A kit for detecting procalcitonin by magnetic microparticle chemiluminescence and a method for detecting procalcitonin

Abstract

This application discloses a chemiluminescence detection kit for acrylonitrile magnetic microparticles and its detection method. The kit includes a magnetic bead working solution and a luminescent label. The magnetic bead working solution includes acrylonitrile monoclonal antibody A and streptavidin. The luminescent label includes acrylonitrile monoclonal antibody B, modified nano-silica, anhydrous toluene, methoxy polyethylene glycol-succinimide ester, N,N-dimethylformamide, acrylonitrile succinimide ester, glutaraldehyde, and ethanolamine. This application uses modified nano-silica as a carrier and grafts a polyethylene glycol hydrophilic layer onto its surface, which amplifies the acrylonitrile ester molecule signal, reduces non-specific adsorption, and improves the sensitivity, specificity, and reliability of the detection.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a peptidyl magnetic microparticle chemiluminescence detection kit and its detection method. Background Technology

[0002] Hypoteptin is a fragment of arginine angiotensinogen, primarily secreted by the hypothalamus and stored in the pituitary gland. During stress, hypopoteptin activates the hypothalamic-pituitary-adrenal axis, promoting the secretion of arginine angiotensin. When the body experiences life-threatening stress or arterial insufficiency, hypopoteptin is released from the pituitary gland and immediately enters the bloodstream. More importantly, hypopoteptin begins to rise in the early stages of myocardial infarction, earlier than other biomarkers such as CK-MB or high-sensitivity troponin T. Therefore, accurate and sensitive detection of hypopoteptin concentration in the blood is crucial for the early diagnosis, severity assessment, treatment monitoring, and prognosis of myocardial infarction.

[0003] Currently, detection methods for leptin include enzyme-linked immunosorbent assay (ELISA) and immunochromatography. These methods are based on the principle of specific binding between antigens and antibodies, using labeled signal molecules (such as enzymes, luciferins, or chemiluminescent substances) for qualitative and quantitative analysis of leptin. However, because the concentration of leptin in samples from myocardial infarction patients may be extremely low, the detection limits of ELISA and chemiluminescent immunoassay may be insufficient, leading to an increased risk of early missed diagnoses.

[0004] Magnetic microparticle chemiluminescence immunoassay is a detection method developed in the last two or three decades. It uses streptavidin magnetic microparticles as the trap, allowing biotin-conjugated antibodies to fully bind to the streptavidin magnetic microparticles and form a magnetic bead suspension. Acridinium ester or acridinium ester derivatives are used to label the antibody as a tracer, and the luminescence intensity of the reaction system is measured using a chemiluminescence analyzer to calculate the concentration of the analyte. However, the steric hindrance caused by the antigen or antibody on the surface of the magnetic beads coated with the analyte-capturing antibody results in the sensitivity of the detection method remaining unsatisfactory. Summary of the Invention

[0005] To improve the sensitivity of peptide detection, this application provides a peptide magnetic microparticle chemiluminescence detection kit and its detection method.

[0006] In a first aspect, this application provides a chemiluminescence detection kit for peptidyl magnetic microparticles, which adopts the following technical solution:

[0007] A chemiluminescence detection kit for peptidyl magnetic microparticles includes a magnetic bead working solution and a luminescent label. The magnetic bead working solution includes peptidyl monoclonal antibody A and streptavidin. The luminescent label includes peptidyl monoclonal antibody B, modified nano-silica, anhydrous toluene, methoxy polyethylene glycol-succinimide ester, N,N-dimethylformamide, acridine succinimide ester, glutaraldehyde, and ethanolamine.

[0008] By employing the above technical solution, this application utilizes modified nano-silica as a carrier to achieve high-capacity loading of acrid ester molecules, thereby generating a signal amplification effect and improving detection sensitivity. Methoxylated polyethylene glycol-succinimide ester is grafted onto the nanoparticle surface to form a hydrophilic protective layer. On the one hand, this layer prevents nanoparticle aggregation through steric stabilization, ensuring reaction uniformity; on the other hand, its flexible polyethylene glycol long chains reduce steric hindrance in the immunoreaction, ensuring efficient binding of the peptide antibody to the antigen. Furthermore, this hydrophilic layer, by forming a hydrated shell, significantly reduces non-specific adsorption, thus achieving a high signal-to-noise ratio. Glutaraldehyde, as a heterogeneous bifunctional crosslinking agent, enables stable covalent bonding between the antibody and the nanoparticles. The synergistic effect of these components significantly improves immunobinding efficiency, enhancing detection sensitivity and reliability.

[0009] Optionally, the preparation steps of the modified nano silica include: soaking nano silica in anhydrous toluene, ultrasonically dispersing it, adding 5% by weight of γ-aminopropyltriethoxysilane to the nano silica, heating under reflux under inert gas protection, washing with ethanol, and drying to obtain modified nano silica.

[0010] By employing the above-described technical solution, γ-aminopropyltriethoxysilane undergoes a dehydration condensation reaction with the silanol groups on the surface of nano-silica, thereby firmly introducing the terminal amino groups of the γ-aminopropyltriethoxysilane molecule into the nano-silica surface through strong Si-O-Si covalent bonds. Simultaneously, the introduction of surface amino groups provides the necessary reaction sites for subsequent covalent coupling of acridine ester molecules and antibodies via amidation reactions. Furthermore, the uniform and dense γ-aminopropyltriethoxysilane monolayer formed under these process conditions effectively ensures the uniformity and reproducibility of subsequent coupling reactions, laying the foundation for constructing high-performance nanolabels.

[0011] Optionally, the particle size of the modified nano-silica is 30-60 nm.

[0012] By adopting the above technical solution, this application uses 30-60nm modified nano-silica, which ensures that the nanoparticles have sufficient specific surface area to covalently load a sufficient number of acrid ester molecules, thereby achieving effective signal amplification and ensuring high sensitivity, while minimizing steric hindrance and improving immune binding efficiency, thus jointly contributing to the realization of high sensitivity.

[0013] Optionally, the preparation steps of the luminescent label include: mixing modified nano-silica and anhydrous toluene, ultrasonically dispersing, adding methoxy polyethylene glycol-succinimide ester for a light-protected reaction, centrifuging to remove the supernatant after the reaction, obtaining polyethylene glycol-modified nanoparticles; mixing the polyethylene glycol-modified nanoparticles with N,N-dimethylformamide, adding acridine succinimide ester for a light-protected reaction, centrifuging to remove the supernatant after the reaction, obtaining acridine ester-modified nanoparticles; mixing the acridine ester-modified nanoparticles with PBS buffer, adding glutaraldehyde for activation, then adding peptide monoclonal antibody B for reaction, adding ethanolamine for blocking reaction, separating and purifying through a dialysis bag to obtain the luminescent label.

[0014] By adopting the above technical solution, this application forms a high-performance composite nanolabel by constructing a polyethylene glycol hydrophilic layer, a high-density acridine ester signaling unit, and a terminal specific antibody. This enables a single nanoparticle to load multiple acridine ester molecules, generating a strong chemiluminescence signal, improving sensitivity, reducing non-specific adsorption, and enhancing the signal-to-noise ratio.

[0015] Optionally, the device may also include a luminescent labeling buffer, which comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin, Proclin 300 antibacterial agent, and polysorbate-20.

[0016] By employing the above technical solutions, the phosphate buffer and sodium chloride work together to maintain a stable physiological pH and osmotic pressure, ensuring the activity and integrity of the labeled substances. Bovine serum albumin and polysorbate-20 work synergistically to occupy potential binding sites on the surface of the labeled substances and the container walls, and to weaken hydrophobic interactions, effectively inhibiting non-specific adsorption and significantly reducing background noise. Proclin 300 antibacterial agent ensures the microbiological safety of the protein-rich buffer during long-term storage. The combined effect of these buffer systems synergistically ensures the high sensitivity and excellent stability of the kit.

[0017] Optionally, it may also include an antigen buffer, which comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin, trehalose, ethylenediaminetetraacetic acid, Proclin 300 antibacterial agent, and polysorbate-20.

[0018] By employing the above technical solutions, trehalose stabilizes the antigen structure through hydration; ethylenediaminetetraacetic acid (EDTA) inhibits enzymatic reactions by chelating metal ions; phosphate and sodium chloride maintain suitable pH and osmotic pressure; bovine serum albumin and polysorbate-20 synergistically reduce non-specific adsorption; and Proclin 300 provides antibacterial protection. The synergistic effect of these components ensures the accuracy and stability of the detection results.

[0019] Optionally, the system also includes calibrators and quality control samples. The calibrators include calibrator 1 and calibrator 2, where calibrator 1 is a 10-50 pmol / L peptide antigen solution and calibrator 2 is a 100-300 pmol / L peptide antigen solution. The quality control samples include quality control sample 1 and quality control sample 2, where quality control sample 1 is a 10-50 pmol / L peptide antigen solution and quality control sample 2 is a 100-300 pmol / L peptide antigen solution.

[0020] By adopting the above technical solutions, calibrators provide precise quantitative standards for detection, ensuring the traceability and accuracy of results; while quality control materials guarantee the reliability of results by monitoring the effectiveness of the entire detection process. By setting calibrators and quality control materials in two concentration ranges (high and low), the key clinical scope of peptide detection can be covered, effectively evaluating the sensitivity and precision of the detection system, and providing a reliable basis for the early diagnosis and risk assessment of myocardial infarction.

[0021] Optionally, the concentration of streptavidin in the working solution of the magnetic beads is 0.1-1 mg / mL, and the concentration of peptidin monoclonal antibody B in the luminescent label is 0.01-5 μg / mL.

[0022] By adopting the above technical solution, the concentration of streptavidin in the magnetic bead working solution is limited to 0.1-1 mg / mL, and the concentration of the luminescent label and peptidyl monoclonal antibody B is limited to 0.01-5 μg / mL. This can minimize non-specific adsorption and reagent consumption while ensuring capture efficiency and signal intensity.

[0023] Secondly, this application provides a method for detecting peptides using a peptidyl magnetic microparticle chemiluminescence detection kit, employing the following technical solution:

[0024] A method for detecting peptides using a peptidyl magnetic microparticle chemiluminescence detection kit includes the following steps:

[0025] S1. Take the sample to be tested, add magnetic bead working solution and luminescent label, mix well, incubate to obtain magnetic bead-coated antibody-antigen-luminescent label antibody complex;

[0026] S2. The magnetic beads coated with the antibody-antigen-luminescent labeled antibody complex are washed, and the intensity of the luminescent signal is detected to obtain the concentration of the sample and peptides.

[0027] In summary, this application includes at least one of the following beneficial technical effects:

[0028] 1. This application utilizes modified nano-silica as a carrier to achieve high-capacity loading of acridinium ester molecules, thereby generating a signal amplification effect and improving detection sensitivity. Methoxylated polyethylene glycol-succinimide ester is grafted onto the nanoparticle surface to form a hydrophilic protective layer. On the one hand, this layer prevents nanoparticle aggregation through steric stabilization, ensuring reaction uniformity; on the other hand, its flexible polyethylene glycol long chains reduce steric hindrance in the immunoreaction, ensuring efficient binding of the peptide antibody to the antigen. Furthermore, this hydrophilic layer, by forming a hydrated shell, significantly reduces non-specific adsorption, thus achieving a high signal-to-noise ratio. Glutaraldehyde, as a heterogeneous bifunctional crosslinking agent, enables stable covalent bonding between the antibody and the nanoparticles. The synergistic effect of these components significantly improves immunobinding efficiency, enhancing detection sensitivity and reliability.

[0029] 2. This application utilizes a dehydration condensation reaction between γ-aminopropyltriethoxysilane and the silanol groups on the surface of nano-silica, thereby firmly introducing the amino groups at the ends of the γ-aminopropyltriethoxysilane molecule into the nano-silica surface through strong Si-O-Si covalent bonds. Simultaneously, the introduction of surface amino groups provides the necessary reaction sites for subsequent covalent coupling of acridine ester molecules and antibodies via amidation reactions. Furthermore, the uniform and dense γ-aminopropyltriethoxysilane monolayer formed under these process conditions effectively ensures the uniformity and reproducibility of subsequent coupling reactions, laying the foundation for constructing high-performance nanolabels.

[0030] 3. This application uses 30-60nm modified nano-silica, which ensures that the nanoparticles have sufficient specific surface area to covalently load a sufficient number of acrid ester molecules, thereby achieving effective signal amplification and ensuring high sensitivity, while minimizing steric hindrance and improving immune binding efficiency, thus jointly contributing to the realization of high sensitivity.

[0031] 4. This application constructs a high-performance composite nanolabel by building a polyethylene glycol hydrophilic layer, a high-density acridine ester signaling unit, and a terminal specific antibody. This enables a single nanoparticle to load multiple acridine ester molecules, generating a strong chemiluminescence signal, improving sensitivity, reducing non-specific adsorption, and enhancing the signal-to-noise ratio. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] This application designs a chemiluminescence detection kit for peptidyl magnetic microparticles, comprising a magnetic bead working solution and luminescent markers. The magnetic bead working solution comprises peptidyl monoclonal antibody A and streptavidin, and the luminescent markers comprise peptidyl monoclonal antibody B, modified nano-silica, anhydrous toluene, methoxy polyethylene glycol-succinimide ester, N,N-dimethylformamide, acridine succinimide ester, glutaraldehyde, and ethanolamine.

[0034] A method for detecting peptides using a peptidyl magnetic microparticle chemiluminescence detection kit includes the following steps:

[0035] S1. Take the sample to be tested, add magnetic bead working solution and luminescent label, mix well, and incubate for 5-10 minutes to obtain magnetic bead-coated antibody-antigen-luminescent label antibody complex.

[0036] S2. The magnetic beads coated with the antibody-antigen-luminescent labeled antibody complex are washed, and the intensity of the luminescent signal is detected to obtain the concentration of the neutralizing peptide in the sample to be tested.

[0037] All raw materials used in the embodiments of this application are commercially available, wherein:

[0038] Magnetic microparticles, 1.0 μm in diameter, Thermo Fisher Scientific (China) Co., Ltd.

[0039] Tris-HCl buffer, pH 7.5, Thermo Fisher Scientific (China) Co., Ltd.

[0040] Nano-silica, G201, Guangdong Slika New Materials Co., Ltd.;

[0041] Methoxylated polyethylene glycol-succinimide ester, mPEG-NHS MW350, Guangzhou Carbon Technology Co., Ltd.

[0042] γ-aminopropyltriethoxysilane, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0043] Heptakin monoclonal antibody A, Qingdao Handerson Biotechnology Co., Ltd.;

[0044] Heptakin monoclonal antibody B, Qingdao Handerson Biotechnology Co., Ltd.;

[0045] Heptakin antigen, Nanjing Ainodi Biotechnology Co., Ltd.;

[0046] Bovine serum albumin, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0047] Proclin 300 antibacterial agent, Beijing Wokai Biotechnology Co., Ltd.;

[0048] Polysorbate-20, Beijing Wokai Biotechnology Co., Ltd.;

[0049] Acridinium ester NSP-DMAE-NHS, Hubei Xindesheng Materials Technology Co., Ltd.;

[0050] Glutaraldehyde, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0051] Ethanolamine, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0052] D-Trehalose, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0053] Ethylenediaminetetraacetic acid (EDTA), Shanghai Aladdin Biochemical Technology Co., Ltd.

[0054] PBS buffer, pH 7.4, Shanghai Kanglang Biotechnology Co., Ltd.

[0055] Preparation Example 1

[0056] Preparation of modified nano-silica: Nano-silica was soaked in anhydrous toluene, ultrasonically dispersed at 400W for 20 min, and 5% by weight of γ-aminopropyltriethoxysilane was added. Under nitrogen protection, the mixture was refluxed at 80℃ for 12 h. After the reaction was completed, the nano-silica was washed three times by centrifugation with ethanol and dried at 70℃ for 12 h to obtain modified nano-silica.

[0057] Preparation Example 2

[0058] Preparation of blocking solution: Bovine serum albumin and Tris-HCl buffer were mixed at a volume ratio of 1:20 and stirred until homogeneous to obtain the blocking solution.

[0059] Preparation Example 3

[0060] Preparation of magnetic bead buffer: Add 6.06g of tris(hydroxymethyl)aminomethane and 8.18g of sodium chloride to 100mL of ultrapure water and stir until homogeneous. Add 1.03g of Proclin300 antibacterial agent and 1g of polysorbate-20 and stir until completely dissolved. Adjust the pH to 7.4, then add 10g of bovine serum albumin and stir until completely dissolved. Make up the volume to 1L to obtain magnetic bead buffer.

[0061] Preparation Example 4

[0062] Preparation of luminescent labeling buffer: 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.29 g of sodium dihydrogen phosphate dihydrate, and 8.77 g of sodium chloride were added to 100 mL of ultrapure water and stirred until homogeneous. 1.03 g of Proclin 300 antibacterial agent and 1 g of polysorbate-20 were added and stirred until completely dissolved. The pH was adjusted to 7.4, and then 10 g of bovine serum albumin was added and stirred until completely dissolved. The volume was adjusted to 1 L to obtain the luminescent labeling buffer.

[0063] Preparation Example 5

[0064] Preparation of antigen buffer: 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.29 g of sodium dihydrogen phosphate dihydrate, 8.77 g of sodium chloride, 20 g of D-trehalose, and 3.72 g of EDTA were added to 100 mL of ultrapure water and stirred until homogeneous. 1.03 mL of Proclin 300 antibacterial agent and 1 mL of polysorbate-20 were added and stirred until completely dissolved. The pH was adjusted to 7.4, and then 30 g of bovine serum albumin was added. The mixture was stirred until homogeneous and the volume was brought to 1 L to obtain the antigen buffer.

[0065] Example 1

[0066] Preparation method of the peptide magnetic microparticle chemiluminescence detection kit:

[0067] (1) Preparation of magnetic bead working solution: Take 10 mg of streptavidin microspheres, wash with PBS buffer, remove the supernatant by magnetic separation, add 0.1 mg of peptidyl monoclonal antibody A, and couple at room temperature. Then, add the blocking solution obtained in Preparation Example 2, and block for 1 h. After magnetic separation and washing, resuspend the magnetic microspheres in the magnetic bead buffer obtained in Preparation Example 3, and adjust the concentration of streptavidin in the magnetic bead working solution to 0.2 mg / mL;

[0068] (2) Preparation of luminescent markers: 100 mg of the modified nano-silica obtained in Preparation Example 1 was mixed with 15 mL of anhydrous toluene and ultrasonically dispersed at 300 W for 20 min. 30 mg of methoxy polyethylene glycol-succinimide ester was added, and the mixture was reacted at room temperature in the dark for 8 h. After centrifugation at 1000 r / min for 10 min, the supernatant was removed to obtain polyethylene glycol-modified nanoparticles. The polyethylene glycol-modified nanoparticles were mixed with 15 mL of N,N-dimethylformamide and 10 mg of acridine ester NSP-DMAE-NHS was added. The mixture was stirred at room temperature for 4 h, and centrifuged at 1000 r / min for 10 min to remove the supernatant, thus obtaining acridine ester-modified silica nanoparticles. Acridinium ester-modified silica nanoparticles were mixed with 5 mL of PBS buffer, 40 mg of glutaraldehyde was added, and the mixture was activated at room temperature for 30 min. Then, 1 mg of peptidyl monoclonal antibody B was added, and the mixture was reacted at room temperature for 2 h. 30 mg of ethanolamine was added, and the mixture was blocked at room temperature for 1 h. The mixture was then separated and purified by dialysis bag, and then mixed with the luminescent label buffer obtained in Preparation Example 4 to adjust the concentration of peptidyl monoclonal antibody B in the luminescent label to 0.2 μg / mL.

[0069] (3) Preparation of calibrators: The peptide antigen was diluted with the antigen buffer obtained in Preparation Example 5 to prepare calibrator 1 (10 pmol / L) and calibrator 2 (100 pmol / L).

[0070] (4) Preparation of quality control samples: The peptide antigen was diluted with the antigen buffer obtained in Preparation Example 5 to prepare quality control sample 1 (10 pmol / L) and quality control sample 2 (100 pmol / L).

[0071] Example 2

[0072] The detection method for the Heptakin magnetic microparticle chemiluminescence detection kit is as follows: Take 25 μL of the sample to be tested, add 20 μL of magnetic bead working solution and 50 μL of luminescent label, mix well, and incubate at 37℃ for 8 min to obtain a magnetic bead-coated antibody-antigen-luminescently labeled antibody complex. Wash the magnetic bead-coated antibody-antigen-luminescently labeled antibody complex with PBS buffer, detect the intensity of the luminescence signal, and calculate the concentration of Heptakin in the sample based on the standard curve plotted by the calibrators and the sample light intensity.

[0073] Example 3

[0074] Stability testing of the Heptacin magnetic microparticle chemiluminescence detection kit: The kit obtained in Example 1 was subjected to routine storage stability tests, and the kit was stored at 2-8℃ for 1, 4, 8, 12, 16, 20, 24, 28, and 32 months. The results showed that the Heptacin magnetic microparticle chemiluminescence detection kit has a shelf life of 24 months when stored at 2-8℃ in a light-protected environment.

[0075] Example 4

[0076] Specificity detection of the Heptakin magnetic microparticle chemiluminescence detection kit: The kit obtained in Example 1 was subjected to additional treatments with 10 μg / ml hepcidin and 100 μg / ml adiponectin, respectively. Experimental results showed that the presence of interfering substances had no effect on the detection of Heptakin, and no cross-reactivity was observed.

[0077] Example 5

[0078] Sensitivity testing of the Heptakin magnetic microparticle chemiluminescence detection kit: Using a zero-concentration calibrator as a sample, the kit obtained in Example 1 was used to repeat the test 20 times, and the luminescence values ​​of the 20 test results were obtained. The mean (M) and standard deviation (SD) were calculated to obtain the luminescence value corresponding to M+2SD. The luminescence values ​​of the zero-concentration calibrator and the adjacent calibrators were fitted to obtain a linear equation. The luminescence value corresponding to M+2SD was then substituted into the linear equation to calculate the corresponding concentration value, which is the limit of detection, as shown in Table 1.

[0079] Table 1. Blank Limit Detection of the Peptide Magnetic Microparticle Chemiluminescence Detection Kit

[0080]

[0081] As shown in Table 1, M is 1316 and SD is 406. According to the linear equation, the limit of detection of the peptide magnetic microparticle chemiluminescence detection kit is 0.180605 pmol / L.

[0082] Example 6

[0083] Linear range detection of the chemiluminescence detection kit for peptidyl magnetic microparticles: The kit obtained in Example 1 was diluted with the antigen buffer obtained in Preparation Example 5 at dilution ratios of 1, 1:2, 1:6, 1:10, 1:50, 1:200, and 1:899. Each concentration of the sample was tested twice to obtain the luminescence value. The measurement results for each sample were recorded, and the average of the two measurements for each sample was calculated. Linear regression analysis was performed with dilution concentration as the independent variable and the mean of the measurement results as the dependent variable. The correlation coefficient was obtained, and the results are shown in Table 2.

[0084] Table 2 shows the linear range of the Peptide Magnetic Particle Chemiluminescence Detection Kit.

[0085]

[0086] As shown in Table 2, the correlation coefficient is 0.99996 within the linear range of 0.670-498.96 pmol / L, indicating that the present application and the Peptide Magnetic Microparticle Chemiluminescence Detection Kit have good linearity in the range of 0.7-490 pmol / L.

[0087] Example 7

[0088] Repeatability testing of the Heptakin magnetic microparticle chemiluminescence detection kit: The kit obtained in Example 1 was used to repeatedly detect 200 pmol / L Heptakin (sample A) and 40 pmol / L Heptakin (sample B). The mean and standard deviation of 10 test results were calculated, and the coefficient of variation was calculated. The results are shown in Table 3. Wherein, coefficient of variation = (standard deviation / mean) × 100%.

[0089] Table 3. Repeatability of the Peptide Magnetic Microparticle Chemiluminescence Detection Kit

[0090]

[0091] As shown in Table 3, when the same concentration of standard was tested 10 times, the coefficient of variation was within 2.99%-4.51%, indicating that the kit of this application has good reproducibility.

[0092] Example 8

[0093] Accuracy testing of the Heptakin magnetic microparticle chemiluminescence detection kit: 200 pmol / L Heptakin (sample A) was added to 40 pmol / L Heptakin (sample B), with a volume ratio of 1:9 between the added Heptakin sample A and sample B. The kit obtained in Example 1 was used for detection, and the results are shown in Table 4.

[0094] Table 4 shows the accuracy of the Peptide Magnetic Particle Chemiluminescence Detection Kit.

[0095]

[0096] As shown in Table 4, the deviations of the chemiluminescence detection kit for the peptide magnetic microparticles in this application are all within -1.65% to 2.69%, indicating good accuracy.

[0097] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A chemiluminescence detection kit for peptidyl magnetic microparticles, characterized in that, The invention comprises a magnetic bead working solution and a luminescent label. The magnetic bead working solution includes peptidyl monoclonal antibody A and streptavidin. The luminescent label includes peptidyl monoclonal antibody B, modified nano-silica, anhydrous toluene, methoxy polyethylene glycol-succinimide ester, N,N-dimethylformamide, acridine succinimide ester, glutaraldehyde, and ethanolamine. The preparation steps of the modified nano-silica include: soaking nano-silica in anhydrous toluene, ultrasonically dispersing it, adding 5% by weight of γ-aminopropyltriethoxysilane to the nano-silica, heating under reflux under inert gas protection, washing with ethanol, and drying to obtain modified nano-silica.

2. The chemiluminescence detection kit for peptidin magnetic microparticles according to claim 1, characterized in that, The modified nano-silica has a particle size of 30-60 nm.

3. The chemiluminescence detection kit for peptidin magnetic microparticles according to claim 1, characterized in that, The preparation steps of the luminescent label include: mixing modified nano-silica and anhydrous toluene, ultrasonically dispersing, adding methoxy polyethylene glycol-succinimide ester for a light-protected reaction, centrifuging to remove the supernatant after the reaction, obtaining polyethylene glycol-modified nanoparticles; mixing the polyethylene glycol-modified nanoparticles with N,N-dimethylformamide, adding acridine succinimide ester for a light-protected reaction, centrifuging to remove the supernatant after the reaction, obtaining acridine ester-modified nanoparticles; mixing the acridine ester-modified nanoparticles with PBS buffer, adding glutaraldehyde for activation, then adding peptide monoclonal antibody B for reaction, adding ethanolamine for blocking reaction, separating and purifying through a dialysis bag to obtain the luminescent label.

4. The chemiluminescence detection kit for peptidin magnetic microparticles according to claim 1, characterized in that, It also includes a luminescent labeling buffer, which comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin, Proclin 300 antibacterial agent, and polysorbate-20.

5. The chemiluminescence detection kit for peptidin magnetic microparticles according to claim 1, characterized in that, It also includes an antigen buffer, which comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin, trehalose, ethylenediaminetetraacetic acid, Proclin 300 antibacterial agent, and polysorbate-20.

6. The chemiluminescence detection kit for peptidin magnetic microparticles according to claim 1, characterized in that, It also includes calibrators and quality control samples. The calibrators include calibrator 1 and calibrator 2. Calibrator 1 is a 10-50 pmol / L Heptakin antigen solution, and calibrator 2 is a 100-300 pmol / L Heptakin antigen solution. The quality control samples include quality control sample 1 and quality control sample 2. Quality control sample 1 is a 10-50 pmol / L Heptakin antigen solution, and quality control sample 2 is a 100-300 pmol / L Heptakin antigen solution.

7. The chemiluminescence detection kit for peptidin magnetic microparticles according to claim 1, characterized in that, The concentration of streptavidin in the working solution of the magnetic beads is 0.1-1 mg / mL, and the concentration of peptidin monoclonal antibody B in the luminescent label is 0.01-5 μg / mL.

8. A method for detecting peptidin using a peptidin magnetic microparticle chemiluminescence detection kit according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Take the sample to be tested, add magnetic bead working solution and luminescent label, mix well, incubate to obtain magnetic bead-coated antibody-antigen-luminescent label antibody complex; S2. The magnetic beads coated with the antibody-antigen-luminescent labeled antibody complex are washed, and the intensity of the luminescent signal is detected to obtain the concentration of the sample and peptides.

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