Formulations for detecting progastrin and uses thereof

Abstract

The present application relates to the technical field of electrochemiluminescence immunoassay, and discloses a preparation for detecting progastrin-releasing peptide and application thereof, which contains a phosphate buffer, an alkali metal inorganic salt, bovine serum albumin, a preservative, a first surfactant and a second surfactant. The preparation has good universality and can be used for the preparation of monoclonal antibody coated magnetic microspheres and a working solution of a ruthenium compound labeled monoclonal antibody. The preparation can not only enhance the luminescence signal intensity of the reagent, but also ensure the stable performance of the reagent during long-term storage, thereby effectively enhancing the clinical applicability of the detection system and the reliability of the detection result.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence immunoassay technology, specifically to a formulation for detecting gastrin-releasing peptide precursors and its application. Background Technology

[0002] Gastrin-releasing peptide (GRP) is a gut-brain peptide with gastrin-stimulating activity, first isolated from the gastric mucosa of pigs. It is mainly expressed in the gastrointestinal tract, respiratory tract, and central nervous system. Progastrin-releasing peptide (proGRP) is the precursor structure of GRP. Elevated proGRP levels are commonly seen in neuroendocrine-derived tumors, including small cell lung cancer (SCLC), carcinoid tumors, undifferentiated large cell lung cancer with neuroendocrine characteristics, medullary thyroid carcinoma, other neuroendocrine carcinomas, and androgen-independent prostate cancer with neuroendocrine characteristics. Serum proGRP levels are significantly elevated in lung cancer patients, showing high sensitivity and specificity for SCLC, and increasing with clinical stage progression. Therefore, it is widely used for the auxiliary diagnosis, treatment monitoring, and prognostic assessment of SCLC. Currently, the main chemiluminescence methods for detecting proGRP in human serum or plasma include: enzyme-catalyzed magnetic microparticle chemiluminescence, acridine ester magnetic microparticle chemiluminescence, and the traditional tripyridine ruthenium electrochemiluminescence (ECL).

[0003] Electrochemiluminescence immunoassay (ECLIA) is a highly sensitive detection method that combines electrochemical reactions with chemiluminescence principles. This technology uses labels such as ruthenium tripyridine (RBI) and electronegatively neutral ruthenium complex (NRC) to trigger the generation of light signals on the electrode surface, achieving precise quantitative detection of antigen-antibody reactions. ECLIA immunoassay reagents typically consist of a working solution prepared by diluting ruthenium-labeled monoclonal antibodies and monoclonal antibody-coated magnetic microspheres. In practical applications, the long-term stability of the reagent components is often a core requirement, directly affecting the reliability of the overall test results. However, the stability of ruthenium complexes and magnetic microsphere complexes is poor during long-term storage, which can lead to abnormal fluctuations, increases, or decreases in the reactive light emission level (RLU) signal generated by the working solution reaction during testing, severely affecting detection accuracy and limiting the effectiveness of the entire reagent system in clinical applications. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a universal electrochemiluminescence immunoassay reagent for detecting progastrin-releasing peptide, as well as a magnetic microsphere assay reagent coated with proGRP monoclonal antibody prepared therefrom and a proGRP monoclonal antibody assay reagent labeled with an electrically neutral ruthenium compound. This reagent not only effectively improves the stability of the working solution but also broadens the universal compatibility of the reagent.

[0005] To achieve the above objectives, the present invention provides a formulation for detecting gastrin-releasing peptide precursor, the formulation 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 nonionic surfactants with different HLB values, the first surfactant having an HLB value of 14-16 and the second surfactant having an HLB value of 13-14.

[0006] A second aspect of the present invention provides a test reagent containing a magnetic microsphere complex, comprising the aforementioned formulation and magnetic microspheres coated with proGRP monoclonal antibody.

[0007] A third aspect of the present invention provides a test reagent containing a ruthenium compound-labeled antibody complex, comprising the aforementioned formulation and a ruthenium compound-labeled proGRP monoclonal antibody.

[0008] A fourth aspect of the present invention provides a kit for detecting progastrin-releasing peptide, the kit comprising: the formulation described in the first aspect, a ruthenium compound-labeled proGRP monoclonal antibody, 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:

[0010] (1) The formulation provided by the present invention can effectively inhibit the degradation of ruthenium compound-labeled antibodies and magnetic microsphere-coated antibodies in the electrochemiluminescence immunoassay reagent system, so that the reagent remains stable during long-term storage and the fluctuation of relative light unit (RLU) value is controlled within ±10%, and better controlled within ±5%. This overcomes the problem of insufficient shelf life caused by abnormal fluctuation of RLU of the test sample during long-term storage, and significantly improves the clinical applicability of the detection system and the reliability of the test results.

[0011] (2) The formulation of the present invention can simultaneously meet the needs of two detection reagents, showing significant advantages in reagent compatibility and application flexibility. Detailed Implementation

[0012] The endpoints and any values ​​of the ranges 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 endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0013] "Pre-gastrin-releasing peptide (PRP)" is generated through the further breakdown of a 148-amino acid preprotein and consists of 68 amino acids. This invention detects the C-terminal region of PRP (31-98), which is commonly found in three types of human PRP splice variants. Serum PRP (31-98) has now been demonstrated to be a reliable biomarker for patients with small cell lung cancer (SCLC).

[0014] The first aspect of this invention provides a formulation for detecting gastrin-releasing peptide precursors. The formulation comprises 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 with different HLB values. The first surfactant has an HLB value of 14-16, and the second surfactant has an HLB value of 13-14. The "HLB value," or hydrophilic-lipophilic balance value, refers to the balance between the size and strength of the hydrophilic and lipophilic groups of a surfactant. A higher HLB value indicates stronger hydrophilicity, while a lower HLB value indicates stronger lipophilicity.

[0015] In this invention, preferably, the weight ratio of the first surfactant and 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.

[0016] 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.

[0017] Preferably, the weight ratio of the alkali metal inorganic salt and 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.

[0018] 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 between the above values.

[0019] Preferably, the weight ratio of the preservative and 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 between the above values.

[0020] According to some preferred embodiments of the present invention, the phosphate, in molar amounts based on phosphate ions, is 6-400 mmol relative to 1 g of the second surfactant, more 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.

[0021] According to some preferred embodiments of the present invention, the formulation further contains water so that it can be used directly for electrochemiluminescence immunoassay.

[0022] According to some preferred embodiments of the present invention, the alkali metal inorganic salt in the preparation is present in a content of 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 between the above values.

[0023] According to some preferred embodiments of the present invention, the alkali metal inorganic salt is selected from NaCl and / or KCl; more preferably NaCl.

[0024] According to some preferred embodiments of the present invention, the pH adjustment range of the phosphate buffer is 7-8; more preferably, the pH adjustment range of the phosphate buffer is 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.

[0025] According to some preferred embodiments of the present invention, the preservative is a biological preservative, a class of highly efficient preservatives that have the effect of inhibiting and killing microorganisms and are stable and low in toxicity, more preferably a biological preservative (ProClin™ 300) whose main component is isothiazolinone (such as 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one). In the biological preservative, the weight ratio of 5-chloro-2-methyl-4-isothiazolin-3-one to 2-methyl-4-isothiazolin-3-one 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.

[0026] According to some preferred embodiments of the present invention, the first surfactant and the second surfactant each independently have a -CH2-CH2-O- structure. More preferably, the number of -CH2-CH2-O- structures in the first surfactant can be 20-25. More preferably, the number of -CH2-CH2-O- structures in the second surfactant can be 8-10.

[0027] 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.

[0028] 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 formulation can be 0.5-1.5 g / L, preferably 0.5-1 g / L.

[0029] According to the present invention, the concentration of bovine serum albumin in the preparation can be 10-50 g / L, preferably 15-30 g / L.

[0030] A second aspect of the present invention provides a test reagent containing a magnetic microsphere complex, comprising the aforementioned formulation and magnetic microspheres coated with proGRP monoclonal antibody.

[0031] According to some preferred embodiments of the present invention, in the magnetic microspheres coated with the proGRP monoclonal antibody, the particle size of the magnetic microspheres is 1-5 μm, and the proGRP monoclonal antibody is coupled to the surface of the magnetic microspheres via amide bonds. The magnetic microspheres have a hydrophilic polymer coating and incorporate carboxyl functional groups for antibody coupling.

[0032] According to some preferred embodiments of the present invention, the content of the magnetic microsphere complex is 0.1-0.5 g, more preferably 0.2-0.3 g, relative to 150 mmol of alkali metal inorganic salt.

[0033] 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.

[0034] In this invention, the proGRP monoclonal antibody can be a common monoclonal antibody in the art capable of specifically binding to proGRP. The amount of antibody coated on each milligram of magnetic microspheres can be 10-50 µg / mg. The preparation method of the proGRP monoclonal antibody-coated magnetic microspheres may include a first mixing of magnetic microspheres with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, followed by a second mixing with the proGRP monoclonal antibody to obtain 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.

[0035] The inventors of this invention have discovered that the formulation of this invention is particularly suitable for the dispersion of magnetic microspheres coated with the aforementioned specific proGRP monoclonal antibody, and can further enhance signal intensity and improve stability when used in electrochemiluminescence immunoassay.

[0036] A third aspect of the present invention provides a test reagent containing a ruthenium compound-labeled antibody complex, comprising the aforementioned formulation and a ruthenium compound-labeled proGRP monoclonal antibody.

[0037] 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.

[0038] According to some preferred embodiments of the present invention, the content of the ruthenium compound-labeled proGRP monoclonal antibody is 0.4-1.5 mg relative to 150 mmol of alkali metal inorganic salt, more preferably 0.6-1 mg.

[0039] According to some preferred embodiments of the present invention, the mass concentration of the ruthenium compound-labeled proGRP monoclonal antibody assay reagent is 0.4-1.5 µg / mL, more preferably 0.6-1 µg / mL.

[0040] According to the present invention, in the ruthenium compound-labeled proGRP monoclonal antibody, the ruthenium compound is preferably an electrically neutral ruthenium compound, and its specific structure is as follows: The labeling link is an amide bond. The preparation method of ruthenium-labeled proGRP monoclonal antibody may include a first mixing of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, N-hydroxythiosuccinimide, and an electrically neutral ruthenium compound, followed by a second mixing with the proGRP monoclonal antibody to obtain the ruthenium-labeled proGRP monoclonal antibody. Preferably, the first mixing is performed at room temperature for 40-60 minutes. More preferably, the second mixing is performed at room temperature for 3-5 hours.

[0041] According to the present invention, it is understood that the ruthenium compound-labeled proGRP monoclonal antibody is different from the monoclonal antibody coated with magnetic microspheres, and the two antibodies recognize different sites of the proGRP antigen.

[0042] The inventors of this invention have discovered that the formulation of this invention is particularly suitable for the dispersion of proGRP monoclonal antibodies labeled with the specific ruthenium compounds described above, and can further enhance signal intensity and improve stability when used in electrochemiluminescence immunoassay.

[0043] A fourth aspect of the present invention provides a kit for detecting proGRP, the kit comprising the aforementioned formulation, a ruthenium compound-labeled proGRP monoclonal antibody, and magnetic microspheres coated with the proGRP monoclonal antibody.

[0044] According to a preferred embodiment of the present invention, the kit includes test reagent I and test reagent II, wherein test reagent I comprises the formulation as described above and a ruthenium compound-labeled proGRP monoclonal antibody, and test reagent II comprises the formulation as described above and magnetic microspheres coated with the proGRP monoclonal antibody.

[0045] The fifth aspect of the present invention provides a method for detecting proGRP, the method comprising:

[0046] (1) Mix the test reagent I described in the third aspect with the test sample to obtain reaction solution I containing antibody-antigen complex;

[0047] (2) The antibody-antigen complex is mixed with the test reagent II described in the second aspect to obtain reaction solution II containing a double antibody sandwich complex;

[0048] (3) The reaction solution II is drawn into the electrochemical reaction cell to carry out the electrochemiluminescence reaction and the electrochemiluminescence signal is collected.

[0049] The sixth aspect of this invention provides the application of the aforementioned formulation or test reagent in the detection of proGRP.

[0050] The applications of the fifth and sixth aspects provided by the present invention can be diagnostic (e.g., for medical testing to further determine and identify subsequent testing / treatment plans) or non-diagnostic (e.g., for research work, for drug screening, disease mechanism research and verification, etc.).

[0051] The present invention will be described in detail below through examples. Unless otherwise specified, all reagents and materials used in the following examples are commercially available products purchased from reputable chemical or biological reagent / material suppliers, and all reagents are of analytical grade. Electrochemiluminescence signal parameters were measured using a YnY 3030 chemiluminescence analyzer from Ansel Diagnostics Technology Co., Ltd.

[0052] "Bias" refers to the change in detection signal value when the proGRP monoclonal antibody-coated magnetic microsphere assay reagent and the proGRP monoclonal antibody assay reagent labeled with an electrically neutral ruthenium compound are stored at 37°C for a certain period of time, compared to when they are stored at 2-8°C for the same period of time for electrochemiluminescence immunoassay.

[0053] Deviation = ((Detection signal value stored at 37℃) - (Detection signal value stored at 2-8℃)) / (Detection signal value stored at 2-8℃) × 100%.

[0054] In the following examples, all test samples were diluted with newborn calf serum (Zhuhai Beso Biotechnology, BS-ZQ0301).

[0055] Example 1

[0056] (I) Effects of the optimized formula

[0057] 1. Buffer solution preparation

[0058] Buffer solution (Formula #1) preparation: Taking 1 L as an example, add 800 mL of purified water, NaH2PO4 (2.340 g), Na2HPO4 (4.330 g), NaCl (8.766 g), BSA (15 g), Proclin 300 (4.8 g), Tween 80 (0.2 g), and Triton X-100 (0.9 g), adjust the pH to 7.2, and add water to a final volume of 1 L. The components in the buffer solution obtained using this method are expressed as their final concentrations (the following formulations also follow this expression): 50 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.

[0059] 2. Preparation of test reagents for magnetic microsphere complex and electrically neutral ruthenium complex

[0060] 2.1 Preparation of proGRP monoclonal antibody-coated magnetic microspheres

[0061] (1) Mix the magnetic microspheres (Thermo Fisher Scientific (China) Co., Ltd., item number: 34310D) with freshly prepared N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide solution (10 mg / mL) at a ratio of 25:1 (mass ratio) and rotate at room temperature for 30 minutes.

[0062] (2) Remove the supernatant by magnetic separation, add proGRP monoclonal antibody 1 (Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number: M3601) at a ratio of antibody to magnetic microspheres of 1:50 (mass ratio), and mix by rotating at room temperature for 4 hours.

[0063] (3) Add an equal volume of 1wt% BSA solution and mix by rotation at room temperature for 30 minutes;

[0064] (4) After removing the supernatant by magnetic separation, add diluent containing 1 wt% BSA, resuspend, and obtain magnetic microspheres coated with proGRP monoclonal antibody.

[0065] 2.2 Preparation of proGRP monoclonal antibodies labeled with neutral ruthenium compounds

[0066] (1) Equal volumes of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide solution (40 mmol / L), N-hydroxythiosuccinimide solution (100 mmol / L), and electrically neutral ruthenium compound solution (10 mmol / L), with purified water as solvent, were mixed at room temperature for 45 minutes.

[0067] Electroneutral ruthenium compound molecular structure

[0068] (2) Incubate at room temperature for 4 hours with a molar ratio of 10:1 for neutral ruthenium compound: proGRP monoclonal antibody 2 (Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number: M3602);

[0069] (3) Remove unbound, electrically neutral ruthenium compounds via a desalting column.

[0070] 2.3 Dilute the electrically neutral ruthenium compound-labeled proGRP monoclonal antibody to 1.0 µg / mL using buffer (Formula #1), and dilute the proGRP monoclonal antibody-coated magnetic microspheres to 0.25 mg / mL to form electrochemiluminescence assay reagent I / II.

[0071] 3. Stability test of working reagents

[0072] The test reagents were divided into four groups. One group was stored at 2-8℃ as a control group, and the other three groups were stored at 37℃ for accelerated testing as experimental groups, which were retrieved at 0, 4, 8, and 12 days, respectively. Antigen (proGRP, Feipeng Biotechnology) was prepared at concentrations of 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, and 2000 pg / mL as test samples. The freshly prepared reagents were used for testing. The control group and the three accelerated groups were also tested simultaneously with the antigen samples. Three replicate tests were performed, and the average values ​​were taken. After the tests were completed, the overall experimental data deviation was calculated.

[0073] The instrument testing steps are as follows:

[0074] (1) Add 85 μL of proGRP monoclonal antibody reagent labeled with neutral ruthenium compound and 30 μL of sample to the detection tube, mix for 3-5 seconds, add 85 μL of magnetic microsphere reagent coated with proGRP monoclonal antibody to the detection tube, mix for 3-5 seconds, and incubate at 37°C for 15 minutes.

[0075] (2) The incubated reaction mixture is sucked into the measuring cell, and the magnet below the measuring cell fixes the magnetic beads in the reaction mixture onto the electrode surface;

[0076] (3) Take out the cleaning solution to clean other substances that are not fixed to the electrode surface, start a specific voltage of 1.5V, and the ruthenium compound undergoes an electrochemical reaction to form luminescence;

[0077] (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 bead, thereby enabling quantitative analysis of proGRP.

[0078] Table 1

[0079]

[0080] As shown in Table 1, the test deviation was still within ±5% after 12 days of acceleration using the preferred combination of buffer solution, indicating that the diluent of this formulation can stabilize the electrochemiluminescence reagent system.

[0081] (II) The effect of the buffer system in the buffer solution on stability

[0082] 1. Replace the phosphate buffer system in formula #1 of step (I) with a different buffer system to obtain a new buffer formula, as follows:

[0083] Formula #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.

[0084] Formula #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.

[0085] Formula #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.

[0086] The preparation methods for the working solution of the electrically neutral ruthenium-labeled proGRP monoclonal antibody and the proGRP monoclonal antibody-coated magnetic microspheres are the same as in step (I).

[0087] 2. Stability test of working reagents

[0088] The test reagents for each dilution formulation were divided into two groups. One group was stored at 2-8℃ for 12 days as a control group (represented by "0" in the table), and the other group was stored at 37℃ for accelerated testing as an experimental group. The test method is described in step (I). The results are shown in the table below.

[0089] Table 2

[0090]

[0091] As shown in Table 2, when the buffer system was changed to different pH values, the deviation of the preferred formulation #1 was within ±5%. Compared with the preferred formulation #1, the signal values ​​of formulations #2 to #4 deviated by more than ±10% under the accelerated conditions of 37℃ for 12 days, indicating poor stability.

[0092] (III) The effect of pH adjustment of buffer solution on stability

[0093] 1. Based on the formula #1 in step (I), the pH value of the buffer solution was adjusted to 6.0, 7.0, 8.0 and 9.0.

[0094] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0095] Table 3

[0096]

[0097] As shown in Table 3, in buffer solutions with different pH values, when the pH is 7-8, the signal value deviation is within ±10% after 12 days of acceleration at 37℃, indicating satisfactory stability.

[0098] (iv) Effect of phosphate concentration on stability

[0099] 1. Based on formula #1 in step (I), adjust the phosphate concentration to obtain a new buffer solution formula, as follows:

[0100] Formula #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.

[0101] Formula #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.

[0102] Formula #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.

[0103] Formula #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.

[0104] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0105] Table 4

[0106]

[0107] 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 accelerated at 37℃ for 12 days is within ±10%, indicating satisfactory stability. When the phosphate concentration is in the range of 20-100 mmol / L, the deviation of the signal value of the reagent accelerated at 37℃ for 12 days is basically within ±5%, indicating better stability.

[0108] (V) The effect of inorganic salt type on stability

[0109] 1. Based on formula #1 in step (I), adjust the types of inorganic salts to obtain a new buffer solution formula, as follows:

[0110] Formula #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.

[0111] Formula #10: 50 mmol / L phosphate, 150 mmol / L ZnCl2, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.02wt% Tween 80, 0.09wt% Triton X-100, pH 7.2.

[0112] Formula #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.

[0113] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0114] Table 5

[0115]

[0116] As shown in Table 5, when the inorganic salts selected in the buffer solution are NaCl and KCl, the test deviation is within ±10%, indicating good stability. Among them, NaCl is the preferred inorganic salt.

[0117] (vi) Effect of NaCl concentration on stability

[0118] 1. Based on formula #1 in step (I), adjust the concentration of NaCl to obtain a new buffer solution formula, as follows:

[0119] Formula #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.

[0120] Formula #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.

[0121] Formula #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.

[0122] Formula #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.

[0123] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0124] Table 6

[0125]

[0126] As shown in Table 6, when the NaCl concentration in the buffer solution is 50-200 mmol / L, the reagent test deviation is within ±10%, indicating good stability.

[0127] (vii) Effects of protein type and concentration on stability

[0128] 1. Based on formula #1 in step (I), adjust the protein types to obtain a new buffer formula, as follows:

[0129] Formula #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.

[0130] Formula #17: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.0 wt% gelatin, 4.8 wt% Proclin 300, 0.02 wt% Tween 80, 0.09 wt% Triton X-100, pH 7.2.

[0131] 2. Based on the formula #1 in step (I), adjust the protein BSA concentration to obtain a new buffer formula, as follows:

[0132] Formula #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.

[0133] Formula #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.

[0134] Formula #20: 50 mmol / L phosphate, 150 mmol / L NaCl, 5.0 wt% BSA, 4.8 wt% Proclin 300, 0.02 wt% Tween 80, 0.09 wt% Triton X-100, pH 7.2.

[0135] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0136] Table 7

[0137]

[0138] As shown in Table 7, when OVA and gelatin were used in the buffer solution, the test deviation of the prepared reagent exceeded ±10%, indicating poor stability; when BSA was used as the protein, with a concentration of 1.0wt%-5.0wt%, the test deviation of the prepared reagent was within ±10%, indicating good stability.

[0139] (viii) The effect of surfactant type on stability

[0140] 1. Based on formulation #1 in step (I), adjust the type and concentration of the surfactant to obtain a new buffer formulation, as follows:

[0141] Formula #21: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Tween 20, pH 7.2.

[0142] Formula #22: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Tween 80, pH 7.2.

[0143] Formula #23: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Triton X-100, pH 7.2.

[0144] Formula #24: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Triton X-450, pH 7.2.

[0145] Formula #25: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Brij-35, pH 7.2.

[0146] Formula #26: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% S9, pH 7.2.

[0147] Formula #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.

[0148] Formula #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.

[0149] Formula #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.

[0150] Formula #30: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Triton X-100, 0.05wt% S9, pH 7.2.

[0151] Formula #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.

[0152] Formula #32: 50 mmol / L phosphate, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 0.05wt% Triton X-100, 0.05wt% Triton X-450, pH 7.2.

[0153] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0154] Table 8

[0155]

[0156] As shown in Table 8, the buffer formulations #22 and #23 exhibit good stability when using 0.05% Tween 80 and 0.05% Triton X-100, respectively. However, the deviations for both formulations #22 and #23 are around ±10%. To achieve higher stability, buffer formulations containing two surfactants were used. Tween 80 and Triton X-100 were combined with Triton X-450, Tween 20, and S9 surfactants, respectively, but the deviations still exceeded ±10%. However, the optimal combination of Tween 80 and Triton X-100 resulted in a buffer with significantly improved stability, with the deviation within ±5%.

[0157] (ix) Effect of surfactant concentration on stability

[0158] 1. Based on formulation #1 in step (I), adjust the concentrations of the two surfactants, Tween80 and Triton X-100, to obtain a new buffer formulation, as follows:

[0159] Formula #33: 0.02wt% Tween80, 0.05wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0160] Formula #34: 0.05wt% Tween80, 0.05wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0161] Formula #35: 0.10wt% Tween80, 0.05wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0162] Formula #36: 0.02wt% Tween80, 0.10wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0163] Formula #37: 0.05wt% Tween80, 0.10wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0164] Formula #38: 0.10wt% Tween80, 0.10wt% Triton X-100, 150 mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0165] Formula #39: 0.02wt% Tween80, 0.15wt% Triton X-100, 150mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0166] Formula #40: 0.05wt% Tween80, 0.15wt% Triton X-100, 150mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0167] Formula #41: 0.10wt% Tween80, 0.15wt% Triton X-100, 150mmol / L NaCl, 1.5wt% BSA, 4.8wt‰ Proclin 300, 50 mmol / L phosphate, pH 7.2.

[0168] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0169] Table 9

[0170]

[0171] The test values ​​of the reagents in the above-mentioned formulation dilution solutions all deviated within ±10%. Using two surfactants in the buffer significantly improved test stability. The concentration range of surfactant Tween 80 was 0.02wt%-0.1wt%, and the concentration range of Triton X-100 was 0.05wt%-0.15wt%. The preferred buffer solution had a Tween 80 concentration range of 0.02wt%-0.05wt% and a Triton X-100 concentration range of 0.05wt%-0.10wt%.

[0172] (x) Effect of preservative Proclin 300 concentration on stability

[0173] 1. Based on formula #1 in step (I), adjust the type of preservative to obtain a new buffer solution formula, as follows:

[0174] Formula #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.

[0175] 2. Based on formula #1 in step (I), adjust the concentration of the preservative Proclin 300 to obtain a new buffer solution formula, as follows:

[0176] Formula #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.

[0177] Formula #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.

[0178] Formula #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.

[0179] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0180] Table 10

[0181]

[0182] As shown in Table 10, the test samples of the kit prepared using the preservative KroVin 100 showed individual deviations exceeding ±10%, indicating poor stability; the test samples of the kit prepared using the preservative Proclin 300 at concentrations of 2.0wt‰-4.8wt‰ showed test deviations within ±10%, indicating good stability.

[0183] (xi) Exploration of the concentration range of each combination

[0184] 1. Based on formulation #1 in step (I), dilutions were prepared by adjusting the concentrations of each component of the buffer solution (the combination of the limiting concentrations of each component and the total limiting concentration) to explore the acceptable range of each concentration. The specific formulation is as follows:

[0185] Formula #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.

[0186] Formula #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.

[0187] Formula #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.

[0188] Formula #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.

[0189] Formula #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.

[0190] Formula #51: 50 mmol / L phosphate, 150 mmol / L NaCl, 5.0 wt% BSA, 4.8 wt% Proclin 300, 0.02 wt% Tween 80, 0.09 wt% Triton X-100, pH 7.2.

[0191] Formula #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.

[0192] Formula #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.

[0193] Formula #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.

[0194] Formula #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.

[0195] Formula #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.

[0196] Formula #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.

[0197] Formula #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.

[0198] Formula #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.

[0199] Formula #60: 200 mmol / L phosphate, 200 mmol / L NaCl, 5.0 wt% BSA, 4.8 wt% Proclin 300, 0.10 wt% Tween 80, 0.15 wt% Triton X-100, pH 8.0.

[0200] 2. Test the samples using the same steps as in step (I), and the results are shown in the table below.

[0201] Table 11

[0202]

[0203] As shown in Table 11, the phosphate concentration range in the buffer formulation is 10-200 mmol / L, the inorganic salt NaCl concentration range is 50-200 mmol / L, the BSA concentration range is 1-5wt%, the preservative Proclin 300 concentration range is 2-4.8wt‰, the Tween 80 concentration range is 0.02wt%-0.1wt%, the Triton X-100 concentration range is 0.05wt%-0.15wt%, and the pH is 7-8. The dilutions prepared from each component within the specified ranges for the proGRP test reagent showed good stability after 12 days of accelerated testing at 37℃ with deviations within ±10%.

[0204] In summary, the buffer formulation of this invention, compared with other formulations, not only ensures that the deviation is within ±10% after 12 days of acceleration at 37°C, but also has the effect of signal enhancement.

[0205] Example 2

[0206] According to the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1175-2010", the finished reagent kit was prepared using formula #1 in step (I) of Example 1. The test method in Example 1 was used to verify the reagent's performance, such as the limit of detection, accuracy, linearity, repeatability, and interference.

[0207] 1. Minimum detection limit

[0208] The zero concentration reference product (newborn calf serum, Zhuhai Biosino Biological, BS-ZQ0301) was repeatedly measured 20 times, and the mean (M) and standard deviation (SD) of the signal values (RLU) of the 20 measurement results were calculated. The RLU value corresponding to (M + 2SD) was obtained. According to the calibration curve equation of the calibration product used in the kit, the RLU value corresponding to M + 2SD was substituted into the above equation to calculate the corresponding concentration value. The corresponding concentration value is the lowest detection limit, and the lowest detection limit should be ≤ 2.00 pg / mL. The test results are shown in the following table:

[0209] Table 12

[0210]

[0211] 2. Accuracy

[0212] The traceable trueness control product was detected and repeatedly measured 3 times, and the measured values were recorded as X i , and the relative deviation B of the measured concentration was calculated according to the following formula i . If the deviations of the 3 results are all within the range of ±10.0%, it is judged as qualified. The test results are shown in the following table:

[0213]

[0214] Table 13

[0215]

[0216] 3. Linearity

[0217] A high-value sample close to the upper limit of the linear range was diluted into 5 concentrations at a certain ratio, and the low-value concentration sample needed to be close to the lower limit of the linear range. Each sample was repeatedly measured 3 times, and the mean of the measurement results was calculated. The average of the measured concentrations and the theoretical concentrations were linearly fitted by the least squares method, and the linear correlation coefficient ( r ) was calculated. In the concentration range of [3.00, 5000] pg / mL, the linear correlation coefficient ( r ) should be ≥ 0.9900. The linear correlation coefficient r of the test reagent is greater than 0.9900 in the range of 2.29 - 5899.62 pg / mL, meeting the standard. As can be seen from Tables 14 and 15, the linear relationship of the detection method within the specified concentration range meets the requirements, indicating that within the specified concentration range, the detection method can accurately calculate the concentration of the analyte through the linear equation, meeting the basic requirements of quantitative analysis.

[0218] Table 14

[0219]

[0220] Table 15

[0221]

[0222] 4. Repeatability

[0223] For samples with concentrations in the range of (60±12) pg / mL and (800±160) pg / mL, each determination was repeated 10 times. The mean (M) and standard deviation (SD) of the 10 determinations for each sample were calculated. The coefficient of variation (CV) of the determination results was calculated according to the following formula. The coefficient of variation (CV) should be ≤8%.

[0224] CV = SD / M × 100%

[0225] In the formula:

[0226] CV—Coefficient of variation;

[0227] SD—the standard deviation of 10 measurements;

[0228] M—The average value of 10 measurements.

[0229] Table 16

[0230]

[0231] The CV for repeatability sample CF1 was 1.21%, and the CV for repeatability sample CF2 was 1.00%, both meeting the standard.

[0232] 5. Anti-interference capability

[0233] High and low value samples were prepared and divided into two groups. One group was prepared by adding a solvent containing interfering substances (hemoglobin, total protein, bilirubin, biotin, triglycerides, and human anti-mouse antibody (HAMA)) to bring the concentration of interfering substances in the sample to the experimental concentration. The other group was prepared by adding a solvent without interfering substances as the control group. The mean value of the test results without interfering substances was used as the standard. The relative deviation of the test results of the samples containing interfering substances was calculated. The relative deviation was ≤ ±10%. See the table below for details.

[0234] Table 17

[0235]

[0236] When testing samples containing triglycerides (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), or human anti-mouse antibody (HAMA concentration ≤50 ng / mL), the relative deviation is within ±10%. The buffer formulation of this test reagent meets the anti-interference requirements and is suitable for testing blood samples.

[0237] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A kit for detecting gastrin-releasing peptide precursor, characterized in that, The kit comprises: a formulation, a ruthenium compound-labeled monoclonal antibody against gastrin-releasing peptide precursor, and magnetic microspheres coated with the monoclonal antibody against gastrin-releasing peptide precursor; the formulation comprises 10-200 mmol / L phosphate buffer, 50-200 mM alkali metal inorganic salt, 10-50 g / L bovine serum albumin, 2-4.8 g / L ProClin 300, 0.2-1 g / L polyoxyethylene sorbitan monooleate, and 0.5-1.5 g / L octylphenyl polyoxyethylene ether, wherein the alkali metal inorganic salt is selected from NaCl and / or KCl; the pH of the phosphate buffer is adjusted to a range of 7-8.

2. The reagent kit according to claim 1, characterized in that, The alkali metal inorganic salt is present in a concentration of 100-150 mM in the formulation; And / or, the alkali metal inorganic salt is NaCl.

3. The reagent kit according to claim 1, characterized in that, The concentration of the phosphate buffer is 20-100 mmol / L; And / or, the pH adjustment range of the phosphate buffer is 7.0-7.

2.

4. The reagent kit according to claim 1, characterized in that, The concentration of bovine serum albumin is 15-30 g / L.

5. The reagent kit according to claim 1, characterized in that, The concentration of ProClin 300 is 3-4.8 g / L.

6. The reagent kit according to claim 1, characterized in that, The concentration of the polyoxyethylene dehydrated sorbitan monooleate is 0.2-0.5 g / L.

7. The kit according to claim 1, characterized in that, The concentration of the octylphenyl polyoxyethylene ether is 0.5-1 g / L.

8. The reagent kit according to claim 1, characterized in that, The content of the magnetic microspheres coated with the gastrin-releasing peptide precursor monoclonal antibody is 0.1-0.5 g relative to 150 mmol of alkali metal inorganic salt. And / or, the mass concentration of the magnetic microspheres coated with the gastrin-releasing peptide precursor monoclonal antibody in the kit is 0.1-0.5 mg / mL.

9. The reagent kit according to claim 1, characterized in that, The amount of the ruthenium compound-labeled gastrin-releasing peptide precursor monoclonal antibody relative to 150 mmol of alkali metal inorganic salt is 0.4-1.5 mg. And / or, the mass concentration of the ruthenium compound-labeled gastrin-releasing peptide precursor monoclonal antibody in the kit is 0.4-1.5 µg / mL.

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