Kit for detecting pepsinogen I and preparation method thereof

Abstract

The invention relates to a kit for detecting pepsinogen I and a preparation method of the kit, and relates to the technical field of biology. The kit comprises an enzyme marker, a luminous marker, a luminous auxiliary agent and a triggering agent, the enzyme marker comprises an anti-PG I monoclonal antibody marked by horse radish peroxidase; the luminous marker comprises an anti-PG I monoclonal antibody marked by 9, 10-dihydroacridine; the light-emitting auxiliary agent comprises H2O2. According to the present invention, the spatial proximity chemiluminescence analysis method is adopted, and the carrier and the washing process are not required, such that the kit cost is reduced, and the detection time is shortened; the method adopts homogeneous reaction, so that the detection result is more real and credible.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit for detecting pepsinogen I and a preparation method thereof. Background Art

[0002] Gastric cancer is a common malignant tumor of the digestive tract. It is characterized by abnormal proliferation of the gastric mucosal epithelium, with a high incidence and severe morbidity. Studies have shown that gastric cancer develops progressively from gastritis, gastric polyps, intestinal metaplasia, and intraepithelial neoplasia under the influence of multiple pathogenic factors. Therefore, improving the detection rate of gastric precancerous lesions is equivalent to improving the detection rate of early gastric cancer.

[0003] Clinical studies suggest that during the development of intestinal gastric cancer, the accompanying inflammatory response worsens, and circulating serum pepsinogen I (PGⅠ) and pepsinogen II (PGⅡ) levels also increase. As the disease progresses, when the chief cells in the gastric mucosa are gradually replaced by pyloric gland cells, the pepsinogen gene in the chief cells becomes damaged, causing PGⅠ levels to decrease. PGⅡ, secreted by various glandular cells, continues to increase, resulting in a decrease in the PGⅠ / PGⅡ (PGR) ratio. Based on this, low PGⅠ can be used clinically as a basis for screening individuals at high risk for gastric cancer. Therefore, the development of a test kit for detecting PGⅠ is essential.

[0004] Currently, common methods for detecting Pepsinogen I include enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay (CLI). ELISA has complex and time-consuming procedures, requires specialized personnel, and is subject to numerous operational factors, which can easily lead to false-positive and false-negative results. Chemiluminescence assays offer high sensitivity, good specificity, and stability, and are non-toxic to the environment and the human body. In light of this, the present invention provides a kit for detecting Pepsinogen I and a method for its preparation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a kit for detecting pepsinogen I and a preparation method thereof. The purpose is to use spatial proximity chemiluminescence analysis, which does not require a carrier and a washing process, reduces the cost of the kit, and shortens the detection time.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In the first aspect, a kit for detecting pepsinogen I is provided, comprising an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger; the enzyme marker comprises an anti-PG I monoclonal antibody labeled with horseradish peroxidase (HRP); the luminescent marker comprises an anti-PG I monoclonal antibody labeled with 9,10-dihydroacridine (Acridan); and the luminescent auxiliary agent comprises H2O2.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the enzyme marker also includes bovine serum albumin, sucrose and trehalose;

[0010] The concentration of the horseradish peroxidase-labeled anti-PGⅠ monoclonal antibody in the enzyme marker is 1 mg / ml;

[0011] The concentration of bovine serum albumin in the enzyme marker is 0.5% to 2%;

[0012] The mass concentration of sucrose in the enzyme marker is 2% to 5%;

[0013] The mass concentration of trehalose in the enzyme marker is 1% to 3%.

[0014] Furthermore, the luminescent marker also includes casein, sucrose and trehalose;

[0015] The concentration of the 9,10-dihydroacridine-labeled anti-PGⅠ monoclonal antibody in the luminescent marker is 0.5 mg / ml;

[0016] The concentration of casein in the luminescent marker is 0.1% to 1%;

[0017] The mass concentration of sucrose in the luminescent marker is 2% to 5%;

[0018] The mass concentration of trehalose in the luminescent marker is 1% to 3%.

[0019] Furthermore, the luminescence auxiliary agent also includes citric acid and sodium citrate;

[0020] Each 1L of the luminescence auxiliary agent includes the following components in the following amounts: 1.5g to 2g of citric acid, 5g to 11g of sodium citrate, and 0.15mL to 0.45mL of 30% hydrogen peroxide.

[0021] Furthermore, each 1L of the triggering agent includes the following components: 3g to 9g of tris(hydroxymethyl)aminomethane hydrochloride, 6g to 12g of NaCl, 1mL to 4mL of concentrated HCl, and 1mL to 4mL of Tween 20;

[0022] The mass content of the concentrated HCl is 36% to 38%.

[0023] In a second aspect, a method for preparing a kit for detecting pepsinogen I comprises the following steps:

[0024] (1) Enzyme markers were prepared using horseradish peroxidase and anti-PGⅠ monoclonal antibodies;

[0025] (2) using 9,10-dihydroacridine and anti-PGⅠ monoclonal antibody to prepare a luminescent marker;

[0026] (3) preparing a luminescence auxiliary agent using citric acid, sodium citrate and hydrogen peroxide;

[0027] (4) The trigger was prepared using tris(hydroxymethyl)aminomethane hydrochloride, NaCl, concentrated HCl and Tween 20.

[0028] Furthermore, the preparation of the enzyme marker in step (1) includes the following specific steps:

[0029] Dissolve horseradish peroxidase in distilled water, add sodium periodate solution, mix well, and dialyze against sodium acetate buffer. Then, add carbonate buffer, anti-PGⅠ monoclonal antibody, and sodium borohydride solution. Mix well, and dialyze again against PBS. Remove the dialyzate, add 50% glycerol, and store at -20°C until use. The amounts of horseradish peroxidase, sodium periodate solution, sodium acetate buffer, anti-PGⅠ monoclonal antibody, and sodium borohydride solution used should meet the requirements.

[0030] For example, 1 mg of horseradish peroxidase (HRP) was dissolved in 200 μL of distilled water, 40 μL of freshly prepared 0.1 M sodium periodate solution was added, and the mixture was shaken at room temperature for 20 min. The above liquid was placed in a dialysis bag, mixed, and then dialyzed in sodium acetate buffer. It was dialyzed overnight at 4°C with 1 mM sodium acetate buffer at pH 4.4. 20 μL of 0.2 M pH 9.5 carbonate buffer was added, and then 1 mg of anti-PGⅠ monoclonal antibody was immediately added. The mixture was gently stirred at room temperature in the dark for 2 h, 10 μL of freshly prepared 4 mg / mL sodium borohydride solution was added, mixed, and placed at 4°C for 2 h. The above liquid was placed in a dialysis bag again in PBS, and dialyzed again with 0.15 M pH 7.4 PBS at 4°C overnight. The dialyzate was removed, added with 50% glycerol, and stored at -20°C for later use.

[0031] Furthermore, the preparation of the luminescent marker in step (2) includes the following specific steps:

[0032] Dissolve 9,10-dihydroacridine, add sodium borate buffer and anti-PGⅠ monoclonal antibody, shake and mix overnight, take out and add 50% glycerol and store in a -20℃ refrigerator.

[0033] The amounts of 9,10-dihydroacridine dissolution, sodium borate buffer, and anti-PGⅠ monoclonal antibody used only need to meet the requirements.

[0034] For example, for 1 mg of luminescent substrate, dissolve 9,10-dihydroacridine (Acridan) in 500 μL of DMF, take out 41.3 μL, add 708.7 μL of 0.05 M sodium borate buffer, then add 250 μL of anti-PGⅠ monoclonal antibody (diluted to 1 mg / mL), invert and mix 5 times, let stand at room temperature for 30 minutes, place the labeled reaction tube on a shaker at 2-8°C and mix overnight, take out and add 50% glycerol and store in a -20°C refrigerator.

[0035] Furthermore, the preparation of the luminescence auxiliary agent includes the following specific steps:

[0036] Take citric acid, sodium citrate and 30% hydrogen peroxide, dissolve them in purified water and adjust the volume to obtain the luminescence auxiliary agent.

[0037] Furthermore, the preparation of the trigger comprises the following specific steps:

[0038] Tris (hydroxymethyl)aminomethane hydrochloride, NaCl, concentrated HCl and Tween 20 were weighed, dissolved and then fixed to volume to obtain the triggering agent.

[0039] The present invention adopts a spatial proximity chemiluminescence analysis method to detect the concentration of PG I. A sample, an anti-PG I monoclonal antibody labeled with horseradish peroxidase (HRP), and an anti-PG I monoclonal antibody labeled with 9,10-dihydroacridine (Acridan) are reacted together to form an antigen-antibody sandwich complex, so that the horseradish peroxidase and 9,10-dihydroacridine (Acridan) are spatially close to each other. A luminescence auxiliary agent and a triggering agent are added to generate flash chemiluminescence. Unbound free HRP-labeled antibody and Acridan-labeled antibody do not emit light. The higher the PG I content in the sample, the greater the measured luminescence value (RLU). Therefore, within a certain concentration range, the luminescence value is positively correlated with the sample concentration. A working curve is drawn by using a calibrator with a known concentration and its luminescence value, and the PG I content in the sample can be calculated based on the luminescence value of the sample.

[0040] The beneficial effects of the present invention are: the present invention adopts spatial proximity chemiluminescence analysis, which does not require a carrier and washing process, which not only reduces the cost of the kit but also shortens the detection time; in addition, the method is a truly homogeneous reaction, making the detection results more real and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the standard curve diagram of the present invention. DETAILED DESCRIPTION

[0042] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0043] Example

[0044] 1. Materials and Reagents

[0045] (1) Antigen and Antibody:

[0046] Anti-PGⅠ monoclonal antibodies were purchased from Nanjing Jingda Biotechnology Co., Ltd., with catalog numbers M9315 and M9316; PGⅠ antigen was purchased from Nanjing Ainuodi Biotechnology Co., Ltd., with catalog number AMP0035.

[0047] (2) Other reagents:

[0048] Horseradish peroxidase (HRP), sodium periodate, sodium borohydride, sodium acetate, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, tris (hydroxymethyl)aminomethane hydrochloride (Tris), hydrochloric acid, citric acid, sodium citrate, glycerol, boric acid, sodium tetraborate, sucrose, aminopyrine, Tween 20, Triton 100, potassium dihydrogen phosphate, hydrogen peroxide, and dimethylformamide were purchased from Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin, casein, Proclin 300, and polyethylene glycol 2000 were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. 9,10-Dihydroacridine (Acridan) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0049] (3) Instruments:

[0050] Guangzhou Jinde Biotechnology Co., Ltd. HomoG 100 fully automatic chemiluminescence analyzer.

[0051] 2. Kit and Preparation Method

[0052] 2.1 Kit for detecting pepsinogen I

[0053] A kit for detecting pepsinogen I, comprising an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger; the enzyme marker comprises an anti-PG I monoclonal antibody labeled with horseradish peroxidase (HRP); the luminescent marker comprises an anti-PG I monoclonal antibody labeled with 9,10-dihydroacridine (Acridan); and the luminescent auxiliary agent comprises H2O2.

[0054] Preferably, the enzyme marker further comprises bovine serum albumin, sucrose and trehalose;

[0055] The concentration of the horseradish peroxidase-labeled anti-PGⅠ monoclonal antibody in the enzyme marker is 1 mg / ml;

[0056] The concentration of bovine serum albumin in the enzyme marker is 0.5%-2%;

[0057] The mass concentration of sucrose in the enzyme marker is 2% to 5%;

[0058] The mass concentration of trehalose in the enzyme marker is 1% to 3%.

[0059] Preferably, in this embodiment, the luminescent marker further comprises casein, sucrose and trehalose;

[0060] The concentration of the 9,10-dihydroacridine-labeled anti-PGⅠ monoclonal antibody in the luminescent marker is 0.5 mg / ml;

[0061] The concentration of casein in the luminescent marker is 0.1% to 1%;

[0062] The mass concentration of sucrose in the luminescent marker is 2% to 5%;

[0063] The mass concentration of trehalose in the luminescent marker is 1% to 3%.

[0064] In this embodiment, the luminescence auxiliary agent further comprises citric acid and sodium citrate;

[0065] Each 1L of the luminescence auxiliary agent includes the following components in the following amounts: 1.5g to 2g of citric acid, 5g to 11g of sodium citrate, and 0.15mL to 0.45mL of 30% hydrogen peroxide.

[0066] In this embodiment, preferably, each 1L of the triggering agent includes the following components: 3g to 9g of tris (hydroxymethyl)aminomethane hydrochloride, 6g to 12g of NaCl, 1mL to 4mL of concentrated HCl, and 1mL to 4mL of Tween 20;

[0067] The mass content of the concentrated HCl is 36% to 38%.

[0068] The following is a detailed description of the preparation process of this kit, and the prepared kit is used for experimental detection.

[0069] 2.2 Preparation of reagents

[0070] (1) Preparation of enzyme markers:

[0071] Weigh 1 mg of horseradish peroxidase (HRP) and dissolve it in 200 μL of distilled water. Add 40 μL of freshly prepared 0.1 M sodium periodate and shake at room temperature for 20 min. Pour the above liquid into a dialysis bag and dialyze it with 1 mM sodium acetate buffer at pH 4.4 at 4°C overnight. Add 20 μL of 0.2 M carbonate buffer at pH 9.5, and then immediately add 1 mg of anti-PGⅠ monoclonal antibody and stir gently at room temperature in the dark for 2 h. Add 10 μL of freshly prepared 4 mg / mL sodium borohydride solution, mix well, and let it stand at 4°C for 2 h. Pour the above liquid into a dialysis bag and dialyze it with 0.15 M pH 7.4 PBS at 4°C overnight. Remove the dialyzate, add 50% glycerol and store it at -20°C for later use.

[0072] (2) Preparation of luminescent markers:

[0073] Dissolve 1 mg of the luminescent substrate 9,10-dihydroacridine (Acridan) in 500 μL of DMF, take out 41.3 μL, add 708.7 μL of 0.05 M sodium borate buffer, then add 250 μL of anti-PGⅠ monoclonal antibody (diluted to 1 mg / mL), invert and mix 5 times, let stand at room temperature for 30 minutes, place the labeled reaction tube on an oscillator at 2-8°C and mix overnight, take out and add 50% glycerol and store in a refrigerator at -20°C.

[0074] (3) Luminescence auxiliary agent:

[0075] Dissolve 1.82 g of citric acid and 10.45 g of sodium citrate in purified water and dilute to 1000 mL. Add 0.3 mL of 30% hydrogen peroxide to the buffer solution, mix well, and place in a 4°C refrigerator for later use (divide into 10 mL / bottle).

[0076] (4) Trigger:

[0077] Weigh 6.06 g of tris (hydroxymethyl)aminomethane hydrochloride (Tris) and 9 g of NaCl, dissolve them in appropriate amount of purified water, add 2.1 mL of concentrated HCl and 2 mL of Tween 20, mix well, and adjust the volume to 1000 mL. Place in a 4°C refrigerator until ready for use.

[0078] (5) Calibrators:

[0079] ① Calibrator diluent: Weigh 14.1 g of potassium dihydrogen phosphate and 3.0 g of sodium dihydrogen phosphate dihydrate, dissolve them in an appropriate amount of purified water, add 0.5 mL of Proclin 300, mix well, and dilute to 1000 mL with purified water. Place in a 4°C refrigerator until ready for use.

[0080] ② Dilute the recombinant PGⅠ to 1.56 ng / mL, 3.13 ng / mL, 12.5 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL with calibrator diluent and place in a 4°C refrigerator for later use.

[0081] 3. Detection method:

[0082] The system requires a sample volume of 10 μL per measurement. Before applying for a test, you should prepare all the materials required for the test and carefully read the chemiluminescence immunoassay user manual to obtain information on system operating procedures, sample management, usage precautions, maintenance, and other information. The main steps for applying for a test are as follows:

[0083] (1) Calibration:

[0084] Perform a calibration test using the included kit and calibrator. Before starting calibration, scan the master curve in the kit's 2D barcode and import it into the system.

[0085] The chemiluminescence immunoassay analyzer uses the results of calibrator tests to adjust the master curve and generate the calibration curve for the current system. The instrument operating software includes a calibration check function that automatically verifies the validity of the calibration curve. If calibration fails, consider the instrument settings, status, location of the calibrators (place low-value calibrators first, then high-value calibrators), and expiration dates.

[0086] A valid calibration curve is required before all tests. Recalibration is required in the following situations:

[0087] ① Use a new batch of test kit;

[0088] ② The same batch of test kits has been used on the analyzer for more than 30 days;

[0089] ③After instrument maintenance;

[0090] ④The quality control value exceeds the specified range;

[0091] (2) Quality Control:

[0092] To ensure the reliability of test results, it is recommended that both high and low level quality control products be tested once every 24 hours. In addition, quality control testing is also recommended after each calibration test, reagent batch change, maintenance and troubleshooting.

[0093] The test values of quality control products should be within the specified range. If they are outside the specified range, the user should check the detection system, such as the location, expiration date, storage method, calibration process, instrument performance and status, and recalibrate if necessary. Sample testing can only be carried out after the quality control is qualified.

[0094] (3) Sample testing:

[0095] ① Enter the sample application interface and enter the sample number, sample rack number, location number, sample type, remarks and other information according to the test requirements;

[0096] ② Select the test item, select the sample type in the item options, enter the number of repetitions and other information;

[0097] ③After preparing the test sample and placing it correctly, click the start button to start the test.

[0098] Among them, the chemiluminescence immunoassay analyzer will perform the following operations in sequence:

[0099] A. The sample handling system transfers the sample rack to the sample aspiration position;

[0100] B. The cuvette loading and scheduling system loads the cuvette from the loading area to the sample loading position;

[0101] C. The sample injection system completes the sample injection and cleaning of the sample needle;

[0102] D. The reagent processing system provides the reagents required for the test, mixes each reagent, and sends it to the reagent aspiration position for aspiration;

[0103] E. The sample injection system completes the reagent injection and cleaning of the reagent needle;

[0104] F. Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0105] G. After incubation is complete, inject substrate solution into the substrate system, mix, incubate, and wait for light detection;

[0106] H. The light measurement reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0107] I. Calculate the amount of analyte in the sample.

[0108] (4) Calculation

[0109] Using stored calibration data, the system software automatically determines the sample test results using a weighted four-parameter logarithmic curve (4PLC, Y-weighted) mathematical method. Results are given in ng / mL.

[0110] 4. Comparison reagents:

[0111] Pepsinogen I (PG I) detection kit (chemiluminescence method) from Ruibo Biotechnology Co., Ltd., Guangdong Medical Device Registration No. 20212400994.

[0112] 5. Experimental Results

[0113] (1) Construction of standard curve

[0114] Detection of 50 gastric acid secretory gland cell function patient samples, and the quantitative correlation coefficient R 2 =0.9779( Figure 1 and Table 1 ).

[0115] Table 1

[0116]

[0117]

[0118] according to Figure 1 As shown in Table 1, the two are well correlated, and the yin-yang coincidence rate is high.

[0119] (2) Minimum detection limit

[0120] Using PBS buffer as a blank sample, the detection and comparison reagents prepared in the present invention were simultaneously measured 25 times. The test strip test and comparison values prepared in the present invention, plus twice the standard deviation, were substituted into the fitted curve for regression. The results showed that the detection kit prepared in the present invention had a minimum detection limit of 0.8, and the comparison reagent had a minimum detection limit of 0.8. The results are shown in Table 2.

[0121] Table 2

[0122]

[0123]

[0124] (3) Accuracy

[0125] A high-concentration reference substance (Solution A) was added to a low-concentration reference substance (Solution B), with the volume ratio between the high-concentration reference substance (Solution A) and the low-concentration reference substance (Solution B) being no greater than 1:9. Each sample was measured three times and the average value was obtained. Calculated according to formula (1), the recovery rate of the detection preparation method prepared by the present invention was 99.52%, indicating good accuracy. The results are shown in Table 3.

[0126]

[0127] Where: R is the recovery rate; V is the volume of solution A added; V0 is the volume of serum sample B; c is the detection concentration of serum sample after adding solution A; c0 is the detection concentration of serum sample B; c s is the concentration of solution A.

[0128] Table 3

[0129]

[0130]

[0131] In summary, the spatial proximity chemical double antibody sandwich method used in this kit is simple to operate and can be fully automatically detected compared to other methods such as enzyme-linked immunosorbent assay and fluorescent immunochromatography. It does not require magnetic beads and washing, which not only reduces reagent costs but also shortens detection time. In addition, it adopts a homogeneous reaction, avoiding the influence of traditional chemiluminescence magnetic separation technology, and the detection results are more accurate and stable.

[0132] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit for detecting pepsinogen I, characterized in that: The kit comprises an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger; the enzyme marker comprises an anti-PG I monoclonal antibody labeled with horseradish peroxidase; the luminescent marker comprises an anti-PG I monoclonal antibody labeled with 9,10-dihydroacridine; and the luminescent auxiliary agent comprises H2O2.

2. A kit for detecting pepsinogen I according to claim 1, characterized in that: The enzyme marker also includes bovine serum albumin, sucrose and trehalose; The concentration of the horseradish peroxidase-labeled anti-PGⅠ monoclonal antibody in the enzyme marker is 1 mg / ml; The concentration of bovine serum albumin in the enzyme marker is 0.5% to 2%; The mass concentration of sucrose in the enzyme marker is 2% to 5%; The mass concentration of trehalose in the enzyme marker is 1% to 3%.

3. A kit for detecting pepsinogen I according to claim 1, characterized in that: The luminescent marker also includes casein, sucrose and trehalose; The concentration of the 9,10-dihydroacridine-labeled anti-PGⅠ monoclonal antibody in the luminescent marker is 0.5 mg / ml; The concentration of casein in the luminescent marker is 0.1% to 1%; The mass concentration of sucrose in the luminescent marker is 2% to 5%; The mass concentration of trehalose in the luminescent marker is 1% to 3%.

4. A kit for detecting pepsinogen I according to claim 1, characterized in that: The luminescence auxiliary agent also includes citric acid and sodium citrate; Each 1L of the luminescence auxiliary agent includes the following components in the following amounts: 1.5g to 2g of citric acid, 5g to 11g of sodium citrate, and 0.15mL to 0.45mL of 30% hydrogen peroxide.

5. A kit for detecting pepsinogen I according to claim 1, characterized in that: Each 1L of the triggering agent includes the following components: 3g to 9g of tris (hydroxymethyl)aminomethane hydrochloride, 6g to 12g of NaCl, 1mL to 4mL of concentrated HCl, and 1mL to 4mL of Tween 20; The mass content of the concentrated HCl is 36% to 38%.

6. A method for preparing a kit for detecting pepsinogen I according to any one of claims 1 to 5, characterized in that: The steps include: (1) Enzyme markers were prepared using horseradish peroxidase and anti-PGⅠ monoclonal antibodies; (2) using 9,10-dihydroacridine and anti-PGⅠ monoclonal antibody to prepare a luminescent marker; (3) preparing a luminescence auxiliary agent using citric acid, sodium citrate and hydrogen peroxide; (4) The trigger was prepared using tris(hydroxymethyl)aminomethane hydrochloride, NaCl, concentrated HCl and Tween 20.

7. The method for preparing a kit for detecting pepsinogen I according to claim 6, characterized in that: The preparation of the enzyme marker in step (1) comprises the following specific steps: Dissolve horseradish peroxidase in distilled water, add sodium periodate solution, mix well, and dialyze against sodium acetate buffer. Then add carbonate buffer, anti-PGⅠ monoclonal antibody, and sodium borohydride solution. Mix well, and dialyze again against PBS. Remove the dialyzate, add 50% glycerol, and store at -20°C until use.

8. The method for preparing a kit for detecting pepsinogen I according to claim 6, characterized in that: The preparation of the luminescent marker in step (2) includes the following specific steps: Dissolve 9,10-dihydroacridine, add sodium borate buffer and anti-PGⅠ monoclonal antibody, shake and mix overnight, take out and add 50% glycerol and store in a -20℃ refrigerator.

9. The method for preparing a kit for detecting pepsinogen I according to claim 6, wherein: The preparation of the luminescence auxiliary agent in step (3) includes the following specific steps: Take citric acid, sodium citrate and 30% hydrogen peroxide, dissolve them in purified water and adjust the volume to obtain the luminescence auxiliary agent.

10. The method for preparing a kit for detecting pepsinogen I according to claim 6, characterized in that: The preparation of the trigger in step (4) includes the following specific steps: Tris (hydroxymethyl)aminomethane hydrochloride, NaCl, concentrated HCl and Tween 20 were weighed, dissolved and then fixed to volume to obtain the triggering agent.

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