Kit and method for detecting human chorionic gonadotropin

Abstract

The invention relates to the field of medical detection, and discloses a kit and a method for detecting human chorionic gonadotropin, the kit comprises a ruthenium complex labeled human chorionic gonadotropin detection antibody, a magnetic microsphere coated by a human chorionic gonadotropin capture antibody, and a diluent; the diluent comprises a bis (2-hydroxyethyl) amino (trihydroxymethyl) methane buffer agent, chloride, bovine serum albumin, gelatin and a surfactant. The method comprises the following steps: mixing a ruthenium complex-labeled human chorionic gonadotropin detection antibody defined in the kit, magnetic microspheres coated with a human chorionic gonadotropin capture antibody, a diluent and a detection sample for reaction; and sucking the obtained reaction liquid into an electrochemical reaction tank to carry out electrochemical luminescence reaction. The method and the kit are high in sensitivity, good in repeatability, wide in linear range and strong in anti-interference capability, detection time can be shortened, and wide application can be realized.

Description

Technical Field

[0001] This invention relates to the field of medical testing, specifically to a kit and method for detecting human chorionic gonadotropin (hCG). Background Technology

[0002] Human chorionic gonadotropin (HCG) is a glycoprotein dimer composed of α and β subunits, with a molecular weight of 37.6 kDa. The α-subunit is structurally similar to other glycoprotein hormones, while the β-subunit is specific to HCG. The main function of HCG is to stimulate the corpus luteum, promoting the sustained secretion of estrogen and progesterone to facilitate the formation of the decidua and placental maturation. Approximately 10 days after implantation, the pregnant woman's body begins to secrete HCG. By detecting HCG levels in blood or urine, pregnancy can be determined or pregnancy outcomes predicted. Clinically, HCG testing is mainly used as an adjunct diagnosis for ectopic pregnancy and early pregnancy.

[0003] To date, the main chemiluminescence methods for detecting HCG in human serum or plasma include: enzyme-catalyzed magnetic microparticle chemiluminescence, acridil ester magnetic microparticle chemiluminescence, and the traditional terpyridine-ruthenium electrochemiluminescence method. However, most of these methods employ a (streptavidin) / biotin coupling mechanism, making HCG detection susceptible to interference from biotin in the sample. Furthermore, the stability of reagent components is a core requirement in practical applications, directly determining the reliability of the overall test results. However, the ruthenium complex-magnetic microsphere complex exhibits insufficient stability during long-term storage, leading to abnormal fluctuations in the signal values ​​generated by the working solution reaction, severely impacting detection accuracy and thus limiting the clinical application efficacy of the reagent system. Therefore, it is necessary to develop new HCG detection technologies to improve the anti-interference capability and reagent stability of the detection methods to meet the actual clinical needs for accurate HCG detection. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of HCG detection being easily interfered with by biotin in the sample and insufficient reagent stability in the prior art, and to provide a kit and method for detecting human chorionic gonadotropin. The kit has high detection sensitivity, good repeatability, wide linear range, strong anti-interference ability, and can effectively shorten the detection time. At the same time, each reagent component in the kit also has excellent detection stability.

[0005] To achieve the above objectives, the first aspect of the present invention provides a kit for detecting human chorionic gonadotropin (hCG), the kit comprising a ruthenium complex-labeled hCG detection antibody, magnetic microspheres coated with hCG capture antibody, and a diluent. The general structural formula of the ruthenium complex is: n = 0-9; The diluent includes bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane buffer, chloride, bovine serum albumin, gelatin, and surfactant.

[0006] A second aspect of the present invention provides a method for detecting human chorionic gonadotropin (hCG), the method comprising: The ruthenium complex-labeled human chorionic gonadotropin detection antibody, human chorionic gonadotropin capture antibody-coated magnetic microspheres, diluent, and detection sample as defined in the kit described in the first aspect are mixed and reacted; the resulting reaction solution is drawn into an electrochemical reaction cell for electrochemiluminescence reaction.

[0007] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The kit and method for detecting human chorionic gonadotropin (hCG) of the present invention have higher sensitivity, better repeatability, wider linear range, and stronger anti-interference ability compared with the prior art. Among them, the anti-interference concentration against biotin can reach up to 1,000,000 ng / mL, which can effectively resist the interference of high concentrations of biotin on the detection results. In addition, each reagent component in the kit of the present invention has excellent detection stability and can significantly shorten the detection cycle and improve detection efficiency, and has broad prospects for clinical detection and practical application. Attached Figure Description

[0008] Figure 1 This is a schematic diagram of the structure of the double antibody sandwich complex formed in the electrochemiluminescence immunoassay of the present invention; Figure 2 The calibration curve is shown for batch 1 of the finished reagent kit containing human chorionic gonadotropin calibrators in the 15-minute incubation time detection procedure (HCG calibrator 1 has an assigned concentration of 4.95 mIU / mL, and HCG calibrator 2 has an assigned concentration of 4998.64 mIU / mL). Figure 3 The calibration curves are for batch 2 of the finished kit human chorionic gonadotropin calibrators in the 15-minute incubation time detection procedure (HCG calibrator 1 has an assigned concentration of 4.95 mIU / mL, and HCG calibrator 2 has an assigned concentration of 4998.64 mIU / mL). Figure 4 The calibration curves are for batches 3 of the finished kit for human chorionic gonadotropin calibrators in the 15-minute incubation time detection procedure (HCG calibrator 1 has an assigned concentration of 4.95 mIU / mL, and HCG calibrator 2 has an assigned concentration of 4998.64 mIU / mL). Detailed Implementation

[0009] 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.

[0010] The first aspect of the present invention provides a kit for detecting human chorionic gonadotropin (hCG), the kit comprising a ruthenium complex-labeled hCG detection antibody, magnetic microspheres coated with hCG capture antibody, and a diluent. The general structural formula of the ruthenium complex is: n = 0-9; The diluent includes bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane buffer, chloride, bovine serum albumin, gelatin, and surfactant.

[0011] In this invention, magnetic microspheres coated with human chorionic gonadotropin (HCG) capture antibodies and ruthenium complex-labeled HCG detection antibodies are used. These two reagent components can undergo an immunobinding reaction with the HCG in the analyte to form a double-antibody sandwich complex structure of magnetic microsphere-antibody-antigen-antibody-ruthenium complex (e.g., ...). Figure 1 (As shown). This invention uses ruthenium complexes to label HCG detection antibodies, which can reduce non-specific interference in immunoassay and generate a strong luminescent signal, effectively improving the sensitivity of HCG detection; at the same time, the accompanying diluent can effectively improve the detection stability of the reagent working solution.

[0012] In this invention, it is understood that the ruthenium complex-labeled HCG detection antibody and the HCG capture antibody coated in the magnetic microspheres are different, and the two antibodies recognize different sites of the HCG antigen respectively.

[0013] In this invention, preferably, n = 3-6.

[0014] In this invention, preferably, the magnetic microspheres coated with HCG capture antibody have a particle size of 1-5 μm.

[0015] More preferably, the magnetic microspheres have a particle size of 2-3 μm.

[0016] In this invention, preferably, the mass ratio of the HCG capturing antibody to the magnetic microspheres is (0.006-0.012):1. In this invention, the fewer the number of antibodies coupled to the magnetic microspheres, the lower the signal value; the more antibodies, the higher the cost. Therefore, controlling the mass ratio of the HCG capturing antibody to the magnetic microspheres within the above range achieves a balance between signal strength and cost.

[0017] More preferably, the mass ratio of the HCG capture antibody to the magnetic microspheres is (0.008-0.01):1.

[0018] In this invention, the formulation of the diluent is not only suitable for the dispersion of magnetic microspheres coated with ruthenium complex-labeled HCG detection antibodies and HCG capture antibodies, but also maintains a stable reaction pH. At the same time, it provides a suitable acid-base environment and appropriate ionic conditions for the specific binding of antigens and antibodies, significantly enhances the detection signal value, and effectively improves the stability of the working solution during storage and use, ensuring the reliability and repeatability of the detection results.

[0019] In this invention, preferably, the chloride in the diluent is selected from sodium chloride and / or potassium chloride, more preferably sodium chloride.

[0020] In this invention, preferably, the surfactant in the diluent is selected from polysorbate-20.

[0021] In this invention, preferably, the mass ratio of bovine serum albumin to gelatin in the diluent is 1:(0.5-3). Controlling the mass ratio of bovine serum albumin to gelatin within this range is beneficial for improving the stability of reagent detection.

[0022] In this invention, preferably, the concentration of bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane) buffer in the diluent is 10-100 mmol / L, more preferably 20-50 mmol / L, such as 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L or any value or range between the above values.

[0023] In this invention, preferably, the pH adjustment range of the bis(2-hydroxyethyl)amino(trihydroxymethyl)methane buffer is 6-7.

[0024] In this invention, preferably, the concentration of chloride in the diluent is 100-200 mmol / L, more preferably 120-180 mmol / L, such as 120 mmol / L, 125 mmol / L, 130 mmol / L, 135 mmol / L, 140 mmol / L, 145 mmol / L, 150 mmol / L, 155 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, or any value or range between the above values.

[0025] In this invention, preferably, the concentration of bovine serum albumin in the diluent is 0.005-0.035 g / mL, more preferably 0.015-0.035 g / mL, such as 0.015 g / mL, 0.0175 g / mL, 0.02 g / mL, 0.0225 g / mL, 0.025 g / mL, 0.0275 g / mL, 0.03 g / mL, 0.0325 g / mL, 0.035 g / mL, or any value or range between the above values.

[0026] In this invention, preferably, the concentration of gelatin in the diluent is 0.01-0.04 g / mL, more preferably 0.02-0.04 g / mL, such as 0.02 g / mL, 0.022 g / mL, 0.024 g / mL, 0.026 g / mL, 0.028 g / mL, 0.03 g / mL, 0.032 g / mL, 0.034 g / mL, 0.036 g / mL, 0.038 g / mL, 0.04 g / mL, or any value or range between the above values.

[0027] In this invention, preferably, the concentration of the surfactant (such as polysorbate-20) in the diluent is 0.001-0.006 g / mL, more preferably 0.003-0.006 g / mL, such as 0.003 g / mL, 0.0035 g / mL, 0.004 g / mL, 0.0045 g / mL, 0.005 g / mL, 0.0055 g / mL, 0.006 g / mL, or any value or range between the above values.

[0028] In this invention, preferably, the kit further includes HCG calibrator 1 and HCG calibrator 2, wherein the HCG antigen concentration range of HCG calibrator 1 is 1-20 mIU / mL; and the HCG antigen concentration range of HCG calibrator 2 is 4000-6000 mIU / mL.

[0029] More preferably, the HCG antigen concentration range in the HCG calibrator 1 is 3-12 mIU / mL, and the HCG antigen concentration range in the HCG calibrator 2 is 4500-5500 mIU / mL.

[0030] A second aspect of the present invention provides a method for detecting human chorionic gonadotropin (hCG), the method comprising: The ruthenium complex-labeled human chorionic gonadotropin detection antibody, human chorionic gonadotropin capture antibody-coated magnetic microspheres, diluent, and detection sample as defined in the kit described in the first aspect are mixed and reacted; the resulting reaction solution is drawn into an electrochemical reaction cell for electrochemiluminescence reaction.

[0031] In this invention, preferably, the mixing method includes first mixing the ruthenium complex-labeled HCG detection antibody and the detection sample, and then adding the magnetic microspheres coated with HCG capture antibody to obtain a reaction solution containing a double antibody sandwich complex; or first mixing the magnetic microspheres coated with HCG capture antibody and the detection sample, and then adding the ruthenium complex-labeled HCG detection antibody to obtain a reaction solution containing a double antibody sandwich complex.

[0032] More preferably, the mixing method involves first mixing the ruthenium complex-labeled HCG detection antibody and the detection sample, and then adding magnetic microspheres coated with HCG capture antibody to obtain a reaction solution containing a double antibody sandwich complex.

[0033] In this invention, preferably, the method further includes: before use, diluting the ruthenium complex-labeled human chorionic gonadotropin detection antibody to a concentration of 0.5-3 μg / mL, more preferably 1-2 μg / mL, using the aforementioned diluent.

[0034] In this invention, preferably, the method further includes: before use, diluting the magnetic microspheres coated with the human chorionic gonadotropin capture antibody to a concentration of 0.1-0.5 mg / mL, more preferably 0.15-0.3 mg / mL, using the aforementioned diluent.

[0035] In this invention, the method involves drawing the above-mentioned reaction solution into an electrochemical reaction cell to perform an electrochemiluminescence reaction and collecting the electrochemiluminescence signal.

[0036] In this invention, preferably, the conditions for the mixing reaction include: a temperature of 35-40°C and an incubation time of 5-15 min.

[0037] In this invention, preferably, the mass ratio of the magnetic microspheres coated with the human chorionic gonadotropin (hCG) capture antibody to the ruthenium complex-labeled hCG detection antibody is (25-900):1, more preferably (150-300):1, such as 150:1, 155:1, 160:1, 170:1, 175:1, 180:1, 185:1, 190:1, 200:1, 210:1, 220:1, 250:1, 300:1, or any value or range between the above values.

[0038] In this invention, preferably, the method for preparing the ruthenium complex-labeled human chorionic gonadotropin (hCG) detection antibody includes: conjugating a ruthenium complex and a hCG detection antibody to obtain the ruthenium complex-labeled hCG detection antibody.

[0039] In this invention, preferably, the preparation method further includes: activating the ruthenium complex and then conjugating it with a human chorionic gonadotropin detection antibody, wherein the reagents used for activation include 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide.

[0040] In this invention, preferably, the molar ratio of the ruthenium complex, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide is 1:(4-15):(10-15).

[0041] More preferably, the molar ratio of the ruthenium complex, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide is 1:(4-6):(10-12).

[0042] In this invention, preferably, the activation time is 20-60 min, more preferably 45-60 min. In this invention, during the activation reaction, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) reacts with the carboxyl group in the ruthenium complex and Sulfo-NHS to form an NHS ester. Controlling the activation time within the above range ensures sufficient activation, avoids incomplete activation due to excessively short activation time, and effectively inhibits the hydrolysis and deactivation of the activation product due to excessively long reaction time.

[0043] In this invention, the activation of the carboxyl group by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride requires a specific pH environment; excessively high or low pH values ​​will affect the activation efficiency. Preferably, the pH of the activation system is 5.5-6.5, more preferably 6-6.5.

[0044] In this invention, room temperature refers to a temperature of 25±5℃.

[0045] Preferably, the activation is performed at room temperature.

[0046] In this invention, preferably, the molar ratio of the ruthenium complex to the HCG detection antibody is (8-12):1. Controlling the molar ratio within this range can both enhance the intensity of the luminescent signal and ensure that the ruthenium complex-labeled HCG detection antibody can fully perform an immunobinding reaction with the HCG in the analyte. In this invention, the molar ratio refers to the molar ratio of each raw material used in the coupling process of the ruthenium complex and HCG.

[0047] More preferably, the molar ratio of the ruthenium complex to the HCG detection antibody is (10-12):1.

[0048] In this invention, preferably, the pH value of the coupling system is 6-10, more preferably 8-9.6.

[0049] In this invention, preferably, the buffer used for coupling is selected from at least one of citrate, carbonate and borate; more preferably, the buffer used for coupling is borate.

[0050] In this invention, preferably, the citrate buffer is mainly composed of citric acid and sodium citrate (represented as citric acid-sodium citrate); the carbonate buffer is mainly composed of sodium carbonate and sodium bicarbonate (represented as sodium carbonate-sodium bicarbonate); and the borate buffer is mainly composed of boric acid and borax (represented as boric acid-borax).

[0051] In this invention, preferably, the conjugation time is 2-5 hours, more preferably 4-5 hours. In this invention, during the conjugation reaction, the conjugation efficiency between the antibody and the ruthenium complex gradually increases with the extension of the conjugation time. However, when the conjugation time reaches a specific threshold, the reaction enters a "plateau phase"; thereafter, further extending the conjugation time does not improve the conjugation efficiency. Therefore, controlling the conjugation time within the above range ensures sufficient conjugation, avoiding problems such as incomplete conjugation and insufficient labeling efficiency caused by excessively short conjugation times. Furthermore, it significantly shortens the experimental cycle and reduces the time cost of the detection process while ensuring the conjugation effect.

[0052] In this invention, preferably, the coupling is performed at room temperature.

[0053] In this invention, preferably, the preparation method of the HCG capture antibody-coated magnetic microspheres may include: activating the magnetic microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and then introducing an HCG capture antibody for coupling, thereby obtaining HCG capture antibody-coated magnetic microspheres. In this invention, the magnetic microspheres have a hydrophilic polymer coating and incorporate carboxyl functional groups for antibody coupling.

[0054] More preferably, the magnetic microspheres are activated by mixing at room temperature for 15-45 minutes, more preferably for 30-45 minutes.

[0055] More preferably, the coupling is performed by mixing at room temperature for 2-4 hours.

[0056] In this invention, preferably, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) to the magnetic microspheres is (0.03-0.05):1. Controlling the mass ratio within this range ensures sufficient activation of the carboxyl groups on the magnetic microspheres. More preferably, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the magnetic microspheres is (0.04-0.05):1.

[0057] In this invention, preferably, the activation of the magnetic microspheres is carried out at a pH of 5-7, more preferably 5.5-6.5.

[0058] More preferably, the buffer for activating the magnetic microspheres is 2-morpholinoethanesulfonic acid.

[0059] In this invention, preferably, the coupling of the HCG capture antibody and the magnetic microspheres is carried out at a pH of 6-7, more preferably 6-6.5.

[0060] In this invention, preferably, the method for preparing the magnetic microspheres coated with HCG capture antibody further includes: blocking the magnetic microspheres coated with HCG capture antibody.

[0061] In this invention, preferably, the sealing and mixing is carried out at room temperature for 2-24 hours, more preferably for 16-24 hours.

[0062] More preferably, the blocking solution used for blocking contains 0.005-0.012 g / mL bovine serum albumin (BSA). More preferably, the pH of the blocking solution is 7-8.

[0063] The present invention will be described in detail below through examples. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents are of analytical grade.

[0064] Both the HCG monoclonal antibody (capture antibody) and the HCG monoclonal antibody (detection antibody) were purchased from Hangzhou Huakui Jinpei Biotechnology Co., Ltd.

[0065] The preparation methods for the freshly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) solution and N-hydroxythiosuccinimide solution used in the following experiments are as follows: 10 mg / mL and 40 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solutions were prepared using 2-morpholinoethanesulfonic acid (MES) buffer (pH=6).

[0066] Prepare a 100 mmol / L solution of N-hydroxythiosuccinimide (Sulfo-NHS) using 2-morpholinoethanesulfonic acid (MES) buffer (pH=6).

[0067] Electrochemiluminescence signal parameters were measured using the YnY 3030 fully automated electrochemiluminescence immunoassay analyzer from Shenzhen Ansai Diagnostics Technology Co., Ltd.

[0068] In the following examples, all test samples were diluted using 2-morpholinoethanesulfonic acid (MES) buffer (pH=6).

[0069] Example 1 This embodiment illustrates the preparation method and condition optimization process of the human chorionic gonadotropin electrochemiluminescence assay kit, as detailed below: I. Preparation method of human chorionic gonadotropin electrochemiluminescence assay kit 1. The preparation of HCG monoclonal antibody (capture antibody) coated magnetic microsphere reagent is as follows: S1. Mix 1 mL of magnetic microspheres with a particle size of 2.7 μm (20 mg / mL) with 80 µL of freshly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) solution (10 mg / mL) and mix by rotation at room temperature for 30 minutes. S2. 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), add 0.4 mg of HCG monoclonal antibody (capture antibody), and mix by rotation at room temperature for 3 hours. S3. Add an equal volume of 0.01 g / mL bovine serum albumin (BSA) solution and mix by rotating at room temperature for 30 minutes; S4. After removing the supernatant by magnetic separation, add 100 mL of diluent containing 30 mM Bis-Tris, 150 mmol / L NaCl, 0.03 g / mL gelatin, 0.006 g / mL Tween-20, and 0.015 g / mL BSA (adjust pH to 6.5) to resuspend the magnetic microspheres, so that the concentration of the magnetic microspheres coated with HCG monoclonal antibody (capture antibody) after dilution is 0.2 mg / mL.

[0070] 2. The preparation of ruthenium complex-labeled HCG monoclonal antibody (detection antibody) reagent is as follows: S1. 7.5 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (40 mmol / L), 6 mL of N-hydroxythiosuccinimide solution (100 mmol / L), and 5 mL of ruthenium complex solution (10 mmol / L, solvent: purified water) were mixed at room temperature for 45 minutes to obtain a ruthenium complex mixed solution. The specific structural formula of the ruthenium complex is as follows: ; S2. Add 5 µmol of HCG monoclonal antibody (detection antibody) to the above ruthenium complex mixture solution and incubate at room temperature for 4 hours; S3. Unbound ruthenium complexes are removed via a desalting column; S4. The labeled antibody was quantified using the quinolinic acid (BCA) protein assay. Based on the extinction coefficient of the labeled molecule, the absorbance of the ruthenium complex at 455 nm was determined to be related to the labeling amount. It was determined that the actual binding molar ratio of the ruthenium complex to the HCG monoclonal antibody (detection antibody) labeled with the ruthenium complex was 10:1. S5. Dilute the ruthenium complex-labeled HCG monoclonal antibody (detection antibody) to 1 μg / mL using a dilution buffer (pH adjusted to 6.5) containing 30 mM Bis-Tris, 150 mmol / L NaCl, 0.03 g / mL gelatin, 0.006 g / mL Tween-20, and 0.015 g / mL BSA.

[0071] 3. The preparation of the human chorionic gonadotropin calibrator is as follows: Preparation of HCG calibrator 1: Weigh 0.05 mL of HCG antigen stock solution (50 IU / mL), and make up to 500 mL with antigen diluent. The concentration of HCG calibrator 1 is 5 mIU / mL. Preparation of HCG calibrator 2: Weigh 50 mL of HCG antigen stock solution (50 IU / mL), and make up to 500 mL with antigen diluent. The concentration of HCG calibrator 2 is 5000 mIU / mL.

[0072] Example 2 This embodiment illustrates the optimal conditions for preparing magnetic microsphere reagents coated with HCG monoclonal antibody (capture antibody).

[0073] I. Test Samples Prepare samples at at least three concentration levels for the antigen: 0 mIU / mL, 0.1 mIU / mL, and 10000 mIU / mL. Test these samples using the pre-set parameters, repeating each concentration test at least three times. All experiments below use ruthenium complexes with n=3.

[0074] II. Signal-to-noise ratio testing methods Using the pre-set parameters for evaluation, samples of three different concentrations were tested. The 0 mIU / mL sample was used to evaluate the background value of the reagent, the 0.1 mIU / mL sample was used to evaluate the sensitivity of the reagent, and the 10000 mIU / mL sample was used to evaluate the maximum detection range of the reagent. The signal-to-noise ratio (S / N) was calculated by dividing the mean signal value (S1) of the 0.1 mIU / mL sample and the mean signal value (S2) of the 10000 mIU / mL sample by the mean signal value (N) of the 0 mIU / mL sample, as shown in Formula 1. Each sample was tested three times, and the mean signal value was calculated.

[0075] Screening criteria: A high signal-to-noise ratio (S1 / N) indicates that the antibody pair has high sensitivity under these conditions, and a high signal-to-noise ratio (S2 / N) indicates that the antibody pair has a wide detection range under these conditions. Therefore, the highest signal-to-noise ratio (S / N) indicates that these are the optimal conditions.

[0076] Signal-to-noise ratio (S / N) = Mean of signal values ​​of samples with positive concentration / Mean of signal values ​​of samples with zero concentration — (Formula 1) III. Main Packaging Optimization Process 1. Concentration of activating reagent EDC•HCl Use 30μg / mg respectively 磁性微球 40μg / mg 磁性微球 50μg / mg 磁性微球 The magnetic microspheres were activated with EDC•HCl and coated with antibodies. The antibody-coated magnetic microspheres were then prepared into a reagent with a concentration of 0.2 mg / mL. The reagent was then used to test HCG detection antibody labeled with ruthenium complex at a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated. The results are shown in Table 1.

[0077] Table 1

[0078] As shown in Table 1, the concentration of EDC•HCl is 30 μg / mg. 磁性微球 The signal value and signal-to-noise ratio are slightly lower; when the concentration of EDC•HCl is 40-50 μg / mg 磁性微球 At this time, the detection signal value and signal-to-noise ratio are relatively high; therefore, the preferred range for EDC•HCl concentration is 40-50 μg / mg. 磁性微球 .

[0079] 2. pH value of activation buffer The activated magnetic microspheres were activated with activation buffers (MES buffer) at pH values ​​of 5.5, 6, and 6.5, respectively, and coated with antibodies. The antibody-coated magnetic microspheres were then prepared into a reagent with a concentration of 0.2 mg / mL, and tested with ruthenium complex-labeled HCG detection antibody at a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 2.

[0080] Table 2

[0081] As can be seen from Table 2, when the pH value of the activation buffer is 5.5, 6 and 6.5, the signal value and signal-to-noise ratio of the test sample are close. Therefore, pH value of 5.5-6.5 is the preferred pH value of the activation buffer.

[0082] 3. Activation reaction time Activation reactions were performed at times of 15 min, 30 min, and 45 min, respectively. The microspheres coated with the antibody were then prepared into a reagent with a concentration of 0.2 mg / mL. The reagent was then used in combination with a ruthenium complex-labeled HCG detection antibody with a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 3.

[0083] Table 3

[0084] As can be seen from Table 3, when the activation reaction time is 30-45 min, the signal-to-noise ratio and signal value are slightly higher. Therefore, the preferred activation reaction time is 30-45 min.

[0085] 4. pH value of coupling buffer The selected coupling buffers (MES buffers) had pH values ​​of 6, 6.5, and 7 for coupling. The antibody-coated magnetic microspheres were prepared into a reagent with a concentration of 0.2 mg / mL and tested with ruthenium complex-labeled HCG detection antibody at a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 4.

[0086] Table 4

[0087] As can be seen from Table 4, when the coupling buffer pH is 6 and 6.5, the signal value and signal-to-noise ratio of the test sample are high and close. Therefore, pH 6-6.5 is the preferred pH value for the activation buffer.

[0088] 5. Coupling reaction time Coupling was performed at reaction times of 2 hours, 3 hours, and 4 hours. The antibody-coated magnetic microspheres were prepared into a reagent with a concentration of 0.2 mg / mL and tested with ruthenium complex-labeled HCG detection antibody at a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 5.

[0089] Table 5

[0090] As can be seen from Table 5, the signal-to-noise ratio is low when the coupling response time is 2 hours, and high and close when the coupling response time is 3 hours and 4 hours. Therefore, the coupling response time is preferably 3-4 hours.

[0091] 6. Coating ratio The antibody coating ratios (antibody mass to magnetic microsphere mass ratio) were selected as 0.006:1, 0.008:1, and 0.01:1. The antibody-coated magnetic microspheres were prepared into a reagent with a concentration of 0.2 mg / mL, and tested with ruthenium complex-labeled HCG detection antibody at a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 6.

[0092] Table 6

[0093] As can be seen from Table 6, when the packet ratio is 0.006:1, the signal value is low and the signal-to-noise ratio is also low. When the packet ratio is (0.008-0.01):1, both the signal value and the signal-to-noise ratio are high. Therefore, the preferred packet ratio is (0.008-0.01):1.

[0094] 7. pH value of the blocking solution The selected blocking solutions had pH values ​​of 7, 7.4, and 8. Antibody-coated magnetic microspheres were prepared into a reagent with a concentration of 0.2 mg / mL, and tested with ruthenium complex-labeled HCG detection antibody at a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 7.

[0095] Table 7

[0096] As can be seen from Table 7, when the pH value of the coupling buffer is 7, 7.4 and 8, the signal value and signal-to-noise ratio of the test sample are close. Therefore, pH value of 7-8 is the preferred pH value of the blocking solution.

[0097] 8. Closure Time Blocking times of 2 h, 4 h, 16 h, and 24 h were selected. Antibody-coated magnetic microspheres were prepared into a reagent with a concentration of 0.2 mg / mL, and tested with ruthenium complex-labeled HCG detection antibody at a concentration of 1 μg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 8.

[0098] Table 8

[0099] As can be seen from Table 8, the signal value and signal-to-noise ratio improve with the increase of the blocking time. The signal value and signal-to-noise ratio of the blocking time of 24h and the blocking time of 16h are high and close. Therefore, the blocking time is preferably 16-24h.

[0100] The results above indicate that the optimal coating conditions are: EDC•HCl concentration of 40-50 μg / mg magnetic microspheres; activation buffer pH of 5.5-6.5; activation reaction time of 30-45 min; coupling buffer pH of 6-6.5; coupling reaction time of 3-4 h; coating ratio of (0.008-0.01):1; blocking buffer pH of 7-8; and blocking time of 16-24 h.

[0101] The optimal coating conditions are a concentration of 40 μg / mg of the activating agent EDC•HCl. 磁性微球 The activation buffer pH was 6, the activation reaction time was 30 min, the coupling buffer pH was 6.5, the coupling reaction time was 3 h, the coating ratio was 0.008:1, the blocking buffer pH was 7.4, and the blocking time was 16 h.

[0102] Example 3 This embodiment illustrates the preferred conditions for the preparation of ruthenium complex-labeled HCG monoclonal antibody (detection antibody) reagents.

[0103] 1. Screening of ruthenium complexes The main difference in the structure of the ruthenium complexes in this invention lies in the number of n. Ruthenium complexes with n=1, 3, 6, and 9 were selected to label antibodies. The antibodies labeled with ruthenium complexes were prepared into a reagent with a concentration of 1 μg / mL. The reagents were then used to test magnetic microspheres coated with HCG capture antibody at a concentration of 0.2 mg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 9.

[0104] Table 9

[0105] As shown in Table 9, the signal value and signal-to-noise ratio of the antibody are slightly lower when n=1 in the structure of the ruthenium complex, while the signal-to-noise ratio of the antibody is relatively higher when n=3 or n=6. Therefore, the preferred structure of the ruthenium complex is selected from n=3-6, and the most preferred is n=3.

[0106] All the following experiments used ruthenium complexes with n=3.

[0107] 2. Molar ratio of ruthenium complex, EDC•HCl and Sulfo-NHS Antibodies were labeled with ruthenium complex: EDC•HCl: Sulfo-NHS molar ratios of 1:4:10, 1:6:10, 1:8:10, 1:10:10, and 1:15:15, respectively. The ruthenium complex-labeled antibodies were prepared into a reagent with a concentration of 1 μg / mL and tested with magnetic microspheres coated with HCG capture antibody at a concentration of 0.2 mg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 10.

[0108] Table 10

[0109] As shown in Table 10, the signal value and signal-to-noise ratio increase with the increase of the molar ratio of EDC•HCl to Sulfo-NHS. The signal-to-noise ratio is slightly higher when the molar ratio is 1:(4-6):(10-12). During the labeling process, the antibody mother liquor labeled with the ratios of 1:8:10, 1:10:10, and 1:15:15 showed precipitation. Therefore, the molar ratio of ruthenium complex:EDC•HCl:Sulfo-NHS = 1:(4-6):(10-12) is the preferred molar ratio.

[0110] 3. Activation reaction time Activation reaction times of 30 min, 45 min, and 60 min were selected. Antibodies labeled with ruthenium complexes were prepared into reagents with a concentration of 1 μg / mL. Magnetic microspheres coated with HCG capture antibody at a concentration of 0.2 mg / mL were tested, and the mean signal value and signal-to-noise ratio were calculated. The results are shown in Table 11.

[0111] Table 11

[0112] As can be seen from Table 11, when the activation reaction time is 45-60 min, the signal-to-noise ratio and signal value are slightly higher. Therefore, the preferred activation reaction time is 45-60 min.

[0113] 4. pH value of activation buffer The ruthenium complex was activated using activation buffers (MES buffer) with pH values ​​of 5.5, 6, and 6.5. Antibodies were then labeled with the ruthenium complex-labeled antibodies. The ruthenium complex-labeled antibodies were prepared into a reagent with a concentration of 1 μg / mL and tested with magnetic microspheres coated with HCG capture antibody at a concentration of 0.2 mg / mL. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 12.

[0114] Table 12

[0115] As can be seen from Table 12, the signal value and signal-to-noise ratio are slightly higher when the pH value of the activation buffer is 6 and 6.5. Therefore, pH value of 6-6.5 is the preferred pH value for the activation buffer.

[0116] 5. Types of coupling buffer solutions Ten coupling buffers were selected for coupling, with the following concentrations and pH values: PBS (20mM, pH 7.0), TRIS (30mM, pH 8.0), citrate-sodium citrate (50mM, pH 6.0), BIS-TRIS (30mM, pH 6.5), PIPES (30mM, pH 6.8), HEPPS (20mM, pH 7.5), EPPS (20mM, pH 8.0), sodium carbonate-sodium bicarbonate (50mM, pH 9.6), boric acid-borax (200mM, pH 8.0), and MES (50mM, pH 6.0). Ruthenium complex-labeled antibodies were prepared to a concentration of 1 μg / mL and tested with magnetic microspheres coated with 0.2 mg / mL HCG capture antibody. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 13.

[0117] Table 13

[0118] As shown in Table 13, the detection signal value and signal-to-noise ratio are relatively high when sodium carbonate-sodium bicarbonate and boric acid-borax are used as the labeling coupling buffers. Among them, the detection signal value and signal-to-noise ratio are the highest when boric acid-borax is used. Therefore, boric acid-borax is used as the optimal coupling buffer.

[0119] 6. Coupling reaction time The coupling reaction times were selected as 2 hours, 3 hours, 4 hours, and 5 hours. The ruthenium complex-labeled antibody was prepared into a reagent with a concentration of 1 μg / mL, and tested with magnetic microspheres coated with 0.2 mg / mL HCG capture antibody. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 14.

[0120] Table 14

[0121] As can be seen from Table 14, the signal value and signal-to-noise ratio show an increasing trend when the coupling response time is between 2 and 4 hours. After 4 and 5 hours, the signal value and signal-to-noise ratio approach a stable period. Therefore, the coupling response time is preferably 4-5 hours.

[0122] 7. Marking Scale Antibodies were labeled using labeling ratios (molar ratio of antibody to ruthenium complex) of 1:8, 1:10, and 1:12. The ruthenium complex-labeled antibodies were prepared into a 1 μg / mL solution and tested with magnetic microspheres coated with 0.25 mg / mL HCG capture antibody. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 15.

[0123] Table 15

[0124] As can be seen from Table 15, when the labeling ratio is 1:(10-12), the signal value of the test antigen is high and the signal-to-noise ratio is also slightly higher. Therefore, the preferred labeling ratio is 1:(10-12).

[0125] The results above indicate that the preferred labeling process is a ruthenium complex, an EDC•HCl and Sulfo-NHS molar ratio of 1:(4-6):(10-12), an activation buffer pH of 6-6.5, an activation reaction time of 45-60 min, a coupling buffer selected from sodium carbonate-sodium bicarbonate and / or boric acid-borax, a coupling reaction time of 4-5 hours, and a labeling ratio of 1:(10-12).

[0126] The optimal labeling process is as follows: the molar ratio of ruthenium complex, EDC•HCl and Sulfo-NHS is 1:6:12, the activation buffer pH is 6, the activation reaction time is 45 min, the coupling buffer is boric acid-borax, the coupling reaction time is 4 h, and the labeling ratio is 1:10.

[0127] Example 4 This example illustrates the screening of reagent diluent formulations.

[0128] 1. Buffer system for reagent dilution The raw materials, namely HCG capture antibody-coated magnetic microspheres and ruthenium complex-labeled HCG detection antibody stock solution prepared through a coating / labeling process, need to be diluted with reagent diluents to form the final working solution. The buffer system of the reagent diluent plays an important role in maintaining the pH of the reagent and the stability of the conjugated antibody. Different reagent buffer systems are as follows: 20 mM PPB (pH 7.0), 30 mM Bis-Tris (pH 6.5), 50 mM MES (pH 6.0), and 20 mM HEPPES (pH 7.5). The four reagent diluent formulations are identical except for the buffer system, consisting of 150 mmol / L NaCl, 0.006 g / mL Tween-20, and 0.015 g / mL BSA. The magnetic microspheres coated with HCG capture antibody were prepared into a working solution with a concentration of 0.2 mg / mL using the four reagent diluents mentioned above. The HCG detection antibody labeled with ruthenium complex was prepared into a reagent with a concentration of 1 μg / mL for testing. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 16.

[0129] Table 16

[0130] As can be seen from Table 16, the signal values ​​of the Bis-Tris and MES buffer systems are relatively high, but the signal-to-noise ratio of the MES buffer system is slightly lower. Therefore, the Bis-Tris buffer system is the optimal buffer system.

[0131] 2. Gelatin concentration During the experiment, it was found that the addition of gelatin could improve the stability of the reagents. Different gelatin concentrations were selected as follows: 0 g / mL gelatin, 0.01 g / mL gelatin, 0.02 g / mL gelatin, 0.03 g / mL gelatin, and 0.04 g / mL gelatin. The five reagent diluents were identical in all components except for the gelatin concentration: 30 mM Bis-Tris, 150 mmol / L NaCl, 0.006 g / mL Tween-20, and 0.015 g / mL BSA. Using the five reagent diluents, a working solution of 0.2 mg / mL was prepared for HCG capture antibody-coated magnetic microspheres, and a reagent of 1 μg / mL was prepared for HCG detection using ruthenium complex-labeled antibodies. The reagents were divided into three portions and stored on day 0, at 4°C for 8 days, and in an incubator at 37°C for 8 days, respectively. After acceleration, samples of four prepared concentrations were tested, and reagents stored at 37℃ and 4℃ for 8 days were also tested. The relative deviations of the 37℃ and 4℃ reagents were compared, and the buffer system with the smallest relative deviation was selected as the reagent dilution. The results are shown in Table 16.

[0132] Table 17

[0133] As can be seen from Table 17, the addition of gelatin can improve the stability of the reagent to a certain extent. When the gelatin concentration is 0.02 g / mL-0.04 g / mL, the acceleration deviation at 37℃ / 4℃ is relatively small. Therefore, the preferred gelatin concentration in the diluent is 0.02 g / mL-0.04 g / mL.

[0134] 3. BSA concentration screening During the experiment, it was found that BSA could enhance the reagent signal value. Different concentrations of BSA were selected as follows: 0 g / mL BSA, 0.005 g / mL BSA, 0.015 g / mL BSA, 0.025 g / mL BSA, and 0.035 g / mL BSA. The five reagent diluents were identical except for the BSA concentration: 30 mM Bis-Tris, 150 mmol / L NaCl, 0.006 g / mL Tween-20, and 0.03 g / mL gelatin. Using these five reagent diluents, a working solution of 0.2 mg / mL was prepared for HCG capture antibody-coated magnetic microspheres. A reagent solution of 1 μg / mL was prepared for HCG detection using ruthenium complex-labeled antibody. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 18.

[0135] Table 18

[0136] As shown in Table 18, increasing the BSA concentration increases the reagent signal value, the S1 / N signal-to-noise ratio, and the reagent sensitivity. However, once the BSA concentration reaches 0.015 g / mL, the reagent signal-to-noise ratio does not improve significantly. Therefore, the preferred BSA concentration in the diluent is 0.015 g / mL to 0.035 g / mL.

[0137] 4. Screening of surfactants Surfactants are an important component of reagents, and different surfactants have different effects on the reagent system. The different surfactants are as follows: no surfactant added, 0.006 g / mL Tween-20, 0.006 g / mL Tween-80, 0.006 g / mL Surfactant S9, and 0.006 g / mL Triton X-100. The diluents for these reagents are identical except for the surfactant type; they consist of 30 mM Bis-Tris, 150 mmol / L NaCl, 0.015 g / mL BSA, and 0.03 g / mL gelatin. Using these reagent diluents, HCG capture antibody-coated magnetic microspheres were prepared into a working solution with a concentration of 0.2 mg / mL. Ruthenium complex-labeled HCG detection antibody was prepared into a reagent with a concentration of 1 μg / mL for testing. The mean signal value and signal-to-noise ratio were calculated, and the results are shown in Table 19.

[0138] Table 19

[0139] As can be seen from Table 19, different surfactants have different effects on the signal value, signal-to-noise ratio and detection sensitivity of the reagent. Among them, the addition of 0.006 g / mL Tween-20 has a significant effect on the signal-to-noise ratio and detection sensitivity of the reagent. Therefore, the surfactant in the diluent is preferably 0.006 g / mL Tween-20.

[0140] Example 5 This example illustrates the performance of the HCG detection kit.

[0141] Based on the selected process conditions and formulations, three batches of reagent kits were produced to verify product performance indicators.

[0142] The calibration procedure using a 15-minute incubation time detection method and human chorionic gonadotropin calibrator is as follows: S1. Add 95 μL of ruthenium complex-labeled HCG monoclonal antibody (detection antibody) reagent and 6 μL of reconstituted calibrator (solvent is purified water) to the detection tube, mix for 3-5 seconds, add 85 μL of HCG monoclonal antibody (capture antibody) coated magnetic microsphere reagent to the detection tube, mix for 3-5 seconds, and incubate at 37°C for 15 minutes; S2. The incubated reaction mixture is drawn into the measuring cell, and the magnet below the measuring cell fixes the magnetic microspheres in the reaction mixture onto the electrode surface; S3. Draw up the cleaning solution to clean other substances not fixed to the electrode surface, apply the starting voltage (0.7V), and the ruthenium complex undergoes an electrochemical reaction to produce luminescence; S4. The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of HCG captured on the surface of the magnetic microspheres, thus enabling quantitative analysis of HCG. S5. Establish a calibration curve using the concentrations of the two calibrators and their corresponding luminescence signal values; The sample detection method for the HCG detection kit is as described above, and the HCG content in the sample is determined by the calibration curve.

[0143] 1. Detection limit assessment The calibration curve was established using a 15-minute incubation time detection procedure and human chorionic gonadotropin calibrator (e.g.) Figure 2-4 As shown in Table 20, take 5 low-value samples with concentrations close to the detection limit (0.1 mIU / mL) and test them with the kit. Each sample should be tested 5 times. The following requirements should be met: the number of test results below the given blank limit (0.07 mIU / mL) should be less than or equal to 3.

[0144] Table 20

[0145] As shown in Table 20, the detection limit assessments for batches 1, 2, and 3 of the reagent kits all meet the requirements.

[0146] 2. Repeatability assessment The calibration curve was established using a 15-minute incubation time detection procedure and human chorionic gonadotropin calibrator (e.g.) Figure 2-4 As shown in Table 21, samples with concentrations in the range of (25±6) mIU / mL and (200±48) mIU / mL were repeatedly measured 10 times. The mean (M) and standard deviation (SD) of the 10 measurements were calculated, and the coefficient of variation (CV) was obtained. The CV should be ≤8%.

[0147] Table 21

[0148] As shown in Table 21, the repeatability assessment of kits in batches 1, 2, and 3 all meet the requirements.

[0149] 3. Linear range evaluation The calibration curve was established using a 15-minute incubation time detection procedure and human chorionic gonadotropin calibrator (e.g.) Figure 2-4 As shown in Table 22, high-value linear samples close to the upper limit of the linear interval (10000 mIU / mL) were diluted proportionally to five concentrations, with low-value linear samples close to the lower limit of the linear interval (0.1 mIU / 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, and the linear correlation coefficient r was calculated. r should satisfy ≥ 0.99.

[0150] Table 22

[0151] As shown in Table 22, the linear range assessment of kits in batches 1, 2, and 3 all meet the requirements.

[0152] 4. Biotin interference assessment The calibration curve was established using a 15-minute incubation time detection procedure and human chorionic gonadotropin calibrator (e.g.) Figure 2-4 As shown in Table 23), biotin was added to a sample with an HCG concentration of (25±6) mIU / mL to achieve a concentration of 1,000,000 ng / mL. The addition ratio did not exceed (1:9). The sample was tested three times. The interference deviation should be within ±10%.

[0153] Table 23

[0154] As shown in Table 23, the biotin interference assessment of batches 1, 2, and 3 all meet the requirements.

[0155] 5. Comparison with imported reagent kits The performance comparison with imported reagent kits is shown in Table 24.

[0156] Table 24

[0157] As shown in Table 24, the kit of the present invention is superior to imported kits in terms of incubation time and biotin interference when detecting HCG, with a significant improvement in biotin interference.

[0158] 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 human chorionic gonadotropin, characterized by, The kit comprises a human chorionic gonadotropin detection antibody labeled with a ruthenium complex, human chorionic gonadotropin capture antibody coated magnetic microspheres, and a diluent; The structure general formula of the ruthenium complex is: ，n=0-9； The diluent comprises bis(2-hydroxyethyl)amino(trishydroxymethyl)methane buffer, chloride, bovine serum albumin, gelatin and surfactant.

2. The kit of claim 1, wherein The n is 3-6. And / or, in the human chorionic gonadotropin capture antibody coated magnetic microspheres, the particle size of the magnetic microspheres is 1-5 μm.

3. The kit according to claim 1 or 2, characterized in that, The chloride in the diluent is selected from sodium chloride and / or potassium chloride; And / or, the surfactant in the diluent is selected from polysorbate-20; And / or, the mass ratio of bovine serum albumin and gelatin in the diluent is 1:(0.5-3).

4. The kit according to claim 1 or 2, characterized in that, The concentration of bis(2-hydroxyethyl)amino(trishydroxymethyl)methane buffer in the diluent is 10-100 mmol / L; And / or, the pH adjustment range of the bis(2-hydroxyethyl)amino(trishydroxymethyl)methane buffer is 6-7; And / or, the concentration of chloride in the diluent is 100-200 mmol / L; And / or, the concentration of bovine serum albumin in the diluent is 0.005-0.035 g / mL; And / or, the concentration of gelatin in the diluent is 0.01-0.04 g / mL; And / or, the concentration of surfactant in the diluent is 0.001-0.006 g / mL.

5. A method for detecting human chorionic gonadotropin, characterized by, The method comprises: Mixing the human chorionic gonadotropin detection antibody labeled with a ruthenium complex, human chorionic gonadotropin capture antibody coated magnetic microspheres, diluent and detection sample defined in the kit of any one of claims 1-4 in a reaction; and sucking the obtained reaction solution into an electrochemical reaction cell to perform electrochemiluminescence reaction.

6. The method of claim 5, wherein, The mass ratio of the human chorionic gonadotropin capture antibody coated magnetic microspheres to the human chorionic gonadotropin detection antibody labeled with a ruthenium complex is (25-900):

1.

7. The method of claim 5, wherein, The preparation method of the human chorionic gonadotropin detection antibody labeled with a ruthenium complex comprises coupling a ruthenium complex and a human chorionic gonadotropin detection antibody to obtain a human chorionic gonadotropin detection antibody labeled with a ruthenium complex.

8. The method of claim 7, wherein, The preparation method further comprises activating the ruthenium complex and then coupling it with the human chorionic gonadotropin detection antibody, wherein the reagents used for the activation include 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride and N-hydroxysuccinimide.

9. The method of claim 8, wherein, The molar ratio of the ruthenium complex, 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride and N-hydroxysuccinimide is 1:(4-15):(10-15); And / or, the activation time is 30-60 min; And / or, the pH value of the activation system is 5.5-6.5; And / or, the molar ratio of the ruthenium complex to the human chorionic gonadotropin detection antibody is (8-12):1; And / or, the pH value of the coupling system is 6-10; And / or, the buffer used for the coupling is selected from at least one of citrate, carbonate and borate; And / or, the coupling time is 2-5 hours.

10. The method according to any one of claims 5-9, characterized in that, The method further comprises diluting the ruthenium complex labeled human chorionic gonadotropin detection antibody to a concentration of 0.5-3 μg / mL with the diluent in the kit of any one of claims 1-4 before use. And / or, the method further comprises diluting the human chorionic gonadotropin capture antibody coated magnetic microspheres to a concentration of 0.1-0.5 mg / mL with the diluent in the kit of any one of claims 1-4 before use.

Images