Reagent composition for detecting neuron-specific enolase and application thereof

Abstract

This invention relates to the field of electrochemiluminescence immunoassay technology, and discloses a reagent composition for detecting neuron-specific enolases and its application. The reagent composition contains a buffer, sodium chloride, bovine serum albumin, a preservative, a first surfactant, and a second surfactant. The reagent composition of this invention is versatile, applicable to both the preparation of monoclonal antibody-coated magnetic microspheres and ruthenium compound-labeled monoclonal antibody working solutions. It also enhances the reagent's luminescent signal and maintains stability during long-term storage, significantly improving the clinical applicability of the detection system and the reliability of the test results.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence immunoassay technology, specifically to a reagent composition for detecting neuron-specific enolases and its application. Background Art

[0002] Neuron-specific enolase (NSE) is a cell-specific isoenzyme of the glycolytic enzyme enolase. In clinical diagnosis, elevated NSE concentrations in serum or cerebrospinal fluid often indicate neuronal damage or malignancy. NSE is a widely used neuroendocrine tumor marker, including neuroblastoma and small cell lung cancer (SCLC). Furthermore, the combined detection of NSE and progastrin-releasing peptide (ProGRP) helps differentiate between SCLC and non-small cell lung cancer (NSCLC). To date, the main chemiluminescence methods used for detecting NSE in human serum include: enzyme-catalyzed magnetic microparticle chemiluminescence, acridine ester magnetic microparticle chemiluminescence, and the traditional triple ruthenium pyridine electrochemiluminescence method.

[0003] Electrochemiluminescence immunoassay (ECLIA) is a highly sensitive detection method that combines electrochemical reactions with chemiluminescence principles. It uses labeled materials (such as ruthenium terpyridine or electronegative ruthenium complex (NRC)) to generate light signals on the electrode surface, enabling quantitative detection of antigen-antibody reactions. It boasts advantages such as high sensitivity, a wide linear range, and a high degree of automation, and is widely used in clinical diagnostics and biological assays.

[0004] The electrochemiluminescence immunoassay assay system consists of two core components: magnetic microspheres coated with monoclonal antibodies and monoclonal antibodies labeled with ruthenium compounds. While both require consideration of antibody degradation, the magnetic microspheres are prone to aggregation and have poor dispersibility. Therefore, the dilution buffers used for the two reagents are often incompatible and difficult to use with other types of reagents. This necessitates frequent changes of the buffer system during the assay, increasing operational complexity and potentially introducing errors due to system switching. Furthermore, there is still room for improvement in the stability of the dilution buffer. These technical shortcomings severely limit the clinical application effectiveness of the assay system. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a universal electrochemiluminescence immunoassay reagent composition for detecting neuron-specific enolases, as well as a working solution of magnetic microspheres coated with NSE monoclonal antibodies prepared therefrom and a working solution of NSE monoclonal antibodies labeled with neutral ruthenium compounds. This reagent composition not only broadens the universality of the reagents but also effectively improves the stability of the working solution.

[0006] To achieve the above objectives, the present invention provides a reagent composition for detecting neuron-specific enolases. This reagent composition contains a buffer, sodium chloride, bovine serum albumin, a preservative, a first surfactant, and a second surfactant. The buffer has a pH range of 6.5-7.5. The first and second surfactants are selected from surfactants having a -CH2-CH2-O- structure. The first surfactant has 20-25 -CH2-CH2-O- structures, and the second surfactant has 8-12 -CH2-CH2-O- structures.

[0007] A second aspect of the present invention provides a reagent working solution in which a magnetic microsphere complex is dispersed, the reagent working solution comprising the reagent composition and magnetic microspheres coated with NSE monoclonal antibody.

[0008] A third aspect of the present invention provides a reagent working solution containing a ruthenium compound-labeled antibody complex, the reagent working solution comprising the reagent composition and a ruthenium compound-labeled NSE monoclonal antibody.

[0009] A fourth aspect of the present invention provides a kit for detecting neuron-specific enolase, the kit comprising: the reagent composition described in the first aspect, a ruthenium compound-labeled NSE monoclonal antibody, and magnetic microspheres coated with the NSE monoclonal antibody.

[0010] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0011] (1) Compared with the limited mode of "one buffer solution can only be adapted to a single detection reagent" in the prior art, the reagent composition of the present invention achieves simultaneous and stable adaptation to two detection reagents by optimizing the component ratio and activity regulation mechanism of the buffer solution. This breakthrough not only fundamentally breaks the technical constraints of single adaptation and greatly improves the compatibility between reagents, enabling multiple detections to be completed efficiently in the same system; it also significantly enhances the flexibility in practical applications, especially in multi-index joint detection scenarios, which can reduce reagent preparation time and reduce operating costs. Its technical advantages and practical value are particularly prominent.

[0012] (2) The reagent composition provided by the present invention can significantly improve the stability of immunoassay reagents, thereby making the immunoassay more sensitive and ensuring that it maintains a stable immunobinding titer during long-term storage, thus extending the shelf life of the test reagents. Attached Figure Description

[0013] Figure 1 It is a visualization graph of the linear fitting equation constructed based on experimental data. Detailed Implementation

[0014] 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.

[0015] Neuron-specific enolase (NSE), also known as γ-enolase or enolase 2 (ENO 2), is a cell-specific isoenzyme (γγ homodimer) of the glycolytic enzyme enolase, in which the γ subunit has a molecular weight of 39 kDα. NSE is encoded by the ENO 2 gene located on chromosome 12p13.31, which consists of 12 exons and 11 introns, distributed across 9213 nucleotides, encoding a total of 434 amino acid residues.

[0016] The first aspect of the present invention provides a reagent composition for detecting neuron-specific enolase, the reagent composition comprising a buffer, sodium chloride, bovine serum albumin, a preservative, a first surfactant, and a second surfactant, wherein the pH of the buffer is adjusted in the range of 6.5-7.5, and the first surfactant and the second surfactant are selected from surfactants having a -CH2-CH2-O- structure, wherein the first surfactant has 20-25 -CH2-CH2-O- structures, and the second surfactant has 8-12 -CH2-CH2-O- structures.

[0017] In this invention, preferably, the weight ratio of the first surfactant and the second surfactant is (0.05-1):1, such as 0.05:1, 0.08:1, 0.1:1, 0.12:1, 0.15:1, 0.18:1, 0.2:1, 0.22:1, 0.25:1, 0.3:1, 0.35:1, 0.38:1, 0.4:1, 0.42:1, 0.45:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, or any value or range between the above values, preferably (0.1-0.5):1.

[0018] According to some preferred embodiments of the present invention, the weight ratio of sodium chloride, bovine serum albumin, preservative and second surfactant is (2-25):(5-100):(0.1-2):1, preferably (4-18):(10-50):(0.2-1):1, more preferably (8-9):(19-21):(0.2-0.5):1.

[0019] Preferably, the weight ratio of the sodium chloride and the second surfactant is (4-18):1, such as 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.

[0020] Preferably, the weight ratio of bovine serum albumin to the second surfactant is (10-50):1, such as 10:1, 15:1, 19:1, 20:1, 21:1, 23:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, or any value or range between the above values.

[0021] Preferably, the weight ratio of the preservative and the second surfactant is (0.2-1):1, such as 0.2:1, 0.23:1, 0.25:1, 0.28:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.48:1, 0.5:1, 0.55:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, or any value or range between the above values.

[0022] According to some preferred embodiments of the present invention, the reagent composition further contains water so that it can be used directly for electrochemiluminescence immunoassay.

[0023] According to some preferred embodiments of the present invention, the sodium chloride content in the reagent composition is 100-200 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.

[0024] According to some preferred embodiments of the present invention, the pH adjustment range of the buffer is 6.8-7.2.

[0025] According to some preferred embodiments of the present invention, the buffer is at least one selected from phosphate buffer (PB), bis(2-hydroxyethyl)amino(tris-hydroxymethyl)methane (Bis-Tris) buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, more preferably a phosphate buffer. According to some preferred embodiments of the present invention, the amount of the buffer relative to 1 g of the second surfactant is 5-200 mmol, more preferably 10-100 mmol (the amount of buffer is based on the total molar amount of all components contained therein).

[0026] According to some preferred embodiments of the present invention, the phosphate buffer, in molar amounts of phosphate relative to 1 g of the second surfactant, is 5-200 mmol, more preferably 10-100 mmol, such as 10 mmol, 15 mmol, 20 mmol, 25 mmol, 30 mmol, 35 mmol, 40 mmol, 45 mmol, 50 mmol, 55 mmol, 60 mmol, 80 mmol, 100 mmol, or any value or range between the above values.

[0027] According to some preferred embodiments of the present invention, the phosphate buffer may be a sodium phosphate buffer (NaH2PO4 & Na2HPO4) and / or a potassium phosphate buffer (K2HPO4 & KH2PO4). The concentration of the phosphate buffer in the reagent composition may be 10-100 mmol / L.

[0028] 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 reagent composition can be 0.2-1 g / L, preferably 0.2-0.5 g / L.

[0029] 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 reagent composition can be 0.1-0.5 g / L, preferably 0.1-0.3 g / L.

[0030] According to some preferred embodiments of the present invention, the second surfactant is a polymer of methyl ethylene oxide, 1,2-ethylenediamine, and ethylene oxide (surfactant S9, CAS No.: 26316-40-5). The EO block (ethylene oxide block) ratio is 40%, and the molecular weight is 700.94 g / mol. The concentration of the second surfactant in the reagent composition can be 0.5-2 g / L, preferably 0.8-1.2 g / L.

[0031] According to some preferred embodiments of the present invention, the concentration of bovine serum albumin in the reagent composition can be 10-50 g / L, preferably 15-30 g / L.

[0032] A second aspect of the present invention provides a reagent working solution in which a magnetic microsphere complex is dispersed, the reagent working solution comprising the reagent composition and magnetic microspheres coated with NSE monoclonal antibody.

[0033] According to some preferred embodiments of the present invention, in the NSE monoclonal antibody-coated magnetic microspheres, the magnetic microspheres have a particle size of 1-5 μm, and the NSE 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.

[0034] According to some preferred embodiments of the present invention, the content of the magnetic microspheres is 0.1-0.5 g relative to 150 mmol of sodium chloride, more preferably 0.2-0.3 g.

[0035] According to some preferred embodiments of the present invention, the mass concentration of magnetic microspheres in the reagent working solution is 0.1-0.5 mg / mL, more preferably 0.2-0.3 mg / mL.

[0036] In this invention, the NSE monoclonal antibody can be a common monoclonal antibody in the art capable of specifically binding to NSE. The amount of antibody coated on each milligram of magnetic microspheres can be 10-50 μg / mg. The preparation method of NSE monoclonal antibody-coated magnetic microspheres may include a first mixing of magnetic microspheres with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide; magnetic separation to remove the supernatant, resuspending the magnetic microspheres with buffer, adding the NSE monoclonal antibody, and performing a second mixing; to obtain NSE 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.

[0037] The inventors of this invention have discovered that the reagent composition of this invention is particularly suitable for dispersing magnetic microspheres coated with the aforementioned specific NSE monoclonal antibody, and can further enhance signal intensity and improve stability when used in electrochemiluminescence immunoassay.

[0038] A third aspect of the present invention provides a reagent working solution containing a ruthenium compound-labeled antibody complex, the reagent working solution comprising the reagent composition and a ruthenium compound-labeled NSE monoclonal antibody.

[0039] According to some preferred embodiments of the present invention, in the ruthenium compound-labeled NSE monoclonal antibody reagent, the molar binding ratio of the ruthenium compound to the antibody is (2-10):1; more preferably, the molar binding ratio of the electrically neutral ruthenium compound to the antibody is (5-8):1.

[0040] According to some preferred embodiments of the present invention, the content of the ruthenium compound-labeled NSE monoclonal antibody is 0.5-3 mg relative to 150 mmol of sodium chloride, more preferably 1-2 mg.

[0041] According to some preferred embodiments of the present invention, the mass concentration of the ruthenium compound-labeled NSE monoclonal antibody reagent working solution is 0.5-3 μg / mL, more preferably 1-2 μg / mL.

[0042] According to the present invention, in the ruthenium compound-labeled NSE monoclonal antibody, the ruthenium compound is preferably an electrically neutral ruthenium compound, and its specific structure is as follows: The labeling linkage is an amide bond. The preparation method of ruthenium-labeled NSE 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 NSE monoclonal antibody to obtain the ruthenium-labeled NSE 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.

[0043] According to the present invention, in the ruthenium compound-labeled NSE monoclonal antibody, the monoclonal antibody is different from the antibody coupled to the surface of the magnetic microspheres, and the two antibodies recognize different sites of the NSE antigen.

[0044] The inventors of this invention have discovered that the reagent composition of this invention is particularly suitable for the dispersion of NSE monoclonal antibodies labeled with the specific ruthenium compounds described above, and can further enhance signal intensity and improve stability when used in electrochemiluminescence immunoassay.

[0045] A fourth aspect of the present invention provides a kit for detecting NSE, the kit comprising the aforementioned reagent composition, a ruthenium compound-labeled NSE monoclonal antibody, and magnetic microspheres coated with the NSE monoclonal antibody.

[0046] According to a preferred embodiment of the present invention, the kit includes reagent working solution I and reagent working solution II, wherein reagent working solution I comprises the reagent composition as described above and a ruthenium compound-labeled NSE monoclonal antibody, and reagent working solution II comprises the reagent composition as described above and magnetic microspheres coated with the NSE monoclonal antibody.

[0047] The fifth aspect of the present invention provides a method for detecting NSE, the method comprising:

[0048] (1) Mix the reagent working solution I described in the third aspect with the test sample to obtain reaction solution I containing antibody-antigen complex;

[0049] (2) The antibody-antigen complex is mixed with the reagent working solution II described in the second aspect to obtain reaction solution II containing a double antibody sandwich complex;

[0050] (3) The reaction solution II is drawn into the electrochemical reaction cell to carry out the electrochemiluminescence reaction and the electrochemiluminescence signal is collected.

[0051] The sixth aspect of the present invention provides the application of the aforementioned reagent composition or reagent working solution in the detection of NSE.

[0052] 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.).

[0053] 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.

[0054] "Relative deviation after acceleration" refers to the change in detection signal value when the working solution of NSE monoclonal antibody-coated magnetic microspheres and the working solution of NSE monoclonal antibody labeled with a neutral ruthenium compound are stored at 37°C for a certain period of time, compared with the value when stored at 2-8°C for the same period of time, and used for electrochemiluminescence immunoassay.

[0055] Relative deviation after acceleration = (Detection signal value stored at 37℃ - Detection signal value stored at 2-8℃) / Detection signal value stored at 2-8℃ × 100%.

[0056] In the following examples, all test samples were diluted with newborn calf serum (Shanghai Unimicron Biotechnology Co., Ltd., abs978).

[0057] Example 1

[0058] 1. Buffer solution preparation

[0059] Taking Formula 2 as an example, to prepare 1 L of solution, add 800 mL of purified water, Na2HPO4 (4.3298 g) and NaH2PO4 (2.3396 g), NaCl (8.765 g), BSA (20 g), ProClin 300 (0.5 g), Tween 80 (0.2 g), and S9 (1 g), adjust the pH to 7.0, and add water to bring the volume to 1 L. The components in the buffer solution (Formula 2) obtained by this method are expressed in their final concentrations (the following formulas are also expressed in this way): 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin 300 (w / v), 0.02% Tween 80 (w / v), and 0.1% S9 (w / v).

[0060] Formula 1: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Tween 20 (w / v).

[0061] Formula 1a: 50 mmol / L MES buffer, pH 6.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Tween 20 (w / v).

[0062] Formula 1b: 50 mmol / L Tris buffer, pH 8.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Tween 20 (w / v).

[0063] Formula 1c: 50 mmol / L Bis-Tris buffer, pH 6.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Tween 20 (w / v).

[0064] Formula 1d: 50 mmol / L HEPES buffer, pH 7.4, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Tween 20 (w / v).

[0065] Formula 2a: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% OVA (w / v), 0.05% ProClin300 (w / v), 0.1% Tween 20 (w / v).

[0066] Formula 2b: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% gelatin (w / v), 0.05% ProClin300 (w / v), 0.1% Tween 20 (w / v).

[0067] Formula 3a: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v).

[0068] Formula 3b: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Triton X 100 (w / v).

[0069] Formula 3c: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Triton X 405 (w / v).

[0070] Formula 3d: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% S9 (w / v).

[0071] Formula 3e: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Brij 35 (w / v).

[0072] Formula 3f: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.04% Tween 80 (w / v).

[0073] Formula 3g: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.2% S9 (w / v).

[0074] Formula 4a: 50 mmol / L MES buffer, pH 6.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0075] Formula 4b: 50 mmol / L Tris buffer, pH 8.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0076] Formula 4c: 50 mmol / L Bis-Tris buffer, pH 6.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0077] Formula 4d: 50 mmol / L HEPES buffer, pH 7.4, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0078] Formula 5a: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% OVA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0079] Formula 5b: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% gelatin (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0080] Formula 6a: 50 mmol / L PB buffer, pH 7.0, 0 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0081] Formula 6b: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.1% BND-10 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0082] Formula 7a: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.1% Triton X 100 (w / v), 0.1% S9 (w / v).

[0083] Formula 7b: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% Triton X 405 (w / v).

[0084] Formula 8a: 10 mmol / L PB buffer, pH 6.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0085] Formula 8b: 50 mmol / L PB buffer, pH 6.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0086] Formula 8c: 100 mmol / L PB buffer, pH 6.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0087] Formula 8d: 10 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0088] Formula 8e: 100 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0089] Formula 8f: 10 mmol / L PB buffer, pH 7.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0090] Formula 8g: 50 mmol / L PB buffer, pH 7.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0091] Formula 8h: 100 mmol / L PB buffer, pH 7.5, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0092] Formula 8i: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 1% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0093] Formula 8j: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 5% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0094] Formula 8k: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.02% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0095] Formula 8L: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.1% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0096] Formula 8m: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.01% Tween 80 (w / v), 0.1% S9 (w / v).

[0097] Formula 8n: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.05% Tween 80 (w / v), 0.1% S9 (w / v).

[0098] Formula 8o: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.05% S9 (w / v).

[0099] Formula 8p: 50 mmol / L PB buffer, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.2% S9 (w / v).

[0100] Formula 8q: 50 mmol / L PB buffer, pH 7.0, 100 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0101] Formula 8r: 50 mmol / L PB buffer, pH 7.0, 200 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v), 0.1% S9 (w / v).

[0102] 2. Preparation of working solution for NSE monoclonal antibody-coated magnetic microspheres and labeled with neutral ruthenium compounds.

[0103] 2.1 Preparation of NSE monoclonal antibody-coated magnetic microspheres

[0104] (1) Mix the magnetic microspheres (Thermo, catalog number: 34310D) with freshly prepared N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide solution (10 mg / mL) at a ratio of 25:1 (mass ratio) and rotate at room temperature for 30 minutes.

[0105] (2) Remove the supernatant by magnetic separation, resuspend the magnetic microspheres in 1 mL of 25 mM 2-morpholine ethanesulfonic acid buffer (MES buffer, pH 6.0), add NSE monoclonal antibody 1 (manufacturer: Feipeng Biotechnology, NSE-REAB-C1-002) at a mass ratio of antibody: magnetic microspheres = 1:50, and mix by rotating at room temperature for 4 hours;

[0106] (3) Add an equal volume of 1wt% BSA (w / v) solution and mix by rotation at room temperature for 30 minutes;

[0107] (4) After removing the supernatant by magnetic separation, add diluent containing 1 wt% BSA, resuspend, and obtain magnetic microspheres coated with NSE monoclonal antibody.

[0108] 2.2 Preparation of labeled NSE monoclonal antibodies against electrically neutral ruthenium compounds

[0109] (1) Equal volumes of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide solution (40 mmol / L), N-hydroxythiosuccinimide solution (100 mmol / L), and electrically neutral ruthenium compound solution (10 mmol / L) were mixed at room temperature for 45 minutes.

[0110] Electroneutral ruthenium compound molecular structure

[0111] (2) Incubate at room temperature for 4 hours with a molar ratio of 10:1 for neutral ruthenium compound: NSE monoclonal antibody 2 (manufacturer: Feipeng Biotechnology, NSE-REAB-C1-003);

[0112] (3) Remove unbound, electrically neutral ruthenium compounds via a desalting column.

[0113] 2.3 The concentration of NSE monoclonal antibody-coated magnetic microspheres was diluted to 0.25 mg / mL using a buffer formulation, and the NSE monoclonal antibody stock solution labeled with an electrically neutral ruthenium compound was diluted to 1 μg / mL to form the electrochemiluminescence working solution.

[0114] 3. Working solution stability: The working solution to be tested was divided into two portions. One portion was stored at 2-8℃, and the other portion was accelerated at 37℃. Then, the test samples (NSE antigen; manufacturer: Tansheng, catalog number: FAP-TE003) were tested simultaneously at 0 days, 8 days and / or 12 days and 15 days using both the 2-8℃ storage reagent and the 37℃ accelerated storage reagent. S0 represents an antigen concentration of 0 ng / mL, S1 represents an antigen concentration of 0.115 ng / mL, and S2 represents an antigen concentration of 254.860 ng / mL. Each test was performed three times, and the average value was calculated. The relative deviation of the signal values ​​before and after acceleration was compared. The results are shown in the table below. The results shown in the same table are from the same batch of experiments.

[0115] The testing method is as follows:

[0116] (1) Add 90 μL of neutral ruthenium compound-labeled NSE monoclonal antibody reagent and 20 μL of sample to the detection tube, mix for 3-5 seconds, add 90 μL of NSE monoclonal antibody-coated magnetic microsphere reagent to the detection tube, mix for 3-5 seconds, and incubate at 37°C for 15 minutes.

[0117] (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;

[0118] (3) Take out the cleaning solution to clean other substances that are not fixed to the electrode surface, start a specific voltage of 1.4V, and the ruthenium compound undergoes an electrochemical reaction to form light;

[0119] (4) The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of NSE captured on the surface of the magnetic bead.

[0120] Table 1

[0121]

[0122] The accelerated stability test results of formulations 1 and 1a-1d in Table 1 show that buffer solutions with different pH values ​​have a significant impact on reagent stability, with PB buffer showing relatively better reagent stability.

[0123] Table 2

[0124]

[0125] Table 2 shows that formulations 1, 1a, 1b, 1c, and 1d have a certain impact on NSE antibody activity. The signal values ​​of formulations 1, 1c, and 1d at various concentration points are significantly higher than those of formulations 1a and 1b. Formulations 1, 1c, and 1d exhibit a higher signal-to-noise ratio when detecting low-concentration samples, indicating low background noise, high target signal, and easier effective identification and amplification. This enables chemiluminescent reagents to detect biomarkers at extremely low concentrations.

[0126] Table 3

[0127]

[0128] Formulas 2a and 2b differ from Formula 1 only in the type of protein. The accelerated stability test results show that BSA is relatively superior in terms of reagent stability.

[0129] Table 4

[0130]

[0131] Table 4 shows that formulations 1, 2a, and 2b have a certain impact on the activity of NSE antibody. When testing samples at various concentration points, the signal value and signal-to-noise ratio of formulation 1 are significantly higher than those of formulations 2a and 2b.

[0132] Table 5

[0133]

[0134] As shown in Table 5, formulations 1 and 3a-3e, compared to formulation 2, contain only one type of surfactant and have different surfactant types, resulting in poorer reagent stability. Similarly, formulations 3f-3g, compared to formulation 2, contain only one of the two required surfactant formulations and have different surfactant amounts, also exhibit poor reagent stability. This demonstrates that the two different surfactants added in this invention synergistically improve reagent stability.

[0135] Table 6

[0136]

[0137] Table 6 shows that, compared to Formula 2, Formulas 4a-4b differ in their buffer systems (different pH values), and the pH of the buffer solution has a greater impact on reagent stability, resulting in poorer stability. Formulas 4c-4d, compared to Formula 2, although changing the type of buffer, still maintain a pH adjustment range of 7.0 ± 0.5, with relative signal deviations within ± 10%, indicating acceptable stability. In Formula 2, the PB buffer (pH 7.0) shows relatively better reagent stability. Formulas 5a and 5b, compared to Formula 2, only change the type of protein; in Formula 2, BSA shows relatively better reagent stability. Formula 6a, compared to Formula 2, only changes the NaCl content; Formula 6a does not contain NaCl, and while signal values ​​at various concentration points increased, its reagent stability is relatively poor. Formula 6b, compared to Formula 2, only changes the type of preservative; in Formula 2, ProClin 300 shows relatively better reagent stability. Compared to Formula 2, Formulas 7a and 7b only changed the type of surfactant. Different surfactant combinations exhibited different reagent stability, with Tween 80 and S9 in Formula 2 showing relatively better reagent stability.

[0138] Table 7

[0139]

[0140]

[0141] Table 7 shows the accelerated stability results of formulations 2 and 8a-8r. After 12 days of accelerated testing at 37℃, the relative deviation of signal values ​​for each concentration point of formulation 2 was within ±5%; the relative deviation of signal values ​​for each concentration point of formulation 8a-8r was within ±10%, both meeting the expected requirements. This indicates that the working solutions of the magnetic microsphere reagent coated with NSE monoclonal antibody and the NSE monoclonal antibody reagent labeled with neutral ruthenium compounds contain buffer solutions of 10-100 mmol / L PB buffer, pH 7.0±0.5, 100 mM-200 mM NaCl, 1%-5% BSA (w / v), 0.02%-0.1% ProClin300 (w / v), 0.01%-0.05% Tween 80 (w / v), and 0.05%-0.2% S9 (w / v). Preferably, the buffer formulation is 50 mmol / L PB buffer salt, pH 7.0, 150 mM NaCl, 2% BSA (w / v), 0.05% ProClin300 (w / v), 0.02% Tween 80 (w / v) and 0.1% S9 (w / v) surfactant.

[0142] 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.

[0143] Table 8

[0144]

[0145] Table 8 shows that for each concentration point tested with Formula 2, the relative deviation of the signal value at 37℃ for 8 and 12 days of acceleration was within ±5%, and the relative deviation of the signal value at 37℃ for 15 days of acceleration was within ±10%, which still met the expected requirements. The diluent of this formula can stabilize the electrochemiluminescence reagent system.

[0146] Example 2

[0147] Assessment of blank limit and limit of detection:

[0148] This experiment referenced YY / T 1789.3-2022 Performance Evaluation Methods for In Vitro Diagnostic Testing Systems - Part 3: Limit of Detection and Limit of Quantitation, and EP17-A2 published by the Clinical and Laboratory Standards Institute (CLSI). Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures And the formulation of experimental methods in industry standards for related chemiluminescence reagent products.

[0149] The finished kit was prepared according to the steps of Example 1. Two blank samples (newborn calf serum, Shanghai Unimicron Biotechnology Co., Ltd., abs978), two samples with near detection limit (0.05 ng / mL) concentrations, three test days, and four repeated measurements were performed on each sample.

[0150] The percentage of blank sample test results that are lower than or equal to the blank limit (0.02 ng / mL) should be greater than the minimum percentage of 87% (corresponding to 24 test samples).

[0151] The percentage of test results for samples with detection limits greater than or equal to the blank limit should be greater than the minimum percentage of 87% (corresponding to 24 test samples).

[0152] Table 9

[0153]

[0154] As shown in Table 9, the percentage of blank sample results ≤ blank limit (0.02 ng / mL) is ≥87%, indicating that the blank limit verification meets the requirements; the percentage of low concentration sample results close to the detection limit ≥ blank limit (0.02 ng / mL) is ≥87%, indicating that the detection limit verification meets the requirements.

[0155] Repeatability assessment:

[0156] The sample concentration is repeated 10 times in the range of (15±5) ng / mL. The mean (M) and standard deviation (SD) of the 10 measurements are calculated to obtain the coefficient of variation (CV), which should meet the requirement of CV≤8%.

[0157] CV = SD / M × 100%

[0158] Table 10

[0159]

[0160] As shown in Table 10, the CV of the repeatable samples is 1.95%, which meets the standard.

[0161] Linear range evaluation:

[0162] High-value linear samples approaching the upper limit of the linear range (370 ng / mL) were proportionally diluted to five concentrations, with low-value linear samples approaching the lower limit of the linear range (0.05 ng / mL). Each concentration was measured three times, and the average value was calculated. The average measured concentration was then fitted to the theoretical concentration using the least squares method to form a linear relationship. Figure 1 ), and calculate the linear correlation coefficient r, which should satisfy r≥0.99.

[0163] Table 11

[0164]

[0165] As shown in Table 11, the linear correlation coefficient r of the test reagent in the range of 0.030-417.743 g / mL is greater than 0.9900, which meets the standard. The linear relationship of this 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.

[0166] Anti-interference assessment:

[0167] Interfering substances: Triglycerides (≤2000 mg / dL), bilirubin (≤72 mg / dL), total protein (≤10 g / dL), biotin (≤1000000 ng / mL), and human anti-mouse antibody (≤50 ng / mL) samples.

[0168] Prepare sample pools containing high and low-value interfering substances;

[0169] Prepare a 50% sample pool, add equal amounts of high-value and low-value samples to a centrifuge tube, and mix thoroughly;

[0170] Prepare a 75% sample pool, add equal amounts of high-value and median-value samples to a centrifuge tube, and mix thoroughly;

[0171] Prepare a 25% sample pool, add equal amounts of low-value and median-value samples to a centrifuge tube, and mix thoroughly;

[0172] Five interference samples were finally obtained: 0% interference sample, 25% interference sample, 50% interference sample, 75% interference sample, and 100% interference sample.

[0173] Using the test mean of the first sample without interference as the standard, calculate the relative deviation of other samples from it. When the relative deviation is within ±10%, the anti-interference requirement is considered to be met.

[0174] Table 12

[0175]

[0176] As shown in Table 12, when the sample contains triglycerides (concentration ≤2000mg / dL), bilirubin (concentration ≤66mg / dL), hemoglobin (concentration ≤1.0 g / dL), total protein (concentration ≤10 g / dL), biotin (concentration up to 1g / L), and human anti-mouse antibody (HAMA concentration ≤50ng / mL), the relative deviation of the test is within ±10%. The buffer formulation of the working solution of this reagent meets the anti-interference requirements and is suitable for testing blood samples.

[0177] 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 neuron-specific enolase, characterized in that, The kit comprises: a reagent composition, a ruthenium compound-labeled neuron-specific enolase monoclonal antibody, and magnetic microspheres coated with the neuron-specific enolase monoclonal antibody; the reagent composition contains a buffer, sodium chloride, bovine serum albumin, a preservative, a first surfactant, and a second surfactant, wherein the pH of the buffer is adjustable in the range of 6.5-7.5; the first surfactant is polyoxyethylene sorbitan monooleate; the second surfactant is a polymer of methyl ethylene oxide with 1,2-ethylenediamine and ethylene oxide; The weight ratio of the first surfactant to the second surfactant is (0.05-1):1; the weight ratio of sodium chloride, bovine serum albumin, preservative, and the second surfactant is (2-25):(5-100):(0.1-2):1; the reagent composition also contains water, the amount of water being such that the sodium chloride content in the reagent composition is 100-200 mM; the reagent composition is used to dilute ruthenium compound-labeled neuron-specific enolase monoclonal antibody and magnetic microspheres coated with neuron-specific enolase monoclonal antibody.

2. The reagent kit according to claim 1, characterized in that, The pH adjustment range of the buffer is 6.8-7.

2.

3. The kit according to claim 1 or 2, characterized in that, The weight ratio of the first surfactant to the second surfactant is (0.1-0.5):1; And / or, the weight ratio of the sodium chloride, bovine serum albumin, preservative, and second surfactant is (4-18):(10-50):(0.2-1):1; And / or, the reagent composition further contains water, the amount of water being such that the sodium chloride content in the reagent composition is 100-150 mM.

4. The kit according to claim 1 or 2, characterized in that, The buffer is a phosphate buffer; And / or, the preservative is a biological preservative.

5. A reagent working solution in which a magnetic microsphere complex is dispersed, characterized in that, The working solution of this reagent comprises a reagent composition and magnetic microspheres coated with a neuron-specific enolase monoclonal antibody. The reagent composition contains a buffer, sodium chloride, bovine serum albumin, a preservative, a first surfactant, and a second surfactant. The pH of the buffer is adjusted to a range of 6.5-7.

5. The first surfactant is polyoxyethylene sorbitan monooleate. The second surfactant is a polymer of methyl ethylene oxide with 1,2-ethylenediamine and ethylene oxide. The weight ratio of the first surfactant to the second surfactant is (0.05-1):

1. The weight ratio of sodium chloride, bovine serum albumin, preservative, and the second surfactant is (2-25):(5-100):(0.1-2):

1. The reagent composition also contains water, the amount of which ensures that the sodium chloride content in the reagent composition is 100-200 mM.

6. The reagent working solution according to claim 5, characterized in that, The pH adjustment range of the buffer is 6.8-7.

2.

7. The reagent working solution according to claim 5 or 6, characterized in that, The weight ratio of the first surfactant to the second surfactant is (0.1-0.5):1; And / or, the weight ratio of the sodium chloride, bovine serum albumin, preservative, and second surfactant is (4-18):(10-50):(0.2-1):1; And / or, the reagent composition further contains water, the amount of water being such that the sodium chloride content in the reagent composition is 100-150 mM.

8. The reagent working solution according to claim 5 or 6, characterized in that, The buffer is a phosphate buffer; And / or, the preservative is a biological preservative.

9. The reagent working solution according to claim 5, characterized in that, The content of the magnetic microspheres is 0.1-0.5 g relative to 150 mmol of sodium chloride; And / or, the mass concentration of the magnetic microspheres coated with neuron-specific enolase monoclonal antibody in the reagent working solution is 0.1-0.5 mg / mL.

10. A reagent working solution dispersing a ruthenium compound-labeled antibody complex, characterized in that, The working solution of this reagent comprises a reagent composition and a ruthenium compound-labeled neuron-specific enolase monoclonal antibody; the reagent composition contains a buffer, sodium chloride, bovine serum albumin, a preservative, a first surfactant, and a second surfactant, wherein the pH of the buffer is adjusted to a range of 6.5-7.5; the first surfactant is polyoxyethylene sorbitan monooleate; the second surfactant is a polymer of methyl ethylene oxide with 1,2-ethylenediamine and ethylene oxide; the weight ratio of the first surfactant to the second surfactant is (0.05-1):1; the weight ratio of sodium chloride, bovine serum albumin, preservative, and second surfactant is (2-25):(5-100):(0.1-2):1; the reagent composition also contains water, the amount of which is such that the sodium chloride content in the reagent composition is 100-200 mM.

11. The reagent working solution according to claim 10, characterized in that, The pH adjustment range of the buffer is 6.8-7.

2.

12. The reagent working solution according to claim 10 or 11, characterized in that, The weight ratio of the first surfactant to the second surfactant is (0.1-0.5):1; And / or, the weight ratio of the sodium chloride, bovine serum albumin, preservative, and second surfactant is (4-18):(10-50):(0.2-1):1; And / or, the reagent composition further contains water, the amount of water being such that the sodium chloride content in the reagent composition is 100-150 mM.

13. The reagent working solution according to claim 10 or 11, characterized in that, The buffer is a phosphate buffer; And / or, the preservative is a biological preservative.

14. The reagent working solution according to claim 10, characterized in that, The amount of the ruthenium compound-labeled neuron-specific enolase monoclonal antibody relative to 150 mmol of sodium chloride is 0.5-3 mg. And / or, the mass concentration of the ruthenium compound-labeled neuron-specific enolase monoclonal antibody in the reagent working solution is 0.5-3 μg / mL.

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