Method for improving chemiluminescence immunoassay sensitivity of magnetic particles, detection kit and application of detection kit

Abstract

The invention relates to a method for improving chemiluminescence immunoassay sensitivity of magnetic particles, a detection kit and application thereof, firstly, magnetic beads are coupled with an antibody, then magnetic beads are sealed to obtain a magnetic bead coated antibody, sealing comprises pre-sealing and second-step sealing, glycine and ethanolamine are added in the pre-sealing process, and in the second-step sealing process, glycine and ethanolamine are added in the second-step sealing process. Casein hydrolysate and dodecyl dimethyl amine ethyl lactone are added; then coupling the antibody with alkaline phosphatase or streptavidin to obtain an alkaline phosphatase labeled antibody or a streptavidin labeled antibody; the magnetic bead coated antibody, the alkaline phosphatase labeled antibody or the streptavidin labeled antibody is used for sample detection. The method is high in sensitivity and wide in detection range, is adaptive to a common immunoassay instrument, does not need expensive equipment, and is also simple to operate; in addition, the AMH concentration with extremely low concentration can be accurately detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of hormone detection, and specifically relates to a method for improving the sensitivity of magnetic particle chemiluminescence immunoassay, a detection kit and an application thereof. Background Art

[0002] Anti-Müllerian hormone (AMH), also known as anti-Mullerian hormone (ATH), is a member of the transforming factor β superfamily. All members of this family are dimeric glycoproteins that play diverse regulatory roles in tissue growth and cell differentiation. During embryonic development, AMH regulates the differentiation and development of the reproductive tract and is crucial for sex differentiation. After birth, AMH regulates the function of Leydig cells in male testes. In adult women, AMH inhibits the recruitment of primordial follicles and the development of antral follicles, preventing premature follicular exhaustion. AMH concentrations increase with the number of small antral follicles in the ovary. Conversely, as follicles are depleted with age and various other factors, AMH concentrations decrease. Approaching menopause, AMH levels gradually approach zero (below the detection limit of 10 pg / mL for current assays), making it a useful marker for predicting ovarian reserve. AMH has been used to predict ovarian hyperstimulation after in vitro fertilization, the timing of menopause (improving assay sensitivity has significantly reduced the timeframe for predicting final menopause from 5 years to 12 months), and assess ovarian reserve.

[0003] Immunodiagnostic reagents are one of the main types of in vitro diagnostic reagents. They utilize the specific binding reaction between antigens and antibodies to perform qualitative or quantitative diagnosis. These reagents are currently experiencing the fastest growth among all diagnostic reagent products, both in terms of technology and market share. Currently, commonly used methods for detecting anti-Müllerian hormone include enzyme-linked immunosorbent assay (ELISA), latex-enhanced immunoturbidimetry, and magnetic microparticle chemiluminescence immunoassay.

[0004] Enzyme-linked immunosorbent assay (ELISA) lacks sensitivity, has a narrow detection range, and is subject to numerous influencing factors, making it prone to false negatives and false positives. Latex-enhanced immunoturbidimetry is simple and rapid, but suffers from low sensitivity and poor reproducibility of low-value results. Magnetic microparticle chemiluminescence immunoassay is the current mainstream method for detecting immune markers. This method uses superparamagnetic microparticles as a solid-phase separation carrier, allowing bound markers to adsorb onto the microsphere surface, while free markers are distributed in the liquid phase. During detection, the solid-phase carrier forms a sandwich structure with the antigen and the detection antibody. Free markers on the magnetic microparticle surface are removed by washing, and then a substrate is added to detect the generated signal, thereby establishing a linear relationship between antigen concentration and signal. Commonly used detection kits include Beckman's Gen II AMH ELISA kit and the Roche fully automated electrochemiluminescence Elecsys AMH kit, all of which have detection limits above 10 pg / mL. This makes it difficult to accurately measure AMH concentrations in a large number of perimenopausal women and patients with premature ovarian insufficiency (POI).

[0005] Therefore, there is an urgent need for a method that is easy to use, has a wide detection range, and is highly sensitive for the detection of daily samples. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention provides a method for improving the sensitivity of magnetic microparticle chemiluminescent immunoassays, a detection kit, and its application. The present invention provides an optimized blocking solution and blocking method for blocking magnetic beads after they are conjugated to antibodies, which effectively reduces the background value of the detection reaction. Furthermore, the present invention provides a labeling method for detecting antibody markers, which effectively increases the signal value of the detection reaction. Combining these two innovations significantly improves the sensitivity of the detection method. Applying these two improvements to the detection of anti-Müllerian hormone (AMH) resulted in the development of a detection kit with a detection limit far lower than existing kits (by two orders of magnitude), capable of accurately detecting extremely low concentrations of AMH.

[0007] To achieve the above objectives, the present invention provides the following technical solutions: A method for improving the sensitivity of magnetic particle chemiluminescence immunoassay, comprising the following steps: (1) First, the magnetic beads are coupled to the antibody and then blocked to obtain the magnetic bead-coated antibody. The blocking includes pre-blocking and second-step blocking. Glycine and ethanolamine are added during the pre-blocking process. During the second-step blocking process, hydrolyzed casein and dodecyldimethylaminoethyl lactone are added. (2) The antibody is then coupled with alkaline phosphatase or streptavidin to obtain an alkaline phosphatase-labeled antibody or a streptavidin-labeled antibody; (3) Magnetic bead-coated antibodies, alkaline phosphatase (ALP)-labeled antibodies, or streptavidin (SA)-labeled antibodies are used for sample detection.

[0008] Preferably, step (1) specifically includes: ① Magnetic bead pretreatment: Place Tosyl magnetic beads in a centrifuge tube and place on a magnetic rack until the supernatant is completely transparent, then discard the supernatant; then add magnetic bead pretreatment solution to the above centrifuge tube, vortex thoroughly to mix, and place on a magnetic rack until the supernatant is completely transparent, then discard the supernatant. Repeat this step once more; ② Magnetic bead coating: Add carbonate buffer, ammonium sulfate, and Tween 20 coating buffer to the magnetic beads treated in step ①, resuspend the magnetic beads to the target volume, add the antibody according to the antibody:magnetic bead ratio and mix thoroughly, and incubate at 30-40°C using a rotary mixer; ③ Magnetic bead washing: After the reaction is completed, place the centrifuge tube on a magnetic rack until the supernatant is completely transparent, discard the supernatant, and add washing buffer to the magnetic beads to wash multiple times; ④ Magnetic bead blocking: Add a pre-blocking solution containing PBS, glycine, ethanolamine, Tween 20, and Proclin 300 and block at room temperature. After the pre-blocking is completed, discard the supernatant and proceed to the second blocking step by adding a second blocking solution containing PBS, BSA, hydrolyzed casein, dodecyldimethylamine betaine, Tween 20, and Proclin 300 and block at room temperature. ⑤ Preparation of working solution: After sealing, place the centrifuge tube on a magnetic rack until the supernatant is completely transparent, discard the supernatant, wash the magnetic beads several times with storage buffer, and dilute for later use.

[0009] Preferably, in step ①, the composition of the magnetic bead pretreatment solution is: 0.1M carbonate buffer, 0.05% Tween20 (v / v), pH 9.5; in step ②, the volume ratio of carbonate buffer to ammonium sulfate is 1:1, and the antibody and magnetic bead feeding ratio is 7~30μg / mg; in step ③, the composition of the washing buffer is: 50mM PBS, 0.05% Tween20 (v / v), 0.05% Proclin 300 (v / v); in step ④, the composition of the pre-blocking solution is: 0.5mL containing 50mM PBS, 50mM glycine, 2M ethanolamine, 0.05% Tween20 (v / v), 0.05% Proclin 300 (v / v); the composition of the re-blocking solution is: 0.5mL containing 50mM PBS, 1% BSA, 1% hydrolyzed casein, 0.05% dodecyldimethylamine betaine, 0.05% Tween 20 (v / v), 0.05% Proclin 300 (v / v); in step ⑤, the composition of the storage buffer is: 50 mM Tris, 0.1% BSA, 150 mM NaCl, 0.05% Tween 20 (v / v), 0.05% Proclin 300 (v / v), pH 7.4.

[0010] Preferably, step (2) specifically includes: (i) Antibody activation 2-Iminothiolane is added to a triethanolamine buffer containing an antibody and reacted at room temperature; after the reaction is completed, glycine is added to the reaction solution and reacted at room temperature to terminate the antibody activation; the activated antibody is desalted for later use; (ii) Activation with alkaline phosphatase or streptavidin Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate is added to a triethanolamine buffer containing alkaline phosphatase or streptavidin and reacted at room temperature; after the reaction is completed, glycine is added to the reaction solution and reacted at room temperature to terminate the activation of alkaline phosphatase or streptavidin; the activated alkaline phosphatase or streptavidin is desalted for later use; (iii) Antibodies conjugated to alkaline phosphatase or streptavidin Mix the activated antibody with alkaline phosphatase or streptavidin, add MgCl2 to the reaction system, and react at room temperature; (iv) Reaction termination After the reaction is completed, maleimide is added to the reaction solution and reacted at room temperature to terminate the coupling reaction; the reaction product is desalted and set aside.

[0011] Preferably, step (3) is: incubating the magnetic bead-coated antibody, the sample to be tested and the alkaline phosphatase-labeled antibody at 37° C. for 10 min, and adding the substrate for signal detection after washing.

[0012] Preferably, step (3) is: incubating the magnetic bead-coated antibody, the sample to be tested and the streptavidin-labeled antibody at 37°C for 10 minutes, washing after the incubation, removing the supernatant, adding 100 μL of biotin-modified alkaline phosphatase (biotinylated alkaline phosphatase) and incubating for 5 minutes, and adding the substrate after washing for signal detection.

[0013] Preferably, in step (iii), the molar ratio of the activated antibody to alkaline phosphatase or streptavidin is 1:1 to 3. More preferably, the molar ratio of the activated antibody to alkaline phosphatase or streptavidin is 1:2.

[0014] The present invention also provides a detection kit for improving the sensitivity of magnetic microparticle chemiluminescence immunoassay, wherein Tosyl magnetic beads coated with anti-AMH mouse monoclonal antibody and SA-modified anti-AMH mouse monoclonal antibody are prepared by the above method, and the detection kit comprises the following components: Reagent R1: Tosyl magnetic beads coated with anti-AMH mouse monoclonal antibody, buffer composition: MES buffer, BSA, ProClin300; Reagent R2: SA-modified anti-AMH mouse monoclonal antibody, buffer composition: Tris buffer, BSA, ProClin300; Reagent R3: biotin-modified alkaline phosphatase, buffer composition: Tris buffer, BSA, ProClin300; Calibrator: recombinant AMH antigen diluted in buffer; Quality control: recombinant AMH antigen diluted in buffer; Luminescent substrates: tris(hydroxymethyl)aminomethane, NaCl, ProClin300, Tween 20, lucigenin, APS-5, sodium sulfite.

[0015] A non-diagnostic application of the detection kit, wherein the kit is used for detecting anti-Mullerian hormone.

[0016] Preferably, the detection limit of anti-Mullerian hormone is 0.085 pg / mL.

[0017] The beneficial effects of the present invention are: 1. The present invention can effectively reduce the background value of the detection reaction by optimizing the blocking solution composition and blocking method; 2. The present invention can effectively increase the signal value of the detection reaction by coupling the antibody with alkaline phosphatase or streptavidin; 3. The present invention can accurately detect extremely low concentrations of AMH; 4. The present invention not only has high sensitivity and a wide detection range, but is also compatible with common immunoassay analyzers, does not require expensive equipment, and is simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The standard curve was obtained by four-parameter nonlinear fitting. DETAILED DESCRIPTION

[0019] In order to better understand the above technical solution, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] The experimental methods described in the examples are conventional methods unless otherwise specified; the percentages involved are by mass unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified, such as alkaline phosphatase purchased from BBI, streptavidin purchased from Thermo, and biotinylated alkaline phosphatase purchased from Thermo.

[0021] Example 1 Magnetic beads coated with antibodies (preparation of Tosyl magnetic beads coated with anti-AMH mouse monoclonal antibodies): ① Magnetic bead pretreatment: Place 5mg of Tosyl magnetic beads in a 2mL centrifuge tube and place on a magnetic stand until the supernatant is completely transparent. Carefully remove the supernatant with a pipette. Next, add 1mL of magnetic bead pretreatment solution (0.1M carbonate buffer, 0.05% Tween 20 (v / v), pH 9.5) to the tube. Vortex thoroughly to mix, then place on a magnetic stand until the supernatant is completely transparent. Carefully remove the supernatant with a pipette. Repeat this step twice.

[0022] ② Magnetic bead coating: Add coating buffer containing 0.1M carbonate buffer (pH 9.5), 3M ammonium sulfate, and 0.05% Tween 20 (v / v) (the volume ratio of 0.1M carbonate buffer to 3M ammonium sulfate is 1:1) to the magnetic beads treated above, resuspend the magnetic beads to the target volume, add the antibody according to the antibody: magnetic bead feed ratio of 10μg / mg and mix thoroughly, and incubate at 37℃ using a rotary mixer for 18h.

[0023] ③ Magnetic bead washing: After the reaction is completed, place the centrifuge tube on a magnetic rack until the supernatant is completely transparent, then carefully discard the supernatant with a pipette, add 1 mL of washing buffer (50 mM PBS (pH 7.5), 0.05% Tween 20 (v / v), 0.05% Proclin 300 (v / v)) to the magnetic beads and wash three times; ④ Magnetic bead blocking: Add 0.5 mL of pre-blocking solution containing 50 mM PBS (pH 7.5), 50 mM glycine, 2 M ethanolamine, 0.05% Tween 20 (v / v), and 0.05% Proclin 300 (v / v) for blocking at room temperature for 30 min; discard the supernatant and add 0.5 mL of re-blocking solution containing 50 mM PBS (pH 7.5), 1% BSA, 1% hydrolyzed casein, 0.05% dodecyldimethylamine betaine, 0.05% Tween 20 (v / v), and 0.05% Proclin 300 (v / v) for blocking at room temperature for 3 h.

[0024] ⑤ Preparation of working solution: After blocking, place the centrifuge tube on a magnetic rack until the supernatant is completely transparent, then carefully discard the supernatant with a pipette, wash the magnetic beads three times with storage buffer (50mM Tris, 0.1% BSA, 150mM NaCl, 0.05% Tween 20 (v / v), 0.05% Proclin 300 (v / v), pH 7.4), and dilute to 10mg / mL for use.

[0025] (2) Alkaline phosphatase (ALP) labeled antibody (i) Antibody activation 1.5% 2-iminothiolane was added to the triethanolamine (1.5%) buffer containing the antibody and reacted at room temperature for 10 minutes. After the reaction, 1M glycine was added to the reaction solution and reacted at room temperature for 5 minutes to terminate the antibody activation. The activated antibody was desalted and set aside.

[0026] (ii) Alkaline phosphatase (ALP) activation 0.7% sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) was added to triethanolamine (0.5%) buffer containing ALP and reacted at room temperature for 10 minutes. After the reaction, 1M glycine was added to the reaction solution and reacted at room temperature for 5 minutes to terminate SA activation. The activated ALP was desalted and set aside.

[0027] (iii) Antibody conjugated to ALP The activated antibody and ALP were mixed at a molar ratio of 1:2, and 1 M MgCl2 (final concentration 2 mM) was added to the reaction system. The mixture was reacted at room temperature for 4 h.

[0028] (iv) Reaction termination After the reaction was completed, 0.1% maleimide was added to the reaction solution and reacted at room temperature for 10 min to terminate the coupling reaction; the reaction product was desalted and set aside.

[0029] (3) Preparation of standard substances: Dilute the AMH standard to 100, 25, 5, 1, 0.1, and 0.01 ng / mL with sample diluent; the sample diluent is Tris buffer (pH 7.5) containing 6% BSA.

[0030] (4) Reaction system and steps A one-step method was used for sample detection: first, the magnetic bead-coated antibody (0.1 mg / mL, 30 μL), the test sample (20 μL), and the ALP-labeled antibody (0.5 μg / mL, 50 μL) were incubated together at 37°C for 10 min. After washing, the substrate was added for signal detection.

[0031] Example 2 Compared with Example 1, the difference is that the antibody is coupled with streptavidin (SA) to obtain an SA-modified anti-AMH mouse monoclonal antibody. The SA activation method is the same as ALP activation, and the antibody-SA coupling molar ratio is 1:2. The reaction system and steps are a two-step method: the magnetic bead-coated antibody (0.1 mg / mL, 30 μL), the test sample (20 μL), and the SA-labeled antibody (0.5 μg / mL, 50 μL) are incubated together at 37°C for 10 minutes. After the incubation, the mixture is washed, the supernatant is removed, and 100 μL of biotin-modified alkaline phosphatase (150 pM, commercially available) is added and incubated for 5 minutes. After washing, the substrate is added for signal detection.

[0032] Comparative Example 1 Compared with Example 1, the difference is that the pre-blocking solution does not contain glycine.

[0033] Comparative Example 2 Compared with Example 1, the difference is that the pre-blocking solution does not contain ethanolamine.

[0034] Comparative Example 3 Compared with Example 1, the difference is that the resealing solution does not contain hydrolyzed casein.

[0035] Comparative Example 4 Compared with Example 1, the difference is that the resealing liquid does not contain dodecyldimethylamine betaine.

[0036] Comparative Example 5 Compared with Example 1, the difference is that no pre-sealing process is involved.

[0037] Comparative Example 6 Compared with Example 1, the difference is that there is no pre-sealing process, and the re-sealing solution does not contain the two components of hydrolyzed casein and sodium laurylaminopropionate.

[0038] Comparative Example 7 Compared with Example 2, the difference is that the molar ratio of the activated antibody to the activated SA is 1:1.

[0039] Comparative Example 8 Compared with Example 2, the difference is that the molar ratio of the activated antibody to the activated SA is 1:3.

[0040] Test results 1. The influence of magnetic bead blocking solution composition and blocking method on the detection background signal value The main difference between Example 1 and Comparative Examples 1 to 6 lies in the composition of the blocking solution and the blocking method. The signal value results of the sample dilution detection using magnetic beads treated by different methods are shown in Table 1: Table 1 Sealing treatment and performance differences By comparing different blocking solutions and blocking methods, it can be seen that pre-blocking of the coated magnetic beads can effectively reduce the background value of the detection system; in addition, hydrolyzed casein in the blocking solution, as a small molecule blocking agent, can more fully block the magnetic beads; sodium dodecylaminopropionate is an amphoteric surfactant that disperses the magnetic beads in the blocking solution, preventing them from agglomerating and promoting more complete blocking.

[0041] 2. Effect of Biotin-Streptavidin Amplification System Streptavidin is a tetrameric protein consisting of four subunits, each with a biotin binding site. One molecule of streptavidin can bind to four molecules of biotin with high specificity, thereby amplifying the signal. The results of the comparison of antibody-labeled SA signal amplification are shown in Table 2: Table 2 Comparison of signal amplification effects of biotin-streptavidin amplification system As can be seen from Table 2, compared with Example 1, the detection antibody in Example 2 was coupled with SA. Although one step was added to the reaction steps, the reaction signal-to-noise ratio and linear range were nearly doubled compared with the conventional antibody-ALP coupling method.

[0042] 3. Effect of antibody-SA coupling ratio on detection sensitivity The present invention optimizes the coupling ratio of antibody to SA, and the results are shown in Table 3: Table 3 Effect of antibody to SA conjugation ratio on detection sensitivity As can be seen in Table 3, the sensitivity and linearity of the reagent did not increase with increasing antibody-to-SA conjugation ratios. Possible reasons for this are: an activated antibody has a limited number of active sites, and the amount of SA that can be conjugated is also limited; on the other hand, as the number of SA conjugated to the antibody increases, the steric hindrance increases, preventing subsequent SA from attaching to the antibody. In the present invention, when the antibody-to-SA conjugation ratio is 1:2, the detection effect is better.

[0043] 4. Detection limit detection A test kit for detecting AMH was prepared using the method of Example 2 of the present invention. The test kit comprises: Reagent R1: Tosyl magnetic beads coated with anti-AMH mouse monoclonal antibody at a concentration of 0.1 mg / mL in a buffer consisting of 0.05 M MES buffer, 3% BSA, and 0.5% ProClin 300 (v / v). Reagent R2: SA-modified anti-AMH mouse monoclonal antibody, concentration 0.5 μg / mL, buffer composition 0.05 M Tris buffer, 3% BSA, 0.5% ProClin 300 (v / v); Reagent R3: biotin-modified alkaline phosphatase, concentration 150 pM, buffer composition 0.05 M Tris buffer, 3% BSA, 0.5% ProClin300 (v / v); Calibrator: recombinant AMH antigen diluted in buffer at concentrations of 0, 0.01, 0.1, 1, 5, 25, and 100 ng / mL; Quality control: recombinant AMH antigen diluted in buffer; Luminescent substrates: tris(hydroxymethyl)aminomethane, NaCl, ProClin300, Tween 20, lucigenin, APS-5, sodium sulfite.

[0044] The test results were fitted with four-parameter nonlinear fitting to obtain the standard curve, such as Figure 1 As shown: Y = (359.25839112 -1845415856.95) / (1 + (x / 2109.41911859) 1.00344788 ) + 1845415856.95.

[0045] Repeat the test 20 times for a sample that does not contain any analyte, and calculate the average concentration of the 20 test results. and standard deviation (SD). The limit of blank (LoB) is defined as +2SD, and the result is 0.00001733 ng / mL. Five low-concentration samples ranging from 1 LoB to 5 LoB were measured four times for each sample over three consecutive days. Analysis of the 60 data points revealed a limit of detection (LOD) of 0.085 pg / mL for the present invention, which is 118 times the detection limit of commonly used test kits (Roche's LOD is 10 pg / mL).

[0046] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for improving the sensitivity of magnetic microparticle chemiluminescence immunoassay for non-diagnostic purposes, characterized in that The steps include: (1) After the magnetic beads are coupled to the antibody, the magnetic beads are blocked to obtain the magnetic bead-coated antibody. The blocking includes pre-blocking and second-step blocking. Glycine and ethanolamine are added during the pre-blocking process. During the second-step blocking process, hydrolyzed casein and dodecyldimethylaminoethyl lactone are added. (2) coupling the antibody with alkaline phosphatase or streptavidin to obtain an alkaline phosphatase-labeled antibody or a streptavidin-labeled antibody; (3) Magnetic bead-coated antibodies, alkaline phosphatase-labeled antibodies, or streptavidin-labeled antibodies are used for sample detection.

2. The method for improving the sensitivity of magnetic microparticle chemiluminescence immunoassay according to claim 1, characterized in that Step (1) specifically includes: ① Magnetic bead pretreatment: Place Tosyl magnetic beads in a centrifuge tube and place on a magnetic rack until the supernatant is completely transparent, then discard the supernatant; then add magnetic bead pretreatment solution to the above centrifuge tube, vortex thoroughly to mix, and place on a magnetic rack until the supernatant is completely transparent, then discard the supernatant. Repeat this step once more; ② Magnetic bead coating: Add carbonate buffer, ammonium sulfate, and Tween 20 coating buffer to the magnetic beads treated in step ①, resuspend the magnetic beads to the target volume, add the antibody according to the antibody:magnetic bead ratio and mix thoroughly, and incubate at 30-40°C using a rotary mixer; ③ Magnetic bead washing: After the reaction is completed, place the centrifuge tube on a magnetic rack until the supernatant is completely transparent, discard the supernatant, and add washing buffer to the magnetic beads to wash multiple times; ④ Magnetic bead blocking: Add a pre-blocking solution containing PBS, glycine, ethanolamine, Tween 20, and Proclin 300 and block at room temperature. After the pre-blocking is completed, discard the supernatant and proceed to the second blocking step by adding a second blocking solution containing PBS, BSA, hydrolyzed casein, dodecyldimethylamine betaine, Tween 20, and Proclin 300 and block at room temperature. ⑤ Preparation of working solution: After sealing, place the centrifuge tube on a magnetic rack until the supernatant is completely transparent, discard the supernatant, wash the magnetic beads several times with storage buffer, and dilute for later use.

3. The method for improving the sensitivity of magnetic microparticle chemiluminescence immunoassay according to claim 2, characterized in that In step ①, the composition of the magnetic bead pretreatment solution is: 0.1M carbonate buffer, 0.05% Tween20, pH 9.5; in step ②, the volume ratio of carbonate buffer to ammonium sulfate is 1:1, and the antibody and magnetic bead feeding ratio is 7-30μg / mg; in step ③, the composition of the washing buffer is: 50mM PBS, 0.05% Tween20, 0.05% Proclin 300; in step ④, the composition of the pre-blocking solution is: 0.5mL containing 50mM PBS, 50mM glycine, 2M ethanolamine, 0.05% Tween20, 0.05% Proclin 300; the composition of the re-blocking solution is: 0.5mL containing 50mM PBS, 1% BSA, 1% hydrolyzed casein, 0.05% dodecyldimethylamine betaine, 0.05% Tween20, 0.05% Proclin 300; in step ⑤, the composition of the storage buffer is: 50mM Tris, 0.1% BSA, 150mM NaCl, 0.05% Tween 20, 0.05% Proclin 300, pH 7.

4.

4. The method for improving the sensitivity of magnetic particle chemiluminescence immunoassay according to claim 1, characterized in that Step (2) specifically includes: (i) Antibody activation 2-Iminothiolane is added to a triethanolamine buffer containing an antibody and reacted at room temperature; after the reaction is completed, glycine is added to the reaction solution and reacted at room temperature to terminate the antibody activation; the activated antibody is desalted for later use; (ii) Activation with alkaline phosphatase or streptavidin Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate is added to a triethanolamine buffer containing alkaline phosphatase or streptavidin and reacted at room temperature; after the reaction is completed, glycine is added to the reaction solution and reacted at room temperature to terminate the activation of alkaline phosphatase or streptavidin; the activated alkaline phosphatase or streptavidin is desalted for later use; (iii) Antibodies conjugated to alkaline phosphatase or streptavidin Mix the activated antibody with alkaline phosphatase or streptavidin, add MgCl2 to the reaction system, and react at room temperature; (iv) Reaction termination After the reaction is completed, maleimide is added to the reaction solution and reacted at room temperature to terminate the coupling reaction; the reaction product is desalted and set aside.

5. The method for improving the sensitivity of magnetic particle chemiluminescence immunoassay according to claim 4, characterized in that Step (3) is: incubate the magnetic beads coated with antibodies, the sample to be tested and the alkaline phosphatase labeled antibody at 37°C for 10 minutes, and then add the substrate for signal detection after washing.

6. The method for improving the sensitivity of magnetic particle chemiluminescence immunoassay according to claim 4, characterized in that Step (3) is: incubate the magnetic bead-coated antibody, the sample to be tested and the streptavidin-labeled antibody at 37°C for 10 minutes, wash after the incubation, remove the supernatant, add 100 μL of biotin-modified alkaline phosphatase and incubate for 5 minutes, and add the substrate after washing for signal detection.

7. The method for improving the sensitivity of magnetic particle chemiluminescence immunoassay according to claim 4, characterized in that In step (iii), the molar ratio of the activated antibody to alkaline phosphatase or streptavidin is 1:1-3.

8. A detection kit for improving the sensitivity of magnetic microparticle chemiluminescence immunoassay, characterized in that: Tosyl magnetic beads coated with anti-AMH mouse monoclonal antibodies and streptavidin-modified anti-AMH mouse monoclonal antibodies are prepared by any of the methods of claims 1-7, and the detection kit comprises the following components: Reagent R1: Tosyl magnetic beads coated with anti-AMH mouse monoclonal antibody, buffer composition: MES buffer, BSA, ProClin300; Reagent R2: streptavidin-modified anti-AMH mouse monoclonal antibody, buffer composition: Tris buffer, BSA, ProClin300; Reagent R3: biotin-modified alkaline phosphatase, buffer composition: Tris buffer, BSA, ProClin300; Calibrator: recombinant AMH antigen diluted in buffer; Quality control: recombinant AMH antigen diluted in buffer; Luminescent substrates: tris(hydroxymethyl)aminomethane, NaCl, ProClin300, Tween 20, lucigenin, APS-5, sodium sulfite.

9. A use of the detection kit according to claim 8 for non-diagnostic purposes, characterized in that: Used for the detection of anti-Mullerian hormone.

10. The use according to claim 10, characterized in that: The detection limit of anti-Mullerian hormone was 0.085 pg / mL.

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