CN115856286A - Preparation method of high-stability magnetic beads - Google Patents

Abstract

The invention discloses a method for preparing magnetic beads with high stability, and relates to the technical field of immunochemical detection. In the present invention, the antibody to be modified is coupled to the magnetic beads by using a double-end functional group cross-linking agent and an inert protein. On the one hand, the physical adsorption of the antibody to the surface of the magnetic bead is reduced. The valence-linked proteins are cross-linked together 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 the double-end functional group cross-linking agent and the method of inert protein coupling, by reducing the physical adsorption of protein on the surface of magnetic beads and enhancing the connection of physical adsorption antibodies and magnetic beads, so that as many antibodies as possible can be coupled Covalently linked and immobilized on the magnetic beads, thereby improving the stability of the magnetic beads. Compared with the magnetic beads obtained by conventional techniques, the method of the invention can significantly improve the stability of the magnetic beads, reduce the shedding of antibodies, and is simple to operate and saves time.

Description

A kind of preparation method of high stability magnetic beads

technical field

The invention relates to the technical field of immunochemical detection, in particular to a preparation method of high-stability magnetic beads.

Background technique

Magnetic particles generally refer to spherical composites with a size of micron and nanometers. They are composite microspheres with certain magnetic properties and special structures formed by combining magnetic inorganic particles and organic polymers by appropriate methods. Its most prominent feature is that it has superparamagnetism, which can be magnetized by an external magnetic field. After the external magnetic field is removed, the magnetism disappears at the same time. This feature enables magnetic micro-nano materials to move and gather under the action of an external magnetic field, and at the same time re-disperse after the external magnetic field is removed, becoming a near-perfect biological separation carrier.

The idea of the invention of magnetic particles originally came from John Ugelstad, a chemist at the Norwegian University of Science and Technology. In 1976, he used polystyrene as the main material to produce uniformly magnetized spherical particles. At present, scientists can not only precisely control the particle size of magnetic beads and develop magnetic beads with a particle size of 1-100 μm, but also have many types of functional groups on the surface of magnetic beads, which are widely used in cell separation, nucleic acid extraction, and target identification of biomedicine And metabolic properties research and protein purification and immunoassay.

At present, magnetic particles are also widely used in various technical fields of in vitro diagnosis. Such as magnetic particle chemiluminescence immunoassay (CLIA). The particle size of the magnetic beads used in CLIA is about 1-3 μm. The magnetic bead preparation technology is mature and well-known magnetic bead manufacturers include Merck of Germany, JSR of Japan, and Dynabeads of the United States. The basic requirements of CLIA on the performance of magnetic beads are as follows: fast magnetic response speed, high magnetic content; good dispersion, slow sedimentation speed; high chemical stability, low non-specific adsorption, high signal-to-noise ratio; high group content, strong bonding force Strong, high sensitivity.

Magnetic particles 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 are widely used in the field of in vitro diagnostics for labeling antigens and antibodies because they do not need to add activators during the antibody coupling process, are simple and convenient to operate, controllable batch-to-batch variation, and easy to scale up the process. However, the hydrophobicity of tosyl-based magnetic beads is relatively strong. When coupling proteins, some proteins will be fixed on the magnetic beads in the form of physical adsorption. As time goes by, this part of physically adsorbed proteins will fall off. The long-term stability of the magnetic beads has an impact, which in turn affects the accelerated and long-term stability of the reagents (Figure 1). At present, people often use multiple washing methods at high temperature of 37-45°C to remove proteins that are not physically adsorbed firmly on the surface of magnetic beads, but this removal method takes a long time and has the problem of incomplete cleaning.

Contents of the invention

The technical problem to be solved by the present invention is how to reduce the physical adsorption of the surface of the magnetic beads to the protein and how to enhance the connection between the physically adsorbed protein and the magnetic beads, so as to improve the stability of the magnetic beads.

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

In a first aspect, the present invention provides a high-stability magnetic bead, the high-stability magnetic bead has the following structure,

The magnetic beads are toluenesulfonyl magnetic beads, the surface of the magnetic beads is coupled with an antibody, the antibody is connected to the magnetic beads through an amino chemical reaction or physical adsorption, and the antibody is connected to the antibody through a cross-linking agent. The crosslinking agent is a bifunctional crosslinking agent.

Further, the high stability magnetic beads provided by the present invention have the following structure,

The magnetic beads are toluenesulfonyl magnetic beads, the surface of the magnetic beads is coupled with an antibody and an inert protein, the antibody is connected to the magnetic bead through an amino chemical reaction or physical adsorption, and the inert protein is connected to the magnetic bead through an amino chemical reaction , the antibody and the antibody, and the antibody and the inert protein are connected through a cross-linking agent, and the cross-linking agent is a bifunctional cross-linking agent.

Further, the bifunctional crosslinking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate, succinimidyl suberate, dimethyl pimelimate or bifunctional PEG.

Further, the inert protein is selected from BSA or casein.

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

In the second aspect, the present invention provides a solution for preparing magnetic beads with high stability, including tosyl magnetic beads, antibodies and bifunctional cross-linking agents.

The present invention utilizes the sulfonyl group on the surface of the toluenesulfonyl magnetic bead to react chemically with the amino group on the antibody for chemical bond connection, without the need of additional antibody coupling process, most of the antibodies are connected to the surface of the magnetic bead through chemical bonds, the connection is stable and not easy to fall off .

Further, the solution also includes inert protein.

The inert protein can bind to redundant sites on the surface of the magnetic bead, thereby reducing the physical adsorption of the antibody on the surface of the magnetic bead and stabilizing the conformation of the antibody, improving the stability of the magnetic bead itself.

Further, the inert protein is selected from BSA or casein.

Further, the bifunctional crosslinking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate (BS3), succinimidyl suberate (DSS), pimelic acid Dimethyl ester (DMP) or difunctional PEG.

Further, the solution also includes a buffer.

On the other hand, the present invention provides a kind of preparation method of high stability magnetic bead, comprises the following steps:

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.

Further, the step S2 also includes putting the inert protein into the washed magnetic beads for coupling.

Further, in the preparation method provided by the present invention, the specific method of step S1 includes: adding boric acid buffer solution 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 ;

Further, in the preparation method provided by the present invention, the specific method of step S2 includes: adding the washed magnetic beads to the borate buffer for resuspension, and putting antibodies into it for coupling;

Further, in the preparation method provided by the present invention, the specific method of step S3 includes: washing the coupled magnetic bead solution with cross-linking buffer for 2-5 times, and then resuspending with cross-linking buffer; Add the bifunctional group cross-linking agent solution, shake and mix, and carry out cross-linking;

Furthermore, in the preparation method provided by the present invention, a blocking step is also included after the cross-linking is completed, and the specific operation is: after the cross-linking reaction is completed, add a blocking buffer to wash, then resuspend, and block at a constant temperature of 37°C for 18-28h , after the blocking is completed, it is stored with a storage buffer to obtain magnetic beads coupled with antibodies.

Further, the bifunctional crosslinking agent solution is prepared by dissolving the bifunctional crosslinking agent in DMSO or water, with a concentration of 5-20 mg/mL.

Further, the cross-linking buffer contains 100mM TEA, 100mMNaCl, pH8.0;

The blocking buffer contains 10mM tris, 0.5% BSA, pH7.4;

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

In one embodiment, the preparation method is specifically as follows:

S1, washing magnetic beads;

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 example, the mass of the tosyl magnetic beads to be coupled is defined as Amg, and the concentration of the magnetic bead stock solution is B mg/mL, then the volume of the magnetic bead stock solution measured is C mL=Amg÷B mg/mL ;

Accurately measure C mL volume of toluenesulfonyl magnetic bead concentrate into the container, add the first coating buffer with a volume of D=(A÷20)mL, oscillate and mix, and discard after magnetic adsorption for a period of time For the supernatant, repeat the above process and wash to obtain magnetic beads that have been washed.

Wherein, the first coating buffer: containing 100mM boric acid, pH9.5;

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

Resuspend the above-washed magnetic beads with the first coating buffer, then add 0.02Amg of antibody and 0.02Amg of inert protein, and add 1/3D mL of the second coating solution, so that the total volume of the final solution is DmL , shake and mix quickly, and carry out the coupling reaction in a 37°C incubator while mixing for 20h.

The inert protein used in this example is BSA.

Second coating buffer: containing 100 mM boric acid, 3M ammonium sulfate, pH 9.5.

S3. Adding a bifunctional cross-linking agent to the magnetic beads coupled in step S2 for cross-linking. Weigh a certain mass of double-end functional group cross-linking agent, and dissolve it in DMSO to obtain a double-end functional group cross-linking agent solution with a concentration of 10 mg/mL.

Wash the coupled magnetic bead solution 3 times with cross-linking buffer, then resuspend with cross-linking buffer, then add 0.002AmL double-end functional group cross-linking agent solution to the solution, oscillate and mix, and keep at 37°C under constant temperature conditions The cross-linking reaction was carried out for 2 h.

S4. Closed

After the cross-linking reaction, add D mL of blocking buffer to the cross-linked magnetic beads to wash, add D mL of blocking buffer to resuspend after washing, and block at 37°C for 18-28 hours. Wash with preservation solution, add D mL of preservation buffer after washing for preservation, and obtain antibody-coupled tosyl magnetic beads.

Compared with the prior art, the technical effects that the present invention can achieve include:

In the present invention, the antibody to be modified is coupled to the magnetic beads by using a double-terminal functional group cross-linking agent and an inert protein. The coupling of the inert protein can reduce the physical adsorption of the antibody to the surface of the magnetic bead. The agent can be cross-linked and immobilized on the magnetic beads with the covalently linked antibody, which finally plays a role in improving the stability of the magnetic beads.

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 double-terminal functional group cross-linking agents and inert proteins. By cross-linking physically adsorbed proteins with covalently linked proteins, antibodies As much as possible is connected and fixed on the magnetic beads, thus improving the stability of the magnetic beads. Compared with the magnetic beads obtained by the conventional process, the method of the invention can significantly improve the stability of the magnetic beads, reduce the shedding of antibodies, and is simple to operate and saves time.

Description of drawings

Fig. 1 is a schematic diagram of the principle of poor stability of tosyl magnetic beads in the prior art.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention, and similar component numbers in the drawings represent similar components. Apparently, the embodiments described below are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be understood that when used in this specification and the appended claims, the terms "comprising" and "comprises" indicate the presence of described features, integers, steps, operations, elements and/or components, but do not exclude one or Presence or addition of multiple other features, integers, steps, operations, elements, components and/or collections thereof.

It should also be understood that the terms used in the 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, the singular forms "a", "an" and "the" are intended to include plural forms unless the context clearly dictates otherwise.

Example 1

The embodiment of the present invention provides a method for preparing magnetic beads with high stability. This method is applicable to all protein-coupled tosyl magnetic beads. This embodiment takes tosyl magnetic beads modified with iPTH antibody as an example, and the specific operation is introduced. as follows:

S1. Washing of magnetic beads

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 example, the mass of tosyl magnetic beads to be coupled is set at 20 mg, and the concentration of the magnetic bead stock solution is 100 mg/mL, then the volume of the magnetic bead stock solution to be measured is 0.2 mL;

Accurately measure 0.2 mL volume of tosyl magnetic bead concentrate and put it into the container, add the first coating buffer with a volume of 1 mL, shake and mix well, discard the supernatant after magnetic adsorption for a period of time, repeat the above process, Wash 3 times in total to obtain magnetic beads that have been washed.

S2. Coupling of Magnetic Beads

Resuspend the above-washed magnetic beads with the first coating buffer, then add 0.4 mg of iPTH antibody and 0.4 mg of inert protein, and add 0.3 mL of the second coating solution, so that the total volume of the final solution is 1 mL. Shake and mix quickly, and carry out the coupling reaction for 20 hours in a 37°C incubator while mixing.

The inert protein used in this example is BSA.

S3. Cross-linking of magnetic beads

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

Wash the coupled magnetic bead solution 3 times with cross-linking buffer, then resuspend with cross-linking buffer, then add 0.04mL double-end functional group cross-linking agent solution to the solution, shake and mix well, and keep at 37°C under constant temperature conditions The cross-linking reaction was carried out for 2 h.

S4. Closed

After the cross-linking reaction, add 1 mL of blocking buffer to the cross-linked magnetic beads to wash 3 times, add 1 mL of blocking buffer to resuspend after washing, and block at 37°C for 18-28 hours. Wash with preservation solution for 3 times, add 1 mL of preservation buffer after washing for preservation, and obtain tosyl magnetic beads coupled with iPTH antibody.

In the embodiment of the present invention, the concrete composition of reagent used is as follows:

The first coating buffer: containing 100mM boric acid, pH9.5;

Second coating buffer: containing 100 mM boric acid, 3M ammonium sulfate, pH 9.5.

Cross-linking buffer: containing 100mM TEA, 100mM NaCl, pH8.0;

Blocking buffer: containing 10mM tris, 0.5% BSA, pH7.4;

Storage buffer: containing 10 mM PBS, 5% sucrose, 0.5% BSA, pH 7.0.

Comparative example 1

Prepare iPTH tosyl magnetic beads by conventional process:

S1. Washing of magnetic beads

Accurately measure 0.2 mL volume of tosyl magnetic bead concentrate and put it into the container, add the first coating buffer with a volume of 1 mL, shake and mix well, discard the supernatant after magnetic adsorption for a period of time, repeat the above process, Wash 3 times in total to obtain magnetic beads that have been washed.

S2. Coupling of Magnetic Beads

Resuspend the above-washed magnetic beads with the first coating buffer, then add 0.2mg of iPTH antibody, add 0.3mL of the second coating solution, make the total volume of the final solution 1mL, shake and mix quickly, The coupling reaction was carried out for 20 hours while mixing in a 37°C incubator.

S3. Closed

After the coupling reaction, add 1 mL of blocking buffer to the magnetic beads to wash for 3 times, add 1 mL of blocking buffer to resuspend after washing, block at 37°C for 18-28 hours, and wash with preservation solution after blocking After washing for 3 times, 1 mL of storage buffer was added for storage to obtain tosyl magnetic beads coupled with iPTH antibody.

Comparative example 2

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

S1. Washing of magnetic beads

Accurately measure 0.2 mL volume of tosyl magnetic bead concentrate and put it into the container, add the first coating buffer with a volume of 1 mL, shake and mix well, discard the supernatant after magnetic adsorption for a period of time, repeat the above process, Wash 3 times in total to obtain magnetic beads that have been washed.

S2. Coupling of Magnetic Beads

Resuspend the above-washed magnetic beads with the first coating buffer, then add 0.4 mg of iPTH antibody and 0.4 mg of inert protein, and add 0.3 mL of the second coating solution, so that the total volume of the final solution is 1 mL. Shake and mix quickly, and carry out the coupling reaction for 20 hours in a 37°C incubator while mixing.

S3. Closed

After the coupling reaction, add 1 mL of blocking buffer to the magnetic beads to wash for 3 times, add 1 mL of blocking buffer to resuspend after washing, block at 37°C for 18-28 hours, and wash with preservation solution after blocking After washing for 3 times, 1 mL of storage buffer was added for storage to obtain tosyl magnetic beads coupled with iPTH antibody.

Select a number of samples of iPTH tosyl magnetic beads obtained in Example 1 and Comparative Example 1-2, and 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 the table 1-6.

Table 1. The light value of each sample of the iPTH tosyl magnetic beads obtained in Comparative Example 1 before and after acceleration at 37°C

sample initial value seventh day 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%

Table 2. The light value of each sample of the iPTH tosyl magnetic beads obtained in Comparative Example 1 after being placed at 2-8°C for one year

Table 3. The light values of each sample of iPTH obtained in Comparative Example 2 before and after acceleration at 37°C

sample initial value seventh day 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%

Table 4. The light value of each sample of the iPTH tosyl magnetic beads obtained in Comparative Example 2 after being placed at 2-8°C for one year

sample Initial value at 4°C Store at 4°C 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%

Table 5. The light value of each sample of the iPTH tosyl magnetic beads obtained in Example 1 before and after acceleration at 37°C

Table 6. The light value of each sample after the iPTH magnetic beads obtained in Example 1 were placed at 2-8°C for one year

sample Initial value at 4°C Store at 4°C 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%

According to the results in Table 1-6, it can be seen that the magnetic beads obtained by the conventional coupling process in Comparative Example 1 have a 20-30% decline in light value before and after acceleration at 37°C, and the drop in light value of the magnetic beads placed at 4°C for one year is also 20-30%. After adding inert protein, the luminous value of magnetic beads decreases by 10-20% before and after acceleration at 37°C, and the decrease in luminous value of magnetic beads placed at 4°C for one year is also 10-20%. It can be seen that adding inert protein can increase the stability of magnetic beads , but still cannot achieve good results. However, in the scheme of the present invention, after cross-linking the magnetic beads by adding an inert protein coupling and cross-linking agent, the light values of each sample 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 stability of the magnetic beads obtained by the method of the present invention is significantly improved.

The above results all indicate that the method provided by the present invention can significantly improve the stability of the tosyl magnetic beads coupled with iPTH antibody.

In the foregoing embodiments, the descriptions of each embodiment have their own emphases, and for parts not described in detail in a certain embodiment, reference may be made to relevant descriptions of other embodiments.

The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technical field can easily think of various equivalent modifications within the technical scope disclosed in the present invention. Or replacement, these modifications or replacements should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (10)

1. A high stability magnetic bead, characterized in that it has the following structure,

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

2. A high stability magnetic bead having a structure,

the magnetic beads are tosyl magnetic beads, the surfaces of the magnetic beads are coupled with antibodies and inert proteins, the antibodies and the magnetic beads are connected through amino chemical reaction or physical adsorption, the inert proteins and the magnetic beads are connected through amino chemical reaction, the antibodies and the inert proteins are connected through cross-linking agents, and the cross-linking agents are bifunctional cross-linking agents.

3. The high stability magnetic bead of claim 1 or 2, wherein the bifunctional crosslinking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate, succinimidyl suberate, dimethyl pimidate, or bifunctional PEG.

4. The high stability magnetic bead of claim 2, wherein the inert protein is selected from BSA or casein.

5. The solution for preparing the high-stability magnetic beads is characterized by comprising tosyl magnetic beads, antibodies and bifunctional cross-linking agents.

6. The solution of claim 5, wherein the solution further comprises an inert protein.

7. The solution of claim 5 or 6, wherein the bifunctional crosslinking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate, succinimidyl suberate, dimethyl pimidate, or bifunctional PEG.

8. The solution of claim 6, wherein the inert protein is selected from BSA or casein.

9. A preparation method of high-stability magnetic beads is characterized by comprising the following steps:

s1, cleaning magnetic beads;

s2, putting the antibody into the washed magnetic beads, and coupling;

and S3, adding a bifunctional cross-linking agent into the magnetic beads coupled in the step S2 for cross-linking.

10. The method of claim 9, wherein the step S2 further comprises coupling an inert protein to the washed magnetic beads.

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