Antibody coating method of small-size magnetic nanoparticles and application of antibody coating method

Abstract

The invention provides an antibody coating method of small-size magnetic nanoparticles and application of the antibody coating method, and belongs to the field of in-vitro detection.The magnetic nanoparticles are nano magnetic beads with surface modification and the size being 20-50 nm. The antibody coating method comprises the following steps that a coating buffer solution is used for conducting liquid changing on the magnetic beads through a G-25 desalination column, and the magnetic beads are subjected to liquid changing; the method comprises the following steps: coating a magnetic bead with an antibody by using a surface modification group, after coating is completed, carrying out liquid exchange by using a cleaning buffer solution through a G-25 desalination column, and sealing and storing a magnetic bead antibody coating object after liquid exchange. Aiming at small-size nano magnetic beads, the magnetic bead antibody coating substance with high coating amount, high stability and high sensitivity is successfully prepared, and the method has important significance in immunodetection of low-abundance samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro detection technology, and particularly relates to an antibody coating method of small size (20-50 nm) magnetic nanoparticles and application thereof in low-abundance immunodetection. BACKGROUND

[0002] Magnetic nanoparticles (MNPs) are a new type of material that has developed rapidly in recent years and has great application value. They are widely used in many fields of modern science, such as biomedicine, magnetic fluid, catalysis, nuclear magnetic resonance imaging, data storage and environmental protection. Magnetic nanoparticles are nanoparticles, generally composed of a magnetic core of metal oxides such as iron, cobalt and nickel, and a shell layer of high polymer / silicon / hydroxyapatite wrapped outside the magnetic core. The most common core is made of Fe3O4 or γ-Fe2O3 with superparamagnetic or ferromagnetic properties, which has magnetic guidance (targeting). Under the action of an external magnetic field, it can move directionally, making it easy to locate and separate from the medium. The most common shell layer is composed of high polymer. The active groups coupled on the shell layer can be combined with various biological molecules, such as proteins, enzymes, antigens, antibodies, nucleic acids, etc., so as to realize its functionalization. The surface modification of magnetic nanoparticles includes non-polymer organic fixation, polymer organic fixation, inorganic molecule fixation, target matching modification, etc. Commonly used substances for modification include polyethylene glycol, dextran, polyvinylpyrrolidone, fatty acid, polyvinyl alcohol, polypeptide, gelatin, chitosan, methylsilane, liposome, etc. Therefore, magnetic nanoparticles have the characteristics of magnetic particles and high polymer particles, and have magnetic guidance, biocompatibility, small size effect, surface effect, active groups and certain biomedical functions.

[0003] Superparamagnetic nanoparticles have shown unique advantages in drug targeting, nuclear magnetic resonance and other biomedical fields. In the direction of immune detection, the specific surface area (surface area to volume ratio) of spherical magnetic nanoparticles (hereinafter referred to as magnetic beads) is inversely proportional to the diameter of the particles. The specific surface area increases significantly as the diameter of the particles decreases. For magnetic beads with a diameter of less than 50 nm, the surface effect is significant, and the number of surface atoms increases rapidly, so the probability of collision with antigens in the sample is higher, that is, compared with ordinary magnetic beads, it can have higher antigen capture efficiency; and because the surface area of a single magnetic bead is small, the ability to non-specifically bind to other substrates in the sample is weak, thereby facilitating the improvement of overall specificity. It can be seen that magnetic beads with a diameter of less than 50 nm have great advantages in the immune detection of many current low-abundance indicators of diseases. However, in actual production, the coating and purification of such small-diameter magnetic beads are more difficult, resulting in no preparation scheme and case of magnetic bead antibody coating with a particle size of less than 100 nm, and also limiting the application of magnetic beads with a particle size of less than 100 nm to cell sorting, and almost no application in the immunochemical luminescence platform.

[0004] Because the smaller the particle size of the nano-magnetic beads, the lower the iron content, the lower the saturation magnetism, and the conventional magnetic force cannot adsorb the magnetic beads below 100 nm, for the magnetic nanoparticles with a particle size of less than 100 nm, the existing technology usually uses a high gradient magnetic field for magnetic adsorption, but the nano-magnetic beads after adsorption by high gradient magnetic separation are easy to agglomerate, which affects the antibody coating. In addition, if a high-speed centrifuge is used, the antibody coated on the surface of the nano-magnetic beads will be damaged, and the nano-magnetic beads will form agglomerates, and the use of ultrasonic separation of the agglomerated nano-magnetic beads will damage the stability of the surface modification and the antibody of the magnetic beads. SUMMARY

[0005] In view of the technical problems in the background art, the present application provides an antibody coating method suitable for small-size magnetic nanoparticles and its application in low-abundance immune detection, aiming to solve the technical problem that small-size magnetic nanoparticles are limited in application in immune detection, and to provide a powerful means for screening of low-abundance indicators of many diseases.

[0006] In a first aspect, the present application provides an antibody coating method for small-size magnetic nanoparticles, wherein the magnetic nanoparticles are nano-magnetic beads with surface modification and a size of 20-50 nm, and the antibody coating method comprises the following steps: S1, using a coating buffer to replace the liquid of the magnetic beads through a G-25 desalting column; S2, using a surface modification group to coat the antibody on the magnetic beads; S3, after the coating is completed, using a washing buffer to replace the liquid through a G-25 desalting column; S4, sealing and storing the magnetic bead antibody coating after replacement.

[0007] In the above scheme, having surface modification means that the magnetic beads are modified with -COOH, -PEG-COOH, -NH2, -PEG-NH2, -OH, -Biotin, -Streptavidin and the like groups; in the prepared magnetic bead antibody coating, the antibody is connected with the above-mentioned modified groups through various coupling agents.

[0008] In the above scheme, the application innovatively uses a gravity desalting column to replace the liquid of the magnetic beads and the magnetic bead antibody coating, which well avoids the damage of mechanical external force to the magnetic beads and the antibody, and fully retains the functional integrity of the antibody magnetic bead coating. The gravity desalting column is commonly used for the purification of proteins, nucleic acids and the like, and its application in magnetic bead coating has not been reported. The inventors accidentally found that the 20-50nm nanometer magnetic beads can be replaced through the G-25 desalting column without affecting the magnetic beads. The G-25 desalting column uses Sephadex G-25 gel composed of cross-linked dextran, has small pore size, moderate flow rate and high resolution, and can better meet the needs of small particle size magnetic beads passing through.

[0009] In some embodiments of the application, for the nanometer magnetic beads (20-50nm) modified with carboxyl groups (including -COOH, -PEG-COOH and the like), step S2 includes the following operations: S21, slowly drop the magnetic beads into the coating buffer containing the monoclonal antibody for pre-reaction, wherein the pH of the reaction solution is less than the PI of the antibody; S22, after the reaction in step S21 is completed, add 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) and N-hydroxysuccinimide (NHS) for reaction.

[0010] In the antibody coating process, although the smaller the particle size of the magnetic beads, the larger the specific surface area, in the actual reaction solution, the antibody has greater steric hindrance relative to the small particle size magnetic beads. If covalent binding occurs rapidly, it is easy to cause the magnetic beads and the antibody to rapidly aggregate, and also reduce the amount of antibody bound to the magnetic beads in the positive direction, resulting in a significant reduction in the final coating efficiency. Therefore, unlike the traditional method of coating magnetic beads with antibodies, in the scheme of the application, the magnetic beads are first mixed with the antibody to form an antibody magnetic bead physical adsorption complex, so that the magnetic beads and the antibody are mixed together in a non-covalent state. Specifically, by limiting the relationship between the pH value of the reaction solution and the PI of the antibody, the surface of the antibody in the reaction solution is positively charged, so that electrostatic adsorption occurs between the positively charged antibody surface and the negatively charged magnetic bead surface, and a large amount of antibody is accumulated on the surface of the magnetic beads in a short time. When the coupling agent is added, covalent binding occurs between the magnetic beads and the antibody, and low temperature can make the covalent binding occur uniformly and slowly, thereby further improving the effective coating efficiency of the antibody.

[0011] Preferably, in the above antibody coating method, the reaction condition of step S22 is 4℃ for 30-90min. The low temperature reaction at 4℃ provides a window period for the antibody on the surface of the magnetic beads to adjust its own antibody orientation, so that the antigen binding site of the antibody faces outward, which can greatly improve the efficiency of antibody capturing antigen; at the same time, the low temperature reaction ensures the stability of the intermediate ester formed after the activation of the carboxyl group, which greatly improves the antibody coating amount of the magnetic beads. More preferably, after the low temperature reaction is completed, the reaction temperature can be restored to room temperature for a period of time (5-30min) to promote the antibody to be saturated as much as possible.

[0012] Preferably, in the above antibody coating method, the coating buffer is 0.01-0.1M MES buffer with pH 5.0-6.3.

[0013] Preferably, in the above antibody coating method, the washing buffer is PBS buffer or Tris buffer with pH 7.4.

[0014] Preferably, in the above antibody coating method, step S4 is specifically: adding a blocking solution to the magnetic bead antibody coating to block, and then directly adding a magnetic bead preservation solution to the reaction solution for preservation after the blocking reaction is completed; wherein the blocking solution contains macromolecular proteins / polymers and small molecule amino acids, and the magnetic bead preservation solution contains sugar alcohol substances and proteins / amino acids.

[0015] In the above scheme, the macromolecular proteins / polymers and small molecule amino acids in the blocking solution are combined to block, so that the gap between the antibodies on the surface of the magnetic beads is more completely blocked, reducing the non-specific binding of the magnetic beads in the later detection.

[0016] More preferably, in the above antibody coating method, the blocking solution is a PBS buffer containing 0.5-8wt% macromolecular proteins / polymers, 0.5-5wt% small molecule amino acids, and 0.1-2% surfactant, wherein the macromolecular proteins / polymers are selected from one or more of BSA, casein, and PVP, the small molecule amino acids are selected from at least one of glycine and lysine, and the surfactant is selected from Tween-20, triton, etc. In some specific embodiments, the above blocking solution can also be mixed with commercial blocking agents.

[0017] More preferably, in the above antibody coating method, the magnetic bead blocking solution is a PBS buffer containing 2-10wt% sugar alcohol substances, 0.5-2wt% proteins / amino acids, and 0.05-1% surfactant, wherein the sugar alcohol substances are selected from one or more of trehalose, sorbitol, and sucrose, and the proteins / amino acids are selected from BSA, glycine, etc. In some specific embodiments, the magnetic bead blocking solution also contains non-protein stabilizers such as biolipidure, etc.

[0018] In a second aspect, the magnetic bead antibody coating prepared according to the antibody coating method described above also belongs to the protection scope of the present application.

[0019] In a third aspect, the present application provides an application of the magnetic bead antibody coating described above in low-abundance immunodetection, specifically: the magnetic bead antibody coating is reacted with a labeled antibody to form a double-antibody sandwich complex, so as to realize immunodetection; wherein the label of the labeled antibody includes but is not limited to acridinium ester, trispyridine ruthenium, adamantane, luminol, horseradish peroxidase and alkaline phosphatase, etc. In view of the fact that the particle size of the magnetic bead in the magnetic bead antibody coating provided by the present application is only 20-50 nm, this greatly increases the specific surface area of the magnetic bead, greatly increases the collision probability of the antigen molecules in the sample with the magnetic bead coated antibody, and can more fully capture the antigen; at the same time, the smaller particle size also avoids a large number of non-specific bindings due to sample matrix effects, and overall improves the sensitivity and specificity of the reagent. Moreover, the experimental data of the present application also very well prove the superiority of the magnetic bead antibody coating described above in low-concentration value detection.

[0020] The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, and the present application can be implemented according to the content of the specification, and in order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following specific embodiments of the present application are described. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the present application, the following will briefly introduce the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0022] Figure 1 The flow chart of the method for coating small-size carboxyl magnetic beads with antibodies in the embodiments of the present application; Figure 2 The magnetic attraction comparison chart when coating micrometer magnetic beads and nanometer magnetic beads by conventional methods in the comparative examples of the present application. DETAILED DESCRIPTION

[0023] The embodiments of the technical solutions of the present application will be described in detail below with reference to the drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terms used herein are intended to have their ordinary meaning in the art which is used by persons skilled in the art; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application; the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion.

[0025] In view of the inherent advantages of magnetic beads with a diameter of ≤50 nm, they have very important value in low-value immune detection. However, the prior art cannot successfully prepare magnetic bead antibody coatings that meet the requirements for detection use for magnetic beads of this particle size, resulting in their limited application in low-value immune detection. In order to solve this technical problem, the present application provides an antibody coating method for small-sized magnetic nanoparticles, which obtains a magnetic bead antibody coating with high coating amount and high stability by changing the liquid exchange method and coating conditions, and experiments show that it has very obvious superiority in low-value sample detection.

[0026] Please refer to Figure 1 The antibody coating method for small-sized magnetic nanoparticles provided by the embodiments of the present application, wherein the magnetic nanoparticles are nanomagnetic beads with carboxyl modification and a size of 20-50 nm, and the antibody coating method comprises the following steps: (1) The magnetic beads are exchanged with a coating buffer solution through a G-25 desalting column; (2) The magnetic beads are dropped into a coating buffer solution containing a monoclonal antibody for pre-reaction, wherein the pH of the reaction solution is less than the PI of the antibody; (3) After the reaction in step (2) is completed, EDC and NHS are added, and the reaction is carried out at low temperature (4℃); (4) After the reaction in step (3) is completed, the magnetic beads are exchanged with a washing buffer solution through a G-25 desalting column; (5) The magnetic bead antibody coating obtained after the exchange is sealed and stored.

[0027] Based on the magnetic bead antibody coating prepared by the present application, the embodiments of the present application provide a low-abundance immune detection method, specifically: the magnetic bead antibody coating is reacted with a labeled antibody to form a double-antibody sandwich complex, so as to use the double-antibody sandwich complex for chemiluminescence detection; wherein the label of the labeled antibody includes acridan ester, trispyridine ruthenium, adamantane, luminol, horseradish peroxidase and alkaline phosphatase.

[0028] Some specific examples are listed below, it should be noted that the examples described below are exemplary and are used to explain the present application, and cannot be understood as limiting the present application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0029] In the following examples, the G-25 gravity desalting column is pre-equilibrated in the following manner: equilibrate with 5-10 column bed volumes of PBS, and use when all liquid has run off.

[0030] Example 1

[0031] This example provides a method for coating 20 nm carboxyl-modified dextran magnetic beads with GFAP antibody, comprising the following steps: (1) After the 20 nm carboxyl-modified dextran magnetic beads are mixed by blowing, three 2.0 mL Ep tubes are labeled, and 0.5 mL (5 mg) of magnetic beads are added to each tube; the three tubes are used as parallel samples, and the following steps are kept the same to verify the stability of the coating method.

[0032] (2) The magnetic beads are exchanged with a pre-equilibrated G-25 gravity desalting column, and the magnetic bead solution is replaced with 0.1M pH 5.5 MES buffer.

[0033] (3) The magnetic bead solution after the exchange is slowly added to 200ug GFAP antibody diluted with pre-cooled 0.1M pH 5.5 MES buffer, mixed thoroughly by blowing, and then placed on a vortex mixer at 1000 rpm and 25°C for 30 min.

[0034] (4) After the reaction in step (3) is completed, 100uL of EDC solution and 100uL of NHS solution are added, and the mixture is mixed again by blowing, then placed on a vortex mixer at 1000 rpm and 4°C for 90 min. The preparation method of EDC solution and NHS solution is as follows: take out 8mg of EDC and 16mg of NHS, both of which are dissolved in 1mL of pre-cooled 0.1M pH 5.5 MES buffer, and placed at 4°C.

[0035] (5) Take out the antibody magnetic bead sample after the reaction, exchange it with a pre-equilibrated G-25 gravity desalting column, and replace the magnetic bead solution with pH 7.4 1x PBS buffer. Take part of the sample for BCA protein quantification detection as a reference value for the amount of magnetic bead coating, and the three parallel sample tubes are numbered as samples 1, 2, and 3.

[0036] (6) Add 100uL of magnetic bead blocking solution containing 5% BSA, 5% glycine, 1% PEG2000, and 1% Tween-20 to the remaining sample, place it on a vortex mixer at 1000 rpm and 25°C for 30 min, and block it.

[0037] (7) The magnetic beads were stored in 1 mL of a magnetic bead storage solution containing 5% trehalose, 2% sorbitol, 0.5% BSA, 0.5% Tween-20, and 2% biolipidure-402, and stored at 4°C.

[0038] The samples subjected to the BCA protein quantitative detection in the above step were subjected to BCA test, and the results are shown in Table 1. In the table, A 562 represents the absorbance of the sample test, and the coating amount represents the amount of ug antibody that can be coated per mg of magnetic beads.

[0039] Table 1 Test data of the amount of GFAP antibody coated on 20 nm magnetic beads

[0040] Example 2

[0041] The present embodiment provides a method for coating GFAP antibody on 50 nm PEG-COOH modified dextran magnetic beads, comprising the following steps: (1) After the 50 nm PEG-COOH modified dextran magnetic beads were mixed by blowing, three 2.0 mL Ep tubes were labeled, and 0.5 mL (5 mg) of magnetic beads were added to each tube; the three tubes were used as parallel samples, and the following steps were operated in the same way to verify the coating stability of the method.

[0042] (2) The magnetic beads were exchanged with a pre-equilibrated G-25 gravity desalting column, and the magnetic bead solution was replaced with 0.025M pH 6.0 MES buffer; (3) The magnetic bead solution after the exchange was slowly added to 150 ug of GFAP antibody diluted with pre-cooled 0.025M pH 6.0 MES buffer, and then mixed by blowing, and placed on a vortex mixer at 1000 rpm and 25°C for 60 min.

[0043] (4) After the reaction in step (3) was completed, 80 uL of EDC solution and 80 uL of NHS solution were added, and then mixed by blowing again, and placed on a vortex mixer at 1000 rpm and 4°C for 60 min, and then restored to 25°C for 30 min. The preparation method of the EDC solution and the NHS solution is as follows: 5 mg of EDC and 12 mg of NHS were dissolved in 1 mL of pre-cooled 0.025M pH 6.0 MES buffer, and placed at 4°C.

[0044] (5) Take out the antibody magnetic bead sample after the reaction is completed, and use the pre-equilibrated G-25 gravity desalting column to replace the solution, and replace the magnetic bead solution with 1x PBS buffer solution with pH 7.4. Take part of the sample for BCA protein quantitative detection as a reference value of the magnetic bead coating amount, and the three parallel samples are sample 1, 2 and 3.

[0045] (6) Add 100 uL of magnetic bead blocking solution containing 5% BSA, 5% glycine, 1% triton X-100 and 2% commercial blocking agent CE510 / CE210 to the remaining sample, and place it on a vortex mixer at 1000 rpm and 25°C for 30 min for blocking. (7) Add 5% sorbitol, 1% sucrose, 0.5% BSA, 0.5% Tween-20 and 2% biolipidure-103 to 1 mL, and store at 4°C.

[0046] The samples taken for BCA protein quantitative detection in the above steps are all subjected to BCA test, and the results are shown in Table 2. In the table, A 562 represents the absorbance of the sample test, and the coating amount represents the amount of antibody that can be coated per mg of magnetic beads.

[0047] Table 2 Test data of 50 nm magnetic bead coating GFAP antibody amount

[0048] Comparative Example 1 In this example, 1.5 um carboxyl magnetic beads are coated with GFAP antibody according to the conventional method, including the following steps: (1) Vortex the 1.5 um carboxyl magnetic beads, label three 2.0 mL Ep tubes, and take 0.5 mL (5 mg) of magnetic beads and add them to each tube; the three tubes are parallel samples, and the following steps are the same.

[0049] (2) Use a magnetic stand to replace the solution of the above magnetic beads, and wash them with 0.05M pH5.8 MES activation buffer for 3 times and resuspend for standby.

[0050] (3) Add 50 uL of EDC solution and 50 uL of NHS solution to the resuspended magnetic beads, and place it on a vortex mixer at 1000 rpm and 25°C for 30 min. The preparation method of EDC solution and NHS solution is as follows: take out 10 mg of EDC and 15 mg of NHS, and dissolve them with 1 mL of pre-cooled 0.05M pH5.8 MES buffer solution.

[0051] (4) After the reaction in step (3) is completed, wash twice with 0.05M pH 6.0 MES coating buffer and resuspend, add 150ug GFAP antibody, place on a vortex mixer at 1000rpm, and react at 25℃ for 120min.

[0052] (5) Take the antibody magnetic bead sample after the reaction is completed and wash it twice with PBST. Reserve a portion of the sample for BCA protein quantitative detection as a reference value for the amount of magnetic bead coating. Number the three parallel tubes of samples 1, 2, and 3.

[0053] (6) Resuspend the magnetic beads in a magnetic bead blocking solution containing 1% BSA and 1% glycine, place them on a vortex mixer at 1000 rpm, and react at 25°C for 120 minutes for blocking.

[0054] (7) After washing twice with PBST, the volume of magnetic bead storage solution containing 5% sorbitol, 1% sucrose, 0.5% BSA and 2% biolipidure-103 was adjusted to 1 mL and stored at 4°C.

[0055] The samples retained for BCA protein quantitative detection in the above steps were all subjected to BCA test. Table 3 shows the test results. 562 It indicates the absorbance of the sample test, and the coating amount indicates how many μg of antibody can be coated per mg of magnetic beads.

[0056] Table 3 Test data of GFAP antibody amount coated on 1.5um magnetic beads

[0057] Comparative Example 2 According to the method in Comparative Example 1, 20 nm carboxyl-modified dextran magnetic beads were coated with GFAP antibodies.

[0058] The experiment found that this method could not coat the above magnetic beads. Figure 2 As shown, the upper picture shows the beginning of magnetic attraction, and the lower picture shows the magnetic attraction ten minutes later. The left side shows the micron magnetic beads in Comparative Example 1, and the right side shows the nano magnetic beads in this example.

[0059] Comparative Example 3 This comparative example provides a method for coating 20 nm carboxyl-modified dextran magnetic beads with GFAP antibodies, comprising the following steps: (1) After mixing the 20 nm carboxyl-modified dextran magnetic beads, label three 2.0 mL Eppendorf tubes and add 0.5 mL (5 mg) of magnetic beads to each tube; the three tubes serve as parallel samples and the following steps are performed identically to verify the coating stability of this method.

[0060] (2) Use a pre-equilibrated G-25 gravity desalting column to replace the magnetic bead solution with 0.1M pH 5.5 MES buffer.

[0061] (3) Add 100 μL each of EDC solution and NHS solution, mix again by pipetting, and then place on a vortex mixer at 1000 rpm and react at 25°C for 20 min. The preparation method of EDC solution and NHS solution is as follows: take out 8 mg of EDC and 16 mg of NHS, and dissolve them in 1 mL of room temperature 0.1 M pH 5.5 MES buffer.

[0062] (4) After the reaction in step (3) is completed, the solution is replaced using the same method as in step (2), and the magnetic bead solution after the replacement solution is slowly added dropwise to 200 μg of GFAP antibody diluted in 0.1 M pH 5.5 MES buffer prepared in advance at room temperature. After being thoroughly blown to mix, the solution is placed on a vortex mixer at 1000 rpm and 25°C for 30 min.

[0063] (5) After the reaction, remove the antibody magnetic bead sample and replace the solution with pH 7.4 1× PBS buffer using a pre-equilibrated G-25 gravity desalting column. A portion of the sample is reserved for BCA protein quantification as a reference value for the amount of magnetic bead coating. Three parallel tubes of samples are numbered as samples 1, 2, and 3.

[0064] (6) Add 100 μL of magnetic bead blocking solution containing 5% BSA, 5% glycine, 1% PEG2000, and 1% Tween-20 to the remaining sample, place on a vortex mixer at 1000 rpm, and react at 25°C for 30 minutes for blocking.

[0065] (7) Use magnetic bead storage solution containing 5% trehalose, 2% sorbitol, 0.5% BSA, 0.5% Tween-20 and 2% biolipidure-402 to position to 1 mL and store at 4°C.

[0066] The samples retained for BCA protein quantitative detection in the above steps were all subjected to BCA test. The results are shown in Table 4. 562 It indicates the absorbance of the sample test, and the coating amount indicates how many μg of antibody can be coated per mg of magnetic beads.

[0067] Table 4 Test data of GFAP antibody amount coated on 20nm magnetic beads

[0068] Example 3

[0069] Based on the magnetic bead antibody coating prepared in Example 1, 2 and Comparative Example 1, the sensitivity of each magnetic bead antibody coating was evaluated by using chemiluminescence to detect the GFAP antigen. It specifically includes the following steps: (1) Preparation of ALP-GFAP enzyme-labeled antibody.

[0070] The alkaline phosphatase (ALP) and GFAP antibody were each ultrafiltrated twice in an ultrafiltration tube through 0.01M pH 7.4 PBS buffer, and each was resuspended to 1 mg / ml for standby.

[0071] Preparation of azide-labeled alkaline phosphatase (ALP-Azide): Take 10 mg of Azide-PEG-NHS and add 1 mL of DMSO to prepare 10 mg / mL; according to the molar ratio of Azide-PEG-NHS:ALP = 20:1, take the above resuspended ALP enzyme and add it to the Azide-PEG-NHS solution, place it on a vortex mixer at 1000 rpm, 25°C, and react for 120 min; after the reaction is completed, ultrafiltrate twice in 0.01M pH 7.4 PBS buffer to remove excess Azide-PEG-NHS, and the product is resuspended to 1 mg / ml using PBS.

[0072] Preparation of DBCO-labeled GFAP antibody (GFAP-DBCO): Take 10 mg of DBCO-PEG-NHS and add 1 mL of DMSO to prepare 10 mg / mL; according to the molar ratio of DBCO-PEG-NHS:GFAP antibody = 20:1, add DBCO-PEG-NHS to the above resuspended GFAP antibody, place it on a vortex mixer at 1000 rpm, 25°C, and react for 120 min; after the reaction is completed, ultrafiltrate twice in 0.01M pH 7.4 PBS buffer to remove excess DBCO-PEG-NHS, and the product is resuspended to 1 mg / ml using PBS.

[0073] Mix the above two products uniformly according to the molar ratio of ALP-Azide:GFAP-DBCO = 3:1, place them on a vortex mixer at 1000 rpm, and react overnight at 2-8°C. The reacted sample is dialyzed in 0.01M pH 7.4 PBS, and an equal volume of glycerol is added for 4°C storage to obtain the final product ALP-GFAP enzyme-labeled antibody.

[0074] (2) Detection of GFAP antigen sample.

[0075] The magnetic bead coating materials obtained from Example 1, 2 and Comparative Example 1 were each diluted to 0.5 mg / mL, and chemiluminescence was used to detect samples of different concentrations of GFAP antigens; the secondary antibody was the ALP-GFAP enzyme-labeled antibody described above, and the luminescent substrate was APS-5. The signal intensities after detection of Comparative Examples 1, 2 and Comparative Example 1 were compared, and the test data are shown in Table 5.

[0076] Table 5 Test data for testing of magnetic bead-coated GFAP antibodies capturing GFAP antigens

[0077] As can be seen from Table 5, in the low-value test, the sensitivity of the nanometer magnetic beads coated with the magnetic beads of Examples 1 and 2 was significantly better than that of the micrometer magnetic beads of Comparative Example 1 and the nanometer magnetic beads coated at room temperature of Comparative Example 3; in the zero-value sample, the non-specific adsorption of the nanometer magnetic beads coated with Examples 1 and 2 was also better than that of the micrometer magnetic beads of Comparative Example 1 and Comparative Example 3. In combination with the magnetic bead coating amount data of Tables 1-4, it can also be seen that the nanometer magnetic beads coated using the method of the present application have a coating amount much higher than that of the micrometer magnetic beads and nanometer magnetic beads coated by conventional methods, and the batch stability of the magnetic bead coating amount is also better than that of the magnetic beads coated by conventional methods, thereby greatly improving the antigen capture performance based on the coating amount and the advantages of nanometer magnetic beads.

[0078] It should be noted that the present application is not limited to the above-described embodiments. The above-described embodiments are merely examples, and embodiments having the same constitution and playing the same effect as the technical idea within the scope of the technical solutions of the present application are all included in the technical scope of the present application. In addition, within the scope of the main idea of the present application, various modifications that can be thought of by those skilled in the art, other ways constructed by combining part of the constitutions of the embodiments are also included in the scope of the present application.

Claims

1. A method for coating small-sized magnetic nanoparticles with antibodies, characterized in that: The magnetic nanoparticles are surface-modified nanomagnetic beads with a size of 20-50 nm. The antibody coating method comprises the following steps: S1. Replace the magnetic beads with coating buffer using a G-25 desalting column. S2, coating the antibody onto magnetic beads using surface modification groups; S3. After coating is completed, the solution is replaced with washing buffer through a G-25 desalting column; S4. After the liquid is exchanged, the magnetic bead antibody coating is blocked and stored.

2. The antibody coating method according to claim 1, characterized in that The nanomagnetic beads are surface carboxyl-modified magnetic beads, and step S2 includes the following operations: S21, slowly dripping the magnetic beads into the coating buffer containing the monoclonal antibody for pre-reaction, wherein the pH of the reaction solution is lower than the antibody PI; S22, after the reaction in step S21 is completed, EDC and NHS are added to react.

3. The antibody coating method according to claim 2, characterized in that The reaction conditions of step S22 are: reaction at 4° C. for 30 to 90 minutes.

4. The antibody coating method according to claim 2, characterized in that The coating buffer is 0.01-0.1 M MES buffer with a pH of 5.0-6.

3.

5. The antibody coating method according to claim 1, characterized in that The washing buffer is a PBS buffer or a Tris buffer at pH 7.

4.

6. The antibody coating method according to claim 1, characterized in that Step S4 specifically includes: adding a blocking solution to the magnetic bead antibody coating for blocking, and directly adding a magnetic bead preservation solution to the reaction solution for preservation after the blocking reaction is completed; wherein, the blocking solution contains large molecular proteins / polymers and small molecular amino acids, and the magnetic bead preservation solution contains sugar alcohol substances and proteins / amino acids.

7. The antibody coating method according to claim 6, characterized in that The blocking solution is a PBS buffer containing 0.5-8% macromolecular protein / polymer, 0.5-5% small molecule amino acid and 0.1-2% surfactant, and the magnetic bead storage solution is a PBS buffer containing 2-10% sugar alcohol, 0.5-2% protein / amino acid and 0.05-1% surfactant.

8. A magnetic bead-antibody coating prepared according to the antibody coating method according to any one of claims 1 to 7.

9. Use of the magnetic bead antibody coating as claimed in claim 8 in low abundance immunoassay.

10. The use according to claim 9, characterized in that The magnetic bead antibody coating reacts with the labeled antibody to form a double antibody sandwich complex, thereby realizing immunoassay; wherein the labeling substance of the labeled antibody includes acridinium ester, terpyridine ruthenium, adamantane, luminol, horseradish peroxidase and alkaline phosphatase.

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