Immunological detection method of analyte in whole blood sample, magnetic microsphere and magnetic microsphere-protein complex

Abstract

The application provides an immunological detection method of a target substance in a whole blood sample, a magnetic microsphere and a magnetic microsphere-protein complex. The immunological detection method comprises the following steps: a) mixing and incubating the whole blood sample and an immunological detection reagent to obtain an incubated complex; wherein the immunological detection 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 capable of specifically binding to the target substance, and the surface of the magnetic microsphere contains methacrylate; and b) performing signal detection on the incubated complex to obtain the amount of the target substance through the signal intensity. The application can solve the problem of low detection accuracy of the whole blood sample in the prior art and is suitable for the field of whole blood sample detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of whole blood sample detection, in particular, to an immunological detection method of a to-be-detected substance in a whole blood sample, a magnetic microsphere and a magnetic microsphere-protein complex. BACKGROUND

[0002] Magnetic microspheres have great potential in the biomedical field, especially in vitro diagnosis and other detection, due to their unique magnetic response and surface modifiability. They are combined by organic polymer materials and inorganic magnetic materials, and can be quickly positioned, guided and separated under the action of an external magnetic field. The surface of the magnetic microspheres can be chemically modified to introduce various functional groups, such as hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), amino (-NH2), thiol (-SH), etc. This not only enhances their biocompatibility, but also provides convenience for the subsequent coupling of antibodies or other biomolecules, and is widely used in magnetic separation immunoassay and other in vitro diagnostic techniques.

[0003] However, in the field of in vitro diagnosis, the detection of whole blood samples is a highly 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, which are much more complex than serum or plasma samples. When using magnetic microspheres for whole blood detection, non-specific adsorption between blood cells and magnetic spheres is a problem that cannot be ignored. Non-specific adsorption refers to the fact that, in immunodetection, in addition to specific recognition and binding between the surface of the magnetic sphere and the target antigen or antibody, it may also bind to non-target molecules in the sample, such as proteins and fragments on the surface of blood cells. This not only increases the background noise of the detection signal, affecting the specificity and sensitivity of the detection, but also can produce false positive results, reducing the accuracy of the detection.

[0004] Currently, there are many magnetic sphere raw materials on the market for whole blood detection, which have made certain progress in improving detection efficiency and simplifying operation procedures. However, these traditional magnetic sphere materials still have deficiencies in dealing with non-specific adsorption problems, and are difficult to meet the requirements of high sensitivity, high specificity and high repeatability for clinical detection. Therefore, developing a new type of magnetic sphere complex to solve the problem of non-specific adsorption in whole blood detection has become a technical problem to be solved in the current biomedical detection field. SUMMARY

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

[0006] In order to achieve the above object, according to a first aspect of the present application, an immunological detection method for a target substance in a whole blood sample is provided, which comprises: a) mixing and incubating the whole blood sample and an immunological detection reagent to obtain an incubation complex; wherein the immunological detection 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 capable of specifically binding to the target substance, and the surface of the magnetic microsphere contains methacrylate; and b) signal detection of the incubation complex to obtain the amount of the target substance by signal intensity.

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

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

[0009] Further, the magnetic microsphere and the first affinity protein are coupled by a crosslinking agent to form the magnetic microsphere-protein complex; preferably, the crosslinking agent is DMTMM, and preferably, the feeding ratio of DMTMM to the magnetic microsphere is (1-4):1.

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

[0011] Further, 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 immunological detection reagent to obtain the incubation complex; preferably, the polyethylene glycol diluent comprises a first polyethylene glycol diluent or a second polyethylene glycol diluent, and the whole blood sample comprises a first whole blood sample or a second whole blood sample, and correspondingly, a) comprises a1) or a2); a1) comprises: 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 immunological detection reagent to obtain the incubation complex; in the first mixed system, the volume ratio of the first whole blood sample is 50%-90%, and the molecular weight of the polyethylene glycol in the first polyethylene glycol diluent is 8000-20000 Da; 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 immunological detection reagent to obtain the incubation complex; in the second mixed system, the volume ratio of the second whole blood sample is 5%-20%, and the molecular weight of the polyethylene glycol in the second polyethylene glycol diluent is 800-5000 Da.

[0012] Further, 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] Further, the immunological detection method is selected from any one of chemiluminescence immunoassay, enzyme-linked immunoassay or radioimmunoassay.

[0014] Further, the immunological detection method is chemiluminescence immunoassay; the signal molecule includes a second affinity protein labeled with a luminescent label.

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

[0016] In order to achieve the above-mentioned purpose, according to the third aspect of the present application, a magnetic microsphere-protein complex is provided, wherein the magnetic microsphere-protein complex comprises the magnetic microsphere and an affinity protein coated on the magnetic microsphere.

[0017] In the immunological detection method of the above-mentioned whole blood sample, the magnetic microsphere-protein complex coated with the first affinity protein is used to capture the analyte to obtain an incubation complex, so as to realize the detection of the amount of the analyte. The surface of the magnetic microsphere used in the immunological detection method contains methacrylate, which can reduce the capture of non-analytes in the whole blood sample, effectively reduce non-specific binding, ensure the specificity and efficiency of the immune reaction, enhance the accuracy and stability of the detection signal, and provide an innovative solution for the accurate detection of the whole blood sample. DETAILED DESCRIPTION

[0018] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0019] Term explanation:

[0020] Magnetic microsphere: the magnetic microsphere is a composite microsphere made by combining magnetic inorganic particles (such as Fe3O4, γ-Fe2O3, etc.) with organic polymer materials (such as polystyrene, polyacrylic acid, etc.), which is also called magnetic sphere.

[0021] As mentioned in the background, the detection of whole blood sample in the prior art is affected by non-specific adsorption, and the detection accuracy is low. In the present application, the inventors have unexpectedly found that the amount of adsorption of interfering components in whole blood sample on the surface of magnetic microspheres coated with methacrylate is small, and the accuracy and stability of the immunological detection method can be improved by reducing non-specific adsorption. Based on this, the inventors have developed a new immunological detection method for the detection of analytes in whole blood sample, and thus proposed a series of protection schemes in the present application.

[0022] In a first typical embodiment of the present application, an immunological detection method for analytes in whole blood sample is provided, which comprises: a) mixing and incubating whole blood sample and immunological detection reagent to obtain an incubation complex; wherein the immunological detection reagent comprises a signal molecule and / or a magnetic microsphere-protein complex, the magnetic microsphere-protein complex comprises magnetic microspheres and a first affinity protein coated on the magnetic microspheres, the first affinity protein is a protein capable of specifically binding to the analyte, and the surface of the magnetic microspheres contains methacrylate; b) signal detection of the incubation complex to obtain the amount of the analyte by signal intensity.

[0023] In the above-mentioned immunological detection method, a magnetic microsphere-protein complex is provided, which comprises: 1) magnetic microspheres with methacrylate on the surface, and 2) a first affinity protein coated on the magnetic microspheres for specific binding to the analyte. The above-mentioned first affinity protein includes but is not limited to antibody or antigen. The magnetic microsphere-protein complex is mixed and incubated with whole blood sample, and the specific binding of the first affinity protein to the analyte is utilized to obtain an incubation complex, i.e., the incubation complex comprises magnetic microspheres, the first affinity protein and the analyte. If the immunological detection reagent also comprises a signal molecule, the signal molecule can also be combined on the incubation mixture by specific binding and other methods commonly used in the prior art.

[0024] Further, the incubation complex is separated by the magnetism of the magnetic microspheres, and signal detection is performed thereon, and the amount of the analyte can be detected by signal intensity. The above-mentioned signal intensity includes but is not limited to the intensity of chemical luminescence, fluorescence, radioactivity, etc.

[0025] In the immunological detection method of the present application, any immunological detection method known in the prior art can be flexibly selected by those skilled in the art, including but not limited to enzyme-linked immunosorbent assay (ELISA), immunofluorescence technology (IFA), radioimmunoassay (RIA), etc.

[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 with the specific particle size can provide a suitable surface area, thereby ensuring the coating amount of the first affinity protein, improving the detection efficiency, and ensuring the stability and repeatability of the signal, and being suitable for various immunological detection methods and automatic detection equipment in the prior art.

[0028] The average particle size of the magnetic microspheres is 1-8 microns, and the “±0.9 μm” indicates that the error of the particle size of 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 above-mentioned magnetic microspheres containing carboxyl groups on the surface with the first affinity protein; preferably, the content of the surface groups of the magnetic microspheres is 10-100 μmol / g; preferably, the surface groups are carboxyl groups.

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

[0030] The magnetic microspheres, as the core carrier, have carboxyl (-COOH) groups on the surface, which have the ability to covalently couple with the first affinity protein, thereby ensuring the stable binding of the protein. Preferably, the content of the surface groups of the magnetic microspheres is 10-100 μmol / g. The group density in this range not only can effectively load a large amount of protein, but also avoids the protein steric hindrance effect that may be caused by the over-dense group arrangement, thereby ensuring the maximization of the 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 feeding ratio of DMTMM to the magnetic microspheres is (1-4):1.

[0032] The use of the 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-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, CAS No. 3945-69-5).

[0033] The principle of using DMTMM as a cross-linking agent to connect the magnetic microspheres and the first affinity protein includes the following: first, the carboxyl group reacts with DMTMM to form an active ester, and releases one molecule of N-methylmorpholine (NMM). Further, the obtained active ester has high reactivity, and can be subjected to nucleophilic attack by an amine, an alcohol or other nucleophilic reagents (groups) derived from the first affinity protein, thereby releasing one molecule of 4,6,-dimethoxy-1,3,5-triazin-2-ol, and forming a stable amide or carboxylic acid derivative. The reaction structure is shown in the following formula:

[0034]

[0035] The magnetic microspheres are connected with the first affinity protein by using the cross-linking agent, which can ensure uniformity and stability of coupling while maintaining biological activity of the protein, and avoids non-specific adsorption and signal loss caused by protein shedding during detection. By using DMTMM as a cross-linking agent, the magnetic microsphere-protein complex of the application exhibits significant beneficial effects in the field of whole blood detection. The problems of protein shedding and non-specific adsorption encountered by traditional magnetic sphere complex in whole blood sample detection are effectively solved, which can improve the sensitivity and specificity of detection.

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

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

[0038] In the application, poly-bull serum albumin (BSA) is preferably used for blocking treatment of the surface of the magnetic microspheres, and the concentration is controlled in the range of 0.3% to 2%(w / v). As a non-specific protein, poly-BSA blocks the active sites on the surface of the 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, poly-BSA can uniformly cover the surface of the magnetic microspheres, form a physical barrier, effectively block the direct contact of non-target proteins, and reduce the opportunity of non-specific binding, thereby optimizing the detection conditions and improving the specificity of whole blood sample detection. The magnetic microspheres blocked by poly-BSA have better effects in the above immunological detection method than the magnetic microspheres blocked by BSA, which can improve the accuracy and sensitivity of detection and improve the repeatability of detection results, making the detection signal more stable and reliable.

[0039] Polymerized BSA (Bovine Serum Albumin) refers to the polymerized form of bovine serum albumin. Bovine serum albumin is a globulin protein that contains 583 amino acid residues and has 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 the present application refers to a molecule formed by the combination of monomers. For bovine serum albumin, polymerization means that multiple bovine serum albumin molecules are connected together to form a larger molecular complex through non-covalent bonds such as hydrogen bonds, van der Waals forces, etc. The formation of such polymers affects its physical and chemical properties and biological functions.

[0040] In a preferred embodiment, 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 an incubated complex; preferably, the polyethylene glycol diluent comprises a first polyethylene glycol diluent or a second polyethylene glycol diluent, and the whole blood sample comprises a first whole blood sample or a second whole blood sample, and a) comprises a1) or a2) accordingly; a1) comprises: 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 an incubated complex; in the first mixed system, the volume percentage 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 polyethylene glycol in the first polyethylene glycol diluent is 8000-20000 Da (including but not limited to 8000 Da, 9000 Da, 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, 15000 Da, 16000 Da, 17000 Da, 18000 Da, 19000 Da, or 20000 Da); 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 an incubated complex; in the second mixed system, the volume percentage of the second whole blood sample is 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 polyethylene glycol in the second polyethylene glycol diluent is 800-5000 Da (including but not limited to 800 Da, 1000 Da, 1500 Da, 200 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, or 5000 Da).

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

[0042] Further, different pre-detection treatments are performed for large sample amount projects (the sample amount of the whole blood sample is more than 50% of the mixed system) and for small sample amount projects (the sample amount of the whole blood sample is less than 20% of the mixed system).

[0043] For large sample amount projects (including but not limited to high-sensitive troponin I (hs-CtnI) detection), a diluent containing a large molecular weight (8000-20000 Da) polyethylene glycol is added to the first whole blood sample. The large molecular weight 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 of blood cells with the detection reagent, significantly reducing the interference of blood cell agglutination, rupture and non-specific adhesion on the immune reaction, and improving the consistency of whole blood and plasma detection.

[0044] For small sample amount projects (including but not limited to procalcitonin (PCT) detection), a diluent containing a small molecular weight (800-5000 Da) polyethylene glycol is added to the second whole blood sample. The small molecular weight PEG can ensure the moderate dispersion of blood cells while not significantly affecting the sedimentation effect of blood cells, thereby avoiding the decrease of detection sensitivity caused by excessive sedimentation of blood cells and maintaining the accuracy of the detection results.

[0045] In actual whole blood sample detection, the components in the whole blood sample can be flexibly adjusted according to the specific operation of the detection project, including but not limited to dilution of the whole blood sample with polyethylene glycol and the like. The addition of such reagents does not affect the binding of the magnetic microsphere-protein complex to the analyte in the present application, nor does it affect the subsequent detection of the analyte-related signal.

[0046] The "first whole blood sample" and "second whole blood sample" in the present application are only used to distinguish the whole blood samples used in large sample amount projects and small sample amount projects. The above "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.

[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] A fresh blood sample (hereinafter referred to as a fresh sample) refers to a whole blood sample stored for no more than 8 hours after blood collection, and an old blood sample (hereinafter referred to as an old blood sample) refers to a whole blood sample stored for more than 5 days under the condition of 2-8℃. The immunological detection method described above can be used for rapid detection of both fresh samples and old blood samples, and the detection results are accurate and reliable.

[0049] The immunological detection method described above can achieve accurate detection of old blood samples, and is convenient for accurate detection of old blood samples in the fields of retrospective research, forensic identification, blood storage quality detection, drug research, and genetic research, thereby avoiding the limitation of detection caused by the necessity of using fresh blood samples for detection.

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

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

[0052] Preferably, the second affinity protein includes but is not limited to an antigen and / or an antibody, and the second affinity protein is connected in the incubation complex by being combined with any one or more of a magnetic microsphere, a first affinity protein, etc. The luminescent label realizes the generation of a light signal 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 a pH buffer, NaCl, bovine serum albumin, glycine, or a preservative; and 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 label includes but is not limited to any one or more of isoluminol and its derivatives, acridinium ester and its derivatives, or trispyridine ruthenium.

[0055] In the immunological detection method of the analyte in the whole blood sample described above, a person skilled in the art can flexibly select known detection methods, detection reagents, and detection parameters in the prior art for testing, and all of them can realize such immunological detection, achieve the detection of the analyte in the whole blood sample, and significantly improve the detection efficiency and accuracy of the analyte 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 closed by poly-BSA.

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

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

[0059] The magnetic microsphere and / or the magnetic microsphere-protein complex can be used for immunological detection of the analyte in a whole blood sample, or for preparation of related reagents for immunological detection of the whole blood sample. By using the magnetic microsphere and / or the magnetic microsphere-protein complex, accurate detection of the analyte in the whole blood sample can be achieved, and detection deviation or even detection error caused by non-specific adsorption of the magnetic sphere to interfering components in the whole blood sample can be reduced.

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

[0061] Example 1 Preparation of magnetic spheres

[0062] I. Preparation method of oleic acid-coated magnetic microparticles

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

[0064] II. Preparation method of magnetic spheres 0

[0065] 1. 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 microparticles were mixed to form an organic phase, which was strongly stirred and ultrasonically treated for 10 min to ensure uniform dispersion of the oleic acid-coated magnetic microparticles.

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

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

[0068] III. Magnetic Sphere 1 Preparation Method

[0069] 1. The organic phase is formed by mixing 3 g of benzoyl peroxide, 36 mL of methyl methacrylate, 20 mL of styrene, 8 mL of divinyl benzene, 38 mL of glycidyl methacrylate, 30 mL of cyclohexane, 16 mL of acrylic acid, and 20 g of the oleic acid-coated magnetic microparticles prepared above, with vigorous stirring and ultrasonic treatment for 10 minutes to ensure uniform dispersion of the oleic acid-coated magnetic microparticles.

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

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

[0072] IV. Magnetic Sphere 2 Preparation Method

[0073] 1. The organic phase is formed by mixing 3 g of benzoyl peroxide, 36 mL of methyl methacrylate, 20 mL of styrene, 8 mL of divinyl benzene, 38 mL of glycidyl methacrylate, 30 mL of cyclohexane, 16 mL of acrylic acid, and 20 g of the oleic acid-coated magnetic microparticles prepared above, with vigorous stirring and ultrasonic treatment for 10 minutes to ensure uniform dispersion of the oleic acid-coated magnetic microparticles.

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

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

[0076] V. Preparation method of the magnetic balls 3

[0077] 1. 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 above-prepared oleic acid-coated magnetic microparticles are mixed to form an organic phase, which is stirred vigorously and ultrasonically treated for 10 minutes to ensure uniform dispersion of the oleic acid-coated magnetic microparticles.

[0078] 2. The aqueous phase is composed of 10 g of polyvinyl alcohol-1788, 15 g of sodium chloride, and 1000 mL of 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 under continuous nitrogen purging. The temperature of the synthesis solution is raised from 45°C to 55°C within 1 h, then maintained at 60°C for 2 h, and finally maintained at 70°C and 80°C for 1 h each.

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

[0080] Example 2: Magnetic ball detection method

[0081] I. Particle size detection method

[0082] The magnetic balls to be tested are ultrasonically mixed for 5 min; 1.5 mL of the magnetic balls are taken into a 20 mL vial, 0.2 mL of Tween is added to the vial, and the mixture is ultrasonically mixed for 1 min at an ultrasonic power of 60%; and then the mixture is detected using a Malvern particle size analyzer.

[0083] II. Carboxyl content detection method

[0084] 1) The magnetic balls to be tested are ultrasonically mixed for 5 min;

[0085] 2) 0.5 g of the magnetic balls are taken into a 250 mL beaker, 3 mL of 2M hydrochloric acid solution is added to the beaker, 150 mL of purified water is added, the mixture is ultrasonically mixed for 1 min at an ultrasonic power of 100%, and the mixture is settled on a magnet with a magnetic field of 300-500 mT for 30 min to remove the supernatant;

[0086] 3) 150 mL of purified water is added again, and the washing is repeated twice;

[0087] 4) 200 mL of purified water is added to the beaker, and the mixture is ultrasonically mixed for 1 min;

[0088] 5) The mixture is detected using an acid-base titrator.

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

[0090] Table 1

[0091]

[0092] Example 3

[0093] 1. Preparation of magnetic ball compound:

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

[0095] Table 2

[0096]

[0097]

[0098] The magnetic ball compound prepared above was stored in a diluent, wherein the components of the diluent were the magnetic ball storage liquid components in the PCT kit produced by Xin Industry, including buffer substances 0.04% potassium dihydrogen phosphate, 0.4% sodium phosphate dibasic, 0.8% NaCl, 0.5% bovine serum albumin, 1M glycine, 0.5mg / mL goat anti-human IgG, preservative 0.15% Proclin300.

[0099] 2. Sample preparation:

[0100] A fresh normal human whole blood sample was collected, numbered as sample, and divided into 3 blood collection tubes. One was not treated and numbered as sample F; one was treated by ultrasonic wave for 20 min and numbered as sample S; the other was centrifuged to obtain blood plasma and numbered as sample P.

[0101] The above steps were repeated to collect 9 groups of samples for standby.

[0102] 3. Detection of sample F and sample S

[0103] The PCT kit produced by Xin Industry is used for detection, the magnetic ball reagent prepared in step 1 is used to replace the magnetic ball reagent, the sample diluent and the marker component in the kit are used, and the detection is carried out in the chemiluminescence immunoassay analyzer MAGLUMI X3 produced by Xin Industry. The detection principle is: chemiluminescence immunoassay.

[0104] A strain of PCT antibody (capture antibody) is used to coat magnetic microspheres, and another strain of PCT antibody (detection antibody) is labeled with ABEI (luminescent agent, N-(4-aminobutyl)-N-ethylisoluminol). The sample, the luminescent label, the buffer and the magnetic microspheres are mixed together and incubated. The analyte in the sample forms an immune complex with the ABEI-labeled PCT antibody 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 automatic immune test system is added, the chemiluminescence reaction is started, and the light signal is generated. The relative light intensity (RLU) measured by the photomultiplier tube is proportional to the PCT concentration in the sample.

[0105] The detection steps are: add 20 μL of sample to the reaction cup, add 20 μL of magnetic ball component, 50 μL of buffer, 50 μL of luminescent label working solution to the reaction cup, mix well, and incubate at 37°C for 14 min. The target analyte in the sample forms a double-antibody sandwich three-way immune complex with the magnetic ball-coated specific antibody in the first reagent and the ABEI-labeled specific antibody. After incubation, the unbound substances are removed by magnetic separation and washing. Finally, hydrogen peroxide and sodium hydroxide are added to the system to excite the label to emit light, and the light signal is detected to obtain RLU.

[0106] The detection results are shown in Tables 3 and 4. Among them, the control group, experimental groups 1-4 represent the detection of whole blood samples using the magnetic balls prepared in Table 2 above.

[0107] Table 3: PCT detection bias results of sample F and sample S

[0108] Sample Control group Experiment group 1 Experiment group 2 Experiment group 3 Experiment group 4 Number Deviation Deviation Deviation Deviation Deviation Mean absolute deviation 122％ 6.49％ 7.23％ 9.14％ 4.55％ Sample Experiment group 5 Experiment group 6 Experiment group 7 Experiment group 8 Experiment group 9 Number Deviation Deviation Deviation Deviation Deviation Mean absolute deviation 4.93％ 3.43％ 3.29％ 2.34％ 2.95％

[0109] In Table 3, bias = (sample S RLU - sample F RLU) / sample F RLU x 100%.

[0110] Each sample is detected 10 times and the CV value is calculated, and the results are shown in Table 4.

[0111] Table 4: Detection repeatability verification of sample F and sample S

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

[0113] The results show that, when the sample F is detected by the magnetic balls in the experimental group, the RLU of the normal human whole blood sample is greatly reduced by 122% compared with the control group, and the RLU deviation of the sample S (with more cell fragments) and the sample F is within 10%, which indicates that the magnetic balls upgraded and modified by the scheme can reduce the interference of blood cell fragments on the immune response, and further using DMTMM as a crosslinking 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 sample S and the sample F in the experimental group 8 is only 2.34%, which indicates that the use of DMTMM crosslinking agent and poly-BSA blocking agent together can further improve the whole blood anti-interference ability of the modified magnetic balls, and the deviation between the experimental group 9 and the experimental group 6 is not large, which indicates that the use of DMTMM alone also has good effect, and it is speculated that DMTMM has a certain blocking effect in addition to being a crosslinking agent; and from the results of the whole blood sample detection repeatability in Table 4, the repeatability CV of the sample S detected by the experimental groups 6, 8 and 9 is within 8%, and the above results comprehensively show that the use of the scheme to modify the magnetic balls and the further use of DMTMM crosslinking agent or poly-BSA blocking agent to treat the magnetic balls can greatly reduce the non-specific adsorption level of the whole blood sample and improve the accuracy of the whole blood detection.

[0114] In the sample S obtained by the above ultrasonic crushing treatment, the whole blood sample is subjected to ultrasonic crushing to simulate the detection environment of the content release caused by the rupture of blood cells under extreme conditions. It is found that the light intensity deviation of the crushed blood sample and the normal blood sample can reach 122% by using the magnetic balls in the prior art for detection, and the deviation of the two samples is greatly reduced when the magnetic balls in the scheme are used for detection, which indicates that the magnetic balls in the scheme have strong anti-non-specific adsorption ability. The use of DMTMM as a crosslinking agent can further reduce the non-specific adsorption of the magnetic ball complex, and the use of poly-BSA for blocking can further reduce the deviation.

[0115] 4. Sample F and sample P detection

[0116] The Roche immunoassay instrument and the matching PCT chemiluminescence kit (item number: (240)09318712190) are used to detect the plasma sample value, and the detection results of the F sample of the application are compared, wherein the whole blood sample is detected by HCT, and the detection results are converted into the concentration value of the target substance in the plasma, and then the absolute deviation is calculated with the detection results of Roche.

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

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

[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 Experiment group 1 Experiment group 2 Experiment group 3 Experiment group 4 Mean absolute deviation 20.3％ 7.8％ 8.65％ 8.96％ 4.98％ Sample Experiment group 5 Experiment group 6 Experiment group 7 Experiment group 8 Experiment group 9 Mean absolute deviation 4.56％ 3.89％ 4.12％ 2.34％ 3.66％

[0124] The results show that the magnetic sphere complex of the present scheme can significantly improve the consistency of whole blood and plasma test results, and the deviation of the magnetic sphere complex prepared by DMTMM crosslinking agent from Roche plasma test results is less than 5%, which has high accuracy.

[0125] Example 4

[0126] 1. Sample diluent preparation

[0127] The sample diluent components of experimental group 9 include 50mM Tris buffer pair, 0.2% NaCl, 0.5% bovine serum albumin, 0.15% Proclin300 preservative, and different molecular weight PEG is further added according to the concentration shown in experimental groups 10-15, to verify the deviation of fresh blood samples and old blood samples from plasma.

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

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

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

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

[0132] Experimental group 14: add PEG with MW = 4000 in the sample diluent of experimental group 9 to a final PEG concentration of 4wt%.

[0133] 2. Sample preparation

[0134] 1 fresh whole blood sample (sample F) was collected, each sample F was divided into 2 blood collection tubes, one was centrifuged to obtain plasma for use, and the PCT concentration value in the plasma was detected, and the other was not treated, and the PCT concentration value in the whole blood was detected;

[0135] Another 1 part of the old sample (sample S) placed for 5 days, also divided into 2 blood collection tubes, centrifuged according to the previous steps to obtain plasma for standby, another part without treatment directly detected.

[0136] Repeat the above steps, collect 9 groups of samples for standby.

[0137] 3. Sample detection

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

[0139] Table 6:

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

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

[0142] Example 5

[0143] The new industry biochemistry luminescence immunoassay analyzer Maglumi X3 and the matching self-produced hs-cTnI luminescence kit were used for detection, and the sample diluent in the kit was replaced with the diluent added with polyethylene glycol, and the magnetic ball and protein complex in the first reagent were replaced with the one prepared according to Example 3, group 9, to detect the consistency of whole blood samples and plasma samples, and the detection repeatability, and the detection steps are as follows:

[0144] 1. Diluent preparation:

[0145] Control group 2: The components are 50mM Tris buffer, 0.2% NaCl, 0.5% bovine serum albumin, 0.015% Proclin300 preservative basic diluent.

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

[0147] Experimental group 17: Add PEG with MW = 20000 to the basic diluent of control group 2 to a final PEG concentration of 0.1wt%;

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

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

[0150] 2. Sample preparation:

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

[0152] Another 1 old sample (sample S) placed for 5 days is also divided into 2 blood collection tubes. The plasma is obtained by centrifugation according to the above steps for use, and the other is directly detected without processing.

[0153] Repeat the above steps to collect 9 groups of samples for use.

[0154] 3. Detection reagent

[0155] In addition to the sample diluent, other components of the hs-cTnI luminescence kit (item number: 130256014M) produced by Xin Industry are selected for detection of the above samples. Other components include:

[0156] First reagent: magnetic ball reagent coated with an antibody specifically binding to hs-cTnI. The preparation method and coating scheme of the magnetic ball are described in experimental group 9. The liquid component contains 0.04% potassium dihydrogen phosphate, 0.4% dipotassium hydrogen phosphate, 0.8% NaCl, 0.5% bovine serum albumin, and 0.15% Proclin300 preservative.

[0157] Second reagent: marker reagent of another antibody specifically binding to hs-cTnI labeled with ABEI, containing 70mM Tris buffer, 0.4% NaCl, 0.5% bovine serum albumin, 1M glycine, and 0.15% Proclin300 preservative.

[0158] 4. Sample detection

[0159] The whole blood sample is detected by using the chemiluminescence immunoassay analyzer Maglumi X3 of Xin Industry. The steps are as follows:

[0160] The sample diluent configured in the foregoing control group and experimental group and the first and second reagents produced by Xin industry are used to detect the whole blood sample in step 2, 150 μL of the sample is added to the reaction cup, and the reagent sampling is performed according to the following sample addition sequence: first reagent-experimental group diluent in step 1-second reagent, which are added to the reaction cup after sampling and mixed, and after mixing, incubation is performed at 37°C for 14 min, so that the target analyte in the sample to be detected forms a triple immunocomplex of double antibody sandwich with the magnetic ball coated specific antibody in the first reagent and the ABEI labeled specific antibody, and after incubation, the unbound substances are removed by magnetic separation and washing. Finally, the substrate solution of the automatic immune detection system, hydrogen peroxide and sodium hydroxide, is added to start the chemiluminescence reaction to generate light signals. The relative light intensity (RLU) measured by the photomultiplier tube; the above steps are repeated to detect the whole blood sample, and the RLU value is obtained, and the concentration value of the target substance hs-cTnI in the sample is calculated, and the detection result is converted into the concentration value of the target substance in the plasma by HCT detection, and the conversion formula is the same as the foregoing embodiment.

[0161] The foregoing plasma samples are detected by Beckman analyzer Access2 81600N and matching reagent hs-CTnI (REF: B52699) to determine the value of the target substance in the plasma.

[0162] According to the above detection results, the absolute deviation of the detection of the 9 groups of whole blood samples and plasma samples is calculated, and the calculation method is the same as the foregoing embodiment.

[0163] The detection results are shown in Table 7.

[0164] Table 7:

[0165]

[0166]

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

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

[0169] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for immunological detection of an analyte in a whole blood sample, characterized in that, The immunological detection method comprises: a) mixing and incubating a whole blood sample and an immunological detection reagent to obtain an incubation complex; wherein the immunological detection reagent comprises a signal molecule and 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 capable of specifically binding to the analyte, and the surface of the magnetic microsphere contains methacrylate; b) signal detection of the incubation complex to obtain the amount of the analyte by signal intensity; the particle size of the magnetic microsphere is (1-8) ± 0.9 μm; the magnetic microsphere-protein complex is obtained by coupling the magnetic microsphere containing carboxyl groups on the surface and the first affinity protein; the content of surface groups of the magnetic microsphere is 10-100 μmol / g; the surface group is a carboxyl group; the magnetic microsphere and the first affinity protein are coupled by a crosslinking agent to form the magnetic microsphere-protein complex; the crosslinking agent is DMTMM; the surface of the magnetic microsphere is blocked with poly-BSA.

2. The immunological detection method according to claim 1, 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 immunological detection reagent to obtain the incubation complex.

3. The immunological detection method according to claim 2, characterized in that, The polyethylene glycol diluent comprises a first polyethylene glycol diluent or a second polyethylene glycol diluent, and the whole blood sample comprises a first whole blood sample or a second whole blood sample, and correspondingly, the a) comprises a1) or a2); The a1) comprises: 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 immunological detection reagent to obtain the incubation complex; in the first mixed system, the volume fraction of the first whole blood sample is 50%-90%, and the molecular weight of polyethylene glycol in the first polyethylene glycol diluent is 8000-20000 Da; The 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 immunological detection reagent to obtain the incubation complex; in the second mixed system, the volume fraction of the second whole blood sample is 5%-20%, and the molecular weight of polyethylene glycol in the second polyethylene glycol diluent is 800-5000 Da.

4. The immunological detection method according to claim 3, wherein: the mass content of polyethylene glycol in the first polyethylene glycol diluent is 0.05-1 wt %; the mass content of polyethylene glycol in the second polyethylene glycol diluent is 0.1-4 wt %.

5. The immunological assay method according to claim 1, characterized by, The whole blood sample comprises a fresh blood sample or an old blood sample.

6. The immunological detection method according to any one of claims 1 to 5, characterized in that, The immunological detection method is selected from any one of chemiluminescence immunoassay, enzyme-linked immunoassay or radioimmunoassay.

7. The immunological assay method according to claim 6, characterized in that, The immunological detection method is chemiluminescence immunoassay; the signal molecule comprises a second affinity protein labeled with a luminescent label.

8. A magnetic microsphere-protein complex, characterized in that, The magnetic microsphere-protein complex comprises the magnetic microsphere of claim 1 and an affinity protein coated on the magnetic microsphere. The magnetic microsphere-protein complex is prepared by adding a cross-linking agent DMTMM and a blocking agent poly-BSA into a magnetic microsphere suspension, and mixing the magnetic microsphere with the affinity protein.

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