Digestive tract tumor fecal protein biomarker detection reagent and application thereof

Abstract

The invention belongs to the field of immunodetection, and particularly relates to a digestive tract tumor fecal protein biomarker detection reagent and application thereof. The marker is specifically hemoglobin, antitrypsin or a fragment thereof; according to the invention, the fecal protein biomarker combination is detected by using an antibody through a single epitope detection method or a mass spectrum mode; the detection result is used for screening or auxiliary diagnosis of digestive tract tumors. It is found for the first time that the hemoglobin fragment can significantly improve the detection rate of upper gastrointestinal tumors and precancerous lesions; the joint detection of the fecal hemoglobin and the antitrypsin fragment can be used for screening the gastrointestinal tumors, the sensitivity of screening the upper gastrointestinal tumors can be remarkably improved by detecting the hemoglobin and the antitrypsin fragment, the detection time is short, the price is easily received by the public, and the method has wide application value.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay, and in particular relates to a digestive tract tumor stool protein biomarker detection reagent and application thereof. Background Art

[0002] In China, the incidence and mortality of digestive system tumors account for about half of the total tumor incidence and mortality, especially gastric cancer, liver cancer, colorectal cancer and esophageal cancer, which often linger in the top few, seriously threatening the health of the general public. How to detect and treat as early as possible is an important issue facing medical workers. Fecal screening for colorectal cancer has achieved great success and is widely accepted, but the detection sensitivity of esophageal cancer (EC) and gastric cancer (GC) is low. The occurrence and development of these two also go through a very long period. However, most patients are already in the middle and late stages when they are discovered, and the treatment effect is far lower than that of patients discovered early. The development of gastric cancer is also a multi-step gradual process; the current blood markers of gastric cancer (gastrin 17, pepsinogen I / II, etc.) have great limitations and are not ideal; WHO regards Helicobacter pylori as a Class I carcinogen; fecal Helicobacter pylori detection has certain indications for gastric cancer, but it is not as closely related as HPV and cervical cancer. Existing fecal occult blood products have achieved clear results in colorectal cancer screening, but the sensitivity for gastric cancer detection is relatively low. This is mainly because occult blood (hemoglobin) is digested from the stomach, passes through the small intestine and large intestine, and there is very little hemoglobin from gastric cancer in the feces, so the positive rate is low. Some scholars believe that proteins such as transferrin, haptoglobin, and calprotectin are more stable than hemoglobin and can play a compensatory role, but the actual effect is limited. Therefore, upper gastrointestinal tumor screening has also become a difficult point in clinical diagnosis.

[0003] The "occult blood bead" method of examining gastric cancer proposed by Professor Qin Dexing of the Cancer Hospital of the Chinese Academy of Medical Sciences in the 1980s can better solve the problem of hemoglobin degradation; the results are relatively ideal; but during the promotion, problems such as high false positives (only 0.5-6% of the positive population are true positives) were also found; gastroscopy provides a good method for screening upper gastrointestinal tumors; but its cost is high and resources are limited; capsule gastroscopy is expensive, and the poor compliance with invasive gastroscopy still restricts its further promotion; other screening methods such as net cytology and occult blood tests in gastric fluid are difficult to promote due to method limitations. Summary of the invention

[0004] In view of the defect of low sensitivity in detecting upper gastrointestinal tumors in the prior art, the inventors unexpectedly discovered that the detection method and marker combination can significantly improve the sensitivity of gastrointestinal tumors (especially upper gastrointestinal tumors) by detecting specific fecal proteins or their fragments (peptides) in the digestive tract. As is well known, since human body's own proteins (proteins derived from the human body itself, proteins from non-food sources) will be degraded by digestive enzymes and enzymes produced by microorganisms in the digestive tract, and since the stomach, especially the small intestine, is the main organ for digestion, the content and activity of digestive enzymes are very high, and the protein in the upper gastrointestinal tract is more severely degraded than that in the lower gastrointestinal tract, the degree of degradation is very high, and even completely degraded; therefore, the detection of upper gastrointestinal tumors is more difficult. Single epitope detection (SED) has higher sensitivity than dual epitopes detection (DED, such as double antibody sandwich method) because it only detects one protein epitope, because SED can obtain more signals of proteins or their protein fragments. SED is often used in immunohistochemical detection of pathology. Soluble protein / peptide detection in solution usually uses double epitope detection. However, like conventional double epitope detection (double antibody sandwich method), SED can also obtain ideal results as long as the specificity of the antibody is good enough.

[0005] AAT is the main protease inhibitor in serum; mature AAT is a single-chain glycoprotein composed of 394aa (containing about 12% sugar), MW52000; it can bind to a variety of proteases (such as trypsin, elastase, etc.) in a 1:1 ratio, so the protease cannot perform its normal function; in the blood, the AAT concentration is much higher than the concentration of various proteases, which effectively protects various components from protein degradation and maintains their functions; a decrease in AAT concentration can lead to the occurrence of various diseases (such as COPD). Under pathological conditions such as inflammation, tissue necrosis, injury or tumor, a1-antitrypsin increases compensatory levels to counteract excessive proteinases released by various cells and bacteria, protecting normal tissue cells from the dissolving damage of proteinases. In digestive tract tumors, on the one hand, cells will secrete excessive amounts of AAT into gastric juice; decreased gastric acidity also weakens protein degradation; on the other hand, esophageal cancer, gastric cancer, and colorectal cancer can lead to increased blood loss, and the already elevated AAT in the blood makes the concentration of AAT entering the digestive tract even higher; although AAT will be slowly degraded by enzymes after combining with various enzymes, due to its strong stability, AAT has a high detection rate in feces.

[0006] Hemoglobin is less stable. Hemoglobin produced by upper gastrointestinal bleeding will be largely degraded in the feces, but there are still a small amount of proteins, fragments or epitopes that can be identified, so that they can be detected by SED (competitive method, not easy to be detected by sandwich method); however, influenced by habitual thinking, people usually use SED to detect small molecule compounds and DED to detect large molecule proteins, so a lot of valuable information will be lost; in addition, tumor patients will secrete more AAT, which is more stable than hemoglobin; the combination of the two will improve the detection sensitivity (this method does not detect the complete molecules of hemoglobin or antitrypsin, but only detects a single epitope). Although some scholars have mentioned the idea of ​​using competitive methods for detection, they have not clearly put forward specific advantages; there is no literature reporting advantages in this regard; and there is no practical application in this regard.

[0007] The first aspect of the present invention discloses a fecal protein biomarker for digestive tract tumors and a combination thereof, specifically hemoglobin or a fragment thereof (Hemoglobin, Hb), and / or antitrypsin or a fragment thereof (alpha-1-antitrypsin, AAT).

[0008] The second aspect of the present invention discloses a product for detecting the above-mentioned fecal protein biomarker combination of digestive tract tumors, wherein the product uses antibodies to detect the fecal protein biomarker combination by single epitope detection or mass spectrometry. The product may be a kit.

[0009] Single epitope detection methods include competitive immunoassay methods such as ELISA, immunochromatography, latex turbidimetry, time-resolved fluorescence, chemiluminescence, etc.

[0010] The above-mentioned immunochromatographic methods include colloidal gold immunochromatography, fluorescence immunochromatography, time-resolved fluorescence immunoassay and quantum dot immunoassay, etc.

[0011] The detection reagents used in the above-mentioned single epitope detection specifically include coated antibodies, enzyme-labeled detection antigens and related auxiliary reagents. Specifically, the coated antibodies include hemoglobin antibodies and anti-trypsin antibodies; enzyme-labeled hemoglobin and enzyme-labeled anti-trypsin; blocking solution, washing solution, detection buffer containing blocking agents, etc.

[0012] The detection product can also be configured as a reagent for detecting haptoglobin or transferrin or the like or their fragments.

[0013] The third aspect of the present invention discloses a kit, comprising the above-mentioned detection reagent, for detecting the above-mentioned digestive tract tumor fecal protein.

[0014] The third aspect of the present invention discloses the use of the above-mentioned detection product in screening or auxiliary diagnosis of digestive tract tumors, especially upper digestive tract tumors.

[0015] The upper digestive tract tumors include gastric cancer and esophageal cancer.

[0016] The present invention achieves the following beneficial technical effects:

[0017] The present invention is the first to find that the use of hemoglobin or its fragments can greatly improve the detection rate of upper gastrointestinal tumors. The present invention is the first to find that the combined detection of fecal hemoglobin and AAT or its fragments can be used for the screening of gastrointestinal tumors. The detection of hemoglobin and its fragments can significantly improve the sensitivity of upper gastrointestinal tumor screening, the detection cycle is short, and the price is acceptable to the public (only a fraction of the molecular detection method), and it has a wide range of application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the DED ELISA standard curve of human hemoglobin in the embodiment; the abscissa is the hemoglobin concentration (ng / ml);

[0019] Figure 2 It is the SED ELISA standard curve of human hemoglobin in the embodiment; The abscissa is the transferrin concentration (ng / ml);

[0020] Figure 3 It is the SED standard curve diagram of human Anti-trypsin (AAT) in the embodiment; the abscissa is the AAT concentration (ng / ml);. DETAILED DESCRIPTION

[0021] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The raw materials and equipment used in the examples are well known to those skilled in the art, and are all available on the market or easily obtained or prepared.

[0022] Example 1 ELISA detection reagent preparation:

[0023] In this example, two ELISA methods, DED and SED, are used to construct standard curves for the index to be tested. The antigen / antibody labeling steps are as follows:

[0024] 1. Take out the HRP labeling kit from -20℃ and equilibrate it at room temperature for 30 minutes.

[0025] 2. Add 1 μl of reaction initiation solution to every 10 μl of commercially available antibody or antigen solution to be labeled and mix thoroughly to avoid bubbles.

[0026] 3. Open the coupled horseradish peroxidase tube, add the activated antibody / antigen solution to the tube, mix thoroughly to avoid bubbles, and place at room temperature away from light for 3 hours.

[0027] 4. Add stop solution to the horseradish peroxidase reaction tube, add 1 μl of stop solution to every 10 μl of antibody / antigen solution, mix thoroughly, and leave at room temperature for 1 hour.

[0028] 5. After termination, add an equal volume of glycerol, mix thoroughly, and store at -20℃ away from light.

[0029] Example 2: ELISA detection method:

[0030] This example uses the labeling reagent described in Example 1 to detect the concentration of gastrointestinal tumor fecal protein or its fragments in the sample. The specific steps of the single epitope ELISA are as follows (see the general information for the double antibody sandwich method. The difference is that a detection antibody incubation and washing process is added after the sample is added and washed):

[0031] 1. Sample preparation and extraction are the same as conventional methods: avoid mixing urine / water when sampling, take 1-2g of feces, add 15ml of preservation solution, and store at -20 degrees Celsius for later use;

[0032] 2. Mix vigorously, take 50ul of liquid and add 1ml of lysis buffer to mix; lyse at room temperature for 20-30 minutes, centrifuge at 12000rpm for 10 minutes; store the lysis supernatant in a -80 degree refrigerator for short-term storage;

[0033] 3. Coating: Coat with 3ug / ml antibody at 4 degrees overnight, the antibodies are human hemoglobin and AAT antibodies; wash 3-5 times with PBST; pat dry;

[0034] 4. Block overnight at 4°C with blocking solution; wash 3 times with PBST; pat dry;

[0035] 5. Add standard, appropriate amount of sample (the above lysis supernatant), enzyme-labeled antigen and detection buffer (add blocking agent 5-15ug / ml) at the same time, and incubate at 37 degrees for 1.5 hours;

[0036] 6. Wash 6 times with PBST washing buffer; pat dry;

[0037] 7. TMB color development, after termination, read the results on a Thermo Multiscan microplate reader at 490 / 630 dual wavelengths.

[0038] 8. Calculate the protein concentration in each sample based on the standard curve.

[0039] The standard curves of human hemoglobin (DED, SED) and AAT (SED) are as follows: Figure 1 , Figure 2 , Figure 3 shown.

[0040] Example 3: Detection of fecal protein or its fragments in digestive tract tumors:

[0041] Fecal specimens (1-2 g) were collected from gastrointestinal surgery patients on the day of admission, the day before endoscopic examination, and normal subjects, and immediately placed in a protein preservation solution (15 ml), and sent to the laboratory within 12-24 hours and stored in a -20 degree refrigerator. The samples were tested in a double-blind manner according to the method of Example 2, and the blind was unblinded after the test results and clinical data were complete. Unqualified specimens were excluded, such as abdominal pain without a clear clinical pathological diagnosis and specimens with a recent history of surgical operations.

[0042] The general information of the sample is as follows:

[0043] Table 1. Sample information

[0044] sample quantity Age Range Median age Average age SD Colorectal cancer 16 55-79 68.5 67.8 8.5 Gastric cancer Esophageal cancer 32 57-90 69 69.7 7.2 Precancerous lesions 3 63-85 73 73.4 11 Benign tumors / polyps 5 54-73 58 60 7.6 Normal / benign disease 22 46-84 60 61.5 11.4

[0045] The sample test results are shown in Table 2:

[0046] Table 2 ELISA test results of hemoglobin, AAT protein or its fragment concentration in each group

[0047] HbSED (ng / ml) HbDED(ng / ml) AATSED (ug / ml) Colorectal cancer (16) 29750.3±64051.4** 1318.8±1480.4** 54.8±130.8** Gastric cancer / esophageal cancer(32) 346.7±676.0** 105.9±198.9** 23.0±41.4** Polyps / dysplasia / adenoma (8) 375.3±665.2 70.7±65.8* 14.8±16.6* Normal / benign (22) 23.1±71.2 15.5±22.6 2.3±2.8

[0048] Note: **: P<0.01 compared with normal / benign samples; *: P<0.05;

[0049] The reference value of Hb is 100ng / ml, and that of AAT is 10ug / ml. If one of them is positive, it is considered positive; if both are negative, it is considered negative. The results are shown in Table 3:

[0050] Table 3: Number of positive results for each index in digestive tract tumor detection

[0051] Number of cases HbDED positive AATSED positive HbSED positive Combined positive Colorectal cancer 16 16 10 16 16 Colorectal precancerous lesions (adenoma) 1 / 1 / 1 Gastric cancer / esophageal cancer 32 7 16 12 19 Gastric precancerous lesions (dysplasia) 2 0 2 1 2 Total cancer and precancerous lesions 51 23 29 29 38 Benign tumors / polyps 5 2 1 2 2 Normal / benign lesions 22 / / 2 2 total 78 / / / /

[0052] In this experiment, 48 cases of gastrointestinal tumors (clinical pathology results were used as the gold standard, including 16 cases of advanced colorectal cancer and 32 cases of gastric cancer / esophageal cancer), 3 cases of precancerous lesions (including adenoma, intraepithelial neoplasia, moderate to severe dysplasia), and 51 cases of cancer and precancerous lesions were detected; 23 cases were detected by conventional occult blood method (DED) (including 16 cases of colorectal cancer and 7 cases of gastric cancer; the overall sensitivity of gastrointestinal tumors was 45%); 29 cases were detected by Hb SED; 38 cases were detected by Hb / AAT SED (including 17 cases of colorectal cancer and adenoma, 21 cases of gastric cancer, esophageal cancer and precancerous lesions; the overall sensitivity was 75%); the sensitivity was greatly improved; the improvement was mainly concentrated in upper gastrointestinal tumors and precancerous lesions, gastric cancer / esophageal cancer (sensitivity increased from 21% to 62%); among the remaining 5 cases of polyps and benign tumors, two cases were positive in Hb DED and SED tests. The overall specificity of DED in 22 cases of benign digestive system lesions and normal people was 100%, while there were two false positives in SED, with a specificity of 91% (one of which was acute pancreatitis, and the other was a normal person of unknown cause). This method not only detected all colorectal cancers (traditional methods had a small number of missed detections); but also greatly improved the detection of upper digestive tract tumors (gastric cancer and esophageal cancer). At the same time, this method tested 3 precancerous lesions (1 colorectal adenoma / moderate-to-high-grade dysplasia, 1 gastric high-grade epithelial neoplasia, and 1 gastric moderate dysplasia) positively, while conventional DED tests were all negative; this shows that this method has a higher sensitivity in the detection of precancerous lesions than conventional DED; it can detect precancerous lesions earlier, so it is very meaningful, because one of the important purposes of screening is to detect precancerous lesions, reduce the incidence of cancer and increase survival rate, and the detection of precancerous lesions (early detection) is conducive to early intervention, so it is very valuable for early screening of digestive tract tumors.

[0053] Because the protein index detected by this method is derived from blood, some benign gastrointestinal lesions can also cause bleeding, thus reducing the specificity. When the number of benign lesion samples related to bleeding is greatly increased (some samples are secondary sampling), such as gastrointestinal ulcers, gastric varicose veins, gallstones, cholecystitis, diverticulitis, Crohn's disease, appendicitis, pancreatitis, etc. (the incidence rate of the general population is low and will not be so high), the specificity is reduced (see Table 4). Of course, the detection of benign lesions also has certain clinical significance, which can be used as the first detection of general screening projects: the initial screening is carried out by automated medium- and high-throughput detection through a biochemical analyzer or a chemiluminescence analyzer. Positive specimens can be clearly distinguished and diagnosed in subsequent endoscopic examinations, and preventing the occurrence of false negatives may be more important for the initial screening. Because for the screening of gastrointestinal tumors, people often pay more attention to not missing detection as much as possible, and try to find early tumors for timely treatment, which is also an advantage of the present invention.

[0054] Table 4: Results of digestive tract tumor detection after increasing the number of samples

[0055]

[0056]

[0057] It should be pointed out that in the real-world screening of digestive tract tumors, as the number of samples increases, the proportion of sample composition will change significantly. For example, as the proportion of normal samples increases, the sensitivity and specificity will change (such as increased specificity), while the sensitivity of SED in upper digestive tract tumors will not change compared to DED (this method has increased by 2-3 times); the same results will appear when related proteins such as haptoglobin, transferrin, and calprotectin are detected. At the same time, due to the limitations of the ELISA method in terms of sensitivity and linearity, the linear range is narrow, and the quantitative deviation of samples near or outside the linear range is often large. If the chemiluminescence method is used instead, the accuracy will be significantly improved. Through the concentration and enrichment of positive objects by this method, the screening efficiency of digestive tract tumors can be significantly improved. Through subsequent gastroscopy and colonoscopy, a high proportion of digestive tract tumors and precancerous lesions can be screened more efficiently, while effectively avoiding the high cost of DNA methylation detection. The scheme of this application has low cost, short detection time, and high detection throughput, and is a detection method with promotion value.

[0058] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A fecal protein biomarker for digestive tract tumors and a combination thereof, specifically hemoglobin or its fragments, and / or antitrypsin or its fragments.

2. The detection product of the composition according to claim 1, characterized in that: Detection of fecal protein biomarkers and their combinations by single epitope immunoassay or mass spectrometry; The detection product is specifically a test kit.

3. A detection product as claimed in claim 2, characterized in that: The single epitope detection method includes competitive immunoassay methods such as ELISA, immunochromatography, latex turbidimetry, time-resolved fluorescence, and chemiluminescence.

4. A detection product as described in claim 3, wherein the immunochromatography includes colloidal gold immunochromatography, fluorescent immunochromatography, time-resolved fluorescent immunoassay and quantum dot immunoassay.

5. A detection product as claimed in claim 2, characterized in that: The detection product includes a detection reagent; The detection reagent contains coated antibodies, enzyme-labeled antigens and related auxiliary reagents.

6. A detection product as claimed in claim 5, characterized in that: The coating antibodies are hemoglobin antibodies and anti-trypsin antibodies; the enzyme-labeled antigens are enzyme-labeled hemoglobin and enzyme-labeled anti-trypsin; and the auxiliary reagents are blocking solution, washing solution and detection buffer containing blocking agent.

7. A detection product as claimed in claim 2, comprising a reagent for detecting haptoglobin or transferrin or the like or their fragments.

8. The detection product according to any one of claims 2 to 6 is used for screening or auxiliary diagnosis of gastrointestinal tumors, and is particularly suitable for screening or auxiliary diagnosis of upper gastrointestinal tumors.

9. The use according to claim 8, characterized in that The upper digestive tract tumors are gastric cancer and esophageal cancer.

Images