A method for preparing high-stability magnetic beads

Abstract

The present invention discloses a method for preparing high-stability magnetic beads, and relates to the field of immunochemical detection technology. The present invention couples the antibody to be modified to the magnetic beads by using a double-end functional group cross-linking agent and an inert protein, which reduces the physical adsorption of the antibody on the surface of the magnetic beads on the one hand, and the double-functional group cross-linking agent can cross-link the physically adsorbed antibody with the covalently linked protein to form a network structure, which ultimately plays a role in improving the stability of the magnetic beads. The method provided by the present invention is based on a method for coupling a double-end functional group cross-linking agent and an inert protein, which reduces the physical adsorption of the protein on the surface of the magnetic beads and enhances the connection between the physically adsorbed antibody and the magnetic beads, so that the coupled antibody is covalently linked and fixed on the magnetic beads as much as possible, thereby improving the stability of the magnetic beads. Compared with the magnetic beads obtained by conventional processes, the method of the present invention can significantly improve the stability of the magnetic beads, reduce the shedding of antibodies, and is simple to operate and time-saving.

Description

Technical Field

[0001] The invention relates to the technical field of immunochemical detection, and in particular to a method for preparing high-stability magnetic beads. Background Art

[0002] Magnetic particles generally refer to spherical composites with sizes in the micrometer and nanometer range. They are composite microspheres with certain magnetism and special structures formed by combining magnetic inorganic particles with organic polymers by appropriate methods. Their most prominent feature is that they have superparamagnetism and can be magnetized by an external magnetic field. When the external magnetic field is removed, the magnetism disappears at the same time. This feature enables magnetic micro-nano materials to move and aggregate under the action of an external magnetic field, and to disperse again after the external magnetic field is removed, making them a nearly perfect biological separation carrier.

[0003] The idea of ​​inventing magnetic microparticles originally came from John Ugelstad, a chemist at the Norwegian University of Science and Technology, who used polystyrene as the main material to create uniformly magnetized spherical particles in 1976. Currently, scientists can not only accurately control the particle size of magnetic beads and develop magnetic beads with a particle size of 1-100μm, but also enrich the types of functional groups on the surface of magnetic beads, which are widely used in cell separation, nucleic acid extraction, biomedical target identification and metabolic property research, protein purification and immunoassay.

[0004] At present, magnetic microparticles are also widely used in various technical fields of in vitro diagnosis. For example, magnetic microparticle chemiluminescence immunoassay technology (CLIA). The particle size of magnetic beads used in CLIA is about 1-3μm. The magnetic bead preparation technology is mature and the relatively well-known magnetic bead manufacturers include Merck in Germany, JSR in Japan, Dynabeads in the United States, etc. CLIA's basic requirements for the performance of magnetic beads are as follows: fast magnetic response speed, high magnetic content; good dispersibility, slow sedimentation speed; high chemical stability, low nonspecific adsorption, high signal-to-noise ratio; high group content, strong bonding force, and high sensitivity.

[0005] Magnetic microparticles can be divided into carboxyl magnetic beads, tosyl magnetic beads, amino magnetic beads, epoxy magnetic beads, streptavidin magnetic beads (SA-MB), etc. according to the different surface functional groups. Among them, tosyl magnetic beads do not require the addition of activators during the antibody coupling process, are simple and convenient to operate, have controllable batch differences, and are easy to scale up, so they are widely used in the field of in vitro diagnosis to label antigens and antibodies. However, tosyl magnetic beads are highly hydrophobic, and while coupling proteins, a portion of the protein will be fixed to the magnetic beads in the form of physical adsorption. Over time, this portion of physically adsorbed protein will fall off, affecting the long-term stability of the magnetic beads, and further affecting the accelerated stability and long-term stability of the reagent ( Figure 1). Currently, people often use a method of multiple washing at high temperature of 37-45°C to remove proteins that are not firmly physically adsorbed on the surface of magnetic beads, but this removal method is time-consuming and has the problem of incomplete cleaning. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to reduce the physical adsorption effect of the magnetic bead surface on the protein and enhance the connection between the physically adsorbed protein and the magnetic beads, thereby improving the stability of the magnetic beads.

[0007] In order to solve the above problems, the present invention proposes the following technical solutions:

[0008] In a first aspect, the present invention provides a high-stability magnetic bead having the following structure:

[0009]

[0010] The magnetic beads are tosyl magnetic beads, the surfaces of the magnetic beads are coupled with antibodies, the antibodies are connected to the magnetic beads through amino chemical reaction or physical adsorption, the antibodies are connected to each other through a cross-linking agent, and the cross-linking agent is a bifunctional cross-linking agent.

[0011] Furthermore, the high stability magnetic beads provided by the present invention have the following structure:

[0012]

[0013] The magnetic beads are tosyl magnetic beads, antibodies and inert proteins are coupled to the surface of the magnetic beads, the antibodies are connected to the magnetic beads through amino chemical reaction or physical adsorption, the inert proteins are connected to the magnetic beads through amino chemical reaction, the antibodies are connected to each other and the antibodies are connected to the inert proteins through a cross-linking agent, and the cross-linking agent is a bifunctional cross-linking agent.

[0014] Furthermore, the bifunctional cross-linking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate, succinimidyl suberate, dimethyl pimelimidate or bifunctional PEG.

[0015] Furthermore, the inert protein is selected from BSA or casein.

[0016] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies, monoclonal antibodies and antibody fragments. The antibody according to the present invention is preferably a goat, sheep, mouse, rabbit or rat antibody, a chimeric antibody or a further genetically engineered antibody, as long as the characteristic properties according to the present invention are retained. "Antibody fragment" comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen binding site. Examples of antibody fragments include diabodies, single-chain antibody molecules and multispecific antibodies formed by antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; diabodies; and multispecific antibodies formed by antibody fragments.

[0017] In a second aspect, the present invention provides a solution for preparing high-stability magnetic beads, comprising tosyl magnetic beads, antibodies and a bifunctional cross-linking agent.

[0018] The present invention utilizes the sulfonyl groups on the surface of tosyl magnetic beads to react with the amino groups on the antibodies to perform chemical bond connection, and does not require an additional antibody coupling process. Most antibodies are chemically bonded to the surface of the magnetic beads, and the connection is stable and not prone to falling off.

[0019] Furthermore, the solution also includes inert protein.

[0020] The inert protein can bind to the redundant sites on the surface of the magnetic beads, thereby reducing the physical adsorption of the antibody on the surface of the magnetic beads and stabilizing the antibody conformation, thereby improving the stability of the magnetic beads themselves.

[0021] Furthermore, the inert protein is selected from BSA or casein.

[0022] Furthermore, the bifunctional cross-linking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate (BS3), succinimidyl suberate (DSS), dimethyl pimelimidate (DMP) or bifunctional PEG.

[0023] Furthermore, the solution also includes a buffer solution.

[0024] In another aspect, the present invention provides a method for preparing high-stability magnetic beads, comprising the following steps:

[0025] S1, washing magnetic beads;

[0026] S2, putting the antibody into the washed magnetic beads for coupling;

[0027] S3. Add a bifunctional cross-linking agent to the magnetic beads coupled in step S2 for cross-linking.

[0028] Furthermore, the step S2 also includes adding the inert protein into the washed magnetic beads for coupling.

[0029] Furthermore, in the preparation method provided by the present invention, the specific method of step S1 includes: adding boric acid buffer to the magnetic bead stock solution, shaking and mixing, discarding the supernatant after magnetic adsorption for a period of time, and repeating the above washing process 2-5 times;

[0030] Furthermore, in the preparation method provided by the present invention, the specific method of step S2 includes: adding the washed magnetic beads to the boric acid buffer to resuspend them, and adding antibodies thereto for coupling;

[0031] Furthermore, in the preparation method provided by the present invention, the specific method of step S3 includes: washing the coupled magnetic bead solution with a cross-linking buffer for 2-5 times, and then resuspending with a cross-linking buffer; then adding a bifunctional cross-linking agent solution to the solution, shaking and mixing, and cross-linking;

[0032] Furthermore, in the preparation method provided by the present invention, a blocking step is also included after the cross-linking is completed. The specific operation is: after the cross-linking reaction is completed, a blocking buffer is added for washing, and then the beads are resuspended and blocked at a constant temperature of 37°C for 18-28 hours. After the blocking is completed, the beads are stored with a storage buffer to obtain antibody-coupled magnetic beads.

[0033] Furthermore, the bifunctional cross-linking agent solution is prepared by dissolving the bifunctional cross-linking agent in DMSO or water, and the concentration is 5-20 mg / mL.

[0034] Further, the cross-linking buffer comprises 100 mM TEA, 100 mM NaCl, pH 8.0;

[0035] The blocking buffer contains 10 mM tris, 0.5% BSA, pH 7.4;

[0036] Storage buffer contained 10 mM PBS, 5% sucrose, 0.5% BSA, pH 7.0.

[0037] In one embodiment, the preparation method is as follows:

[0038] S1, washing magnetic beads;

[0039] It should be noted that the magnetic bead stock solution contains magnetic beads and preservation solution, and the amount of magnetic beads needs to be determined during coupling. In this embodiment, the mass of the tosyl magnetic beads to be coupled is set as A mg, and the concentration of the magnetic bead stock solution is B mg / mL. The volume of the magnetic bead stock solution measured is C mL = A mg ÷ B mg / mL;

[0040] Accurately measure C mL of tosyl magnetic bead concentrate and put it into a container, add D = (A ÷ 20) mL of the first coating buffer, shake and mix, discard the supernatant after magnetic adsorption for a period of time, repeat the above process, wash, and obtain cleaned magnetic beads.

[0041] Wherein, the first coating buffer comprises: 100 mM boric acid, pH 9.5;

[0042] S2, putting the antibody into the washed magnetic beads for coupling;

[0043] The washed magnetic beads were resuspended in the first coating buffer, and then 0.02A mg of antibody and 0.02A mg of inert protein were added, and 1 / 3D mL of the second coating solution was added to make the total volume of the final solution D mL. The mixture was quickly shaken and mixed, and the coupling reaction was carried out in a 37°C incubator for 20 h while mixing.

[0044] The inert protein used in this example is BSA.

[0045] Secondary coating buffer: Contains 100 mM boric acid, 3 M ammonium sulfate, pH 9.5.

[0046] S3, adding a bifunctional crosslinking agent to the magnetic beads after coupling in step S2 for crosslinking. Weigh a certain mass of the bifunctional crosslinking agent, dissolve it with DMSO, and obtain a bifunctional crosslinking agent solution with a concentration of 10 mg / mL.

[0047] The coupled magnetic bead solution was washed three times with cross-linking buffer and then resuspended with cross-linking buffer. Subsequently, 0.002AmL of double-end functional group cross-linking agent solution was added to the solution and shaken to mix. The cross-linking reaction was carried out at a constant temperature of 37°C for 2h.

[0048] S4. Closed

[0049] After the cross-linking reaction is completed, D mL of blocking buffer is added to the cross-linked magnetic beads for washing. After washing, D mL of blocking buffer is added for resuspending, and the beads are blocked at 37°C for 18-28 hours. After blocking, the beads are washed with preservation solution. After washing, D mL of preservation buffer is added for preservation to obtain antibody-coupled tosyl magnetic beads.

[0050] Compared with the prior art, the technical effects achieved by the present invention include:

[0051] The present invention couples the antibody to be modified to the magnetic beads by using a double-end functional group cross-linking agent and an inert protein. The coupling of the inert protein can reduce the physical adsorption of the antibody on the surface of the magnetic beads. For the physically adsorbed antibody, the double-functional group cross-linking agent can cross-link it with the covalently linked antibody and fix it on the magnetic beads, thereby ultimately improving the stability of the magnetic beads.

[0052] The preparation method of the high-stability magnetic beads provided by the present invention is a method for obtaining antibody-containing magnetic beads based on the coupling of a double-end functional group cross-linking agent and an inert protein, by cross-linking physically adsorbed proteins with covalently linked proteins, so that as many antibodies as possible are connected and fixed on the magnetic beads, thereby improving the stability of the magnetic beads. Compared with the magnetic beads obtained by conventional processes, the method of the present invention can significantly improve the stability of the magnetic beads, reduce the shedding of antibodies, and is simple to operate and time-saving. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the principle that tosyl magnetic beads have poor stability in the prior art. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Similar component numbers in the drawings represent similar components. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0056] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0057] Example 1

[0058] The embodiment of the present invention provides a method for preparing high-stability magnetic beads. This method is applicable to all tosyl magnetic beads coupled to proteins. This embodiment takes tosyl magnetic beads modified with iPTH antibodies as an example. The specific operation is described as follows:

[0059] S1. Washing of magnetic beads

[0060] It should be noted that the magnetic bead stock solution contains magnetic beads and preservation solution, and the amount of magnetic beads needs to be determined during coupling. In this embodiment, the mass of the tosyl magnetic beads to be coupled is set to 20 mg, the concentration of the magnetic bead stock solution is 100 mg / mL, and the volume of the magnetic bead stock solution measured is 0.2 mL;

[0061] Accurately measure 0.2 mL of tosyl magnetic bead concentrate and put it into a container, add 1 mL of the first coating buffer, shake and mix, discard the supernatant after magnetic adsorption for a period of time, repeat the above process, wash 3 times in total, and obtain cleaned magnetic beads.

[0062] S2. Coupling of magnetic beads

[0063] The washed magnetic beads were resuspended in the first coating buffer, and then 0.4 mg of iPTH antibody and 0.4 mg of inert protein were added, and 0.3 mL of the second coating solution was added to make the total volume of the final solution 1 mL. The solution was quickly shaken and mixed, and the coupling reaction was carried out in a 37°C incubator for 20 h while mixing.

[0064] The inert protein used in this example is BSA.

[0065] S3. Cross-linking of magnetic beads

[0066] A certain mass of a double-end functional group cross-linking agent DSS was weighed and dissolved with DMSO to obtain a double-end functional group cross-linking agent solution with a concentration of 10 mg / mL.

[0067] The coupled magnetic bead solution was washed three times with cross-linking buffer and then resuspended with cross-linking buffer. Subsequently, 0.04 mL of double-end functional group cross-linking agent solution was added to the solution and shaken to mix. The cross-linking reaction was carried out at a constant temperature of 37°C for 2 h.

[0068] S4. Closed

[0069] After the cross-linking reaction is completed, 1 mL of blocking buffer is added to the cross-linked magnetic beads for washing 3 times. After washing, 1 mL of blocking buffer is added for resuspending, and the beads are blocked at 37°C for 18-28 hours. After blocking, the beads are washed 3 times with preservation solution. After washing, 1 mL of preservation buffer is added for preservation to obtain tosyl magnetic beads coupled with iPTH antibody.

[0070] In the embodiments of the present invention, the specific components of the reagents used are as follows:

[0071] First coating buffer: containing 100 mM boric acid, pH 9.5;

[0072] Secondary coating buffer: Contains 100 mM boric acid, 3 M ammonium sulfate, pH 9.5.

[0073] Cross-linking buffer: contains 100 mM TEA, 100 mM NaCl, pH 8.0;

[0074] Blocking buffer: contains 10 mM tris, 0.5% BSA, pH 7.4;

[0075] Storage buffer: Contains 10 mM PBS, 5% sucrose, 0.5% BSA, pH 7.0.

[0076] Comparative Example 1

[0077] iPTH tosyl magnetic beads were prepared using conventional techniques:

[0078] S1. Washing of magnetic beads

[0079] Accurately measure 0.2 mL of tosyl magnetic bead concentrate and put it into a container, add 1 mL of the first coating buffer, shake and mix, discard the supernatant after magnetic adsorption for a period of time, repeat the above process, wash 3 times in total, and obtain cleaned magnetic beads.

[0080] S2. Coupling of magnetic beads

[0081] The washed magnetic beads were resuspended in the first coating buffer, and then 0.2 mg of iPTH antibody was added, and 0.3 mL of the second coating solution was added to make the total volume of the final solution 1 mL. The beads were quickly shaken and mixed, and the coupling reaction was carried out in a 37°C incubator while mixing for 20 h.

[0082] S3. Closed

[0083] After the coupling reaction is completed, 1 mL of blocking buffer is added to the magnetic beads for washing 3 times. After washing, 1 mL of blocking buffer is added for resuspending, and the beads are blocked at 37°C for 18-28 hours. After blocking, the beads are washed 3 times with preservation solution. After washing, 1 mL of preservation buffer is added for preservation to obtain tosyl magnetic beads coupled with iPTH antibody.

[0084] Comparative Example 2

[0085] iPTH tosyl magnetic beads prepared by adding only inert protein without adding bifunctional cross-linking agent:

[0086] S1. Washing of magnetic beads

[0087] Accurately measure 0.2 mL of tosyl magnetic bead concentrate and put it into a container, add 1 mL of the first coating buffer, shake and mix, discard the supernatant after magnetic adsorption for a period of time, repeat the above process, wash 3 times in total, and obtain cleaned magnetic beads.

[0088] S2. Coupling of magnetic beads

[0089] The washed magnetic beads were resuspended in the first coating buffer, and then 0.4 mg of iPTH antibody and 0.4 mg of inert protein were added, and 0.3 mL of the second coating solution was added to make the total volume of the final solution 1 mL. The solution was quickly shaken and mixed, and the coupling reaction was carried out in a 37°C incubator for 20 h while mixing.

[0090] S3. Closed

[0091] After the coupling reaction is completed, 1 mL of blocking buffer is added to the magnetic beads for washing 3 times. After washing, 1 mL of blocking buffer is added for resuspending, and the beads are blocked at 37°C for 18-28 hours. After blocking, the beads are washed 3 times with preservation solution. After washing, 1 mL of preservation buffer is added for preservation to obtain tosyl magnetic beads coupled with iPTH antibody.

[0092] Multiple iPTH tosyl magnetic bead samples obtained in Example 1 and Comparative Examples 1-2 were selected to test their light values ​​after being accelerated at 37° C. for 7 days and placed at 2-8° C. for one year. The test results are shown in Tables 1-6.

[0093] Table 1. Light values ​​of samples of iPTH tosyl magnetic beads obtained in Comparative Example 1 before and after acceleration at 37°C

[0094] sample Initial Value Day 7 Decline A 7848 5530 -29.54％ B 46520 32871 -29.34％ C 274523 198425 -27.72％ D 1348450 965220 -28.42％ E 5794989 4186300 -27.76％ F 9997122 7014980 -29.83％ G 13033746 9496387 -27.14％

[0095] Table 2. Light values ​​of the iPTH tosyl magnetic beads obtained in Comparative Example 1 after being placed at 2-8°C for one year

[0096]

[0097]

[0098] Table 3. Light values ​​of samples of iPTH obtained in Comparative Example 2 before and after acceleration at 37°C

[0099] sample Initial Value Day 7 Decline A 8849 7146 -19.24％ B 46630 38171 -18.14％ C 269525 223652 -17.02％ D 1378485 1150759 -16.52％ E 5801990 4881794 -15.86％ F 9899192 8342049 -15.73％ G 13135748 10897417 -17.04％

[0100] Table 4. Light values ​​of the iPTH tosyl magnetic beads obtained in Comparative Example 2 after being placed at 2-8°C for one year

[0101] sample 4℃ initial value 4℃ for one year Decline A 7959 6367 -20.00％ B 47089 39079 -17.01％ C 274026 230127 -16.02％ D 1370997 1160960 -15.32％ E 5804998 4812924 -17.09％ F 10214325 8711798 -14.71％ G 13357754 11535756 -13.64％

[0102] Table 5. Light values ​​of samples of iPTH tosyl magnetic beads obtained in Example 1 before and after acceleration at 37°C

[0103]

[0104]

[0105] Table 6. Light values ​​of the iPTH magnetic beads obtained in Example 1 after being stored at 2-8°C for one year

[0106] sample 4℃ initial value 4℃ for one year Decline A 7845 7744 -1.29％ B 45837 45333 -1.10％ C 249909 243486 -2.57％ D 1349769 1366911 1.27％ E 5644868 5723332 1.39％ F 9931786 9746062 -1.87％ G 11739358 11469353 -2.30％

[0107] According to the results of Tables 1-6, the light value of the magnetic beads obtained by the conventional coupling process in Comparative Example 1 decreased by 20-30% before and after acceleration at 37°C, and the light value of the magnetic beads placed at 4°C for one year also decreased by 20-30%. After adding inert protein, the light value of the magnetic beads decreased by 10-20% before and after acceleration at 37°C, and the light value of the magnetic beads placed at 4°C for one year also decreased by 10-20%. It can be seen that adding inert protein can increase the stability of the magnetic beads, but still cannot achieve better results. The scheme of the present invention, after adding inert protein coupling and cross-linking agent to the magnetic beads for cross-linking, the light values ​​of the obtained magnetic beads before and after acceleration at 37°C and after being placed at 4°C for one year are all within 10%. Obviously, the magnetic beads obtained by the method of the present invention have significantly improved stability.

[0108] The above results all indicate that the method provided by the present invention can significantly improve the stability of tosyl magnetic beads coupled to iPTH antibodies.

[0109] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0110] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. High stability magnetic beads, characterized in that: The high stability magnetic beads have the following structure: The magnetic beads are tosyl magnetic beads, the surfaces of the magnetic beads are coupled with antibodies and inert proteins, the antibodies are connected to the magnetic beads through amino chemical reaction or physical adsorption, the inert proteins are connected to the magnetic beads through amino chemical reaction, the antibodies are connected to each other and to the inert proteins through a crosslinker, and the crosslinker is a bifunctional crosslinker; the inert protein is selected from BSA or casein.

2. The high stability magnetic beads according to claim 1, characterized in that: The bifunctional cross-linking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate, succinimidyl suberate, dimethyl pimelimidate or bifunctional PEG.

3. A method for preparing high-stability magnetic beads, characterized in that: The following steps are involved: S1, washing magnetic beads; S2, putting the antibody into the washed magnetic beads for coupling; S3, adding a bifunctional cross-linking agent to the magnetic beads coupled in step S2 for cross-linking; The step S2 also includes putting the inert protein into the washed magnetic beads for coupling; The inert protein is selected from BSA or casein.

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