Preparation for detecting gastrin-releasing peptide precursor and application thereof

Abstract

The invention relates to the technical field of electrochemiluminescence immunoassay, and discloses a preparation for detecting a gastrin-releasing peptide precursor and application thereof, the preparation contains a phosphate buffer, an alkali metal inorganic salt, bovine serum albumin, a preservative, a first surfactant and a second surfactant. The preparation provided by the invention has good universality, and can be synchronously suitable for preparation of monoclonal antibody coated magnetic microspheres and ruthenium compound labeled monoclonal antibody working solutions. The preparation not only can enhance the luminous signal intensity of the reagent, but also can ensure that the reagent maintains stable performance in a long-term storage process, and finally effectively enhances the clinical applicability of a detection system and the reliability of a detection result.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence immunoassay, and in particular to a preparation for detecting gastrin-releasing peptide precursor and application thereof. Background Art

[0002] Gastrin-releasing peptide (GRP), a brain-gut peptide first isolated from porcine gastric mucosa, is expressed primarily in the gastrointestinal tract, respiratory tract, and central nervous system. Pro-gastrin-releasing peptide (proGRP) is the precursor of GRP. Elevated proGRP levels are commonly seen in neuroendocrine tumors, including small cell lung cancer (SCLC), carcinoid tumors, undifferentiated large cell lung cancer with neuroendocrine features, medullary thyroid carcinoma, other neuroendocrine carcinomas, and androgen-independent prostate cancer with neuroendocrine features. Serum proGRP is significantly elevated in lung cancer patients and has high sensitivity and specificity for SCLC. It increases with clinical stage, making it widely used in the diagnosis, treatment response monitoring, and prognosis of SCLC. Currently, the main chemiluminescence methods used for the detection of proGRP in human serum or plasma include enzymatic magnetic microparticle chemiluminescence, acridinium ester magnetic microparticle chemiluminescence, and traditional terpyridine ruthenium electrochemiluminescence (ECL).

[0003] Electrochemiluminescence immunoassay (ECLIA) is a highly sensitive detection method that combines electrochemical reactions with chemiluminescence. This technique uses markers such as ruthenium terpyridine and neutrally charged ruthenium complexes (NRCs) to trigger light signals on the electrode surface, enabling precise quantitative detection of antigen-antibody reactions. ECLIA immunoassay reagents typically consist of a monoclonal antibody labeled with a ruthenium compound and a monoclonal antibody-coated magnetic microsphere reagent, prepared using a diluent to create a working solution. In practical applications, the long-term stability of the reagent components is a key requirement, directly impacting the reliability of the overall test results. However, the long-term stability of the ruthenium complex and magnetic microsphere complex is poor during long-term storage. This can cause abnormal fluctuations in the signal (RLU) generated by the working solution reaction, either increasing or decreasing, severely impacting test accuracy. This can limit the effectiveness of the entire reagent system in clinical applications. Summary of the Invention

[0004] The present invention aims to overcome the problems of the prior art by providing a universal electrochemiluminescent immunoassay preparation for detecting progastrin-releasing peptide (PGRP), as well as a proGRP monoclonal antibody-coated magnetic microsphere test reagent and a proGRP monoclonal antibody test reagent labeled with a neutral ruthenium compound, prepared using the preparation. This preparation not only effectively improves the stability of the working solution but also broadens the reagent's universal compatibility.

[0005] To achieve the above objectives, the present invention provides, on one hand, a preparation for detecting gastrin-releasing peptide precursor, comprising a phosphate buffer, an alkali metal inorganic salt, bovine serum albumin, a preservative, a first surfactant and a second surfactant, wherein the first surfactant and the second surfactant are each independently selected from non-ionic surfactants with different HLB values, the HLB value of the first surfactant is 14-16, and the HLB value of the second surfactant is 13-14.

[0006] The second aspect of the present invention provides a test reagent dispersed with a magnetic microsphere complex, comprising the preparation and magnetic microspheres coated with a proGRP monoclonal antibody.

[0007] The third aspect of the present invention provides a test reagent dispersed with a ruthenium compound-labeled antibody complex, comprising the above-mentioned preparation and a proGRP monoclonal antibody labeled with a ruthenium compound.

[0008] A fourth aspect of the present invention provides a kit for detecting progastrin-releasing peptide, comprising: the preparation described in the first aspect, a proGRP monoclonal antibody labeled with a ruthenium compound, and magnetic microspheres coated with the proGRP monoclonal antibody.

[0009] Through the above technical solution, the present invention can achieve at least the following beneficial effects: (1) The preparation provided by the present invention can effectively inhibit the degradation of ruthenium compound-labeled antibodies and magnetic microsphere-coated antibodies in the electrochemiluminescence immunoassay system, so that the reagent remains stable during long-term storage, and the fluctuation range of the relative light unit (RLU) value is controlled within ±10%, and can be better controlled within ±5%, thereby overcoming the problem of insufficient shelf life caused by abnormal fluctuations in the RLU value of the test sample during long-term storage of the reagent, and significantly improving the clinical applicability of the detection system and the reliability of the test results.

[0010] (2) The preparation of the present invention can simultaneously meet the requirements of two detection reagents and exhibits significant advantages in reagent compatibility and application flexibility. DETAILED DESCRIPTION

[0011] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0012] Progastrin-releasing peptide (PGRP) is produced by further decomposition of a 148-amino acid preproprotein, consisting of 68 amino acids. The present invention detects the carboxyl-terminal region of PGRP (31-98), which is commonly found in three types of human PGRP splice variants. Serum PGRP (31-98) has been shown to be a reliable marker for patients with small cell lung cancer (SCLC).

[0013] In a first aspect, the present invention provides a preparation for detecting gastrin-releasing peptide precursor, comprising a phosphate buffer, an alkali metal inorganic salt, bovine serum albumin, a preservative, a first surfactant, and a second surfactant. The first and second surfactants are each independently selected from nonionic surfactants having different HLB values, with the first surfactant having an HLB value of 14-16 and the second surfactant having an HLB value of 13-14. "HLB value," also known as the hydrophilic-lipophilic balance, refers to the balance in size and strength between the hydrophilic and lipophilic groups of a surfactant. A larger HLB value indicates a more hydrophilic surfactant, while a smaller HLB value indicates a more lipophilic surfactant.

[0014] In the present invention, preferably, the weight ratio of the first surfactant to the second surfactant is (0.1-2):1, such as 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 1.8:1, 2:1 or any value or range between the above values, preferably (0.2-1):1.

[0015] According to some preferred embodiments of the present invention, the weight ratio of the alkali metal inorganic salt, bovine serum albumin, preservative and second surfactant is (1.5-25):(6-100):(1-10):1, preferably (3-18):(10-56):(2-10):1, and more preferably (6-18):(11-34):(3-10):1.

[0016] Preferably, the weight ratio of the alkali metal inorganic salt to the second surfactant is (3-18):1, such as 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1 or any value or range between the above values.

[0017] Preferably, the weight ratio of bovine serum albumin to the second surfactant is (10-56):1, such as 10:1, 11:1, 13:1, 15:1, 16:1, 17:1, 18:1, 20:1, 23:1, 25:1, 28:1, 30:1, 33:1, 35:1, 38:1, 40:1, 45:1, 50:1, 53:1, 56:1 or any value or range therebetween.

[0018] Preferably, the weight ratio of the preservative to the second surfactant is (2-10):1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or any value or range therebetween.

[0019] According to some preferred embodiments of the present invention, relative to 1 g of the second surfactant, the molar amount of the phosphate calculated as phosphate group is 6-400 mmol, further preferably 10-223 mmol, such as 10 mmol, 15 mmol, 20 mmol, 25 mmol, 30 mmol, 35 mmol, 40 mmol, 45 mmol, 50 mmol, 60 mmol, 80 mmol, 100 mmol, 120 mmol, 150 mmol, 180 mmol, 200 mmol, 220 mmol, 223 mmol or any value or range between the above values.

[0020] According to some preferred embodiments of the present invention, the preparation further contains water, so that it can be directly used in electrochemiluminescence immunoassay.

[0021] According to some preferred embodiments of the present invention, the content of the alkali metal inorganic salt in the preparation is 50-200 mM, more preferably 100-150 mM, such as 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM or any value or range therebetween.

[0022] According to some preferred embodiments of the present invention, the alkali metal inorganic salt is selected from NaCl and / or KCl; more preferably NaCl.

[0023] According to some preferred embodiments of the present invention, the pH of the phosphate buffer is adjusted to a range of 7-8; further preferably, the pH of the phosphate buffer is adjusted to a range of 7.0-7.2. More specifically, the phosphate buffer can be a sodium phosphate buffer (NaH2PO4 & Na2HPO4) and / or a potassium phosphate buffer (K2HPO4 & KH2PO4). The concentration of the phosphate buffer in the formulation can be 10-200 mmol / L, preferably 20-100 mmol / L.

[0024] According to some preferred embodiments of the present invention, the preservative is a biopreservative, a highly effective, stable, and low-toxic preservative that inhibits and kills microorganisms. More preferably, it is a biopreservative (ProClin™ 300) whose primary components are isothiazolinones (such as 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one). The weight ratio of 5-chloro-2-methyl-4-isothiazolin-3-one to 2-methyl-4-isothiazolin-3-one in this biopreservative can be (2-5):1. More specifically, the content of 5-chloro-2-methyl-4-isothiazolin-3-one is 2.1-2.8 wt%, and the content of 2-methyl-4-isothiazolin-3-one is 0.6-1 wt%. The concentration of the preservative in the formulation can be 2-4.8 g / L, preferably 3-4.8 g / L.

[0025] According to some preferred embodiments of the present invention, the first surfactant and the second surfactant further independently have a -CH2-CH2-O- structure. More preferably, the number of -CH2-CH2-O- structures in the first surfactant may be 20 to 25. More preferably, the number of -CH2-CH2-O- structures in the second surfactant may be 8 to 10.

[0026] According to a particularly preferred embodiment of the present invention, the first surfactant is polyoxyethylene sorbitan monooleate (Tween 80, CAS No.: 9005-65-6). The concentration of the first surfactant in the formulation can be 0.2-1 g / L, preferably 0.2-0.5 g / L.

[0027] According to a particularly preferred embodiment of the present invention, the second surfactant is octylphenyl polyoxyethylene ether (Triton x-100, CAS No.: 9002-93-1). The concentration of the second surfactant in the preparation can be 0.5-1.5 g / L, preferably 0.5-1 g / L.

[0028] According to the present invention, the concentration of bovine serum albumin in the preparation may be 10-50 g / L, preferably 15-30 g / L.

[0029] The second aspect of the present invention provides a test reagent dispersed with a magnetic microsphere complex, comprising the preparation and magnetic microspheres coated with a proGRP monoclonal antibody.

[0030] According to some preferred embodiments of the present invention, the proGRP monoclonal antibody-coated magnetic microspheres have a particle size of 1-5 μm, and the proGRP monoclonal antibody is coupled to the surface of the magnetic microspheres via an amide bond. The magnetic microspheres have a hydrophilic polymer coating and incorporate carboxyl functional groups for antibody coupling.

[0031] According to some preferred embodiments of the present invention, the content of the magnetic microsphere composite is 0.1-0.5 g, more preferably 0.2-0.3 g, relative to 150 mmol of the alkali metal inorganic salt.

[0032] According to some preferred embodiments of the present invention, the mass concentration of the magnetic microsphere complex in the test reagent is 0.1-0.5 mg / mL, more preferably 0.2-0.3 mg / mL.

[0033] In the present invention, the proGRP monoclonal antibody can be a commonly used monoclonal antibody in the art that specifically binds to proGRP. The amount of antibody coated per milligram of magnetic microspheres can be 10-50 µg / mg. The method for preparing proGRP monoclonal antibody-coated magnetic microspheres can include first mixing the magnetic microspheres with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, followed by a second mixing with the proGRP monoclonal antibody, to produce the proGRP monoclonal antibody-coated magnetic microspheres. Preferably, the first mixing is performed at room temperature for 20-50 minutes. More preferably, the second mixing is performed at room temperature for 3-5 hours.

[0034] The inventors of the present invention have found that the preparation of the present invention is particularly suitable for dispersing the magnetic microspheres coated with the above-mentioned specific proGRP monoclonal antibody, and can further enhance the signal intensity and improve the stability when used in electrochemiluminescence immunoassay.

[0035] The third aspect of the present invention provides a test reagent dispersed with a ruthenium compound-labeled antibody complex, comprising the above-mentioned preparation and a proGRP monoclonal antibody labeled with a ruthenium compound.

[0036] According to some preferred embodiments of the present invention, in the ruthenium compound-labeled proGRP monoclonal antibody reagent, the molar binding ratio of the ruthenium compound to the antibody is (1-20):1; more preferably, the molar binding ratio of the electrically neutral ruthenium compound to the antibody is (2-10):1.

[0037] According to some preferred embodiments of the present invention, the content of the proGRP monoclonal antibody labeled with the ruthenium compound is 0.4-1.5 mg, more preferably 0.6-1 mg, relative to 150 mmol of the alkali metal inorganic salt.

[0038] According to some preferred embodiments of the present invention, the mass concentration of the ruthenium compound-labeled proGRP monoclonal antibody test reagent is 0.4-1.5 µg / mL, more preferably 0.6-1 µg / mL.

[0039] According to the present invention, in the proGRP monoclonal antibody labeled with a ruthenium compound, the ruthenium compound is preferably an electrically neutral ruthenium compound, and its structure is specifically: The labeling linker position is an amide bond. A method for preparing a ruthenium compound-labeled proGRP monoclonal antibody can include uniformly mixing N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, N-hydroxysulfosuccinimide, and an electrically neutral ruthenium compound for a first mixing step, followed by adding the proGRP monoclonal antibody for a second mixing step, to obtain the ruthenium compound-labeled proGRP monoclonal antibody. Preferably, the first mixing step is performed at room temperature for 40-60 minutes. More preferably, the second mixing step is performed at room temperature for 3-5 hours.

[0040] According to the present invention, it is understood that the proGRP monoclonal antibody labeled with a ruthenium compound is different from the monoclonal antibody coated with magnetic microspheres, and the two antibodies recognize different sites on the proGRP antigen.

[0041] The inventors of the present invention have found that the preparation of the present invention is particularly suitable for the dispersion of the proGRP monoclonal antibody labeled with the above-mentioned specific ruthenium compound, and can further enhance the signal intensity and improve the stability when used in electrochemiluminescence immunoassay.

[0042] In a fourth aspect, the present invention provides a kit for detecting proGRP, comprising the aforementioned preparation, a proGRP monoclonal antibody labeled with a ruthenium compound, and magnetic microspheres coated with the proGRP monoclonal antibody.

[0043] According to a preferred embodiment of the present invention, the kit comprises a test reagent I and a test reagent II, wherein the test reagent I comprises the preparation as described above and a proGRP monoclonal antibody labeled with a ruthenium compound, and the test reagent II comprises the preparation as described above and magnetic microspheres coated with a proGRP monoclonal antibody.

[0044] A fifth aspect of the present invention provides a method for detecting proGRP, the method comprising: (1) mixing the test reagent I described in the third aspect with the test sample to obtain a reaction solution I containing an antibody-antigen complex; (2) mixing the antibody-antigen complex with the test reagent II described in the second aspect to obtain a reaction solution II containing a double antibody sandwich complex; (3) The reaction solution II is aspirated into the electrochemical reaction cell to carry out an electrochemiluminescence reaction and collect the electrochemiluminescence signal.

[0045] A sixth aspect of the present invention provides use of the aforementioned preparation or test reagent in detecting proGRP.

[0046] The applications of the fifth and sixth aspects provided by the present invention can be diagnostic (for example, used for medical testing to further judge and determine subsequent testing / treatment plans) or non-diagnostic (for example, used for research work, for drug screening, disease mechanism research and verification, etc.).

[0047] The present invention will be described in detail below through the examples. In the following examples, unless otherwise specified, the reagents and materials used were all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents were analytically pure. Electrochemiluminescence signal parameters were measured using a chemiluminescence analyzer YnY 3030 from Ansai Diagnostics Technology Co., Ltd.

[0048] "Deviation" refers to the change in the detection signal value of the proGRP monoclonal antibody-coated magnetic microsphere test reagent and the proGRP monoclonal antibody test reagent labeled with an electrically neutral ruthenium compound after being stored in an accelerated test at 37°C for a certain period of time, compared to the change in the detection signal value when stored at 2-8°C for the same period of time for electrochemiluminescence immunoassay detection, namely: Deviation = ((detection signal value stored at 37°C) - (detection signal value stored at 2-8°C)) / detection signal value stored at 2-8°C × 100%.

[0049] In the following examples, the test samples were diluted with newborn calf serum (Zhuhai Beisuo Biological, BS-ZQ0301).

[0050] Example 1 (1) Effect of the optimal formula 1. Buffer preparation To prepare a 1 L buffer solution (Recipe #1), add 800 mL of purified water, 2.340 g of NaH2PO4, 4.330 g of Na2HPO4, 8.766 g of NaCl, 15 g of BSA, 4.8 g of Proclin 300, 0.2 g of Tween 80, and 0.9 g of Triton X-100. Adjust the pH to 7.2 and bring the volume to 1 L with water. The following buffer solution is expressed as follows (the following recipe also uses this formula): 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ of Proclin 300, 0.02 wt% of Tween 80, and 0.09 wt% of Triton X-100, with a pH of 7.2.

[0051] 2. Preparation of magnetic microsphere complex and electrically neutral ruthenium complex test reagents 2.1 Preparation of proGRP monoclonal antibody-coated magnetic microspheres (1) Magnetic microspheres (Thermo Fisher Scientific (China) Co., Ltd., Catalog No.: 34310D) were mixed with freshly prepared N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide solution (10 mg / mL) at a mass ratio of 25:1 and rotated at room temperature for 30 minutes; (2) Remove the supernatant by magnetic separation, add proGRP monoclonal antibody 1 (Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number: M3601) at a mass ratio of antibody to magnetic microspheres = 1:50, and rotate and mix at room temperature for 4 hours; (3) Add an equal volume of 1 wt% BSA solution and rotate and mix at room temperature for 30 minutes; (4) After removing the supernatant by magnetic separation, add diluent containing 1 wt% BSA and resuspend to obtain magnetic microspheres coated with proGRP monoclonal antibodies.

[0052] 2.2 Preparation of proGRP monoclonal antibodies labeled with electrically neutral ruthenium compounds (1) Equal volumes of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide solution (40 mmol / L), N-hydroxysulfosuccinimide solution (100 mmol / L), and electrically neutral ruthenium compound solution (10 mmol / L) in purified water were mixed at room temperature for 45 minutes. Molecular structure of electrically neutral ruthenium compounds (2) Incubate at room temperature for 4 hours at a molar ratio of 10:1 (neutral ruthenium compound: proGRP monoclonal antibody 2 (Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number: M3602); (3) Remove unbound electrically neutral ruthenium compounds through a desalting column.

[0053] 2.3 Use buffer (Recipe #1) to dilute the neutrally charged ruthenium compound-labeled proGRP monoclonal antibody to 1.0 µg / mL and the proGRP monoclonal antibody-coated magnetic microspheres to 0.25 mg / mL to form electrochemiluminescence assay reagents I / II.

[0054] 3. Stability test of working reagents The test reagents were divided into four groups: one group was stored at 2-8°C as the control group, and the other three groups were stored at 37°C to simulate accelerated growth, serving as the experimental groups. The samples were removed at 0, 4, 8, and 12 days, respectively. Antigen (proGRP, Feipeng Bio) was prepared at concentrations of 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, and 2000 pg / mL. The freshly prepared reagents were used for testing. The control group and the accelerated group were then tested simultaneously with the antigen samples. Three replicates were performed, and the mean values ​​were calculated. After the test was completed, the overall experimental data was analyzed for deviation.

[0055] The instrument detection steps are as follows: (1) Pipette 85 μL of proGRP monoclonal antibody reagent labeled with a neutral ruthenium compound and 30 μL of sample into the test tube, mix for 3-5 seconds, and then pipette 85 μL of magnetic microsphere reagent coated with proGRP monoclonal antibody into the test tube, mix for 3-5 seconds, and incubate at 37°C for 15 minutes. (2) The reaction mixture after incubation is aspirated into the measuring cell, and the magnet under the measuring cell fixes the magnetic beads in the reaction mixture on the electrode surface; (3) Absorb the cleaning solution to clean other substances not fixed to the electrode surface, start the specific voltage of 1.5V, and the ruthenium compound undergoes an electrochemical reaction to generate light; (4) The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of proGRP captured on the surface of the magnetic beads, thereby performing quantitative analysis of proGRP.

[0056] Table 1

[0057] As shown in Table 1, the test deviation of the reagent after 12 days of accelerated testing using the preferred combination of buffer solutions is still within ±5%. This diluent formula can stabilize the electrochemiluminescence reagent system.

[0058] (II) Effect of the buffer system in the buffer on stability 1. Replace the phosphate buffer system in step (1) formulation #1 with a different buffer system to obtain a new buffer formulation as follows: Formulation #2: 50 mmol / L Tris, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin300, 0.02wt% Tween80, 0.09wt% Triton X-100, pH 8.0.

[0059] Formulation #3: 50 mmol / L HEPES, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin300, 0.02wt% Tween80, 0.09wt% Triton X-100, pH 6.8.

[0060] Formulation #4: 50 mmol / L MES, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin300, 0.02wt% Tween80, 0.09wt% Triton X-100, pH 6.0.

[0061] The preparation method of the electrically neutral ruthenium-labeled proGRP monoclonal antibody and the proGRP monoclonal antibody-coated magnetic microsphere working solution is the same as step (1).

[0062] 2. Stability test of working reagents The test reagents of each dilution formula were divided into two groups. One group was stored at 2-8℃ for 12 days as the control group (indicated by "0" in the table), and the other group was stored at 37℃ to simulate accelerated temperature as the experimental group and then taken out after 12 days. The test method is shown in step (1). The results are shown in the table below.

[0063] Table 2

[0064] As shown in Table 2, when the buffer system with different pH values ​​was changed, the deviation of the preferred formula #1 was within ±5%. Compared with the preferred formula #1, the signal value deviation of formula #2 to formula #4 exceeded ±10% under the accelerated conditions of 37°C for 12 days, indicating poor stability.

[0065] (III) Effect of buffer pH adjustment on stability 1. Based on step (1) recipe #1, the pH value of the buffer was adjusted to 6.0, 7.0, 8.0, and 9.0.

[0066] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0067] Table 3

[0068] As shown in Table 3, in buffers with different pH values, when the pH is 7-8, the signal value deviations after 12 days of acceleration at 37°C are all within ±10%, indicating acceptable stability.

[0069] (IV) Effect of phosphate concentration on stability 1. Based on step (1) formula #1, adjust the phosphate concentration to obtain a new buffer solution formula as follows: Formulation #5: 10 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0070] Formulation #6: 20 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0071] Formulation #7: 100 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0072] Formulation #8: 200 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.02 wt% Tween 80, 0.09 wt% Triton X-100, pH 7.2.

[0073] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0074] Table 4

[0075] As shown in Table 4, when the phosphate concentration is in the range of 10-200 mmol / L, the deviation of the signal value of the reagent at 37°C for 12 days is within ±10%, and the stability is qualified. When the phosphate concentration is in the range of 20-100 mmol / L, the deviation of the signal value of the reagent at 37°C for 12 days is basically within ±5%, and the stability is better.

[0076] (V) Effect of inorganic salt types on stability 1. Based on step (1) formula #1, adjust the type of inorganic salt to obtain a new buffer solution formula as follows: Formulation #9: 50 mmol / L phosphate, 150 mmol / L KCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0077] Formulation #10: 50 mmol / L phosphate, 150 mmol / L ZnCl2, 1.5wt% BSA, 4.8wt‰ Proclin300, 0.02wt% Tween80, 0.09wt% Triton X-100, pH 7.2.

[0078] Formulation #11: 50 mmol / L phosphate, 150 mmol / L MgCl2, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0079] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0080] Table 5

[0081] As shown in Table 5, when the inorganic salt in the buffer solution is selected as NaCl or KCl, the test deviation is within ±10%, which indicates good stability, and NaCl is the preferred inorganic salt.

[0082] (VI) Effect of NaCl concentration on stability 1. Based on step (1) formula #1, adjust the concentration of NaCl to obtain a new buffer solution formula as follows: Formulation #12: 50 mmol / L phosphate, 50 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0083] Formulation #13: 50 mmol / L phosphate, 100 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0084] Formulation #14: 50 mmol / L phosphate, 200 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0085] Formulation #15: 50 mmol / L phosphate, 0 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0086] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0087] Table 6

[0088] From the results in Table 6, it can be seen that when the NaCl concentration in the buffer solution is 50-200 mmol / L, the reagent test deviation is within ±10%, and the stability is good.

[0089] (VII) Effect of protein type and concentration on stability 1. Based on step (1) formula #1, adjust the protein type to obtain a new buffer formula as follows: Formulation #16: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.0wt% OVA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0090] Formulation #17: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.0wt% gelatin, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0091] 2. Based on step (1) formula #1, adjust the BSA concentration to obtain a new buffer formula as follows: Formulation #18: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.0wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0092] Formulation #19: 50 mmol / L phosphate, 150 mmol / L NaCl, 3.0wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0093] Formulation #20: 50 mmol / L phosphate, 150 mmol / L NaCl, 5.0wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0094] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0095] Table 7

[0096] As shown in the results of Table 7, when OVA and gelatin are used in the buffer solution, the deviation of the prepared reagent test exceeds ±10%, and the stability is poor; when the protein is BSA with a concentration of 1.0wt%-5.0wt%, the deviation of the prepared reagent test is within ±10%, and the stability is good.

[0097] (8) Effect of surfactant type on stability 1. Based on step (1) formula #1, adjust the surfactant type and concentration to obtain a new buffer solution formula as follows: Formulation #21: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.05 wt% Tween 20, pH 7.2.

[0098] Formulation #22: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Tween 80, pH 7.2.

[0099] Formulation #23: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.05 wt% Triton X-100, pH 7.2.

[0100] Formulation #24: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.05 wt% Triton X-450, pH 7.2.

[0101] Formulation #25: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.05 wt% Brij-35, pH 7.2.

[0102] Formulation #26: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.05 wt% S9, pH 7.2.

[0103] Formulation #27: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Tween 20, 0.05wt% Tween 80, pH 7.2.

[0104] Formulation #28: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Tween 80, 0.05wt% Triton X-450, pH 7.2.

[0105] Formulation #29: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Triton X-100, 0.05wt% Tween 20, pH 7.2.

[0106] Formulation #30: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.05 wt% Triton X-100, 0.05 wt% S9, pH 7.2.

[0107] Formulation #31: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Tween 80, 0.05wt% S9, pH 7.2.

[0108] Formulation #32: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.05 wt% Triton X-100, 0.05 wt% Triton X-450, pH 7.2.

[0109] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0110] Table 8

[0111] As shown in Table 8, the buffer formulation #22 with 0.05% Tween80 and the buffer formulation #23 with 0.05% Triton X-100 exhibited good stability. However, the deviations between formulations #22 and #23 were around ±10%. To achieve higher stability, a buffer formulation containing two surfactants was used. Tween80 and Triton X-100 were combined with Triton X-450, Tween20, and S9 surfactants, respectively, but the deviations still exceeded ±10%. However, the combination of Tween80 and Triton X-100 resulted in a buffer formulation with an optimal combination, which significantly improved stability and was within ±5%.

[0112] (IX) Effect of surfactant concentration on stability 1. Based on step (1) formula #1, adjust the concentrations of Tween 80 and Triton X-100 to obtain a new buffer solution formula as follows: Formulation #33: 0.02wt% Tween 80, 0.05wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0113] Formulation #34: 0.05wt% Tween 80, 0.05wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0114] Formulation #35: 0.10wt% Tween 80, 0.05wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0115] Formulation #36: 0.02wt% Tween 80, 0.10wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0116] Formulation #37: 0.05wt% Tween 80, 0.10wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0117] Formulation #38: 0.10wt% Tween 80, 0.10wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0118] Formulation #39: 0.02wt% Tween 80, 0.15wt% Triton X-100, 150mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50mmol / L phosphate, pH 7.2.

[0119] Formulation #40: 0.05wt% Tween 80, 0.15wt% Triton X-100, 150mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50mmol / L phosphate, pH 7.2.

[0120] Formulation #41: 0.10wt% Tween 80, 0.15wt% Triton X-100, 150mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50mmol / L phosphate, pH 7.2.

[0121] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0122] Table 9

[0123] The test value deviations of the above-mentioned diluent reagents were all within ±10%. The use of dual surfactants in the buffer significantly improved the test stability. The concentration range of the surfactant Tween 80 was 0.02wt%-0.1wt%, and the concentration range of Triton X-100 was 0.05wt%-0.15wt%. The preferred buffer surfactant concentration range was 0.02wt%-0.05wt% for Tween 80 and 0.05wt%-0.10wt% for Triton X-100.

[0124] (10) Effect of preservative Proclin 300 concentration on stability 1. Based on step (1) formula #1, adjust the preservative type to obtain a new buffer solution formula as follows: Formulation #42: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 2.4wt‰ KroVin100, 0.02wt% Tween80, 0.09wt% Triton X-100, pH 7.2.

[0125] 2. Based on step (1) formula #1, adjust the concentration of the preservative Proclin 300 to obtain a new buffer solution formula as follows: Formulation #43: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 2.0wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0126] Formulation #44: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 3.5wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0127] Formulation #45: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 6.0wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0128] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0129] Table 10

[0130] As shown in Table 10, the test samples in the kits prepared with the preservative KroVin 100 had individual deviations exceeding ±10%, indicating poor stability. However, the test samples in the kits prepared with the preservative Proclin 300 at concentrations of 2.0 wt‰ to 4.8 wt‰ had test deviations within ±10%, indicating good stability.

[0131] (11) Exploration of the concentration range of each combination 1. Based on step (1) formula #1, dilutions were prepared by adjusting the concentrations of each component of the buffer (the combination of the limiting concentrations of each component and the full limiting concentration) to explore the acceptable range of each concentration. The specific formula is as follows: Formulation #46: 10 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0132] Formulation #47: 200 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0133] Formulation #48: 50 mmol / L phosphate, 50 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0134] Formulation #49: 50 mmol / L phosphate, 200 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0135] Formulation #50: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.0wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0136] Formulation #51: 50 mmol / L phosphate, 150 mmol / L NaCl, 5.0wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0137] Formulation #52: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 2.0wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0138] Formulation #53: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0139] Formulation #54: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5 wt% BSA, 4.8 wt‰ Proclin 300, 0.10 wt% Tween 80, 0.09 wt% Triton X-100, pH 7.2.

[0140] Formulation #55: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.05wt% Triton X-100, pH 7.2.

[0141] Formulation #56: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.15wt% Triton X-100, pH 7.2.

[0142] Formulation #57: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.0.

[0143] Formulation #58: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 8.0.

[0144] Formulation #59: 10 mmol / L phosphate, 50 mmol / L NaCl, 1.0wt% BSA, 2.0wt‰ Proclin 300, 0.02wt% Tween 80, 0.15wt% Triton X-100, pH 7.0.

[0145] Formulation #60: 200 mmol / L phosphate, 200 mmol / L NaCl, 5.0wt% BSA, 4.8wt‰ Proclin 300, 0.10wt% Tween 80, 0.15wt% Triton X-100, pH 8.0.

[0146] 2. Test the sample in the same way as step (1). The results are shown in the table below.

[0147] Table 11

[0148] As shown in Table 11, the phosphate concentration in the buffer solution formula ranges from 10 to 200 mmol / L, the inorganic salt NaCl concentration ranges from 50 to 200 mmol / L, the BSA concentration ranges from 1 to 5 wt%, the preservative Proclin 300 concentration ranges from 2 to 4.8 wt‰, the Tween 80 concentration ranges from 0.02 wt% to 0.1 wt%, the Triton X-100 concentration ranges from 0.05 wt% to 0.15 wt%, and the pH is 7 to 8. The dilutions prepared with each component within the range are used to prepare the proGRP test reagent. The deviations after accelerated incubation at 37°C for 12 days are all within ±10%, indicating good stability.

[0149] In summary, the buffer solution formulation of the present invention has a signal enhancement effect compared with other formulations while ensuring that the deviation of the 12-day accelerated reaction at 37°C is within ±10%.

[0150] Example 2 According to the Pharmaceutical Industry Standard of the People's Republic of China YY / T 1175-2010, a finished test kit was prepared using Formula #1 in step (1) of Example 1. The detection reagents were used to verify the minimum detection limit, accuracy, linearity, repeatability, interference, and other performance of the reagents according to the test method in Example 1.

[0151] 1. Minimum detection limit Repeat the measurement of the zero-concentration reference substance (newborn calf serum, Zhuhai Beisuo Biological, BS-ZQ0301) 20 times, calculate the mean (M) and standard deviation (SD) of the signal value (RLU) of the 20 measurement results, and obtain the RLU value corresponding to (M+2SD). According to the calibration curve equation of the calibrator used in the kit, substitute the RLU value corresponding to M+2SD into the above equation to calculate the corresponding concentration value. The corresponding concentration value is the minimum detection limit, which should be ≤2.00 pg / mL. The test results are shown in the following table: Table 12

[0152] 2. Accuracy The accuracy control product with traceability is tested and repeated 3 times. The measured value is recorded as X i , calculate the relative deviation B of the measured concentration according to the following formula i If the deviation of the three results is within the range of ±10.0%, it is considered qualified. The test results are shown in the following table:

[0153] Table 13

[0154] 3. Linear The high-value sample close to the upper limit of the linear range was diluted to 5 concentrations according to a certain ratio, among which the low-value sample was required to be close to the lower limit of the linear range. Each sample was measured three times, and the mean of the measurement results was calculated. The mean of the measured concentration was fitted with the theoretical concentration using the least squares method, and the linear correlation coefficient was calculated ( r ), in the concentration range of [3.00, 5000] pg / mL, the linear correlation coefficient ( r ) should be ≥0.9900. The test reagent's linear correlation coefficient, r, is greater than 0.9900 within the range of 2.29-5899.62 pg / mL, meeting the standard. Tables 14 and 15 show that the test method's linear relationship within the specified concentration range meets the requirements, indicating that within the specified concentration range, the test method can accurately infer the concentration of the analyte using a linear equation, meeting the basic requirements for quantitative analysis.

[0155] Table 14

[0156] Table 15

[0157] 4. Repeatability For samples with a concentration between (60±12) pg / mL and (800±160) pg / mL, the measurement was repeated 10 times. The mean (M) and standard deviation (SD) of the 10 measurement results for each sample were calculated. The coefficient of variation (CV) of the measurement results was calculated according to the following formula. The coefficient of variation (CV) should be ≤8%.

[0158] CV=SD / M×100% Where: CV—coefficient of variation; SD—standard deviation of 10 measurement results; M—the average value of 10 measurement results.

[0159] Table 16

[0160] The CV of the test reagent repeatability sample CF1 was 1.21%, and the CV of the repeatability sample CF2 was 1.00%, both meeting the standards.

[0161] 5. Anti-interference Prepare high and low value samples and divide them into two groups. One group is added with a solvent containing interfering substances (hemoglobin, total protein, bilirubin, biotin, triglycerides, human anti-mouse antibody (HAMA)) to make the concentration of interfering substances in the sample reach the experimental concentration as the experimental group; the other group is added with a solvent without interfering substances as the control group. The test mean without interfering substances is used as the standard. The relative deviation of the test results of samples containing interfering substances is calculated. The relative deviation is ≤±10%. See the table below for details.

[0162] Table 17

[0163] When testing samples of triglyceride (concentration ≤ 2000 mg / dL), bilirubin (concentration ≤ 66 mg / dL), hemoglobin (concentration ≤ 1.0 g / dL), total protein (concentration ≤ 10 g / dL), biotin (concentration up to 1 g / L), and human anti-mouse antibody (HAMA concentration ≤ 50 ng / mL), the relative deviation of the test is within ±10%. The buffer formula of this test reagent meets the anti-interference requirements and is suitable for testing blood samples.

[0164] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A preparation for detecting gastrin-releasing peptide precursor, characterized in that: The preparation contains a phosphate buffer, an alkali metal inorganic salt, bovine serum albumin, a preservative, a first surfactant and a second surfactant, wherein the first surfactant and the second surfactant are each independently selected from non-ionic surfactants with different HLB values, the HLB value of the first surfactant is 14-16, and the HLB value of the second surfactant is 13-14.

2. The preparation according to claim 1, characterized in that The weight ratio of the first surfactant to the second surfactant is (0.1-2):1; and / or, the weight ratio of the alkali metal inorganic salt, bovine serum albumin, preservative and second surfactant is (1.5-25):(6-100):(1-10):1; and / or, relative to 1 g of the second surfactant, the molar amount of the phosphate as phosphate radical is 6-400 mmol; And / or, the preparation further contains water, and the content of the alkali metal inorganic salt in the preparation is 50-200 mM; And / or, the alkali metal inorganic salt is selected from NaCl and / or KCl.

3. The preparation according to claim 1 or 2, characterized in that The weight ratio of the first surfactant to the second surfactant is (0.2-1):1; and / or, the weight ratio of the alkali metal inorganic salt, bovine serum albumin, preservative and second surfactant is (3-18):(10-56):(2-10):1; and / or, relative to 1 g of the second surfactant, the molar amount of the phosphate as phosphate radical is 10-223 mmol; And / or, the preparation further contains water, and the content of the alkali metal inorganic salt in the preparation is 100-150 mM; And / or, the alkali metal inorganic salt is NaCl.

4. The preparation according to claim 1 or 2, characterized in that The pH adjustment range of the phosphate buffer is 7-8; And / or, the preservative is a biological preservative.

5. The preparation according to claim 1 or 2, characterized in that The first surfactant is polyoxyethylene sorbitan monooleate; And / or, the second surfactant is octylphenyl polyoxyethylene ether.

6. A test reagent dispersed with a magnetic microsphere complex, characterized in that: The test reagent comprises the preparation according to any one of claims 1 to 5 and a magnetic microsphere complex, wherein the magnetic microsphere complex is a magnetic microsphere coated with a gastrin-releasing peptide precursor monoclonal antibody.

7. The test reagent according to claim 6, characterized in that The content of the magnetic microsphere composite is 0.1-0.5 g relative to 150 mmol of the alkali metal inorganic salt; And / or, the mass concentration of the magnetic microsphere complex in the test reagent is 0.1-0.5 mg / mL.

8. A test reagent containing a ruthenium compound-labeled antibody complex, characterized in that: The test reagent comprises the preparation according to any one of claims 1 to 5 and a gastrin-releasing peptide precursor monoclonal antibody labeled with a ruthenium compound.

9. The test reagent according to claim 8, characterized in that The content of the gastrin-releasing peptide precursor monoclonal antibody labeled with the ruthenium compound is 0.4-1.5 mg relative to 150 mmol of the alkali metal inorganic salt; And / or, the mass concentration of the ruthenium compound-labeled gastrin-releasing peptide precursor monoclonal antibody in the test reagent is 0.4-1.5 μg / mL.

10. A kit for detecting gastrin-releasing peptide precursor, characterized in that: The kit comprises: the preparation according to any one of claims 1 to 5, a gastrin-releasing peptide precursor monoclonal antibody labeled with a ruthenium compound, and magnetic microspheres coated with the gastrin-releasing peptide precursor monoclonal antibody.

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