Gastrin-releasing peptide precursor determination kit and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly discloses a gastrin-releasing peptide precursor determination kit and application, the gastrin-releasing peptide precursor determination kit comprises an anti-reagent A and an anti-reagent B; according to the invention, the antibody is labeled by the enhanced nano material, the detection sensitivity is obviously improved by the signal amplification effect, and the detection limit can be effectively detected.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a gastrin-releasing peptide precursor assay kit and application thereof. Background Art

[0002] Gastrin-releasing peptide (GRP) is an important regulatory molecule involved in many physiological functions and pathological conditions in the human body. The human GRP gene encodes a 148-amino acid precursor of the gastrin-releasing peptide (preproGRP), which consists of a signal peptide, GRP1-27, and the C-terminal GRP31-125. PreproGRP is then converted to pro-GRP1-125, which undergoes endogenous proteolysis and amidation to generate mature GRP1-27, GRP18-27, and a C-terminal extension peptide. The C-terminal extension peptide then generates molecules containing the C-terminal proGRP. proGRP, the precursor structure of gastrin-releasing peptide, has a long half-life, is more stable than GRP, and is easily detected.

[0003] Studies have confirmed that proGRP can represent GRP levels and gene expression, and therefore can be used as a tumor marker for SCLC. ProGRP is very useful for differentiating small cell lung cancer from non-small cell lung cancer (NSCLC). Although proGRP is a specific tumor marker for SCLC, elevated levels can also be seen in a small percentage of NSCLC patients. These patients have significantly lower serum proGRP concentrations than those in SCLC patients. Several researchers have reported that proGRP is helpful in monitoring SCLC patients undergoing treatment and in detecting recurrences. NSE can serve as a supplemental tumor marker for SCLC. Combining NSE and proGRP results can provide additional evidence for histological diagnosis, prognosis, and follow-up. Summary of the Invention

[0004] The present invention provides a gastrin-releasing peptide precursor assay kit and its application. The present invention uses enhanced nanomaterials to label antibodies, and the signal amplification effect significantly improves the detection sensitivity, which can effectively detect the detection limit.

[0005] The present invention solves its technical problems by adopting the following technical solutions: A progastrin-releasing peptide (proGRP) assay kit, comprising anti-reagent A and anti-reagent B; The preparation method of the anti-reagent A is as follows: (1) tetrachloroauric acid, vitamin C, citric acid, and polyvinylpyrrolidone are uniformly dispersed in an ethanol solution, and then RGDT (arginine-glycine-aspartic acid-threonine) is added and stirred to obtain a mixed solution; (2) Biotin and proGRP antibody were evenly dispersed in phosphate buffer, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and the pH was adjusted to 5.4-5.8. The reaction was carried out (temperature 40-45°C, time 2-6 hours) to obtain a precursor solution; (3) Add the precursor solution to the mixed solution, then add sodium borohydride, adjust the pH to 3.5-4.2, stir evenly, filter, and then dilute with a diluent to obtain anti-reagent A.

[0006] As a preferred embodiment of the present invention, the mass ratio of tetrachloroauric acid, vitamin C, citric acid, polyvinyl pyrrolidone, ethanol solution, and RGDT is 1: (0.05-0.2): (0.05-0.2): (0.05-0.12): (4-12): (0.02-0.06).

[0007] As a preferred embodiment of the present invention, the mass ratio of biotin, proGRP antibody, phosphate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(2-5):(4-10):(0.1-0.25):(0.1-0.25).

[0008] As a preferred embodiment of the present invention, the mass ratio of the precursor solution, the mixed solution, and sodium borohydride is 1: (0.2-0.5): (0.05-0.12).

[0009] As a preferred embodiment of the present invention, the preparation method of the anti-reagent B is: Alkaline phosphatase was dissolved in carbonate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and then proGRP antibody, potassium dichromate and sodium borohydride were added, stirred evenly, and then diluted with diluent to obtain anti-reagent B.

[0010] As a preferred embodiment of the present invention, the mass ratio of the alkaline phosphatase to the carbonate buffer is 1:(5-100).

[0011] As a preferred embodiment of the present invention, the mass ratio of alkaline phosphatase, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, proGRP antibody, potassium dichromate, and sodium borohydride is 1:(0.1~0.12):(0.1~0.15):(1~2):(0.2~0.6):(0.2~0.6).

[0012] As a preferred embodiment of the present invention, the diluent includes the following components in parts by mass: 0.2-0.6% Tween 20, 0.4-1.2% polyvinyl alcohol, 0.2-0.8% polyvinyl pyrrolidone, 0.4-1% L-cysteine, 0.4-1% calcium oxide, 0.05-0.2% diethylenetriaminepentaacetic acid, 2-4% bovine serum albumin, 0.2-0.8% reduced glutathione, and the balance phosphate buffer.

[0013] As a preferred embodiment of the present invention, it also includes a magnetic particle reagent, which is magnetic particles coupled with streptavidin. The preparation method of the magnetic particles coupled with streptavidin is as follows: 10 mg of zinc oxide ferromagnetic particles are resuspended with MES buffer, and then 100-200 μL of 5-15 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution is added and stirred evenly. 0.5-2 mg of streptavidin is added and reacted at room temperature for 1-3 hours. The supernatant is magnetically separated and washed, and then a blocking agent is added for blocking. Finally, the volume is adjusted to 500 mL with a stabilizing diluent to obtain magnetic particles coupled with streptavidin.

[0014] As a preferred embodiment of the present invention, it further comprises a substrate solution, which is an enzymatic luminescent substrate.

[0015] As a preferred embodiment of the present invention, it further comprises a cleaning solution, which is a Tris buffer.

[0016] As a preferred embodiment of the present invention, the kit of the present invention may further include calibrators and quality control products conventional in the art.

[0017] The present invention also provides the use of the gastrin-releasing peptide precursor assay kit in detecting the concentration of gastrin-releasing peptide precursor in human serum and / or plasma.

[0018] The beneficial effects of the present invention are as follows: (1) The present invention uses enhanced nanomaterials to label antibodies, and the signal amplification effect significantly improves the detection sensitivity, which can effectively detect the detection limit; (2) The anti-reagent A and anti-reagent B of the present invention can effectively improve the selectivity and stability, eliminate non-specific interference, and by optimizing the binding efficiency of antibodies and antigens, the kit can provide more accurate quantitative analysis results, which is particularly suitable for the screening and monitoring of early cancer; (3) The kit method of the present invention is stable, has good repeatability, high tightness, and a good linear range. DETAILED DESCRIPTION

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0020] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0021] In the present invention, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0022] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0023] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained commercially. The raw materials used in the comparative examples and the raw materials used in the parallel experiments of the examples are the same commercially available products unless otherwise specified.

[0024] The gastrin-releasing peptide precursor assay kit of the present invention adopts the sandwich method principle for detection: (1) The sample, anti-reagent A and anti-reagent B are reacted under incubation conditions to form an antibody-antigen-antibody complex which is then bound to the magnetic particles through the reaction of biotin and streptavidin.

[0025] (2) Detection reading: After the incubation is completed, a magnetic field is applied to precipitate the solution, the supernatant is removed, the precipitated complex is washed with a cleaning solution, the waste liquid is aspirated to remove the substances not bound to the magnetic particles, and the reaction cup is then placed into the measurement chamber. The instrument automatically pumps in the chemiluminescent substrate 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane disodium salt (AMPPD). The luminescent substrate generates a chemiluminescent signal under the catalysis of alkaline phosphatase, and the luminescence intensity is measured by an optical detection system.

[0026] (3) The instrument automatically calculates the test results through the working curve.

[0027] The detection instrument can be the POClia chemiluminescence analyzer (models: POClia 8, POClia minus, POClia plus, POClia auto) produced by Taizhou Zecheng Biotechnology Co., Ltd., or the CIA 600, CIA 1200, CIA 1200M, CIA 1800, CIA 2800 produced by Taizhou Zecheng Biotechnology Co., Ltd., or the Shine i1900 and Shine i2000 produced by Shenzhen Yingkai Biotechnology Co., Ltd.

[0028] The samples are human serum and plasma (anticoagulant: sodium heparin, lithium heparin, or EDTA).

[0029] The unopened test kit is shelf life for 15 months when stored sealed and protected from light at 2°C–8°C. Once opened, it is stable for 28 days when stored at 2°C–8°C and protected from light. Calibrators can be stored at 2°C–8°C for 28 days after reconstitution. Controls can be stored at 2°C–8°C for 28 days after reconstitution.

[0030] The test kit should be transported at low temperature (2℃~8℃) and the transportation time should not exceed 8 days.

[0031] Sample collection and processing: (1) Sample collection: Use collection tubes without anticoagulants or collection tubes containing anticoagulants (sodium heparin anticoagulant, lithium heparin anticoagulant, EDTA anticoagulant) to collect blood samples according to standard procedures.

[0032] (2) Sample processing The collected blood samples were allowed to stand at room temperature and then centrifuged (3000 rpm for 5 minutes) to separate the serum or plasma fraction.

[0033] If the serum or plasma sample is turbid or contains visible flocculent fibrin, centrifuge the sample at 3000 rpm for 15 minutes and use the supernatant.

[0034] Sample transportation and storage: Samples should be centrifuged immediately after blood collection and tested the same day. If immediate testing is not possible, samples should be stored in cryotubes without rubber stoppers at 2°C to 8°C for 7 days and at -20±5°C for 6 months. Avoid repeated freezing and thawing. Samples should be transported at -20±5°C for no more than 5 days. Samples should be equilibrated to room temperature before testing.

[0035] Example 1 A gastrin-releasing peptide precursor assay kit comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a substrate solution, and a cleaning solution; The preparation method of the anti-reagent A is as follows: (1) tetrachloroauric acid, vitamin C, citric acid, and polyvinyl pyrrolidone are uniformly dispersed in a 65wt% ethanol solution, and then RGDT is added and stirred to obtain a mixed solution; the mass ratio of the tetrachloroauric acid, vitamin C, citric acid, polyvinyl pyrrolidone, ethanol solution, and RGDT is 1:0.05:0.05:0.05:4:0.02.

[0036] (2) Biotin and proGRP antibody were evenly dispersed in phosphate buffer, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and the pH was adjusted to 5.4-5.8. The reaction was carried out (temperature was 40°C, time was 4 hours) to obtain a precursor solution; the mass ratio of biotin, proGRP antibody, phosphate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide was 1:5:4:0.1:0.1.

[0037] (3) Add the precursor solution to the mixed solution, then add sodium borohydride, adjust the pH to 3.5-4.2, stir evenly, filter, and then dilute 100 times with a diluent to obtain anti-reagent A. The mass ratio of the precursor solution, mixed solution, and sodium borohydride is 1:0.2:0.05.

[0038] The anti-reagent B is prepared by dissolving alkaline phosphatase in carbonate buffer (pH 9), adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, then adding proGRP antibody, potassium dichromate, and sodium borohydride, stirring evenly, and then diluting 100-fold with a diluent to obtain anti-reagent B. The mass ratio of alkaline phosphatase to carbonate buffer is 1:50:0.1:0.1:1:0.2:0.2.

[0039] The diluent includes the following components in parts by weight: 0.5% Tween 20, 1% polyvinyl alcohol, 0.6% polyvinyl pyrrolidone, 0.8% L-cysteine, 0.7% calcium oxide, 0.1% diethylenetriaminepentaacetic acid, 3% bovine serum albumin, 0.5% reduced glutathione, and the balance phosphate buffer.

[0040] Preparation of magnetic particle reagent: Resuspend 10 mg of zinc oxide ferromagnetic particles in MES buffer (pH 5), then add 150 μL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and stir evenly. Add 1 mg of streptavidin and react at room temperature for 2 hours. After magnetic separation, the supernatant is washed and blocked with a blocking agent. Finally, the volume is adjusted to 500 mL with a stabilizing diluent to obtain magnetic particles coupled with streptavidin.

[0041] The blocking agent is a phosphate buffer with a pH of 7.5 containing 5% BSA and 0.1% ProClin300.

[0042] The substrate solution is an enzymatic luminescent substrate, wherein the luminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane disodium salt.

[0043] The cleaning solution is Tris buffer.

[0044] Calibrator Preparation: Dilute the antigen calibrator in a series of concentrations using Calibrator Diluent (Tris diluent, pH 7.4, containing 0.05% prolin 300 and 0.5% BSA). Lyophilize and use as the kit calibrator. Calibrator concentrations are: 0, 20, 40, 80, 200, and 500 pg / mL.

[0045] Example 2 A gastrin-releasing peptide precursor assay kit comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a substrate solution, and a cleaning solution; The preparation method of the anti-reagent A is as follows: (1) tetrachloroauric acid, vitamin C, citric acid, and polyvinyl pyrrolidone are uniformly dispersed in a 65wt% ethanol solution, and then RGDT is added and stirred to obtain a mixed solution; the mass ratio of the tetrachloroauric acid, vitamin C, citric acid, polyvinyl pyrrolidone, ethanol solution, and RGDT is 1:0.2:0.2:0.12:12:0.06.

[0046] (2) Biotin and proGRP antibody were evenly dispersed in phosphate buffer, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and the pH was adjusted to 5.4-5.8. The reaction was carried out (temperature was 40°C, time was 4 hours) to obtain a precursor solution; the mass ratio of biotin, proGRP antibody, phosphate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide was 1:2:10:0.25:0.25.

[0047] (3) Add the precursor solution to the mixed solution, then add sodium borohydride, adjust the pH to 3.5-4.2, stir evenly, filter, and then dilute 100 times with a diluent to obtain anti-reagent A. The mass ratio of the precursor solution, mixed solution, and sodium borohydride is 1:0.2:0.05.

[0048] The anti-reagent B is prepared by dissolving alkaline phosphatase in carbonate buffer (pH 9), adding ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, then adding proGRP antibody, potassium dichromate, and sodium borohydride, stirring evenly, and then diluting 100-fold with a diluent to obtain anti-reagent B. The mass ratio of alkaline phosphatase to carbonate buffer is 1:50:0.1:0.1:1:0.2:0.2.

[0049] The diluent includes the following components in parts by weight: 0.5% Tween 20, 1% polyvinyl alcohol, 0.6% polyvinyl pyrrolidone, 0.8% L-cysteine, 0.7% calcium oxide, 0.1% diethylenetriaminepentaacetic acid, 3% bovine serum albumin, 0.5% reduced glutathione, and the balance phosphate buffer.

[0050] Preparation of magnetic particle reagent: Resuspend 10 mg of zinc oxide ferromagnetic particles in MES buffer (pH 5), then add 150 μL of 10 mg / mL ethyl-(3-dimethylaminopropyl)carbodiimide solution and stir evenly. Add 1 mg of streptavidin and react at room temperature for 2 hours. After magnetic separation, the supernatant is washed and blocked with a blocking agent. Finally, the volume is adjusted to 500 mL with a stabilizing diluent to obtain magnetic particles coupled with streptavidin.

[0051] The blocking agent is a phosphate buffer with a pH of 7.5 containing 5% BSA and 0.1% ProClin300.

[0052] The substrate solution is an enzymatic luminescent substrate, wherein the luminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane disodium salt.

[0053] The cleaning solution is Tris buffer.

[0054] Calibrator Preparation: Dilute the antigen calibrator in a series of concentrations using Calibrator Diluent (Tris diluent, pH 7.4, containing 0.05% prolin 300 and 0.5% BSA). Lyophilize and use as the kit calibrator. Calibrator concentrations are: 0, 20, 40, 80, 200, and 500 pg / mL.

[0055] Comparative Example 1

[0056] The difference between Comparative Example 2 and Example 1 is that the diluent is pure water, and the other aspects are the same.

[0057] Comparative Example 2

[0058] Comparative Example 2 differs from Example 1 in that the diluent is a phosphate buffer solution containing 0.5% Tween 20 and 1% polyvinyl alcohol, and all other conditions are the same.

[0059] Comparative Example 3

[0060] The difference between Comparative Example 3 and Example 1 is that the preparation method of anti-reagent A is different, and the other aspects are the same.

[0061] The preparation method of the anti-reagent A is as follows: biotin and proGRP antibody are uniformly dispersed in a phosphate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, the pH is adjusted to 5.4-5.8, the reaction is carried out (temperature is 40°C, time is 4 hours), and then diluted with a diluent to obtain anti-reagent A; the mass ratio of biotin, proGRP antibody, phosphate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:2-5:4:0.1:0.1.

[0062] Test Case 1. Limit of Detection: Repeat the test 20 times using a zero-concentration calibrator as the sample to obtain the RLU values (relative luminescence values) of the 20 measurements. Calculate the mean (M) and standard deviation (SD) to obtain the RLU value corresponding to M+2SD. Perform a linear equation based on the calibration curve equation of the calibrator used in the kit or the concentration-RLU value results between the zero concentration and the adjacent calibrator by performing a two-point regression fit. Substitute the RLU value corresponding to M+2SD into the above equation to obtain the corresponding concentration value, which is the limit of detection. The result should be less than 2 pg / mL.

[0063] Among them, the detection limit of Example 1 is 1 pg / mL, and the detection limit of Example 2 is 1.5 pg / mL; the detection limit of Comparative Example 1 is 20.8 pg / mL, the detection limit of Comparative Example 2 is 18.7 pg / mL, and the detection limit of Comparative Example 3 is 10.9 pg / mL.

[0064] 2. Accuracy Test: Select enterprise accuracy reference materials with concentrations of (60.00±12.00) pg / mL and (1000.00±200.00) pg / mL for measurement. Repeat the test three times. Record the test results as (M). Calculate the relative deviation (B) according to the following formula. If all three results meet the requirements of 2.1.4, the test is considered qualified. If two or more results fail, the test is considered unqualified. If even one result fails to meet the requirements, retest 20 times continuously and calculate the relative deviation for each test. If the results of 19 or more tests are within 10%, the test is considered qualified.

[0065] ; Where: B--relative deviation; M--measurement results; T--calibrated concentration.

[0066] Among them, the accuracy of Examples 1 and 2 is within the range of 10%, which meets the requirements, while the accuracy of Comparative Examples 1 to 3 is outside the range of 10%, which does not meet the requirements.

[0067] Specifically, the content of Example 1 is 5.8%, that of Example 2 is 6.2%, that of Comparative Example 1 is 16.8%, that of Comparative Example 2 is 15.4%, and that of Comparative Example 3 is 10.6%.

[0068] 3. Linearity Test: Dilute a high-value reference substance near the upper limit of the linear range at a specific ratio to create five sample concentrations. The low-value sample concentration must be close to the lower limit of the linear range. Repeat the test three times for each concentration. Calculate the average value of each sample concentration. Use the least squares method to fit a straight line between the average measured concentration and the theoretical concentration. Calculate the linear correlation coefficient, r, according to the formula. A value of 0.9900 or higher is considered acceptable.

[0069] ; The linearity of Examples 1 and 2 meets the requirements, and the correlation coefficient r is not less than 0.9900 in the range of 3.00 pg / mL to 5000.00 pg / mL.

[0070] Comparative Examples 1 to 3 do not meet the requirements.

[0071] Among them, r of Example 1 is 0.994, r of Example 2 is 0.992, r of Comparative Example 1 is 0.944, r of Comparative Example 2 is 0.949, and r of Comparative Example 3 is 0.959.

[0072] 4. Repeatability test: Use the same batch number test kit to measure two samples with different concentration levels, repeat the measurement 10 times, calculate the mean (M) and standard deviation (SD) of the 10 measurement values, and calculate the coefficient of variation (CV) according to the formula. If the calculated value is within 10%, it is qualified.

[0073] ; Where: CV - coefficient of variation; SD - standard deviation of 10 measurements; M--the average value of 10 measurement results.

[0074] Among them, Examples 1-2 and Comparative Examples 1-3 all meet the requirements, and the coefficient of variation (CV) is no more than 10.0%.

[0075] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A gastrin-releasing peptide precursor assay kit, characterized in that: Including anti-reagent A and anti-reagent B; The preparation method of the anti-reagent A is as follows: (1) uniformly dispersing tetrachloroauric acid, vitamin C, citric acid, and polyvinyl pyrrolidone in an ethanol solution, then adding RGDT, and stirring uniformly to obtain a mixed solution; (2) Biotin and proGRP antibody are evenly dispersed in phosphate buffer, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, and the pH is adjusted to 5.4-5.8, and the reaction is carried out to obtain a precursor solution; (3) Add the precursor solution to the mixed solution, then add sodium borohydride, adjust the pH to 3.5-4.2, stir evenly, filter, and then dilute with a diluent to obtain anti-reagent A.

2. The gastrin-releasing peptide precursor assay kit according to claim 1, characterized in that: The mass ratio of the tetrachloroauric acid, vitamin C, citric acid, polyvinyl pyrrolidone, ethanol solution, and RGDT is 1: (0.05-0.2): (0.05-0.2): (0.05-0.12): (4-12): (0.02-0.06).

3. The gastrin-releasing peptide precursor assay kit according to claim 1, characterized in that: The mass ratio of the biotin, proGRP antibody, phosphate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(2-5):(4-10):(0.1-0.25):(0.1-0.25).

4. The gastrin-releasing peptide precursor assay kit according to claim 1, characterized in that: The mass ratio of the precursor solution, the mixed solution and sodium borohydride is 1: (0.2-0.5): (0.05-0.12).

5. The gastrin-releasing peptide precursor assay kit according to claim 1, characterized in that: The preparation method of the anti-reagent B is: Alkaline phosphatase was dissolved in carbonate buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and then proGRP antibody, potassium dichromate and sodium borohydride were added, stirred evenly, and then diluted with diluent to obtain anti-reagent B.

6. The gastrin-releasing peptide precursor assay kit according to claim 5, characterized in that: The mass ratio of the alkaline phosphatase to the carbonate buffer is 1:(5-100).

7. The gastrin-releasing peptide precursor assay kit according to claim 5, characterized in that: The mass ratio of the alkaline phosphatase, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, proGRP antibody, potassium dichromate, and sodium borohydride is 1:(0.1-0.12):(0.1-0.15):(1-2):(0.2-0.6):(0.2-0.6).

8. The gastrin-releasing peptide precursor assay kit according to any one of claims 1 to 7, characterized in that: The diluent comprises the following components in parts by weight: 0.2-0.6% Tween 20, 0.4-1.2% polyvinyl alcohol, 0.2-0.8% polyvinyl pyrrolidone, 0.4-1% L-cysteine, 0.4-1% calcium oxide, 0.05-0.2% diethylenetriaminepentaacetic acid, 2-4% bovine serum albumin, 0.2-0.8% reduced glutathione, and the balance phosphate buffer.

9. The gastrin-releasing peptide precursor assay kit according to claim 1, characterized in that: The preparation method of the magnetic particle reagent is as follows: 10 mg of zinc oxide ferromagnetic particles are resuspended in MES buffer, 100 to 200 μL of 5 to 15 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution is added, and the mixture is stirred evenly. 0.5 to 2 mg of streptavidin is added and reacted at room temperature for 1 to 3 hours. The supernatant is magnetically separated and washed, and then a blocking agent is added for blocking. Finally, the volume is adjusted to 500 mL with a stabilizing diluent to obtain magnetic particles coupled with streptavidin.

10. Use of the progastrin-releasing peptide assay kit according to any one of claims 1 to 9 for detecting the concentration of progastrin-releasing peptide in human serum and / or plasma.