Immunological detection method for substance to be detected in whole blood sample, magnetic microsphere and magnetic microsphere-protein compound

Abstract

The invention provides an immunological detection method for a to-be-detected substance in a whole blood sample, a magnetic microsphere and a magnetic microsphere-protein compound. The immunological detection method comprises the following steps: a) mixing a whole blood sample and an immunodetection reagent, and incubating to obtain an incubated compound; wherein the immunodetection reagent comprises a signal molecule and / or a magnetic microsphere-protein compound, the magnetic microsphere-protein compound comprises a magnetic microsphere and a first affinity protein coated on the magnetic microsphere, the first affinity protein is a protein capable of being specifically combined with an object to be detected, and the surface of the magnetic microsphere contains methacrylate; and b) carrying out signal detection on the incubation compound, and obtaining the quantity of the to-be-detected substance through the signal intensity. The problem of low detection accuracy of the whole blood sample in the prior art can be solved, and the method is suitable for the field of whole blood sample detection.

Description

Technical Field

[0001] The present invention relates to the field of whole blood sample detection, and in particular to an immunological detection method for an object to be detected in a whole blood sample, a magnetic microsphere and a magnetic microsphere-protein complex. Background Art

[0002] Magnetic microspheres, due to their unique magnetic responsiveness and surface modifiability, have shown great potential in the biomedical field, especially in in vitro diagnostics and other tests. They are made of organic polymer materials combined with inorganic magnetic materials, and can achieve rapid positioning, guidance and separation under the action of an external magnetic field. The surface of magnetic microspheres can be chemically modified to introduce a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), amino (-NH2), thiol (-SH), etc., which not only enhances their biocompatibility, but also facilitates the subsequent coupling of antibodies or other biomolecules. They are widely used in in vitro diagnostic technologies such as magnetic separation immunoassays.

[0003] However, in the field of in vitro diagnosis, the detection of whole blood samples is an extremely challenging task. Whole blood samples contain various components in blood, such as red blood cells, white blood cells, platelets, and various proteins and cytokines in plasma, and their complexity is far greater than that of serum or plasma samples. When using magnetic microspheres for whole blood testing, nonspecific adsorption between blood cells and magnetic microspheres is a problem that cannot be ignored. Nonspecific adsorption means that in immunoassays, in addition to specific recognition and binding with the target antigen or antibody, the surface of the magnetic microsphere may also bind to non-target molecules in the sample, such as proteins and fragments on the surface of blood cells. This not only leads to an increase in the background noise of the detection signal, affecting the specificity and sensitivity of the test, but may also produce false positive results and reduce the accuracy of the test.

[0004] There are many magnetic sphere materials for whole blood testing on the market, and these materials have made certain progress in improving detection efficiency and simplifying operation procedures. However, these traditional magnetic sphere materials are still insufficient in dealing with nonspecific adsorption problems and are difficult to meet the requirements of clinical testing for high sensitivity, high specificity and high repeatability. Therefore, the development of a new type of magnetic sphere complex to solve the problem of nonspecific adsorption in whole blood testing has become a technical problem that needs to be solved urgently in the current biomedical testing field. Summary of the invention

[0005] The main purpose of the present invention is to provide an immunological detection method for an analyte in a whole blood sample, a magnetic microsphere and a magnetic microsphere-protein complex, so as to solve the problem of low detection accuracy of whole blood samples in the prior art.

[0006] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, there is provided an immunological detection method for an analyte in a whole blood sample, the above-mentioned immunological detection method comprising: a) mixing and incubating the whole blood sample and an immunodetection reagent to obtain an incubation complex; wherein the immunodetection reagent comprises a signal molecule and / or a magnetic microsphere-protein complex, the magnetic microsphere-protein complex comprises a magnetic microsphere and a first affinity protein coated on the magnetic microsphere, the first affinity protein is a protein that can specifically bind to the analyte, and the surface of the magnetic microsphere contains methyl acrylate; b) performing signal detection on the incubation complex, and obtaining the amount of the analyte through the signal intensity.

[0007] Furthermore, the particle size of the magnetic microspheres is (1-8)±0.9 μm.

[0008] Furthermore, the magnetic microsphere-protein complex is obtained by coupling the magnetic microspheres containing carboxyl groups on the surface and the first affinity protein; preferably, the surface group content of the magnetic microspheres is 10-100 μmol / g; preferably, the surface group is carboxyl.

[0009] Furthermore, the magnetic microspheres and the first affinity protein are coupled via a cross-linking agent to form a magnetic microsphere-protein complex; preferably, the cross-linking agent is DMTMM, and preferably, the feed ratio of DMTMM to the magnetic microspheres is (1-4):1.

[0010] Furthermore, the surface of the magnetic microspheres is blocked with polymerized BSA; preferably, the concentration of the polymerized BSA during blocking is 0.3%-2% (w / v).

[0011] Further, a) includes: mixing a whole blood sample with a polyethylene glycol diluent to obtain a mixed system, and then mixing and incubating the mixed system with an immunodetection reagent to obtain an incubation complex; preferably, the polyethylene glycol diluent includes a first polyethylene glycol diluent or a second polyethylene glycol diluent, and the whole blood sample includes a first whole blood sample or a second whole blood sample, and correspondingly, a) includes a1) or a2); a1) includes: mixing the first whole blood sample with the first polyethylene glycol diluent to obtain a first mixed system, and then mixing and incubating the first mixed system with an immunodetection reagent to obtain Incubation complex; in the first mixed system, the volume of the first whole blood sample accounts for 50%-90%, and the molecular weight of the polyethylene glycol in the first polyethylene glycol diluent is 8000-20000Da; a2) includes: mixing the second whole blood sample with the second polyethylene glycol diluent to obtain a second mixed system, and then mixing and incubating the second mixed system with an immunoassay reagent to obtain an incubation complex; in the second mixed system, the volume of the second whole blood sample accounts for 5%-20%, and the molecular weight of the polyethylene glycol in the second polyethylene glycol diluent is 800-5000Da.

[0012] Furthermore, the mass content of polyethylene glycol in the first polyethylene glycol diluent is 0.05-1wt%; the mass content of polyethylene glycol in the second polyethylene glycol diluent is 0.1-4wt%; preferably, the whole blood sample includes a fresh blood sample or an old blood sample.

[0013] Furthermore, the immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

[0014] Furthermore, the immunological detection method is a chemiluminescent immunoassay; the signal molecule includes a second affinity protein labeled with a luminescent marker.

[0015] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a magnetic microsphere is provided, wherein the surface of the magnetic microsphere contains methacrylate, preferably, the particle size of the magnetic microsphere is (1-8)±0.9μm; preferably, the particle size of the magnetic microsphere is (1-2)±0.9μm, preferably, the surface group content of the magnetic microsphere is 10-100μmol / g; preferably, the surface group is carboxyl; preferably, the surface of the magnetic microsphere is enclosed with poly-BSA.

[0016] In order to achieve the above object, according to the third aspect of the present invention, a magnetic microsphere-protein complex is provided. The magnetic microsphere-protein complex comprises the magnetic microspheres and affinity proteins coated on the magnetic microspheres.

[0017] By applying the technical solution of the present invention, in the immunological detection method of the analyte in the whole blood sample, the analyte is captured by a magnetic microsphere-protein complex coated with a first affinity protein to obtain an incubation complex, thereby realizing the subsequent detection of the amount of the analyte. The surface of the magnetic microspheres used in the above immunological detection method contains methyl acrylate, which can reduce the capture of non-analytes in the whole blood sample, effectively reduce nonspecific binding, ensure the specificity and efficiency of the immune response, enhance the accuracy and stability of the detection signal, and provide an innovative solution for the accurate detection of whole blood samples. DETAILED DESCRIPTION

[0018] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0019] Terminology explanation:

[0020] Magnetic microspheres: Magnetic microspheres are composite microspheres made by combining magnetic inorganic particles (such as ferroferric oxide, γ-Fe2O3, etc.) with organic polymer materials (such as polystyrene, polyacrylic acid, etc.), and are referred to as magnetic microspheres.

[0021] As mentioned in the background technology, the detection of whole blood samples in the prior art is affected by nonspecific adsorption, and the detection accuracy is low. In the present application, the inventor unexpectedly discovered that the magnetic microspheres with methacrylate bound to the surface have less adsorption of interfering components in whole blood samples, and can improve the accuracy and stability of immunological detection methods by reducing nonspecific adsorption. Based on this, the inventor has developed a new immunological detection method for the analyte in whole blood samples, and then proposed a series of protection schemes of the present application.

[0022] In a first typical embodiment of the present application, an immunological detection method for an analyte in a whole blood sample is provided, the immunological detection method comprising: a) mixing and incubating the whole blood sample and an immunodetection reagent to obtain an incubation complex; wherein the immunodetection reagent comprises a signal molecule and / or a magnetic microsphere-protein complex, the magnetic microsphere-protein complex comprises a magnetic microsphere and a first affinity protein coated on the magnetic microsphere, the first affinity protein is a protein that can specifically bind to the analyte, and the surface of the magnetic microsphere contains methyl acrylate; b) performing signal detection on the incubation complex, and obtaining the amount of the analyte through the signal intensity.

[0023] In the above-mentioned immunological detection method, a magnetic microsphere-protein complex is provided, including: 1) magnetic microspheres containing methyl methacrylate on the surface, 2) a first affinity protein coated on the magnetic microspheres for specific binding to the object to be detected. The above-mentioned first affinity protein includes but is not limited to antibodies or antigens. The magnetic microsphere-protein complex is mixed and incubated with a whole blood sample, and the specific binding of the first affinity protein to the object to be detected is used to obtain an incubation complex, that is, the incubation complex includes: magnetic microspheres, the first affinity protein and the object to be detected. If the immunodetection reagent also includes a signal molecule, the signal molecule can also be bound to the incubation mixture by methods commonly used in the prior art such as specific binding.

[0024] Furthermore, the incubation complex is separated by the magnetism of the magnetic microspheres, and the signal is detected, and the amount of the analyte can be detected by the signal intensity. The above signal intensity includes but is not limited to the intensity of chemiluminescence, fluorescence, radioactivity and other signals.

[0025] In the immunological detection method of the present application, those skilled in the art can flexibly select any immunological detection method known in the prior art, including but not limited to enzyme-linked immunosorbent assay (ELISA); immunofluorescence technique (IFA), radioimmunoassay (RIA) and the like.

[0026] In a preferred embodiment, the particle size of the magnetic microspheres is (1-8)±0.9 μm. In a more preferred embodiment, the particle size of the magnetic microspheres is (1-2)±0.9 μm.

[0027] The magnetic microspheres of this specific particle size can provide a suitable surface area, thereby ensuring the coating amount of the first affinity protein and improving the detection efficiency; and ensuring the stability and repeatability of the signal, and are suitable for various immunological detection methods and automated detection equipment in the prior art.

[0028] The average particle size of the magnetic microspheres is 1 micron to 8 microns, and "±0.9 μm" means that the error of the particle size in the magnetic microspheres is ±0.9 μm. That is, the average particle size of each magnetic microsphere in the magnetic microspheres fluctuates by 0.9 microns. In a preferred embodiment, the magnetic microsphere-protein complex is obtained by coupling the magnetic microspheres having carboxyl groups on the surface and the first affinity protein; preferably, the surface group content of the magnetic microspheres is 10-100 μmol / g; preferably, the surface group is a carboxyl group.

[0029] The high density of carboxyl groups can bind affinity proteins more firmly, reduce nonspecific binding, and improve the accuracy and reliability of detection, especially in high-throughput detection.

[0030] Magnetic microspheres, as core carriers, have carboxyl (-COOH) groups on their surface, which have the ability to covalently couple with the first affinity protein, ensuring stable binding of the protein. The preferred surface group content of the magnetic microspheres is between 10 and 100 μmol / g. The group density within this range can not only effectively load a large amount of protein, but also avoid the protein steric hindrance effect that may be caused by overly dense group arrangement, ensuring the maximization of protein function.

[0031] In a preferred embodiment, the magnetic microspheres and the first affinity protein are coupled by a cross-linking agent to form a magnetic microsphere-protein complex; preferably, the cross-linking agent is DMTMM, and preferably, the feed ratio of DMTMM to magnetic spheres is (1-4):1.

[0032] The use of a cross-linking agent can help the groups (such as carboxyl groups) on the magnetic microspheres to better couple with the first affinity protein to form a magnetic microsphere-protein complex. Preferably, the cross-linking agent is DMTMM (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, CAS No. 3945-69-5).

[0033] The principle of using DMTMM as a cross-linking agent to connect magnetic microspheres and first affinity proteins includes: first, the carboxyl group reacts with DMTMM to form an active ester, releasing a molecule of N-methylmorpholine (NMM). Further, the obtained active ester is highly reactive and can be nucleophilically attacked by amines, alcohols or other nucleophiles (groups) derived from the first affinity protein, releasing a molecule of 4,6-dimethoxy-1,3,5-triazine-2-ol to form a stable amide or carboxylic acid derivative. The reaction structure is shown below:

[0034]

[0035] The use of the above cross-linking agent to connect the magnetic microspheres to the first affinity protein can ensure the uniformity and stability of the coupling while maintaining the biological activity of the protein, and avoid nonspecific adsorption and signal loss caused by protein shedding during the detection process. By using DMTMM as a cross-linking agent, the magnetic microsphere-protein complex of the present invention has shown significant beneficial effects in the field of whole blood detection. It effectively solves the protein shedding and nonspecific adsorption problems encountered by traditional magnetic ball complexes in whole blood sample detection, and can improve the sensitivity and specificity of detection.

[0036] Preferably, in the process of using DMTMM as a cross-linking agent for the reaction, the concentration of DMTMM is preferably 3-20 mg / mL.

[0037] In a preferred embodiment, the surface of the magnetic microspheres is blocked with polymerized BSA (polymerized bovine serum albumin, Roche, catalog number: 11866737103); preferably, the concentration of the polymerized BSA used in blocking is 0.3%-2% (w / v).

[0038] In the present invention, polymeric bovine serum albumin (BSA) is preferably used for the sealing treatment of the surface of magnetic microspheres, and its concentration is controlled in the range of 0.3% to 2% (w / v). As a non-specific protein, the blocking effect of polymeric BSA is to shield the active sites on the surface of magnetic microspheres that are not involved in antibody coupling, thereby reducing the adsorption of blood cell components and other non-specific molecules in the subsequent detection process. With its rich amino acid sequence and good stability, polymeric BSA can evenly cover the surface of magnetic spheres to form a physical barrier, effectively blocking the direct contact of non-target proteins, reducing the chance of non-specific binding, thereby optimizing the detection conditions and improving the specificity of whole blood sample detection. Compared with the magnetic microspheres sealed with BSA, the above-mentioned magnetic microspheres sealed with polymeric BSA have better effects in the above-mentioned immunological detection method, can improve the accuracy and sensitivity of the detection, and improve the repeatability of the detection results, making the detection signal more stable and reliable.

[0039] Poly-BSA (Bovine Serum Albumin) refers to the polymer form of bovine serum albumin. Bovine serum albumin is a globulin containing 583 amino acid residues and a molecular weight of approximately 66.43 kDa. In biochemical experiments, bovine serum albumin is widely used as a stabilizer, carrier protein or as a control group for other experiments due to its rich amino acid composition and relatively stable structure. "Polymer" in this application refers to a molecule formed by the combination of monomers. For bovine serum albumin, a polymer means a larger molecular complex formed by multiple bovine serum albumin molecules connected together by non-covalent bonds (such as hydrogen bonds, van der Waals forces, etc.). The formation of this polymer will affect its physicochemical properties and biological functions.

[0040] In a preferred embodiment, a) includes: mixing a whole blood sample with a polyethylene glycol diluent to obtain a mixed system, and then mixing and incubating the mixed system with an immunoassay reagent to obtain an incubation complex; preferably, the polyethylene glycol diluent includes a first polyethylene glycol diluent or a second polyethylene glycol diluent, and the whole blood sample includes a first whole blood sample or a second whole blood sample, and correspondingly, a) includes a1) or a2); a1) includes: mixing the first whole blood sample with the first polyethylene glycol diluent to obtain a first mixed system, The first mixed system is then mixed and incubated with an immunodetection reagent to obtain an incubation complex; in the first mixed system, the volume proportion of the first whole blood sample is 50%-90% (including but not limited to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%), and the molecular weight of the polyethylene glycol in the first polyethylene glycol diluent is 8000-20000Da (including but not limited to 8000Da, 9000Da, 10000Da, 11000Da, 1 2000Da, 13000Da, 14000Da, 15000Da, 16000Da, 17000Da, 18000Da, 19000Da or 20000Da); a2) comprising: mixing a second whole blood sample with a second polyethylene glycol diluent to obtain a second mixed system, and then mixing and incubating the second mixed system with an immunoassay reagent to obtain an incubation complex; in the second mixed system, the volume of the second whole blood sample accounts for 3%-20% (including but not limited to 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%), and the molecular weight of the polyethylene glycol in the second polyethylene glycol dilution is 800-5000Da (including but not limited to 800Da, 1000Da, 1500Da, 200Da, 2500Da, 3000Da, 3500Da, 4000Da, 4500Da or 5000Da).

[0041] In the above immunological detection method, preferably, before mixing the whole blood sample with the immunodetection reagent, the whole blood sample is first diluted with polyethylene glycol diluent to obtain a mixed system, and then the mixed system is mixed and incubated with the immunodetection reagent.

[0042] Furthermore, different pre-test treatments are performed for items with large sample volumes (the sample volume of the whole blood sample exceeds 50% of the mixed system) and items with small sample volumes (the sample volume of the whole blood sample is less than 20% of the mixed system).

[0043] For projects with large sample volumes (including but not limited to high-sensitivity troponin I (hs-CtnI) detection), a diluent containing high molecular weight (8000-20000Da) polyethylene glycol is added to the first whole blood sample. The large molecule PEG can effectively promote the rapid sedimentation of blood cells in the reaction system through its strong hydrophilicity and steric hindrance effect, thereby reducing the direct contact between blood cells and detection reagents, significantly reducing the interference of blood cell aggregation, rupture and nonspecific adhesion on the immune response, and improving the consistency of whole blood and plasma detection.

[0044] For projects with small sample volumes (including but not limited to procalcitonin (PCT) detection), a diluent containing small molecular weight (800-5000Da) polyethylene glycol is added to the second whole blood sample. The small molecule PEG will not significantly affect the sedimentation effect of blood cells while ensuring appropriate dispersion of blood cells, thereby avoiding a decrease in detection sensitivity caused by excessive sedimentation of blood cells, thereby maintaining the accuracy of the test results.

[0045] In actual whole blood sample testing, the components in the whole blood sample can be flexibly adjusted according to the specific operations of the test items, including but not limited to diluting the whole blood sample using reagents such as polyethylene glycol. The addition of such reagents does not affect the binding of the magnetic microsphere-protein complex with the analyte in the present application, nor does it affect the subsequent detection of signals related to the analyte.

[0046] The "first whole blood sample" and "second whole blood sample" in this application are only used to distinguish the whole blood samples used in large sample addition projects and small sample addition projects. The above-mentioned "first whole blood sample" and "second whole blood sample" include but are not limited to blood samples from the same individual or different individuals, or whole blood samples obtained from the same individual in the same batch or different batches of blood collection. The "first whole blood sample" and "second whole blood sample" are each independently selected from a fresh blood sample or an old blood sample.

[0047] In a preferred embodiment, the mass content of polyethylene glycol in the first polyethylene glycol diluent is 0.05-1wt%; the mass content of polyethylene glycol in the second polyethylene glycol diluent is 0.1-4wt%; preferably, the whole blood sample includes a fresh blood sample or an old blood sample.

[0048] Fresh blood samples (referred to as fresh samples) refer to whole blood samples that have been stored for no more than 8 hours after blood collection, and old blood samples (referred to as old blood samples) refer to whole blood samples that have been stored for more than 5 days at 2-8°C. The above-mentioned immunological detection method can be used to quickly detect both fresh samples and old blood samples, and the test results are accurate and reliable.

[0049] The above-mentioned immunological detection method can realize accurate detection of old blood samples, which is convenient for accurate detection of old blood samples in the fields of retrospective research, forensic identification, stored blood quality detection, drug research, genetic research, etc., avoiding the limitations of detection caused by the need to use fresh blood samples for testing.

[0050] In a preferred embodiment, the immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

[0051] In a preferred embodiment, the immunological detection method is a chemiluminescent immunoassay; the signal molecule includes a second affinity protein labeled with a luminescent marker.

[0052] Preferably, the second affinity protein includes but is not limited to antigens and / or antibodies, and the second affinity protein is connected to the incubation complex by binding to, including but not limited to, magnetic microspheres, the first affinity protein, etc. The luminescent marker realizes the generation of light signals in the subsequent signal detection of the incubation mixture.

[0053] In a preferred embodiment, the polyethylene glycol diluent further includes, but is not limited to, any one or more of the following: pH buffer, NaCl, bovine serum albumin, glycine or a preservative; preferably, the pH buffer includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or Tris-HCl.

[0054] In a preferred embodiment, the luminescent marker includes, but is not limited to, any one or more of the following: isoluminol and its derivatives, acridinium ester and its derivatives, or terpyridine ruthenium.

[0055] In the above-mentioned immunological detection method of the analyte in the whole blood sample, technicians in this field can flexibly select detection methods, detection reagents, detection parameters, etc. known in the prior art to conduct experiments, and can realize this immunological detection, realize the detection of the analyte in the whole blood sample, and significantly improve the detection efficiency and accuracy of the components to be detected in the whole blood sample.

[0056] In a second typical embodiment of the present application, a magnetic microsphere is provided, wherein the surface of the magnetic microsphere contains methacrylate; preferably, the particle size of the magnetic microsphere is (1-8) ± 0.9 μm; preferably, the surface group content of the magnetic microsphere is 10-100 μmol / g; preferably, the surface group is carboxyl; preferably, the surface of the magnetic microsphere is encapsulated with poly-BSA.

[0057] In a third typical embodiment of the present application, a magnetic microsphere-protein complex is provided. The magnetic microsphere-protein complex comprises the magnetic microspheres and affinity proteins coated on the magnetic microspheres.

[0058] The above affinity protein includes but is not limited to the above first affinity protein capable of specifically binding to the analyte.

[0059] The magnetic microspheres and / or magnetic microsphere-protein complexes can be used for immunological detection of analytes in whole blood samples, or for preparing relevant reagents for immunological detection of whole blood samples. The magnetic microspheres and / or magnetic microsphere-protein complexes can be used to accurately detect analytes in whole blood samples, and reduce detection deviations and even detection errors caused by nonspecific adsorption of magnetic spheres and interfering components in whole blood samples.

[0060] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0061] Example 1 Preparation of magnetic spheres

[0062] 1. Preparation method of oleic acid coated magnetic particles:

[0063] Oleic acid-coated magnetic microparticles were prepared by coprecipitation. Under continuous nitrogen purge, 23.5 g FeCl3·6H2O and 8.6 g FeCl2·4H2O were dissolved in 500 mL deionized water, the solution temperature was maintained at 85 °C, and then 27.8 mL NH4OH was quickly added. Oleic acid was then gradually added dropwise to the solution within 10 min. The stirring speed was controlled at 600 rpm until a blocky magnetite (Fe3O4) gel appeared. The obtained magnetite gel was used as an aggregate of OMP, cooled to room temperature, washed 8 times with deionized water, and stored in a vacuum after removing the supernatant.

[0064] 2. Preparation method of magnetic ball 0

[0065] 1. Mix 2 g of benzoyl peroxide, 95 mL of styrene, 9 mL of divinylbenzene, 33 mL of acrylic acid and 20 g of oleic acid-coated magnetic particles to form an organic phase, stir vigorously, and perform ultrasonic treatment for 10 minutes to ensure that the oleic acid-coated magnetic particles are evenly dispersed.

[0066] 2. The aqueous phase consists of 25 g polyvinyl alcohol-1788, 30 g sodium chloride and 1000 mL deionized water. The aqueous phase is added to the organic phase and the two phases are mixed at a stirring speed of 600 rpm and continuous nitrogen purge. The temperature of the synthetic solution is maintained at 80°C for 15 hours.

[0067] 3. After the synthesis process is completed, the magnetic microspheres 0 are separated from the solution using a magnet and washed with deionized water for 5 times to remove the stabilizer and other impurities attached to the surface of the particles.

[0068] 3. Preparation method of magnetic ball 1

[0069] 1. Mix 3 g of benzoyl peroxide, 36 mL of methyl methacrylate, 20 mL of styrene, 8 mL of divinylbenzene, 38 mL of glycidyl methacrylate, 30 mL of cyclohexane, 16 mL of acrylic acid and 20 g of the oleic acid-coated magnetic particles prepared above to form an organic phase, stir vigorously, and perform ultrasonic treatment for 10 minutes to ensure that the oleic acid-coated magnetic particles are evenly dispersed.

[0070] 2. The aqueous phase consists of 10 g polyvinyl alcohol-1788, 15 g sodium chloride and 1000 mL deionized water. The aqueous phase is added to the organic phase and the two phases are mixed at a stirring speed of 600 rpm and continuous nitrogen purge. The temperature of the synthetic solution is increased from 45°C to 55°C within 1 hour, then maintained at 60°C for 2 hours, and finally maintained at 70°C and 80°C for 1 hour each.

[0071] 3. After the synthesis process is completed, the magnetic ball 1 is separated from the solution by a magnet and washed with deionized water for 5 times to remove the stabilizer and other impurities attached to the surface of the particles.

[0072] 4. Preparation method of magnetic ball 2

[0073] 1. Mix 3 g of benzoyl peroxide, 32 mL of methyl methacrylate, 18 mL of styrene, 6 mL of divinylbenzene, 45 mL of glycidyl methacrylate, 30 mL of cyclohexane, 15 mL of acrylic acid and 20 g of the oleic acid-coated magnetic particles prepared above to form an organic phase, stir vigorously, and perform ultrasonic treatment for 10 minutes to ensure that the oleic acid-coated magnetic particles are evenly dispersed.

[0074] 2. The aqueous phase consists of 10 g polyvinyl alcohol-1788, 15 g sodium chloride and 1000 mL deionized water. The aqueous phase is added to the organic phase and the two phases are mixed at a stirring speed of 600 rpm and continuous nitrogen purge. The temperature of the synthetic solution is increased from 45°C to 55°C within 1 hour, then maintained at 60°C for 2 hours, and finally maintained at 70°C and 80°C for 1 hour each.

[0075] 3. After the synthesis process is completed, the magnetic ball 2 is separated from the solution by a magnet and washed with deionized water for 5 times to remove the stabilizer and other impurities attached to the surface of the particles.

[0076] 5. Preparation method of magnetic ball 3

[0077] 1. Mix 3 g of benzoyl peroxide, 40 mL of methyl methacrylate, 18 mL of styrene, 8 mL of divinylbenzene, 30 mL of glycidyl methacrylate, 30 mL of cyclohexane, 13 mL of acrylic acid and 20 g of the oleic acid-coated magnetic particles prepared above to form an organic phase, stir vigorously, and perform ultrasonic treatment for 10 minutes to ensure that the oleic acid-coated magnetic particles are evenly dispersed.

[0078] 2. The aqueous phase consists of 10 g polyvinyl alcohol-1788, 15 g sodium chloride and 1000 mL deionized water. The aqueous phase is added to the organic phase and the two phases are mixed at a stirring speed of 600 rpm and continuous nitrogen purge. The temperature of the synthetic solution is increased from 45°C to 55°C within 1 hour, then maintained at 60°C for 2 hours, and finally maintained at 70°C and 80°C for 1 hour each.

[0079] 3. After the synthesis process is completed, the magnetic balls 3 are separated from the solution by a magnet and washed with deionized water for 5 times to remove the stabilizer and other impurities attached to the surface of the particles.

[0080] Embodiment 2 Magnetic ball detection method

[0081] 1. Particle size detection method

[0082] Ultrasonic mixing of the magnetic spheres to be tested was performed for 5 minutes; 1.5 mL of the magnetic spheres were taken into a 20 mL vial, and 0.2 mL of Tween was added into the vial, and ultrasonic mixing was performed at 60% power for 1 minute; and detection was performed using a Malvern particle size analyzer.

[0083] 2. Carboxyl content detection method

[0084] 1) Mix the magnetic spheres to be tested under ultrasound for 5 minutes;

[0085] 2) Take 0.5g of magnetic ball solution into a 250mL beaker, add 3mL of 2M hydrochloric acid solution to the beaker, add 150mL of purified water, ultrasonically mix at 100% power for 1min, and settle on a magnet with a magnetic field of 300-500 millitesla (mT) for 30min to remove the supernatant;

[0086] 3) Add 150 mL of purified water again and repeat the washing twice;

[0087] 4) Add 200 mL of purified water to the beaker and mix by ultrasonic stirring for 1 min;

[0088] 5) Test with an acid-base titrator.

[0089] The parameters of the four magnetic balls were obtained by the above detection method, as shown in Table 1 below:

[0090] Table 1

[0091]

[0092] Example 3

[0093] 1. Preparation of magnetic sphere complex:

[0094] The above 20 mg magnetic microspheres were mixed with 5 mL of pH 5.0 phosphate buffer and stirred for 2 to 3 minutes. The magnetic microspheres were adsorbed by an external magnetic field and the supernatant was removed. The washing step was repeated three times. After washing, the magnetic microspheres were resuspended to 20 mg / mL by adding pH 5.0 phosphate buffer. Cross-linking agent and blocking agent were added to the above suspension at the concentration shown in Table 2 and mixed evenly. Finally, the magnetic microspheres and antibodies (antibodies produced by Xinye Biotechnology) were mixed evenly at a mass ratio of 1 mg:10 μg. The mixture was placed in a constant temperature shaker at 40°C and shaken for reaction for 2 hours. After the reaction was completed, the magnetic microspheres were magnetically separated and washed three times with buffer, and suspended to 20 mg / mL with a magnetic microsphere diluent for storage.

[0095] Table 2

[0096]

[0097]

[0098] The magnetic sphere complex prepared as above is stored in a diluent, wherein the components of the diluent are the liquid components of the magnetic sphere preservation solution in the PCT kit produced by Xinye Biotechnology, including buffer substances 0.04% potassium dihydrogen phosphate, 0.4% disodium hydrogen phosphate, 0.8% NaCl, 0.5% bovine serum albumin, 1M glycine, 0.5mg / mL sheep anti-human IgG, and preservative 0.15% Proclin300.

[0099] 2. Sample preparation:

[0100] A fresh normal human whole blood sample was collected and numbered as sample. The sample was divided into three blood collection tubes, one of which was not processed and numbered as sample F; one was processed with ultrasonic wave for 20 minutes and numbered as sample S; and the other was centrifuged to obtain plasma for later use and numbered as sample P.

[0101] Repeat the above steps to collect 9 sets of samples for later use.

[0102] 3. Sample F and Sample S Testing

[0103] The PCT kit produced by Xinye Biotechnology was used for detection. The magnetic ball reagent was replaced by the magnetic ball reagent prepared in the experimental group of step 1. The sample diluent and marker components in the kit were used to perform the detection in the chemiluminescence immunoassay analyzer MAGLUMI X3 produced by Xinye Biotechnology. The detection principle was: chemiluminescence immunoassay sandwich method.

[0104] A PCT antibody (capture antibody) is used to coat magnetic microspheres, and another PCT antibody (detection antibody) is labeled with ABEI (luminescent agent, N-(4-aminobutyl)-N-ethylisoluminol). The sample, luminescent marker, buffer and magnetic microspheres are mixed and incubated together. The analyte in the sample forms an immune complex with the PCT antibody labeled with ABEI and the PCT antibody on the magnetic microspheres. After incubation, the unbound substances are removed by magnetic separation and washing. Finally, the substrate solution for the fully automatic immunoassay system is added to start the chemiluminescent reaction and generate a light signal. The relative light intensity (RLU) measured by the photomultiplier tube is proportional to the PCT concentration in the sample.

[0105] The detection steps are as follows: add 20 μL of sample to the reaction cup, add 20 μL of magnetic ball component, 50 μL of buffer solution, 50 μL of luminescent marker working solution, add them to the reaction cup and mix well, incubate at 37°C for 14 minutes after mixing, so that the target analyte in the sample to be tested forms a double antibody sandwiched ternary immune complex with a specific antibody coated with magnetic balls in the first reagent and another specific antibody labeled with ABEI, and remove unbound substances by magnetic separation and washing after incubation. Finally, add hydrogen peroxide and sodium hydroxide to the system to excite the marker to emit light, detect the light signal, and obtain RLU.

[0106] The test results are shown in Tables 3 and 4. The control group and experimental groups 1-4 represent whole blood sample tests using the magnetic balls prepared in Table 2 above.

[0107] Table 3 PCT detection deviation results of samples F and S

[0108] sample Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 Experimental Group 4 serial number deviation deviation deviation deviation deviation Mean absolute deviation 122％ 6.49％ 7.23％ 9.14％ 4.55％ sample Experimental Group 5 Experimental Group 6 Experimental Group 7 Experimental Group 8 Experimental Group 9 serial number deviation deviation deviation deviation deviation Mean absolute deviation 4.93％ 3.43％ 3.29％ 2.34％ 2.95％

[0109] In Table 3, Deviation=(Sample S RLU−Sample F RLU) / Sample F RLU×100%.

[0110] The test was repeated 10 times for each sample and the CV value was calculated. The results are shown in Table 4 below.

[0111] Table 4: Verification of test repeatability of samples F and S

[0112] Sample F Control group Experimental Group 6 Experimental Group 8 Experimental Group 9 Average CV (%) 11.34 4.26 1.91 3.24 Sample S Control group Experimental Group 6 Experimental Group 8 Experimental Group 9 Average CV (%) 19.42 6.57 2.99 7.37

[0113] The results showed that when the magnetic balls in the experimental group were used to detect sample F, the RLU of the normal whole blood sample was significantly reduced compared with 122% of the control group, and the RLU deviations of the detected sample S (more cell debris) and sample F were within 10%, indicating that the magnetic balls upgraded by this scheme can reduce the interference of blood cell debris on the immune response, and further using DMTMM as a cross-linking agent can reduce the non-specific adsorption of the magnetic ball complex in the whole blood sample reaction system; further, the RLU deviation of the experimental group 8 detected samples S and sample F was only 2.34%, indicating that the DMTMM cross-linking agent and the poly BSA blocking agent The joint use can further improve the whole blood anti-interference ability of the modified magnetic spheres, and the deviations between experimental group 9 and experimental group 6 are not much different, indicating that DMTMM alone also has a good effect. It is speculated that DMTMM has a certain blocking effect in addition to being a cross-linking agent; and according to the results of the repeatability of whole blood sample detection in Table 4, the repeatability CV of sample S detected in experimental groups 6, 8, and 9 is all within 8%. The above results comprehensively show that the use of this scheme to modify the magnetic spheres and further use of DMTMM cross-linking agent or poly-BSA blocking agent to treat the magnetic spheres can greatly reduce the level of nonspecific adsorption of whole blood samples and improve the accuracy of whole blood detection.

[0114] In the sample S obtained by ultrasonic crushing, the whole blood sample was ultrasonically crushed to simulate the detection environment in which the blood cells were ruptured and the contents were released under extreme conditions. The magnetic balls in the prior art were used for detection, and it was found that the light intensity deviation between the crushed blood sample and the normal blood sample could reach up to 122%. When the magnetic balls in this scheme were used for detection, the deviation of the two samples was greatly reduced, indicating that the magnetic balls in this scheme have a strong ability to resist nonspecific adsorption. Using DMTMM as a cross-linking agent can further reduce the nonspecific adsorption of the magnetic ball complex, and the deviation is further reduced after blocking with poly-BSA.

[0115] 4. Sample F and Sample P Testing

[0116] The plasma sample values ​​were tested using a Roche immunoassay analyzer and a matching PCT chemiluminescence kit (Cat. No.: (240)09318712190) and compared with the test results of sample F of the present application. The whole blood sample was tested by HCT, and the test results were converted to the concentration values ​​of the target substance in plasma and then the absolute deviation was calculated with the Roche test results.

[0117] Plasma concentration = whole blood test concentration / (1-HCT);

[0118] HCT was detected by spectrophotometry, and the calculation formula was:

[0119]

[0120] Absolute deviation = |plasma sample test value - whole blood sample test value| / plasma sample test value.

[0121] The test results are shown in Table 5.

[0122] Table 5:

[0123] sample Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 Experimental Group 4 Mean absolute deviation 20.3％ 7.8％ 8.65％ 8.96％ 4.98％ sample Experimental Group 5 Experimental Group 6 Experimental Group 7 Experimental Group 8 Experimental Group 9 Mean absolute deviation 4.56％ 3.89％ 4.12％ 2.34％ 3.66％

[0124] The results showed that the magnetic sphere complex prepared by this scheme could significantly improve the consistency of whole blood and plasma test results. The deviation between the magnetic sphere complex prepared by DMTMM cross-linker and Roche plasma test results was less than 5%, which had high accuracy.

[0125] Example 4

[0126] 1. Preparation of sample diluent

[0127] The sample diluent components of experimental group 9 included 50 mM Tris buffer, 0.2% NaCl, 0.5% bovine serum albumin, and 0.15% Proclin 300 preservative. PEG of different molecular weights was further added at the concentrations shown in experimental groups 10-15 to verify the deviation of fresh blood samples and old blood samples from plasma.

[0128] Experimental group 10: PEG with MW (molecular weight) = 800 was added to the sample dilution of experimental group 9 to a final PEG concentration of 0.1 wt%;

[0129] Experimental group 11: PEG with MW = 4000 was added to the sample dilution of experimental group 9 to a final PEG concentration of 0.1 wt%;

[0130] Experimental group 12: PEG with MW = 5000 was added to the sample dilution of experimental group 9 to a final PEG concentration of 0.1 wt%;

[0131] Experimental group 13: PEG with MW = 4000 was added to the sample dilution of experimental group 9 to a final PEG concentration of 2 wt%;

[0132] Experimental group 14: PEG with MW=4000 was added to the sample dilution solution of experimental group 9 to a final PEG concentration of 4 wt%.

[0133] 2. Sample Preparation

[0134] Collect 1 fresh whole blood sample (sample F), and divide each sample F into 2 blood collection tubes. One sample is centrifuged to extract plasma for later use and the PCT concentration in plasma is tested. The other sample is not processed and the PCT concentration in whole blood is tested.

[0135] Take another old sample (sample S) that has been stored for 5 days and divide it into two blood collection tubes. Centrifuge it according to the above steps to obtain plasma for later use. The other sample is tested directly without treatment.

[0136] Repeat the above steps and collect 9 sets of samples for later use.

[0137] 3. Sample testing

[0138] The PCT values ​​in the plasma and whole blood of the above samples were detected according to the steps in Example 3, and the deviations were calculated. The results are shown in Table 6 below.

[0139] Table 6:

[0140] Sample F Experimental Group 9 Experimental Group 10 Experimental Group 11 Experimental Group 12 Experimental Group 13 Experimental Group 14 Mean absolute deviation 3.76％ 3.54％ 3.21％ 3.09％ 2.85％ 3.16％ Sample S Experimental Group 9 Experimental Group 10 Experimental Group 11 Experimental Group 12 Experimental Group 14 Experimental Group 15 Mean absolute deviation 5.97％ 5.21％ 4.96％ 4.87％ 4.21％ 4.36％

[0141] The results show that when the amount of whole blood sample is less than 20% of the volume ratio of the detection system, adding polyethylene glycol with a molecular weight of 800-5000Da to the sample diluent can further effectively reduce the detection deviation of fresh whole blood samples, old whole blood samples and plasma.

[0142] Example 5

[0143] The detection was performed using the Xinye Biochemiluminescent Immunoassay Maglumi X3 and the matching self-produced hs-cTnI luminescent kit. The sample diluent in the kit was replaced with a diluent added with polyethylene glycol, and the magnetic sphere and protein complex in the first reagent were replaced with those prepared according to Experimental Group 9 in Example 3. The consistency of the whole blood sample and the plasma sample, as well as the repeatability of the detection, were detected. The detection steps were as follows:

[0144] 1. Preparation of diluent:

[0145] Control group 2: The composition was a basic dilution solution consisting of 50 mM Tris buffer, 0.2% NaCl, 0.5% bovine serum albumin, and 0.015% Proclin 300 preservative.

[0146] Experimental group 16: PEG with MW (molecular weight, Da) = 8000 was added to the basic diluent of control group 2 to a final concentration of PEG of 0.1 wt%;

[0147] Experimental group 17: PEG with MW = 20,000 was added to the basic dilution of control group 2 to a final concentration of PEG of 0.1 wt%;

[0148] Experimental group 18: PEG with MW = 10000 was added to the basic dilution of control group 2 to a final concentration of PEG of 0.05 wt%;

[0149] Experimental group 19: PEG with MW=10000 was added to the basic diluent of control group 2 to a final concentration of PEG of 1 wt%.

[0150] 2. Sample preparation:

[0151] Collect 1 fresh whole blood sample (sample F), and divide each sample F into 2 blood collection tubes. One sample is centrifuged (3000 rpm, 5 min) to obtain plasma for later use and to detect the hs-cTnI concentration in plasma. The other sample is not processed and is used to detect the hs-CTnI concentration in whole blood.

[0152] Take another old sample (sample S) that has been stored for 5 days and divide it into two blood collection tubes. Centrifuge it according to the above steps to obtain plasma for later use. The other sample is tested directly without treatment.

[0153] Repeat the above steps and collect 9 sets of samples for later use.

[0154] 3. Detection Reagents

[0155] In addition to the sample diluent, other components of the hs-cTnI luminescence kit (Cat. No.: 130256014M) produced by New Industry Biotechnology were selected to detect the above samples. The other components include:

[0156] The first reagent: a magnetic sphere reagent coated with an antibody that specifically binds to hs-cTnI. The preparation method and coating scheme of the magnetic sphere refer to the method described in Experimental Group 9. The liquid component contains 0.04% potassium dihydrogen phosphate, 0.4% potassium dihydrogen phosphate, 0.8% NaCl, 0.5% bovine serum albumin, and 0.15% Proclin300 preservative;

[0157] The second reagent: a labeling reagent for labeling another antibody of ABEI that specifically binds to hs-cTnI, comprising 70 mM Tris buffer, 0.4% NaCl, 0.5% bovine serum albumin, 1 M glycine, and 0.15% Proclin 300 preservative.

[0158] 4. Sample testing

[0159] The whole blood sample was tested using the Maglumi X3 biochemiluminescence immunoassay analyzer from Xinye. The steps are as follows:

[0160] The whole blood sample in step 2 was tested using the sample diluent configured in the control group and the experimental group and the first and second reagents produced by Xinye Biotechnology. 150 μL of the sample was added to the reaction cup, and the reagent sampling was performed according to the following order of adding samples: the first reagent-the experimental group diluent in step 1-the second reagent, and the samples were added to the reaction cup and mixed together, and incubated at 37°C for 14 minutes after mixing, so that the target analyte in the sample to be tested and the specific antibody coated with the magnetic ball in the first reagent and the other specific antibody labeled with ABEI formed a double antibody sandwiched ternary immune complex, and the unbound substances were removed by magnetic separation and washing after incubation. Finally, hydrogen peroxide and sodium hydroxide, the substrate liquid for the fully automatic immunoassay system, were added to start the chemiluminescent reaction and generate a light signal. The relative light intensity (RLU) measured by the photomultiplier tube; the above steps were repeated to detect the whole blood sample to obtain the RLU value, and the concentration value of the target substance hs-cTnI in the sample was calculated, and the test result was converted to the concentration value of the target substance in plasma by HCT detection, and the conversion formula was the same as the above embodiment.

[0161] The aforementioned plasma samples were tested using Beckman Analyzer Access2 81600N and the supporting reagent hs-CTnI (REF: B52699) to determine the values ​​of the target substances in the plasma.

[0162] Based on the above test results, the absolute deviations of the 9 groups of whole blood samples and plasma samples were calculated in the same way as in the above embodiment.

[0163] The test results are shown in Table 7 below.

[0164] Table 7:

[0165]

[0166]

[0167] The results show that when the amount of whole blood sample exceeds 50% of the volume ratio of the detection system, adding polyethylene glycol with a molecular weight of 8000-20000Da to the sample diluent can further effectively reduce the detection deviation of fresh whole blood samples, old whole blood samples and plasma.

[0168] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the immunological detection method and magnetic microsphere-protein complex provided by the present application improve the sensitivity and specificity of detection by utilizing magnetic microspheres containing methacrylate on the surface. The specific particle size and surface group content of the magnetic microspheres, as well as the surface-enclosed poly-BSA, can further effectively reduce nonspecific binding (adsorption) and enhance the stability of the detection signal. In addition, by adjusting the composition of the polyethylene glycol diluent, the pretreatment of whole blood samples can be optimized, and the detection efficiency and accuracy can be further improved, which is applicable to a variety of detection methods and detection schemes in the prior art. This scheme is particularly suitable for the rapid and accurate detection of fresh or old blood samples, and provides a powerful tool for clinical diagnosis and disease monitoring.

[0169] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An immunological detection method for an analyte in a whole blood sample, characterized in that: The immunological detection method comprises: a) mixing and incubating the whole blood sample and the immunoassay reagent to obtain an incubation complex; Wherein, the immunoassay reagent includes a signal molecule and / or a magnetic microsphere-protein complex, The magnetic microsphere-protein complex comprises a magnetic microsphere and a first affinity protein coated on the magnetic microsphere, The first affinity protein is a protein that can specifically bind to the analyte, and the surface of the magnetic microsphere contains methacrylate; b) performing signal detection on the incubation complex and obtaining the amount of the analyte according to the signal intensity.

2. The immunological detection method according to claim 1, characterized in that: The particle size of the magnetic microspheres is (1-8)±0.9 μm.

3. The immunological detection method according to claim 1, characterized in that: The magnetic microsphere-protein complex is obtained by coupling the magnetic microsphere containing carboxyl groups on the surface with the first affinity protein; Preferably, the surface group content of the magnetic microspheres is 10-100 μmol / g; Preferably, the surface groups are carboxyl groups.

4. The immunological detection method according to any one of claims 1 to 3, characterized in that: The magnetic microspheres and the first affinity protein are coupled via a cross-linking agent to form the magnetic microsphere-protein complex; Preferably, the cross-linking agent is DMTMM.

5. The immunological detection method according to any one of claims 1 to 4, characterized in that: The surface of the magnetic microspheres is enclosed with poly-BSA; 6. The immunological detection method according to any one of claims 1 to 5, characterized in that: The a) comprises: mixing the whole blood sample with a polyethylene glycol diluent to obtain a mixed system, and then mixing and incubating the mixed system with the immunoassay reagent to obtain the incubation complex; Preferably, the polyethylene glycol diluent includes a first polyethylene glycol diluent or a second polyethylene glycol diluent, the whole blood sample includes a first whole blood sample or a second whole blood sample, and correspondingly, a) includes a1) or a2); The a1) includes: Mixing the first whole blood sample with the first polyethylene glycol diluent to obtain a first mixed system, and then mixing and incubating the first mixed system with the immunoassay reagent to obtain the incubation complex; In the first mixed system, the volume proportion of the first whole blood sample is 50%-90%, and the molecular weight of the polyethylene glycol in the first polyethylene glycol diluent is 8000-20000Da; a2) comprises: Mixing the second whole blood sample with the second polyethylene glycol diluent to obtain a second mixed system, and then mixing and incubating the second mixed system with the immunoassay reagent to obtain the incubation complex; In the second mixed system, the volume proportion of the second whole blood sample is 5%-20%, and the molecular weight of the polyethylene glycol in the second polyethylene glycol diluent is 800-5000Da.

7. The immunological detection method according to claim 6, characterized in that: The mass content of polyethylene glycol in the first polyethylene glycol diluent is 0.05-1wt%; The mass content of polyethylene glycol in the second polyethylene glycol diluent is 0.1-4wt%; Preferably, the whole blood sample includes a fresh blood sample or an old blood sample.

8. The immunological detection method according to any one of claims 1 to 7, characterized in that: The immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

9. The immunological detection method according to claim 8, characterized in that: The immunological detection method is the chemiluminescent immunoassay; The signal molecule includes a second affinity protein labeled with a luminescent marker.

10. A magnetic microsphere, characterized in that: The surface of the magnetic microspheres contains methacrylate, Preferably, the particle size of the magnetic microspheres is (1-8) ± 0.9 μm; Preferably, the surface group content of the magnetic microspheres is 10-100 μmol / g; Preferably, the surface group is a carboxyl group; Preferably, the surface of the magnetic microspheres is capped with polymerized BSA.

11. A magnetic microsphere-protein complex, characterized in that: The magnetic microsphere-protein complex comprises the magnetic microsphere according to claim 10 and an affinity protein coated on the magnetic microsphere.

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