Diagnostic preparation for measuring immunological level and method for preparing the same

Abstract

This application relates to the field of biomedical detection, specifically disclosing a diagnostic reagent for measuring immune levels and its preparation method. The diagnostic reagent comprises the following raw materials: magnetic nanoparticle-antibody complex, acoustically sensitive hollow microsphere-antibody complex, composite stabilizer, and buffer solution. The magnetic nanoparticle-antibody complex is prepared by co-incubating an antibody with γ-Fe₂O₃ after carboxyl activation. The acoustically sensitive hollow microsphere-antibody complex is prepared by encapsulating perfluoropentane in a lipid film, followed by ultrasonic treatment, gradient centrifugation, and then binding it to an antibody. The composite stabilizer is composed of trehalose, pullulan, green tea EGCG, and thyme oil in a specific ratio. This application also provides a method for preparing the diagnostic reagent, including steps such as raw material mixing, ultrasonic dispersion, and stabilizer addition. The diagnostic reagent of this application is characterized by its ease of operation, high stability, and excellent sensitivity, making it suitable for various fields.

Description

Technical Field

[0001] This application relates to the field of biomedical testing, specifically a diagnostic reagent for measuring immune levels and its preparation method. Background Technology

[0002] Immunoassay, as an important component of modern medical diagnostics, plays a crucial role in the diagnosis of infectious diseases, monitoring of autoimmune diseases, detection of tumor markers, and evaluation of vaccination efficacy. With the development of precision medicine and personalized treatment, higher demands are being placed on immunoassay technologies, requiring more sensitive, specific, and rapid detection methods.

[0003] Currently, the most widely used immunoassay techniques in clinical practice mainly include the following categories:

[0004] (1) Enzyme-linked immunosorbent assay (ELISA): As the most classic immunoassay method, ELISA technology has been developed and matured. Its basic principle is that enzyme-labeled antibodies or antigens specifically bind to the analyte, and then the substrate is detected by enzyme catalysis. Although ELISA has the advantages of simple operation and low cost, its detection sensitivity is usually at the ng / mL level, which is difficult to meet the detection needs of low abundance biomarkers. In addition, ELISA detection time is long, operation is cumbersome and easy to introduce errors.

[0005] (2) Chemiluminescent immunoassay (CLIA): CLIA technology uses chemiluminescent substances to label antibodies or antigens, and has higher sensitivity than ELISA. However, CLIA equipment is expensive, requires professional operators, and has limited throughput, making it unsuitable for primary healthcare institutions. Furthermore, the chemiluminescent signal is easily affected by environmental factors, and its stability needs improvement.

[0006] (3) Immunochromatographic technology: Rapid detection technology, represented by colloidal gold immunochromatography, is simple to operate and has a short detection time, making it suitable for on-site testing and primary healthcare. However, it has low sensitivity and poor quantitative ability, and is mainly used for qualitative or semi-quantitative detection.

[0007] In recent years, the development of nanomaterials technology has brought new opportunities to immunoassay. For example, magnetic nanoparticles can rapidly separate target analytes using an external magnetic field, but simple magnetic separation technology lacks an effective signal amplification mechanism, limiting the improvement in detection sensitivity; certain special liposomes can generate strong acoustic signals under ultrasound, but lack targeted enrichment capabilities. Based on the above, this application provides a diagnostic reagent for determining immune levels and a method for preparing the same. Summary of the Invention

[0008] To address the problems of errors easily introduced by multi-step operations in the existing detection process, poor reagent stability, poor specificity, and low sensitivity, this application provides a diagnostic reagent for measuring immune levels and its preparation method.

[0009] In a first aspect, this application provides a diagnostic reagent for measuring immune levels, employing the following technical solution:

[0010] A diagnostic reagent for measuring immune levels comprises the following raw materials in parts by weight: 4-6 parts magnetic nanoparticle-antibody complex, 2-4 parts acoustically sensitive hollow microsphere-antibody complex, 3-5 parts composite stabilizer, and 60-80 parts buffer solution.

[0011] Preferably, the diagnostic reagent for measuring immune levels comprises the following raw materials in parts by weight: 5 parts magnetic nanoparticle-antibody complex, 3 parts acoustic hollow microsphere-antibody complex, 4 parts composite stabilizer, and 70 parts buffer solution.

[0012] Preferably, the preparation method of the magnetic nanoparticle-antibody complex is as follows: γ-Fe2O3 is ultrasonically dispersed in MES buffer, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added for carboxyl activation to obtain activated magnetic nanoparticles; the activated magnetic nanoparticles, antibody, bromelain, and papain are co-incubated; BSA solution is added to block unreacted sites, and then washed with PBS buffer to obtain the magnetic nanoparticle-antibody complex.

[0013] Preferably, the preparation method of the magnetic nanoparticle-antibody complex includes the following steps:

[0014] A1. Mix 50-100 nm γ-Fe2O3 with MES buffer at a mass-to-volume ratio of 1:10-20 and disperse by ultrasonication at 100-200 W and 30-50 kHz for 10-20 min. Add EDC and NHS to make the final concentrations 10-20 mM and 5-10 mM respectively, and react at 20-30 °C with shaking for 30-40 min to obtain activated magnetic nanoparticles.

[0015] A2. Mix the activated magnetic nanoparticles with PBS buffer at a mass-to-volume ratio of 1:10-20, then add the antibody at a mass ratio of 1:20-30 to the activated magnetic nanoparticles to obtain mixture A. Add 10-15% of the mass of mixture A with a concentration of 2-4 U / mL of bromelain solution and 8-12% of the mass of mixture A with a concentration of 2-4 U / mL of papain solution. Incubate at 4-6℃ and 10-15 rpm for 3-5 hours to obtain complex solution A.

[0016] A3. Add 4-6% of the mass of complex solution A with a concentration of 1-3 mg / mL of BSA solution, block unreacted sites at 35-40℃ for 50-70 min, and wash 2-4 times with PBS buffer to obtain magnetic nanoparticle-antibody complex.

[0017] Preferably, the preparation method of the acoustically sensitive hollow microsphere-antibody complex is as follows: SPC, DSPC, and phytosterol are dissolved in an ethanol-ethyl acetate solution, and the mixture is rotary evaporated and then vacuum dried to obtain a lipid film; PBS buffer containing perfluoropentane is added to the lipid film, followed by the addition of DSPE-PEG2000 and cucurbituril hydrate, and the mixture is vortexed to hydrate and detach the lipid film to obtain a primary emulsion; the primary emulsion is placed in an ice bath, sonicated, and then centrifuged to obtain hollow microspheres; after resuspending in PBS buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to activate the carboxyl groups on the surface of the hollow microspheres, and then the antibody is added for co-incubation; after adding BSA solution to block unreacted sites, the mixture is washed with PBS buffer to obtain the acoustically sensitive hollow microsphere-antibody complex.

[0018] Preferably, the preparation method of the acoustically sensitive hollow microsphere-antibody complex includes the following steps:

[0019] B1. Mix SPC, DSPC and phytosterols in a molar ratio of 5-7:2:1 to obtain mixture B, then dissolve it in an ethanol-ethyl acetate solution with a volume ratio of 1.5-2.5:1 and a volume ratio of 1:8-12 between mixture B and the ethanol-ethyl acetate solution. Rotary evaporate the mixture at 55-65℃ for 50-70 min, and then vacuum dry it at 40-50℃ for 3-5 h to obtain a lipid film.

[0020] B2. Mix the lipid film with PBS buffer (containing 4-6 wt% perfluoropentane) that has been pre-degassed and heated to 50-60℃ at a mass ratio of 1:10-16 to obtain mixture C; add DSPE-PEG2000 and cucurbita hydrate to mixture C to make final concentrations of 1-2M and 0.3-0.5mM, respectively, and vortex for 4-6 min to fully hydrate and detach the lipid film to obtain the primary emulsion;

[0021] B3. Transfer the colostrum to an ice bath environment and sonicate it at 100-200W, 20-30kHz for 30-50s. At this time, the solution turns into a light blue semi-transparent state, and hollow microspheres are obtained.

[0022] B4. First, centrifuge at 300-400 rpm for 4-6 min to remove excessively large hollow microspheres, then centrifuge at 800-1200 rpm for 8-12 min to obtain the target hollow microspheres;

[0023] B5. Resuspend the target hollow microspheres in pre-degassed PBS buffer to obtain a hollow microsphere suspension. The mass ratio of PBS buffer to hollow microspheres is 10-20:1. Add EDC and NHS to make the final concentrations 10-20mM and 5-10mM, respectively. After reacting for 30-40 min, add antibody at a concentration of 50-70 μg antibody / mL hollow microsphere suspension. React for 50-70 min to obtain complex solution B.

[0024] B6. Add 4-6% of BSA solution with a concentration of 1-3 mg / mL to the complex solution B. Block unreacted sites at 35-40℃ for 15-25 min. Wash 2-4 times with PBS buffer to obtain acoustic hollow microsphere-antibody complex.

[0025] Preferably, the composite stabilizer comprises trehalose, pullulan, green tea EGCG, and thyme oil in a mass ratio of 1-2:1:0.3-0.5:0.01-0.03.

[0026] Preferably, the buffer solution is PBS buffer, wherein 0.3-0.5 wt% BSA is added.

[0027] Secondly, this application provides a method for preparing a diagnostic reagent for measuring immune levels, employing the following technical solution:

[0028] A method for preparing a diagnostic reagent for measuring immune levels includes the following steps:

[0029] S1. Weigh the magnetic nanoparticle-antibody complex, acoustic hollow microsphere-antibody complex, composite stabilizer and buffer solution according to the weight ratio.

[0030] S2. Mix the magnetic nanoparticle-antibody complex and the acoustically sensitive hollow microsphere-antibody complex, and then ultrasonically disperse them in a buffer solution to obtain a dispersion.

[0031] S3. Mix the dispersion with the composite stabilizer and vortex for 3-5 minutes to ensure uniform dispersion and obtain the mixture.

[0032] S4. Adjust the pH of the mixture and let it stand to stabilize, thus obtaining the diagnostic reagent for measuring immune levels.

[0033] Preferably, the conditions for ultrasonic dispersion in step S2 are: power 100-200W, frequency 20-30kHz, time 5-10min, and temperature 15-25℃.

[0034] Preferably, in step S4, the pH is adjusted to 7.0-7.4, the settling temperature is 4-8℃, and the settling time is 1-2 hours.

[0035] Thirdly, this application provides the application of diagnostic reagents for measuring immune levels in the detection of infectious disease antibodies, diagnosis of autoimmune diseases, evaluation of vaccination efficacy, or monitoring of tumor immune status.

[0036] In summary, this application has the following beneficial effects:

[0037] (1) During the preparation of the magnetic nanoparticle-antibody complex, the EDC / NHS-mediated carboxyl activation reaction forms uniform active carboxyl sites on the surface of the γ-Fe2O3 magnetic nanoparticles, improving the coupling efficiency of the antibody molecules. Co-incubation with bromelain and papain effectively removes impurity proteins from the antibody surface through their specific proteolytic action, ensuring sufficient exposure of the antibody epitopes and enhancing the specificity of subsequent immunoassays. Blocking with BSA solution completely shields unreacted carboxyl sites, minimizing non-specific binding.

[0038] (2) In the preparation process of the acoustically sensitive hollow microsphere-antibody complex, an optimized lipid formulation combined with rotary evaporation-hydration method was used to form a lipid film with excellent monodispersity and uniform particle size. The introduction of perfluoropentane gave the hollow microspheres super acoustic responsiveness, while the post-modification with DSPE-PEG2000 avoided the degradation of PEG during rotary evaporation and significantly reduced the aggregation of hollow microspheres. It could also synergize with cucurbituril hydrate to form a supramolecular complex, enhancing the stability of the hollow microspheres. Through EDC / NHS activation and gradient centrifugation purification, the antibody was directionally coupled to the surface of the hollow microspheres. Combined with BSA blocking treatment, the complex had both high targeting and low background noise.

[0039] (3) The synergistic effect of the magnetic nanoparticle-antibody complex and the acoustically sensitive hollow microsphere-antibody complex significantly improves the sensitivity and specificity of immunoassay. The dual-target recognition mechanism enables the antibodies modified on the surface of the magnetic nanoparticles and the acoustically sensitive hollow microspheres to bind to different epitopes of the target antigen, achieving dual recognition. At the same time, the superparamagnetism of the magnetic nanoparticles allows them to rapidly enrich the target antigen under the action of an external magnetic field, reducing non-specific binding interference; the acoustically sensitive hollow microspheres generate a cavitation effect under the action of ultrasound, enhancing the local signal intensity and achieving cascade amplification of the signal. The combination of the two enables efficient capture and detection of low-concentration antigens, further improving the detection sensitivity.

[0040] (4) This diagnostic reagent is compatible with multiple detection technologies, combining magnetic and acoustic properties. In terms of magnetic detection, the magnetic response characteristics of the magnetic nanoparticles allow for magnetic separation and magnetic signal detection, enabling quantitative analysis of target antibodies by detecting changes in the magnetic signal. In terms of acoustic detection, the acoustically sensitive hollow microspheres vibrate, expand, and rupture under ultrasound, generating unique acoustic signals that can be monitored in real time using ultrasound imaging equipment. Furthermore, the perfluoropentane in the acoustically sensitive hollow microspheres produces a photoacoustic effect under laser irradiation, which can be used for photoacoustic imaging, providing more options for clinical diagnosis.

[0041] (5) The addition of composite stabilizers significantly improved the stability of the formulation. Trehalose and pullulan formed a protective film on the surface of nanoparticles and hollow microspheres, preventing particle aggregation and fusion, while reducing physical and chemical damage to the particles from the external environment. Green tea EGCG has antioxidant properties, which can inhibit the oxidative damage of free radicals to nanoparticles and acoustically sensitive hollow microspheres; thyme oil has antibacterial properties, preventing formulation degradation caused by microbial contamination. In addition, the BSA blocking agent and lipid film material used in the formulation have good biocompatibility, which can reduce non-specific interactions with other components in biological samples, reduce contamination of detection equipment, and extend the shelf life of the formulation.

[0042] (6) The preparation method of the diagnostic reagent is simple and low-cost. The sample to be tested is simply mixed with the diagnostic reagent once, incubated for a short time, and then a magnetic field is applied to enrich the target complex, allowing for direct magnetic signal or ultrasonic / photoacoustic detection. During ultrasonic dispersion, the magnetic nanoparticles and acoustically sensitive hollow microspheres form a stable dispersion system due to the differences in their surface charge and physical properties, eliminating the need for additional surfactants or stabilizers. The components in the composite stabilizer work synergistically, simultaneously providing physical protection, chemical stability, and biocompatibility, simplifying the formulation and preparation process. Furthermore, this reagent can be used in various immunoassay scenarios, reducing the types and quantities of reagents required for different testing items, lowering testing costs and operational complexity, and improving testing efficiency and economy. Detailed Implementation

[0043] The present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0046] PBS buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: R26274.

[0047] MES buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: R28967.

[0048] Preparation Examples 1-3 and Comparative Preparation Examples 1-3 provide methods for preparing magnetic nanoparticle-antibody complexes.

[0049] Preparation Example 1

[0050] The magnetic nanoparticle-antibody complex was prepared by the following method:

[0051] A1. Mix 50 nm γ-Fe2O3 with MES buffer at a mass-to-volume ratio of 1:10, and disperse by ultrasonication at 100 W and 30 kHz for 10 min; add EDC and NHS to make the final concentrations 10 mM and 5 mM respectively, and react at 20 °C with shaking for 30 min to obtain activated magnetic nanoparticles.

[0052] A2. Mix the activated magnetic nanoparticles with PBS buffer at a mass-to-volume ratio of 1:10, then add human IgG antibody at a mass ratio of 1:20 to the activated magnetic nanoparticles to obtain mixture A. Add 10% of the mass of mixture A and 2U / mL of bromelain solution and 8% of the mass of mixture A and 2U / mL of papain solution. Incubate at 4°C and 10 rpm for 3 hours to obtain complex solution A.

[0053] A3. Add 1 mg / mL BSA solution, accounting for 4% of the mass of complex solution A, block unreacted sites at 35°C for 50 min, and wash twice with PBS buffer to obtain magnetic nanoparticle-antibody complex.

[0054] Preparation Example 2

[0055] The magnetic nanoparticle-antibody complex was prepared by the following method:

[0056] A1. Mix 75nm γ-Fe2O3 with MES buffer at a mass-to-volume ratio of 1:15, and disperse by ultrasonication at 150W and 40kHz for 15min. Add EDC and NHS to make the final concentrations 15mM and 8mM respectively, and react at 25℃ for 35min to obtain activated magnetic nanoparticles.

[0057] A2. Mix the activated magnetic nanoparticles with PBS buffer at a mass-to-volume ratio of 1:15, then add human IgG antibody at a mass ratio of 1:25 to the activated magnetic nanoparticles to obtain mixture A. Add 12.5% ​​of the mass of mixture A and 3U / mL of bromelain solution and 10% of the mass of mixture A and 3U / mL of papain solution. Incubate at 5°C and 12 rpm for 4 hours to obtain complex solution A.

[0058] A3. Add 2 mg / mL BSA solution, accounting for 5% of the mass of complex solution A, block unreacted sites at 37°C for 60 min, and wash three times with PBS buffer to obtain magnetic nanoparticle-antibody complex.

[0059] Preparation Example 3

[0060] The magnetic nanoparticle-antibody complex was prepared by the following method:

[0061] A1. Mix 100 nm γ-Fe2O3 with MES buffer at a mass-to-volume ratio of 1:20, and disperse by ultrasonication at 200 W and 50 kHz for 20 min; add EDC and NHS to make the final concentrations 20 mM and 10 mM respectively, and react at 30 °C with shaking for 40 min to obtain activated magnetic nanoparticles.

[0062] A2. Mix the activated magnetic nanoparticles with PBS buffer at a mass-to-volume ratio of 1:20, then add human IgG antibody at a mass ratio of 1:30 to the activated magnetic nanoparticles to obtain mixture A. Add 15% of the mass of mixture A and 12% of the mass of mixture A and 4U / mL of the mass of mixture A and papain solution. Incubate at 6°C and 15 rpm for 5 hours to obtain complex solution A.

[0063] A3. Add 3 mg / mL BSA solution, accounting for 6% of the mass of complex solution A, block unreacted sites at 40°C for 70 min, and wash 4 times with PBS buffer to obtain magnetic nanoparticle-antibody complex.

[0064] Comparative Preparation Example 1

[0065] The magnetic nanoparticle-antibody complex was prepared by the following method:

[0066] A1. Mix 75nm γ-Fe2O3 with MES buffer at a mass-to-volume ratio of 1:15, and disperse by ultrasonication at 150W and 40kHz for 15min. Add EDC and NHS to make the final concentrations 15mM and 8mM respectively, and react at 25℃ for 35min to obtain activated magnetic nanoparticles.

[0067] A2. Mix the activated magnetic nanoparticles with PBS buffer at a mass-to-volume ratio of 1:15, then add human IgG antibody at a mass ratio of 1:25 to the activated magnetic nanoparticles to obtain mixture A. Add 22.5% of the mass of mixture A with a concentration of 3 U / mL of bromelain solution, and incubate at 5°C and 12 rpm for 4 hours to obtain complex solution A.

[0068] A3. Add 2 mg / mL BSA solution, accounting for 5% of the mass of complex solution A, block unreacted sites at 37°C for 60 min, and wash three times with PBS buffer to obtain magnetic nanoparticle-antibody complex.

[0069] Comparative Preparation Example 2

[0070] The magnetic nanoparticle-antibody complex was prepared by the following method:

[0071] A1. Mix 75nm γ-Fe2O3 with MES buffer at a mass-to-volume ratio of 1:15, and disperse by ultrasonication at 150W and 40kHz for 15min. Add EDC and NHS to make the final concentrations 15mM and 8mM respectively, and react at 25℃ for 35min to obtain activated magnetic nanoparticles.

[0072] A2. Mix the activated magnetic nanoparticles with PBS buffer at a mass-to-volume ratio of 1:15, then add human IgG antibody at a mass ratio of 1:25 to the activated magnetic nanoparticles to obtain mixture A. Add 22.5% of the mass of mixture A with a concentration of 3 U / mL of papain solution, and incubate at 5°C and 12 rpm for 4 hours to obtain complex solution A.

[0073] A3. Add 2 mg / mL BSA solution, accounting for 5% of the mass of complex solution A, block unreacted sites at 37°C for 60 min, and wash three times with PBS buffer to obtain magnetic nanoparticle-antibody complex.

[0074] Preparation Examples 4-6 and Comparative Preparation Examples 3-5 provide methods for preparing acoustically sensitive hollow microsphere-antibody complexes.

[0075] Preparation Example 4

[0076] The acoustically sensitive hollow microsphere-antibody complex was prepared by the following method:

[0077] B1. Mix SPC, DSPC and phytosterol in a molar ratio of 5:2:1 to obtain mixture B, then dissolve it in an ethanol-ethyl acetate solution with a volume ratio of 1.5:1 and a volume ratio of 1:8 between mixture B and the ethanol-ethyl acetate solution. The mixture is then rotary evaporated in a water bath at 55°C for 50 min and then vacuum dried at 40°C for 3 h to obtain a lipid film.

[0078] B2. Mix the lipid film with PBS buffer (containing 4 wt% perfluoropentane) that has been pre-degassed and heated to 50°C at a mass ratio of 1:10 to obtain mixture C; add DSPE-PEG2000 and cucurbituril hydrate to mixture C to make final concentrations of 1M and 0.3mM, respectively, and vortex for 4 min to fully hydrate and detach the lipid film to obtain the primary emulsion.

[0079] B3. Transfer the colostrum to an ice bath environment and sonicate at 100W, 20kHz for 30s. At this time, the solution turns into a light blue semi-transparent state, and hollow microspheres are obtained.

[0080] B4. First, centrifuge at 300 rpm for 4 min to remove excessively large hollow microspheres, then centrifuge at 800 rpm for 8 min to obtain the target hollow microspheres;

[0081] B5. Resuspend the target hollow microspheres in pre-degassed PBS buffer to obtain a hollow microsphere suspension. The mass ratio of PBS buffer to hollow microspheres is 10:1. Add EDC and NHS to make the final concentrations 10mM and 5mM, respectively. After reacting for 30 min, add human IgG antibody at a concentration of 50 μg antibody / mL hollow microsphere suspension. React for 50 min to obtain complex solution B.

[0082] B6. Add 1 mg / mL BSA solution, accounting for 4% of the mass of complex solution B, block unreacted sites at 35°C for 15 min, wash twice with PBS buffer to obtain acoustically sensitive hollow microsphere-antibody complex.

[0083] Preparation Example 5

[0084] The acoustically sensitive hollow microsphere-antibody complex was prepared by the following method:

[0085] B1. Mix SPC, DSPC and phytosterol in a molar ratio of 6:2:1 to obtain mixture B, then dissolve it in an ethanol-ethyl acetate solution with a volume ratio of 2:1 and a volume ratio of 1:10 between mixture B and the ethanol-ethyl acetate solution. The mixture is then rotary evaporated in a water bath at 60°C for 60 min and then vacuum dried at 45°C for 4 h to obtain a lipid film.

[0086] B2. Mix the lipid film with PBS buffer (containing 5 wt% perfluoropentane) that has been pre-degassed and heated to 55°C at a mass ratio of 1:13 to obtain mixture C; add DSPE-PEG2000 and cucurbituril hydrate to mixture C to make final concentrations of 1.5M and 0.4mM, respectively, and vortex for 5 min to fully hydrate and detach the lipid film to obtain the primary emulsion;

[0087] B3. Transfer the colostrum to an ice bath environment and sonicate it at 150W and 25kHz for 40s. At this time, the solution turns into a light blue semi-transparent state, and hollow microspheres are obtained.

[0088] B4. First, centrifuge at 350 rpm for 5 min to remove excessively large hollow microspheres, then centrifuge at 1000 rpm for 10 min to obtain the target hollow microspheres.

[0089] B5. Resuspend the target hollow microspheres in pre-degassed PBS buffer to obtain a hollow microsphere suspension. The mass ratio of PBS buffer to hollow microspheres is 15:1. Add EDC and NHS to make the final concentrations 15mM and 7.5mM, respectively. After reacting for 35 min, add human IgG antibody at a concentration of 60 μg antibody / mL hollow microsphere suspension. React for 60 min to obtain complex solution B.

[0090] B6. Add 5% of the mass of complex solution B with a concentration of 2 mg / mL BSA solution, block unreacted sites at 37℃ for 20 min, wash 3 times with PBS buffer to obtain acoustic hollow microsphere-antibody complex.

[0091] Preparation Example 6

[0092] The acoustically sensitive hollow microsphere-antibody complex was prepared by the following method:

[0093] B1. Mix SPC, DSPC and phytosterol in a molar ratio of 7:2:1 to obtain mixture B, then dissolve it in an ethanol-ethyl acetate solution with a volume ratio of 2.5:1 and a volume ratio of 1:12 between mixture B and the ethanol-ethyl acetate solution. The mixture is then rotary evaporated in a water bath at 65°C for 70 min and then vacuum dried at 50°C for 5 h to obtain a lipid film.

[0094] B2. Mix the lipid film with PBS buffer (containing 6 wt% perfluoropentane) that has been pre-degassed and heated to 60°C at a mass ratio of 1:16 to obtain mixture C; add DSPE-PEG2000 and cucurbituril hydrate to mixture C to make final concentrations of 2M and 0.5mM, respectively, and vortex for 6 min to fully hydrate and detach the lipid film to obtain the primary emulsion.

[0095] B3. Transfer the colostrum to an ice bath environment and sonicate it at 200W and 30kHz for 50s. At this time, the solution turns into a light blue semi-transparent state, and hollow microspheres are obtained.

[0096] B4. First, centrifuge at 400 rpm for 6 min to remove excessively large hollow microspheres, then centrifuge at 1200 rpm for 12 min to obtain the target hollow microspheres;

[0097] B5. Resuspend the target hollow microspheres in pre-degassed PBS buffer to obtain a hollow microsphere suspension. The mass ratio of PBS buffer to hollow microspheres is 20:1. Add EDC and NHS to make the final concentrations 20mM and 10mM, respectively. After reacting for 40 min, add human IgG antibody at a concentration of 70 μg antibody / mL hollow microsphere suspension. React for 70 min to obtain complex solution B.

[0098] B6. Add 3 mg / mL BSA solution, accounting for 6% of the mass of complex solution B, block unreacted sites at 40°C for 25 min, and wash 4 times with PBS buffer to obtain acoustically sensitive hollow microsphere-antibody complex.

[0099] Comparative preparation example 3

[0100] Antibody complexes are prepared by the following methods:

[0101] B1. Mix SPC, DSPC and phytosterol in a molar ratio of 6:2:1 to obtain mixture B, then dissolve it in an ethanol-ethyl acetate solution with a volume ratio of 2:1 and a volume ratio of 1:10 between mixture B and the ethanol-ethyl acetate solution. The mixture is then rotary evaporated in a water bath at 60°C for 60 min and then vacuum dried at 45°C for 4 h to obtain a lipid film.

[0102] B2. Mix the lipid film with PBS buffer (containing 5 wt% perfluoropentane) that has been pre-degassed and heated to 55°C at a mass ratio of 1:13 to obtain mixture C; add DSPE-PEG2000 and cucurbituril hydrate to mixture C to make final concentrations of 1.5M and 0.4mM, respectively, and vortex for 5 min to fully hydrate and detach the lipid film to obtain the primary emulsion;

[0103] B3. Add EDC and NHS to the colostrum to make the final concentrations 15mM and 7.5mM respectively. After reacting for 35 min, add human IgG antibody at a concentration of 60 μg antibody / mL colostrum and react for 60 min to obtain complex solution B.

[0104] B4. Add 5% of the mass of complex solution B with a concentration of 2 mg / mL BSA solution, block unreacted sites at 37℃ for 20 min, and wash three times with PBS buffer to obtain antibody complex.

[0105] Comparative preparation example 4

[0106] The acoustically sensitive hollow microsphere-antibody complex was prepared by the following method:

[0107] B1. Mix SPC, DSPC, phytosterols and DSPE-PEG2000 in a molar ratio of 6:2:1:1.5 to obtain mixture B, which is then dissolved in an ethanol-ethyl acetate solution with a volume ratio of 2:1 and a volume ratio of 1:10 between mixture B and the ethanol-ethyl acetate solution. The mixture is then rotary evaporated in a water bath at 60°C for 60 min and then vacuum dried at 45°C for 4 h to obtain a lipid film.

[0108] B2. Mix the lipid film with PBS buffer (containing 5 wt% perfluoropentane) that has been pre-degassed and heated to 55°C at a mass ratio of 1:13 to obtain mixture C; add cucurbituril hydrate to mixture C to a final concentration of 0.4 mM, vortex for 5 min to fully hydrate and detach the lipid film, and obtain the primary emulsion;

[0109] B3. Transfer the colostrum to an ice bath environment and sonicate it at 150W and 25kHz for 40s. At this time, the solution turns into a light blue semi-transparent state, and hollow microspheres are obtained.

[0110] B4. First, centrifuge at 350 rpm for 5 min to remove excessively large hollow microspheres, then centrifuge at 1000 rpm for 10 min to obtain the target hollow microspheres.

[0111] B5. Resuspend the target hollow microspheres in pre-degassed PBS buffer at a mass ratio of 15:1. Add EDC and NHS to make the final concentrations 15mM and 7.5mM, respectively. After reacting for 35 min, add human IgG antibody at a concentration of 60 μg antibody / mL hollow microspheres and react for 60 min to obtain complex solution B.

[0112] B6. Add 5% of the mass of complex solution B with a concentration of 2 mg / mL BSA solution, block unreacted sites at 37℃ for 20 min, wash 3 times with PBS buffer to obtain acoustic hollow microsphere-antibody complex.

[0113] Comparative preparation example 5

[0114] The acoustically sensitive hollow microsphere-antibody complex was prepared by the following method:

[0115] B1. Mix SPC, DSPC and phytosterol in a molar ratio of 6:2:1 to obtain mixture B, then dissolve it in an ethanol-ethyl acetate solution with a volume ratio of 2:1 and a volume ratio of 1:10 between mixture B and the ethanol-ethyl acetate solution. The mixture is then rotary evaporated in a water bath at 60°C for 60 min and then vacuum dried at 45°C for 4 h to obtain a lipid film.

[0116] B2. Mix the lipid film with PBS buffer (containing 5 wt% perfluoropentane) that has been pre-degassed and heated to 55°C at a mass ratio of 1:13 to obtain mixture C; add DSPE-PEG2000 to mixture C to make a final concentration of 1.5M, vortex for 5 min to fully hydrate and detach the lipid film, and obtain the primary emulsion.

[0117] B3. Transfer the colostrum to an ice bath environment and sonicate it at 150W and 25kHz for 40s. At this time, the solution turns into a light blue semi-transparent state, and hollow microspheres are obtained.

[0118] B4. First, centrifuge at 350 rpm for 5 min to remove excessively large hollow microspheres, then centrifuge at 1000 rpm for 10 min to obtain the target hollow microspheres.

[0119] B5. Resuspend the target hollow microspheres in pre-degassed PBS buffer at a mass ratio of 15:1. Add EDC and NHS to make the final concentrations 15mM and 7.5mM, respectively. After reacting for 35 min, add human IgG antibody at a concentration of 60 μg antibody / mL hollow microspheres and react for 60 min to obtain complex solution B.

[0120] B6. Add 5% of the mass of complex solution B with a concentration of 2 mg / mL BSA solution, block unreacted sites at 37℃ for 20 min, wash 3 times with PBS buffer to obtain acoustic hollow microsphere-antibody complex.

[0121] Example 1

[0122] A diagnostic reagent for measuring immune levels comprises the following raw materials in parts by weight: 4 parts magnetic nanoparticle-antibody complex, 2 parts acoustically sensitive hollow microsphere-antibody complex, 3 parts composite stabilizer, and 60 parts buffer solution.

[0123] The magnetic nanoparticle-antibody complex was prepared in Preparation Example 1.

[0124] The acoustically sensitive hollow microsphere-antibody complex was prepared in Preparation Example 4.

[0125] The composite stabilizer comprises trehalose, pullulan, green tea EGCG, and thyme oil in a mass ratio of 1:1:0.3:0.01.

[0126] The buffer was a PBS buffer with 0.3 wt% BSA added.

[0127] A method for preparing a diagnostic reagent for measuring immune levels includes the following steps:

[0128] S1. Weigh the magnetic nanoparticle-antibody complex, acoustic hollow microsphere-antibody complex, composite stabilizer and buffer solution according to the weight ratio.

[0129] S2. Mix the magnetic nanoparticle-antibody complex and the acoustically sensitive hollow microsphere-antibody complex, add buffer solution, and sonicate at 100W, 20kHz, 15℃ for 5min to obtain a dispersion.

[0130] S3. Mix the dispersion with the composite stabilizer and vortex for 3 minutes to ensure uniform dispersion and obtain the mixture.

[0131] S4. Adjust the pH of the mixture to 7.0, and then let it stand at 4°C for 1 hour to obtain the diagnostic reagent for measuring immune levels.

[0132] Example 2

[0133] A diagnostic reagent for measuring immune levels comprises the following raw materials in parts by weight: 5 parts magnetic nanoparticle-antibody complex, 3 parts acoustically sensitive hollow microsphere-antibody complex, 4 parts composite stabilizer, and 70 parts buffer solution.

[0134] The magnetic nanoparticle-antibody complex was prepared in Preparation Example 2.

[0135] The acoustically sensitive hollow microsphere-antibody complex was prepared in Preparation Example 5.

[0136] The composite stabilizer consists of trehalose, pullulan, green tea EGCG, and thyme oil in a mass ratio of 1.5:1:0.4:0.02.

[0137] The buffer was a PBS buffer with 0.4 wt% BSA added.

[0138] A method for preparing a diagnostic reagent for measuring immune levels includes the following steps:

[0139] S1. Weigh the magnetic nanoparticle-antibody complex, acoustic hollow microsphere-antibody complex, composite stabilizer and buffer solution according to the weight ratio.

[0140] S2. Mix the magnetic nanoparticle-antibody complex and the acoustically sensitive hollow microsphere-antibody complex, add buffer solution, and sonicate at 150W, 25kHz, 20℃ for 8min to obtain a dispersion.

[0141] S3. Mix the dispersion with the composite stabilizer and vortex for 4 minutes to ensure uniform dispersion and obtain the mixture.

[0142] S4. Adjust the pH of the mixture to 7.2, and then let it stand at 6°C for 1.5 hours to obtain the diagnostic reagent for measuring immune levels.

[0143] Example 3

[0144] A diagnostic reagent for measuring immune levels comprises the following raw materials in parts by weight: 6 parts magnetic nanoparticle-antibody complex, 4 parts acoustic hollow microsphere-antibody complex, 5 parts composite stabilizer, and 80 parts buffer solution.

[0145] The magnetic nanoparticle-antibody complex was prepared in Preparation Example 3.

[0146] The acoustically sensitive hollow microsphere-antibody complex was prepared in Preparation Example 6.

[0147] The composite stabilizer consists of trehalose, pullulan, green tea EGCG, and thyme oil in a mass ratio of 2:1:0.5:0.03.

[0148] The buffer was a PBS buffer with 0.5 wt% BSA added.

[0149] A method for preparing a diagnostic reagent for measuring immune levels includes the following steps:

[0150] S1. Weigh the magnetic nanoparticle-antibody complex, acoustic hollow microsphere-antibody complex, composite stabilizer and buffer solution according to the weight ratio.

[0151] S2. Mix the magnetic nanoparticle-antibody complex and the acoustically sensitive hollow microsphere-antibody complex, add buffer solution, and sonicate at 200W, 30kHz, 25℃ for 10min to obtain a dispersion.

[0152] S3. Mix the dispersion with the composite stabilizer and vortex for 5 minutes to ensure uniform dispersion and obtain the mixture.

[0153] S4. Adjust the pH of the mixture to 7.4, and then let it stand at 8°C for 2 hours to stabilize, thus obtaining the diagnostic reagent for measuring immune levels.

[0154] Comparative Example 1

[0155] Same as Example 2, except that the magnetic nanoparticle-antibody complex was prepared in Comparative Preparation Example 1.

[0156] Comparative Example 2

[0157] Same as Example 2, except that the magnetic nanoparticle-antibody complex was prepared in Comparative Preparation Example 2.

[0158] Comparative Example 3

[0159] Same as Example 2, except that the acoustically sensitive hollow microsphere-antibody complex was prepared in Comparative Preparation Example 3.

[0160] Comparative Example 4

[0161] Same as Example 2, except that the acoustically sensitive hollow microsphere-antibody complex was prepared in Comparative Preparation Example 4.

[0162] Comparative Example 5

[0163] Same as Example 2, except that the acoustically sensitive hollow microsphere-antibody complex was prepared in Comparative Preparation Example 5.

[0164] Comparative Example 6

[0165] Same as Example 2, except that the composite stabilizer includes trehalose and pullulan in a mass ratio of 1.5:1.

[0166] Comparative Example 7

[0167] Same as Example 2, except that the composite stabilizer includes trehalose, pullulan, and green tea EGCG in a mass ratio of 1.5:1:0.42.

[0168] Comparative Example 8

[0169] Same as Example 2, except that the composite stabilizer includes trehalose, pullulan, and thyme oil in a mass ratio of 1.5:1:0.42.

[0170] Sensitivity test

[0171] Sample preparation: Using human IgG antigen as the target molecule, prepare 100 μL of serially diluted standard solutions (concentration range: 0.1 pg / mL-100 pg / mL). Add 100 μL of the diagnostic reagent prepared in Example 2 to each sample, vortex to mix, and incubate for 30 min.

[0172] Detection steps: Magnetic signal detection: Place the mixture in a magnetic separator and separate for 5 minutes. Measure the signal value using a magnetic relaxation time (T2) detector; Ultrasonic signal detection: Measure the acoustic signal intensity of the hollow microspheres using a high-frequency ultrasonic probe (10 MHz).

[0173] Data analysis: The minimum detection limit was determined based on a signal-to-noise ratio (S / N) ≥ 3, and the results are shown in Table 1.

[0174] Table 1 Sensitivity Test Results

[0175]

[0176] Conclusion: The limit of detection (LOD) of the diagnostic reagent for measuring immune levels in this application is 1 pg / mL.

[0177] Specificity test

[0178] Sample preparation: Prepare a blank control (100 μL): PBS buffer;

[0179] Prepare 100 μL of target antigen: 10 pg / mL human IgG;

[0180] Prepare 100 μL of each of the following cross antigens: 10 pg / mL human IgM, 10 pg / mL bovine serum albumin, and 10 pg / mL lysozyme.

[0181] Detection steps: 100 μL of target antigen and cross antigen were vortexed with 100 μL of the diagnostic reagent prepared in Example 2, incubated for 30 min, and the magnetic signal was detected after magnetic separation and the acoustic signal was detected by ultrasound.

[0182] Data analysis: Cross-reactivity rate = (cross antigen magnetic signal intensity / target antigen magnetic signal intensity) × 100%, and the results are shown in Table 2.

[0183] Table 2 Specificity test results

[0184]

[0185] Conclusion: The diagnostic reagents used in this application to measure immune levels showed a cross-reactivity rate of <12% to non-target antigens, demonstrating their high specificity.

[0186] Stability test

[0187] Storage conditions: The diagnostic reagent prepared in Example 2 was stored in a simulated cold chain at 4°C and in a simulated room temperature at 25°C;

[0188] Testing time points: 0, 7, 14, and 30 days;

[0189] Detection indicators: Physical stability: particle size change (dynamic light scattering, DLS); Functional stability: retention rate of magnetic and ultrasonic signals compared to initial values.

[0190] Data analysis: Signal retention rate = (current signal / initial signal) × 100%, the results are shown in Table 3.

[0191] Table 3 Stability Test Results

[0192]

[0193] Conclusion: The diagnostic reagent for measuring immune levels in this application exhibits excellent stability with a signal retention rate of >90% and a particle size increase of <10nm after 30 days of storage at 4℃; however, the signal decreases significantly after 30 days of storage at 25℃, making cold chain storage a stronger recommendation.

[0194] Performance Comparison

[0195] The diagnostic preparations prepared in Examples 1-3 and Comparative Examples 1-8 were subjected to the above three tests according to the sensitivity test, specificity test and stability test methods described above.

[0196] In the sensitivity test, human IgG antigen with a concentration gradient range of 1 pg / mL to 1000 pg / mL was used as the detection limit of the target molecule. In the specificity test, 10 pg / mL human IgG antigen was used as the target antigen and 10 pg / mL human IgM antigen was used as the cross-reactivity antigen to determine the cross-reactivity rate. In the stability test, the magnetic signal retention rate and ultrasonic signal retention rate were measured at 4℃ for 30 days. The experimental data were statistically analyzed, and the results are shown in Table 4.

[0197] Table 4 Performance Comparison of Examples and Comparative Examples

[0198]

[0199] Analysis of the comprehensive performance data of the diagnostic reagents in Examples 1-3 and Comparative Examples 1-8 in Table 4 leads to the following conclusions: Examples 1-3 utilize the synergistic detection system based on magnetic nanoparticle-antibody complexes and acoustically sensitive hollow microsphere-antibody complexes provided in this application. By optimizing key aspects such as material preparation, composite stabilizer formulation, and detection process, the detection sensitivity, specificity, and stability are significantly improved. In contrast, Comparative Examples 1-8 underwent modifications in certain aspects, resulting in a decrease in overall performance, thus fully validating the necessity of the technical features of this application.

[0200] Regarding the core diagnostic reagent materials, Examples 1-3 employed a dual-signal system of carboxyl-activated γ-Fe2O3 magnetic nanoparticles and perfluoropentane acoustically sensitive hollow microspheres, achieving an ultra-high detection sensitivity of 1 pg / mL. In contrast, Comparative Example 1 used only bromelain and Comparative Example 2 used only papain, resulting in reduced antibody conjugation efficiency and a sensitivity drop to 10 pg / mL. Comparative Example 3 did not employ ultrasonic treatment and gradient centrifugation to purify the hollow microspheres, leading to a significant decrease in targeting efficiency and signal amplification, and a substantial reduction in sensitivity to 1000 pg / mL.

[0201] Regarding specificity control, Examples 1-3 controlled the cross-reactivity rate at 10.8-12.5% ​​by blocking with BSA and optimizing the antibody conjugation process; while Comparative Examples 1 and 2, due to insufficient exposure of antibody epitopes, had cross-reactivity rates of 14.7% and 13.9%, respectively; Comparative Example 4 had a cross-reactivity rate of 12.9% due to insufficient stability of hollow microspheres caused by co-evaporation of DSPE-PEG2000 and lipid film; and Comparative Example 5, due to the absence of cucurbituril hydrate, showed increased aggregation of hollow microspheres, resulting in a cross-reactivity rate of 13.0%.

[0202] Regarding the formulation of the composite stabilizer, Examples 1-3 employed a composite system of trehalose, pullulan, green tea EGCG, and thyme oil, which synergistically exerted physical protection, antioxidant, and antibacterial effects, resulting in magnetic and ultrasonic signal retention rates exceeding 90% after storage at 4°C for 30 days. In contrast, Comparative Example 6 used only trehalose and pullulan, lacking antioxidant and antibacterial components, and its signal retention rate significantly decreased to below 65%. Comparative Example 7 did not add thyme oil, resulting in insufficient antibacterial performance and a decrease in ultrasonic signal retention rate to 77.4%. Comparative Example 8 did not add green tea EGCG, weakening its antioxidant performance and reducing its magnetic signal retention rate to 82.9%.

[0203] The above specific embodiments are merely explanations of this application and are not intended to limit this application. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A diagnostic reagent for measuring immune levels, characterized in that, The raw materials include the following parts by weight: 4-6 parts magnetic nanoparticle-antibody complex, 2-4 parts acoustically sensitive hollow microsphere-antibody complex, 3-5 parts composite stabilizer, and 60-80 parts buffer solution; The preparation method of the magnetic nanoparticle-antibody complex is as follows: γ-Fe2O3 is ultrasonically dispersed in MES buffer, and EDC and NHS are added for carboxyl activation to obtain activated magnetic nanoparticles; the activated magnetic nanoparticles, antibody, bromelain and papain are co-incubated; BSA solution is added to block unreacted sites, and then washed with PBS buffer to obtain the magnetic nanoparticle-antibody complex. The method for preparing the acoustically sensitive hollow microsphere-antibody complex is as follows: SPC, DSPC, and phytosterol are dissolved in an ethanol-ethyl acetate solution, and after rotary evaporation and vacuum drying, a lipid film is obtained; PBS buffer containing perfluoropentane is added to the lipid film, followed by the addition of DSPE-PEG2000 and cucurbituril hydrate, and the mixture is vortexed to hydrate and detach the lipid film, resulting in a primary emulsion; the primary emulsion is placed in an ice bath, sonicated, and then centrifuged to obtain hollow microspheres; after resuspending in PBS buffer, EDC and NHS are added to activate the carboxyl groups on the surface of the hollow microspheres, and then the antibody is added for co-incubation; after adding BSA solution to block unreacted sites, the mixture is washed with PBS buffer to obtain the acoustically sensitive hollow microsphere-antibody complex; The composite stabilizer comprises trehalose, pullulan, green tea EGCG, and thyme oil in a mass ratio of 1-2:1:0.3-0.5:0.01-0.

03.

2. The diagnostic reagent for measuring immune levels according to claim 1, characterized in that, The raw materials include the following parts by weight: 5 parts magnetic nanoparticle-antibody complex, 3 parts acoustically sensitive hollow microsphere-antibody complex, 4 parts composite stabilizer, and 70 parts buffer solution.

3. The diagnostic reagent for measuring immune levels according to claim 1, characterized in that, The buffer solution is PBS buffer with 0.3-0.5 wt% BSA added.

4. A method for preparing a diagnostic reagent for measuring immune levels according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Weigh the magnetic nanoparticle-antibody complex, acoustic hollow microsphere-antibody complex, composite stabilizer and buffer solution according to the weight ratio. S2. Mix the magnetic nanoparticle-antibody complex and the acoustically sensitive hollow microsphere-antibody complex, and then ultrasonically disperse them in a buffer solution to obtain a dispersion. S3. Mix the dispersion with the composite stabilizer and vortex for 3-5 minutes to ensure uniform dispersion and obtain the mixture. S4. Adjust the pH of the mixture and let it stand to stabilize, thus obtaining the diagnostic reagent for measuring immune levels.

5. The method for preparing the diagnostic reagent for measuring immune levels according to claim 4, characterized in that, The conditions for ultrasonic dispersion in step S2 are: power 100-200W, frequency 20-30kHz, time 5-10min, and temperature 15-25℃.

6. The method for preparing the diagnostic reagent for determining immune levels according to claim 4, characterized in that, In step S4, the pH is adjusted to 7.0-7.4, the settling temperature is 4-8℃, and the settling time is 1-2 hours.

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