma, including 160 serum samples from normal people (control group) and 160 serum samples from patients with early esophageal squamous cell carcinoma (esophageal squamous cell carcinoma group). Among the 160 normal subjects, there were 83 males and 77 females, with an average age of 55.8±9.1 years and an age range of 39-80 years. Among the 160 patients with esophageal squamous cell carcinoma, there were 86 males and 74 females, with an average age of 58.9±7.3 years and an age range of 48-80 years. All esophageal squamous cell carcinoma sera were collected when the patients were initially diagnosed with early-stage esophageal squamous cell carcinoma and had not received any chemoradiotherapy, and were diagnosed between January 2017 and December 2018. Normal human serum was obtained from outpatient gastroscopy and pathologically confirmed non-cancerous or non-precancerous healthy people.

2. Experimental method

The test strips prepared in Example 1 of the present invention were used to detect the serum samples of the esophageal squamous cell carcinoma group and the precancerous lesion group respectively. The specific detection method was as follows: drop 100 μL of the serum sample to be tested on the sample pad of the test strip, Lay the test strip flat, observe the color change of the detection line and the quality control line within 5 to 15 minutes, and record the results.

At the same time, in order to compare the detection performance with the test strip prepared in Example 1 of the present invention, two kinds of comparative test strips were prepared in the present invention, and the detection indexes of the two kinds of comparative test strips were MPST protein and NME1 protein respectively. Two kinds of comparative test strips were used to detect serum samples of esophageal squamous cell carcinoma group and precancerous lesion group respectively.

According to the judgment criteria of the results, the positive rates of these two antigens in the esophageal cancer group and the control group were calculated respectively (the number of positive objects detected in each group divided by the total number of detected objects in this group is the positive rate); using spss26 .0 software was used for statistical test, and the two independent samples chi-square test method was used to compare the positive rate of antigen in the esophageal squamous cell carcinoma group and the control group. The diagnostic value of autoantibodies in detecting esophageal squamous cell carcinoma was evaluated by the method of detection test (Table 1).

3. Analysis of results

According to the test results of the test strips, the screening and diagnosis value of esophageal squamous cell carcinoma was evaluated, and the results are shown in Table 1.

Table 1 Authenticity evaluation of the diagnostic value of different antigen combinations for esophageal squamous cell carcinoma

It can be seen from Table 1 that the positive rates of MPST protein and NME1 protein in the esophageal squamous cell carcinoma group were 57.5% and 61.9%, respectively, and the positive rates of MPST protein and NME1 protein in the control group were 10.0% and 9.4%, respectively. The positive rates in the esophageal squamous cell carcinoma group were higher than those in the control group, and the difference between the esophageal squamous cell carcinoma group and the control group was statistically significant (P<0.05). It can be seen that MPST protein and NME1 protein can be used as indicators for the diagnosis and detection of early esophageal squamous cell carcinoma, and have diagnostic value for the detection of early esophageal squamous cell carcinoma.

Moreover, it can also be seen from Table 1 that with the increase of the detection index (antigen) of the test strip, in the serum of patients with early esophageal squamous cell carcinoma, the positive rate of the antigen (that is, the sensitivity) gradually increases, while the specificity gradually decreases. Small; when the test strip combined to detect MPST protein and NME1 protein, the detection sensitivity of early esophageal squamous cell carcinoma reached 86.3%, which means that the detection method can correctly diagnose esophageal squamous cell carcinoma in patients with esophageal squamous cell carcinoma. The percentage is 86.3%. With the increase of the detection index of the test strip, the specificity of the detection has decreased, but when the two antigens of MPST protein and NME1 protein are detected in combination, the specificity of the detection can still reach 83.1%, which means that the non-esophageal squamous cell carcinoma patients use this method. When tested by this method, the percentage of correctly diagnosed healthy people was 83.1%.

Moreover, compared with the test strip for detecting one tumor-associated antigen alone, when the test strip combining the detection of MPST protein and NME1 protein was used for the diagnosis of early esophageal squamous cell carcinoma, the sensitivity was 100% for the detection of a single indicator MPST protein, respectively. , NME1 protein 1.5 times, 1.39 times.

Therefore, the combined detection of MPST protein and NME1 protein in serum samples for the early screening and diagnosis of esophageal squamous cell carcinoma can greatly improve the sensitivity of diagnosis on the premise of ensuring the specificity of diagnosis; the risk assessment of esophageal squamous cell carcinoma of the subjects to be tested has better Diagnostic and applied value.

In addition, the Youden index refers to the sum of sensitivity and specificity minus 1 in statistics, and its range is 0 to 1. The closer the Youden index is to 1, the higher the diagnostic value. In the present invention, with the increase of the antigen index detected by the test strip, the Youden index keeps increasing and gradually tends to 1, indicating that the combination of the two antigens has good diagnostic value for diagnosing and screening early esophageal squamous cell carcinoma.

To sum up, the combined detection of MPST protein and NME1 protein in serum samples can ensure high specificity and sensitivity for early esophageal squamous cell carcinoma screening and diagnosis, and the risk assessment of esophageal squamous cell carcinoma of the subject to be detected has better diagnosis and diagnosis. Value.

The above are only preferred embodiments of the present invention, but are not limited to the above examples. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention. within.

Claims (9)

1. A serum marker for screening early esophageal squamous carcinoma, which is a combination of MPST protein and NME1 protein.

2. The use of the detection reagent for the serum marker according to claim 1 in the preparation of a product for screening early esophageal squamous carcinoma, wherein the detection sample of the product is serum.

3. The use of claim 2, wherein the detection reagent is an antibody that specifically binds to the serum marker.

4. The use of claim 2, wherein the product is used for detection of the serum markers in a sample by immunochromatography or enzyme-linked immunoassay.

5. The use of any one of claims 2 to 4, wherein the product is a test strip or kit.

6. The test strip for screening early esophageal squamous carcinoma is characterized by comprising a binding pad and a chromatographic pad, wherein the binding pad is coated with a capture antibody, the capture antibody is a mixture of MPST antibody A, NME1 antibody A, and MPST antibody A, NME1 antibody A is provided with a detectable marker; the chromatography pad is provided with two detection lines and a quality control line, detection antibodies are fixed on the detection lines, the detection antibodies fixed on the two detection lines are respectively an MPST antibody B, NME1 antibody B, and an antibody A and an antibody B of the same antigen recognize different epitopes of the antigen; and goat anti-mouse IgG is fixed on the quality control line.

7. The test strip for screening early esophageal squamous carcinoma according to claim 6, wherein the marker is colloidal gold particles.

8. The test strip for screening early esophageal squamous carcinoma according to claim 7, wherein the test strip further comprises a sample pad, a sample sucking pad and a bottom plate, and the sample pad, the binding pad, the chromatography pad and the sample sucking pad are fixed on the bottom plate in sequence.

9. A kit comprising the test strip for screening early esophageal squamous carcinoma according to any one of claims 6-8.

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