Pepsinogen Ⅰ and pepsinogen Ⅱ combined detection kit and preparation method thereof

Abstract

The present invention relates to the field of medical testing technology, and discloses a pepsinogen I and pepsinogen II joint detection kit and a preparation method thereof, comprising a PVC plate, a sample pad, a quantum pad, a nitrocellulose membrane and blotting paper, the quantum pad is fixed with quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody, the nitrocellulose membrane is fixed with a detection line 1, a detection line 2 and a quality control line composed of goat anti-mouse IgG composed of a secondary antibody for recognizing pepsinogen I antibody and a secondary antibody for pepsinogen II antibody. The present invention can jointly detect pepsinogen I and pepsinogen II in serum with high sensitivity, and can be applied to rapid diagnosis in clinical practice and rapid diagnosis on site, which is not only conducive to promotion in tertiary and large hospitals, but also conducive to use in primary medical units.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, in particular to a pepsinogen I and pepsinogen II combined testing kit and a preparation method thereof. Background Art

[0002] Pepsinogen is an inactive precursor of pepsin in gastric juice. It is secreted by the chief cells and mucous cells of the gastric body. Most of it is secreted into the gastric cavity, and a small amount can be detected in the blood. According to its biochemical properties and immunogenicity, it is divided into two subgroups. Components 1-5 have the same immunogenicity and are called pepsinogen I, which is mainly secreted by the chief cells and mucous neck cells of the fundic glands; components 6 and 7 are called pepsinogen II. In addition to being secreted by the chief cells and mucous neck cells of the fundic glands, the mucous neck cells of the pyloric glands of the cardia gland and the antrum, and the upper duodenum can also produce pepsinogen II. The decrease in the ratio of pepsinogen I to pepsinogen II in serum and plasma is positively correlated with the severity of gastric body atrophy. In moderate and severe atrophic gastritis, the ratio is <3, and the incidence of gastric cancer is significantly increased. Therefore, the combined determination of the ratio of pepsinogen I to pepsinogen II can play a role in early gastric cancer risk indication. Through the different serum measurement values, there are different degrees of changes in various gastric diseases, providing reliable diagnostic value for clinical practice.

[0003] The current diagnostic methods for gastric diseases and gastric cancer involve endoscopy, B-ultrasound, X-ray and tumor marker detection, each of which has its own limitations: X-ray examination is valuable for the diagnosis of advanced gastric cancer, but it has radiation risks, is expensive, and is not suitable for early detection; although endoscopy is accurate, it is expensive, complicated to operate, and uncomfortable for patients, making it difficult to popularize; tumor markers are of limited effectiveness in early diagnosis. Although the existing enzyme-linked immunosorbent assay and chemiluminescence assay have a certain degree of accuracy, they are cumbersome to operate, time-consuming, and require high equipment. They are not suitable for large-scale screening or instant testing, and they are insufficient in terms of cost, sensitivity, and specificity. In contrast, serum PGⅠ / PGⅡ combined detection has advantages in its non-invasive, convenient, and economical characteristics, while the fluorescent immunochromatography method simplifies the operation process and reduces costs while maintaining high sensitivity and high accuracy, making it more suitable for promotion, especially in an environment with limited medical resources, showing higher practical value and industrialization potential.

[0004] Quantum dots (QDs) are a class of attractive semiconductor nanomaterials derived from a combination of group II-VI or group III-V elements, with particle sizes shrunk to near or below the Bohr radius of the laser. These tiny nanoparticles can emit fluorescence after absorbing specific excitation light. These unique properties include high-intensity fluorescence emission, a broad excitation spectrum, and a narrow and symmetrical emission spectrum, providing an ideal tool for multicolor labeling and simultaneous analysis of multiple components, greatly broadening the application prospects of biomedical detection and imaging.

[0005] However, current labeling strategies mostly rely on simply mixing quantum dots with antibodies to achieve labeling. This method has some limitations: the stability of the labeling is poor, resulting in low effective coupling efficiency of the antibody, which in turn affects the accuracy and repeatability of quantum dot fluorescence labeling. Therefore, developing more efficient and stable labeling technology to improve the performance of quantum dot fluorescence labeling has become a key issue that needs to be solved in this field. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a pepsinogen I and pepsinogen II joint detection kit and a preparation method thereof, so as to jointly detect pepsinogen I and pepsinogen II in serum with high sensitivity.

[0007] The present invention solves the above technical problems by the following technical means:

[0008] In the first aspect, the present invention provides a pepsinogen I and pepsinogen II joint detection kit, comprising a PVC plate, a sample pad, a quantum pad, a nitrocellulose membrane and absorbent paper, the quantum pad being fixed with quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody, the nitrocellulose membrane being fixed with a secondary antibody for recognizing pepsinogen I antibody and a secondary antibody for recognizing pepsinogen II antibody, a detection line 1, a detection line 2 and a quality control line composed of goat anti-mouse IgG.

[0009] In a second aspect, the present invention provides a method for preparing a pepsinogen I and pepsinogen II combined detection kit, comprising the following steps:

[0010] S1. Preparation of quantum pad:

[0011] A mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody is prepared; the mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody is coated on a glass cellulose membrane to prepare a quantum pad, and dried;

[0012] S2. Preparation of nitrocellulose membrane: Paste nitrocellulose membrane on PVC base plate, coat secondary antibody recognizing pepsinogen I antibody and secondary antibody recognizing pepsinogen II antibody on detection line 1 and detection line 2 of nitrocellulose membrane, coat goat anti-mouse IgG on quality control line of nitrocellulose membrane, and then put the coated nitrocellulose membrane into drying oven to dry to form a large plate;

[0013] S3, assembly: stick the sample pad, quantum pad and absorbent paper on the large board.

[0014] Preferably, the specific operation method for preparing the mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody in step S1 includes:

[0015] S1.1, add ZnSO4 solution, MnCl2 solution and CTAB solution into a flask respectively, stir continuously under nitrogen protection, slowly drop Na2S2O3, stir for 25-35 minutes, then precipitate in a water bath to obtain CTAB-modified Mn-ZnS quantum dots, and then wash with anhydrous ethanol and ultrapure water respectively;

[0016] S1.2, prepare a water-ethanol mixed solution, add ammonia water, and slowly add the Mn-ZnS quantum dots in step S1.1 into the water-alcohol-ammonia mixed solution under stirring;

[0017] S1.3, adding TEOS solution dropwise under stirring, continuing to react under stirring at room temperature for 3.5-4.5 hours, transferring the solution to a centrifuge tube, centrifuging at 7000-9000 rpm for 8-12 minutes, collecting the precipitate, repeatedly washing the precipitate with ethanol and ultrapure water for 3 times, drying to obtain quantum dots, and redispersing the quantum dots in ultrapure water to prepare a quantum dot solution;

[0018] S1.4, adding an activator to the quantum dot solution prepared in step S1.3, activating for 10-20 minutes, purifying, then adding pepsinogen I antibody, mixing for 2-4 hours, centrifuging to obtain precipitate particles, resuspending the precipitate particles with blocking solution, mixing at room temperature for 1-1.5 hours, and then purifying to obtain quantum dot-labeled pepsinogen I antibody;

[0019] S1.5. Prepare quantum dot-labeled pepsinogen II antibody in the same manner as step S1.4, and then mix the quantum dot-labeled pepsinogen I antibody and the quantum dot-labeled pepsinogen II antibody.

[0020] Preferably, in step S1.1, the concentration of the ZnSO4 solution is 0.1-0.2 mol / L, the concentration of the MnCl2 solution is 0.01-0.02 mol / L, the concentration of CTAB is 0.1-0.2 mol / L, and the concentration of Na2S2O3 is 0.1-0.2 mol / L.

[0021] Preferably, the water bath temperature of the water bath precipitation in step S1.1 is 45-55° C., and the precipitation time is 1.5-2.5 h.

[0022] Preferably, the volume ratio of water to ethanol in the water-ethanol mixed solution in step S1.2 is 1:9, and the concentration of ammonia water is 25%-28%.

[0023] Preferably, the blocking solution in step S1.4 comprises glycine with a molar concentration of 150 mmol / L and BSA with a mass concentration of 5%.

[0024] Preferably, in step S1.5, the volume mixing ratio of the quantum dot-labeled pepsinogen I antibody and the quantum dot-labeled pepsinogen II antibody is 1:1.

[0025] Preferably, the specific method for preparing the nitrocellulose membrane in step S2 includes respectively taking a secondary antibody of pepsinogen I antibody with a concentration of 0.5-2.0 mg / ml, a secondary antibody of pepsinogen II antibody with a concentration of 0.5-1.5 mg / ml, and spraying them onto the detection line 1, the detection line 2 and the quality control line of the nitrocellulose membrane at a speed of 1 μL / cm and 10 cm / s, respectively.

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention detects PGⅠ and PGⅡ in the human body, and the detection linear range is PGⅠ: 10-300μg / L, PGⅡ: 5-100μg / L, thereby improving the detection efficiency and realizing the quantitative analysis of pepsinogen Ⅰ (PGⅠ) and pepsinogen Ⅱ (PGⅡ), and the specific contents of these two key indicators can be accurately obtained from a single sampling.

[0028] (2) The present invention is easy to prepare, small in size, convenient to transport and carry, with relatively low detection cost, small intra-batch and inter-batch differences in mass production, and can be applied to rapid clinical diagnosis and rapid on-site diagnosis; it can be stored at room temperature, which is not only conducive to promotion in tertiary and large hospitals, but also conducive to use in primary medical institutions.

[0029] (3) The present invention utilizes silicon dioxide to coat zinc sulfide quantum dots (Mn 2+ The use of zinc sulfide quantum dots (Mn2) to label pepsinogen I and pepsinogen II antibodies can precisely control the antibody coupling density, thereby greatly improving the antibody loading and labeling efficiency, ensuring that each quantum dot can carry a sufficient number of antibodies, and enhancing the strength and specificity of the detection signal. 2+ Doping) itself has the fluorescence characteristics of narrow bandwidth and high brightness. After being coated with silica, the fluorescence self-extinguishing phenomenon is further reduced, making the fluorescence signal purer and the background noise lower, which is beneficial to improve the signal-to-noise ratio and sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the partial side structure of the kit of the present invention;

[0031] Figure 2 It is a schematic diagram of the side structure of the kit of the present invention;

[0032] Figure 3 It is a schematic diagram of the structure of the test kit of the present invention from a top view.

[0033] Among them, 1 is a PVC plate, 2 is a nitrocellulose membrane, 3 is absorbent paper, 4 is a quantum pad, 5 is a sample pad, 6 is a card shell, 60 is an upper shell, 61 is a lower shell, 7 is an observation and detection port; 8 is a sample addition hole, T1 is a detection line 1, T2 is a detection line 2, and C line is a quality control line. DETAILED DESCRIPTION

[0034] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention:

[0035] The present invention discloses a pepsinogen I and pepsinogen II combined detection kit and a preparation method thereof, wherein the kit is as follows Figure 1-3 As shown, it includes a PVC plate 1, a sample pad 5, a quantum pad 4, a nitrocellulose membrane 2 and an absorbent paper 3, the quantum pad is coated with quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody in a mass ratio of 1:1; a detection line 1, a detection line 2 and a control line are arranged on the nitrocellulose membrane, the detection line 1 is coated with a secondary antibody that recognizes the pepsinogen I antibody, the detection line 2 is coated with a secondary antibody that recognizes the pepsinogen II antibody, and the quality control line is coated with goat anti-mouse IgG.

[0036] The reagent kit further comprises a card shell 6 , which comprises an upper shell 60 and a lower shell 61 . The upper shell 60 is provided with an observation and detection port 7 and a sample addition hole 8 .

[0037] Example 1

[0038] This example is a preparation of a pepsinogen I and pepsinogen II combined detection kit.

[0039] A method for preparing a pepsinogen I and pepsinogen II combined detection kit comprises the following steps:

[0040] S1. Preparation of quantum pad:

[0041] 1) Preparing a mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody;

[0042] S1.1. Add 5 mL of 0.1 mol / L ZnSO4 solution, 2 mL of 0.01 mol / L MnCl2 solution, and 20 ml of 0.1 mol / L CTAB to a flask, stir the mixture magnetically under nitrogen protection, slowly drop 5 mL of 0.1 mol / L Na2S2O3, stir for 25 min, and place the flask in a 45 °C water bath for 2.5 h to obtain CTAB-modified Mn-ZnS quantum dots, which are then washed three times with anhydrous ethanol and ultrapure water respectively.

[0043] S1.2, prepare 50 mL of a water-ethanol mixed solution (5 mL of water, 45 mL of ethanol), add 2 mL of 25%-28% ammonia water, and slowly add the Mn-ZnS quantum dots in step S1.1 to the water-alcohol-ammonia mixed solution under magnetic stirring;

[0044] S1.3, add 10 mL of TEOS solution dropwise under magnetic stirring, continue stirring the reaction at room temperature for 3.5 hours, transfer the solution to a centrifuge tube, centrifuge at 9000 rpm for 8 minutes, collect the precipitate; repeatedly wash the precipitate with ethanol and deionized water for 3 times, dry to obtain quantum dots, and redisperse the quantum dots in ultrapure water to prepare a quantum dot solution;

[0045] S1.4, add 15 mL of activator to the quantum dot solution prepared in step S1.3, activate for 10 minutes, purify, then add pepsinogen I antibody, mix for 2 hours, centrifuge to obtain precipitate particles, resuspend the precipitate particles with blocking solution, mix at room temperature for 1 hour, and then purify to obtain quantum dot-labeled pepsinogen I antibody;

[0046] S1.5. Prepare quantum dot-labeled pepsinogen II antibody in the same manner as step S1.4, and then mix the quantum dot-labeled pepsinogen I antibody and the quantum dot-labeled pepsinogen II antibody.

[0047] 2) coating a mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody on a glass cellulose membrane to prepare a quantum pad, and drying;

[0048] S2. Preparation of nitrocellulose membrane: Paste nitrocellulose membrane on PVC base plate, take 0.5 mg / ml secondary antibody of pepsinogen I antibody, 0.5 mg / ml secondary antibody of pepsinogen II antibody and 0.5 mg / ml goat anti-mouse IgG, spray them on detection line 1, detection line 2 and quality control line of nitrocellulose membrane at speeds of 1 μL / cm and 10 cm / s respectively, then put the coated nitrocellulose membrane into a drying oven and dry it to form a large plate.

[0049] S3, assembly: stick the sample pad, quantum pad and absorbent paper on the large board.

[0050] Example 2

[0051] This example is a preparation of a pepsinogen I and pepsinogen II combined detection kit.

[0052] A method for preparing a pepsinogen I and pepsinogen II combined detection kit comprises the following steps:

[0053] S1. Preparation of quantum pad:

[0054] 1) Preparing a mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody;

[0055] S1.1. Add 5 mL of 0.15 mol / L ZnSO4 solution, 2 mL of 0.015 mol / L MnCl2 solution, and 20 ml of 0.15 mol / L CTAB to a flask, and continuously stir the mixture with magnetic force under nitrogen protection; slowly drop 5 mL of 0.15 mol / L Na2S2O3, stir for 30 min, and place the flask in a 50°C water bath for 2 h to obtain CTAB-modified Mn-ZnS quantum dots, which are then washed three times with anhydrous ethanol and ultrapure water respectively;

[0056] S1.2, prepare 50 mL of a water-ethanol mixed solution (5 mL of water, 45 mL of ethanol), add 2 mL of 25%-28% ammonia water, and slowly add the Mn-ZnS quantum dots in step S1.1 to the water-alcohol-ammonia mixed solution under magnetic stirring;

[0057] S1.3, add 10 mL of TEOS solution dropwise under magnetic stirring, continue stirring and reacting at room temperature for 4 hours, transfer the solution to a centrifuge tube, centrifuge at 8000 rpm for 10 minutes, collect the precipitate; repeatedly wash the precipitate with ethanol and deionized water for 3 times, dry to obtain quantum dots, and redisperse the quantum dots in ultrapure water to prepare a quantum dot solution;

[0058] S1.4, add 15 mL of activator to the quantum dot solution prepared in step S1.3, activate for 15 minutes, purify, then add pepsinogen I antibody, mix for 3 hours, centrifuge to obtain precipitate particles, resuspend the precipitate particles with blocking solution, mix at room temperature for 1.25 hours, and then purify to obtain quantum dot-labeled pepsinogen I antibody;

[0059] S1.5. Prepare quantum dot-labeled pepsinogen II antibody in the same manner as step S1.4, and then mix the quantum dot-labeled pepsinogen I antibody and the quantum dot-labeled pepsinogen II antibody.

[0060] 2) coating a mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody on a glass cellulose membrane to prepare a quantum pad, and drying;

[0061] S2. Preparation of nitrocellulose membrane: Paste nitrocellulose membrane on PVC base plate, take 1.25 mg / ml secondary antibody of pepsinogen I antibody, 1.25 mg / ml secondary antibody of pepsinogen II antibody and 1 mg / ml goat anti-mouse IgG, spray them on detection line 1, detection line 2 and quality control line of nitrocellulose membrane at speeds of 1 μL / cm and 10 cm / s, respectively, then put the coated nitrocellulose membrane into a drying oven and dry it to form a large plate;

[0062] S3, assembly: stick the sample pad, quantum pad and absorbent paper on the large board.

[0063] Example 3

[0064] This example is a preparation method of a pepsinogen I and pepsinogen II combined detection kit.

[0065] A method for preparing a pepsinogen I and pepsinogen II combined detection kit comprises the following steps:

[0066] S1. Preparation of quantum pad:

[0067] 1) Preparing a mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody;

[0068] S1.1. Add 5 mL of 0.2 mol / L ZnSO4 solution, 2 mL of 0.02 mol / L MnCl2 solution and 20 ml of 0.2 mol / L CTAB into a flask, and continuously stir the mixture with magnetic force under nitrogen protection. Slowly drop 5 mL of 0.2 mol / L Na2S2O3 into the flask, stir for 35 min, and place the flask in a 55°C water bath for 1.5 h to obtain CTAB-modified Mn-ZnS quantum dots, which are then washed three times with anhydrous ethanol and ultrapure water respectively.

[0069] S1.2, prepare 50 mL of a water-ethanol mixed solution (5 mL of water, 45 mL of ethanol), add 2 mL of 25%-28% ammonia water, and slowly add the Mn-ZnS quantum dots in step S1.1 to the water-alcohol-ammonia mixed solution under magnetic stirring;

[0070] S1.3, add 10 mL of TEOS solution dropwise under magnetic stirring, continue stirring the reaction at room temperature for 4.5 hours, transfer the solution to a centrifuge tube, centrifuge at 7000 rpm for 12 minutes, collect the precipitate; repeatedly wash the precipitate with ethanol and deionized water for 3 times, dry to obtain quantum dots, and redisperse the quantum dots in ultrapure water to prepare a quantum dot solution;

[0071] S1.4, add 15 mL of activator to the quantum dot solution prepared in step S1.3, activate for 20 minutes, purify, then add pepsinogen I antibody, mix for 4 hours, centrifuge to obtain precipitate particles, resuspend the precipitate particles with blocking solution, mix at room temperature for 1.5 hours, and then purify to obtain quantum dot-labeled pepsinogen I antibody;

[0072] S1.5. Prepare quantum dot-labeled pepsinogen II antibody in the same manner as step S1.4, and then mix the quantum dot-labeled pepsinogen I antibody and the quantum dot-labeled pepsinogen II antibody.

[0073] 2) coating a mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody on a glass cellulose membrane to prepare a quantum pad, and drying;

[0074] S2. Preparation of nitrocellulose membrane: Paste nitrocellulose membrane on PVC base plate, take 2.0 mg / ml secondary antibody of pepsinogen I antibody, 2.0 mg / ml secondary antibody of pepsinogen II antibody and 1.5 mg / ml goat anti-mouse IgG, spray them on detection line 1, detection line 2 and quality control line of nitrocellulose membrane at speeds of 1 μL / cm and 10 cm / s, respectively, then put the coated nitrocellulose membrane into a drying oven and dry it to form a large plate;

[0075] S3, assembly: stick the sample pad, quantum pad and absorbent paper on the large board.

[0076] Comparative Example 1 (Example 4)

[0077] This example is a preparation method of a pepsinogen I and pepsinogen II combined detection kit.

[0078] The difference between this embodiment and embodiment 2 is that, in step S1, the method of S1.1-S1.3 is omitted, and 2.5 mol / L zinc sulfide quantum dot solution is directly used to label pepsinogen I antibody and pepsinogen II antibody, and the other steps are the same as those in embodiment 2.

[0079] Comparative Example 2 (Example 5)

[0080] This example is a preparation method of a pepsinogen I and pepsinogen II combined detection kit.

[0081] The difference between this embodiment and embodiment 2 is that step S1 cancels the method of S1.3 and directly uses the zinc sulfide quantum dots (Mn 2+ Doping) is dispersed with pure water to make a solution, and the other steps are the same as Example 2.

[0082] 10 clinical samples were randomly selected and the concentrations of pepsinogen I and pepsinogen II were tested using the kits prepared in Examples 1-3 and Comparative Examples 1-2. The specific steps of the test include:

[0083] Take 100uL of the sample to be tested and drop it onto the sample pad of the kit. After 3-5 minutes, put the color-developed standard and the test strip of the sample to be tested into the fluorescent immunochromatography reader, read the luminescence value (T / C) of the corresponding detection line and record the data. Input the obtained T / C value into the corresponding standard curve, and calculate the concentration of pepsinogen I and pepsinogen II. The specific results are shown in Table 1.

[0084] Table 1

[0085]

[0086] As can be seen from Table 1, the test data of the kit prepared by the method of Examples 1-3 and the test data of the kit prepared by the method of Comparative Examples 1-2 for pepsinogen I and pepsinogen II are more accurate, indicating that the test results of the kit prepared by the present invention for pepsinogen I and pepsinogen II are accurate. The test data of the kit prepared by the method of Comparative Examples 1-2 have a certain deviation in the detection of low-concentration pepsinogen II, while the test data of the kit prepared by the present invention are more accurate in the detection of low-concentration pepsinogen II, making it more competitive in trace detection.

[0087] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A kit for the combined detection of pepsinogen I and pepsinogen II, comprising a PVC plate, a sample pad, a quantum pad, a nitrocellulose membrane and absorbent paper, characterized in that: The quantum pad is fixed with quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody, and the nitrocellulose membrane is fixed with detection line 1 and detection line 2 composed of secondary antibodies recognizing pepsinogen I antibody and secondary antibodies recognizing pepsinogen II antibody, and a quality control line composed of goat anti-mouse IgG; The preparation method of the labeled quantum dots comprises the following steps: S1.1, add ZnSO4 solution, MnCl2 solution and CTAB solution into a flask respectively, stir continuously under nitrogen protection, slowly drop Na2S2O3, stir for 25-35 minutes, then precipitate in a water bath to obtain CTAB-modified Mn-ZnS quantum dots, and then wash with anhydrous ethanol and ultrapure water respectively; S1.2, prepare a water-ethanol mixed solution, add ammonia water, and slowly add the Mn-ZnS quantum dots in step S1.1 into the water-alcohol-ammonia mixed solution under stirring; S1.3, adding TEOS solution dropwise under stirring, continuing to react under stirring at room temperature for 3.5-4.5 hours, transferring the solution to a centrifuge tube, centrifuging at 7000-9000 rpm for 8-12 minutes, collecting the precipitate, repeatedly washing the precipitate with ethanol and ultrapure water for 3 times, drying to obtain quantum dots, and redispersing the quantum dots in ultrapure water to prepare a quantum dot solution; In step S1.1, the concentration of the ZnSO4 solution is 0.1-0.2 mol / L, the concentration of the MnCl2 solution is 0.01-0.02 mol / L, the concentration of the CTAB is 0.1-0.2 mol / L, the concentration of the Na2S2O3 is 0.1-0.2 mol / L, the water bath temperature of the water bath precipitation is 45-55°C, and the precipitation time is 1.5-2.5h; The volume ratio of water to ethanol in the water-ethanol mixed solution in step S1.2 is 1:9, and the concentration of ammonia water is 25%-28%.

2. A method for preparing the combined detection kit for pepsinogen I and pepsinogen II as claimed in claim 1, characterized in that: The following steps are included: S1. Preparation of quantum pad: A mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody is prepared; the mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody is coated on a glass cellulose membrane to prepare a quantum pad, and dried; S2. Preparation of nitrocellulose membrane: Paste nitrocellulose membrane on PVC base plate, coat secondary antibody recognizing pepsinogen I antibody and secondary antibody recognizing pepsinogen II antibody on detection line 1 and detection line 2 of nitrocellulose membrane, coat goat anti-mouse IgG on quality control line of nitrocellulose membrane, and then put the coated nitrocellulose membrane into drying oven to dry to form a large plate; S3, assembly: stick the sample pad, quantum pad and absorbent paper on the large board.

3. The method for preparing the combined detection kit for pepsinogen I and pepsinogen II according to claim 2, characterized in that: The specific operation method for preparing the mixture of quantum dot-labeled pepsinogen I antibody and quantum dot-labeled pepsinogen II antibody in step S1 includes: S1.4, adding an activator to the quantum dot solution prepared in step S1.3, activating for 10-20 minutes, purifying, then adding pepsinogen I antibody, mixing for 2-4 hours, centrifuging to obtain precipitate particles, resuspending the precipitate particles with blocking solution, mixing at room temperature for 1-1.5 hours, and then purifying to obtain quantum dot-labeled pepsinogen I antibody; S1.

5. Prepare quantum dot-labeled pepsinogen II antibody in the same manner as step S1.4, and then mix the quantum dot-labeled pepsinogen I antibody and the quantum dot-labeled pepsinogen II antibody.

4. The method for preparing the combined detection kit for pepsinogen I and pepsinogen II according to claim 3, characterized in that: The blocking solution in step S1.4 includes glycine with a molar concentration of 150 mmol / L and BSA with a mass concentration of 5%.

5. The method for preparing the combined detection kit for pepsinogen I and pepsinogen II according to claim 3, characterized in that: In the step S1.5, the volume mixing ratio of the quantum dot-labeled pepsinogen I antibody and the quantum dot-labeled pepsinogen II antibody is 1:

1.

6. The method for preparing the combined detection kit for pepsinogen I and pepsinogen II according to claim 2, characterized in that: The specific method for preparing the nitrocellulose membrane in step S2 includes respectively taking a secondary antibody of pepsinogen I antibody with a concentration of 0.5-2.0 mg / ml, a secondary antibody of pepsinogen II antibody and a goat anti-mouse IgG with a concentration of 0.5-1.5 mg / ml, and spraying them onto the detection line 1, the detection line 2 and the quality control line of the nitrocellulose membrane at a speed of 1 μL / cm and 10 cm / s respectively.

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