CN115856286A - Preparation method of high-stability magnetic beads - Google Patents
Preparation method of high-stability magnetic beads Download PDFInfo
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Abstract
The invention discloses a preparation method of high-stability magnetic beads, and relates to the technical field of immunochemistry detection. According to the invention, the antibody to be modified is coupled on the magnetic beads by using the double-end functional group cross-linking agent and the inert protein, so that physical adsorption of the antibody on the surfaces of the magnetic beads is reduced, and meanwhile, the double-functional group cross-linking agent can cross-link the physically adsorbed antibody and the covalently connected protein together to form a network structure, thereby finally playing a role in improving the stability of the magnetic beads. The method provided by the invention is based on a coupling method of a double-end functional group cross-linking agent and inert protein, and the coupled antibodies are covalently connected and fixed on magnetic beads as much as possible by reducing the physical adsorption of the protein on the surfaces of the magnetic beads and enhancing the connection between the physically adsorbed antibodies and the magnetic beads, so that the stability of the magnetic beads is improved. Compared with the magnetic beads obtained by the conventional process, the method provided by the invention can obviously improve the stability of the magnetic beads, reduce the falling of the antibody, and is simple to operate and time-saving.
Description
Technical Field
The invention relates to the technical field of immunochemistry detection, in particular to a preparation method of a high-stability magnetic bead.
Background
The magnetic particles are generally spherical composites with micron and nanometer sizes, and are composite microspheres with certain magnetism and special structures formed by combining magnetic inorganic particles and organic polymers by a proper method. The magnetic material has the most prominent characteristics of superparamagnetism, can be magnetized by an external magnetic field, and the magnetism disappears simultaneously after the external magnetic field is removed. The characteristic enables the magnetic micro-nano material to have the ability of moving and gathering under the action of an external magnetic field and re-dispersing after the external magnetic field is removed, thus becoming a nearly perfect biological separation carrier.
The inventive concept of magnetic particles was originally derived from the chemist of norwegian science and technology university, john ugelstad who produced uniformly magnetized spherical particles in 1976 using polystyrene as the main material. At present, scientists can accurately control the particle size of magnetic beads and develop the magnetic beads with the particle size of 1-100 mu m, and the types of functional groups on the surfaces of the magnetic beads are rich, so the magnetic beads are widely used for cell separation, nucleic acid extraction, target identification and metabolic property research of biological medicines and protein purification and immunoassay.
Magnetic particles are also widely used in various technical fields of in vitro diagnosis. Such as magnetic particle chemiluminescence immunoassay (CLIA). The CLIA beads have a particle size of about 1 to 3 μm, and the magnetic bead production technology is well-established and relatively well-known from German Merck, JSR, dynabeads, USA. The performance of the CLIA on magnetic beads basically requires the following: the magnetic response speed is high, and the magnetic content is high; the dispersibility is good, and the sedimentation speed is slow; the chemical stability is high, the non-specific adsorption is low, and the signal-to-noise ratio is high; high radical content, strong bonding force and high sensitivity.
The magnetic particles may be classified into carboxyl magnetic beads, tosyl magnetic beads, amino magnetic beads, epoxy magnetic beads, streptavidin magnetic beads (SA-MB), and the like according to the difference in surface functional groups. The tosyl magnetic beads are free from adding an activating agent in the process of coupling the antibody, so the method is simple and convenient to operate, controllable in process batch difference and easy to amplify, and thus the tosyl magnetic beads are widely applied to the field of in vitro diagnosis for labeling antigen and antibody. However, the tosyl magnetic beads have a relatively strong hydrophobicity, and a part of proteins are physically adsorbed and fixed on the magnetic beads while being coupled with proteins, and the physically adsorbed proteins fall off with the lapse of time, so that the long-term stability of the magnetic beads is affected, and the accelerated stability and the long-term stability of the reagent are affected (fig. 1). At present, people often use a method of cleaning for many times at the high temperature of 37-45 ℃ to remove proteins which are physically and insecure adsorbed on the surfaces of magnetic beads, but the removal method is long in time consumption and has the problem of incomplete cleaning.
Disclosure of Invention
The invention aims to solve the technical problems of reducing the physical adsorption effect of the surfaces of magnetic beads on proteins, enhancing the connection between physically adsorbed proteins and the magnetic beads and improving 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 having a 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 and the antibodies are connected through a cross-linking agent, and the cross-linking agent is a bifunctional cross-linking agent.
Further, the high-stability magnetic bead provided by the invention has 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 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.
Further, the bifunctional crosslinking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate, succinimidyl suberate, dimethyl pimidate, 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 antibody according to the 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 invention are retained. An "antibody fragment" comprises a portion of a full-length antibody, preferably a variable domain thereof, or at least an antigen-binding site 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; a single chain antibody molecule; scFv, sc (Fv) 2; a diabody; and multispecific antibodies formed from antibody fragments.
In a second aspect, the present invention provides a solution for preparing high-stability magnetic beads, comprising tosyl magnetic beads, antibodies, and bifunctional cross-linking agents.
The invention utilizes the chemical reaction between the sulfonyl on the surface of the tosyl magnetic bead and the amino on the antibody to carry out chemical bond connection, does not need an additional antibody coupling process, and has stable connection and difficult shedding because most of the antibody is connected to the surface of the magnetic bead through the chemical bond.
Further, the solution also includes an inert protein.
The inert protein can be combined with redundant sites on the surface of the magnetic bead, so that the physical adsorption of the surface of the magnetic bead on the antibody is reduced, the antibody conformation is stabilized, and the stability of the magnetic bead is improved.
Further, the inert protein is selected from BSA or casein.
Further, the bifunctional crosslinking agent is selected from glutaraldehyde, disulfosuccinimidyl suberate (BS 3), succinimidyl suberate (DSS), dimethyl pimidate (DMP), or bifunctional PEG.
Further, the solution also includes a buffer.
On the other hand, the invention provides a preparation method of high-stability magnetic beads, which comprises the following steps:
s1, cleaning magnetic beads;
s2, putting the antibody into the washed magnetic beads for coupling;
and S3, adding a bifunctional cross-linking agent into the magnetic beads coupled in the step S2 for cross-linking.
Further, the step S2 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 into the magnetic bead stock solution, shaking and uniformly mixing, carrying out magnetic adsorption for a period of time, then removing the supernatant, and repeating the washing process for 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 into the boric acid buffer solution for resuspension, and adding antibodies into the boric acid buffer solution for coupling;
further, in the preparation method provided by the present invention, the specific method in step S3 includes: washing the coupled magnetic bead solution with a cross-linking buffer solution for 2-5 times, and then re-suspending with the cross-linking buffer solution; adding a bifunctional cross-linking agent solution into the solution, shaking and uniformly mixing, and crosslinking;
further, in the preparation method provided by the invention, a blocking step is further included after the crosslinking is completed, and the specific operations are as follows: and adding a blocking buffer solution for cleaning after the crosslinking reaction is finished, then carrying out heavy suspension, blocking for 18-28h at the constant temperature of 37 ℃, and preserving by using a preservation buffer solution after the blocking is finished to obtain the magnetic beads coupled with the antibody.
Furthermore, the bifunctional cross-linking agent solution is prepared by dissolving the bifunctional cross-linking agent in DMSO or water, and the concentration is 5-20mg/mL.
Further, the crosslinking buffer comprises 100mM TEA,100mM NaCl, pH8.0;
the blocking buffer contained 10mM tris,0.5% BSA, pH7.4;
the preservation buffer contained 1 mM PBS,5% sucrose, 0.5% BSA, pH7.0.
In one embodiment, the preparation method is as follows:
s1, cleaning magnetic beads;
the stock solution of magnetic beads includes magnetic beads and a preservation solution, and the amount of the magnetic beads needs to be determined during coupling. In this embodiment, 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 measured magnetic bead stock solution is C mL = Amg ÷ B mg/mL;
accurately measuring tosyl magnetic bead concentrated solution with the volume of C mL, putting the tosyl magnetic bead concentrated solution into a container, adding a first coating buffer solution with the volume of D = (A/20) mL, oscillating and uniformly mixing, carrying out magnetic adsorption for a period of time, discarding a supernatant, repeating the process, and washing to obtain the washed magnetic beads.
Wherein the first coating buffer: comprising 100mM boric acid, pH9.5;
s2, putting the antibody into the washed magnetic beads for coupling;
and (2) resuspending the washed magnetic beads with a first coating buffer solution, adding 0.02Amg of antibody and 0.02Amg of inert protein, adding 1/3D mL of a second coating solution to enable the total volume of the final solution to be DmL, rapidly shaking and uniformly mixing, and carrying out coupling reaction for 20 hours while uniformly mixing in a 37 ℃ incubator.
The inert protein used in this example was BSA.
Second coating buffer: comprising 100mM boric acid, 3M ammonium sulphate, pH9.5.
And S3, adding a bifunctional crosslinking agent into the magnetic beads coupled in the step S2 for crosslinking. Weighing a certain mass of the double-end functional group cross-linking agent, and dissolving the double-end functional group cross-linking agent by using DMSO (dimethylsulfoxide) to obtain a double-end functional group cross-linking agent solution with the concentration of 10 mg/mL.
And (3) washing the coupled magnetic bead solution for 3 times by using a crosslinking buffer solution, then re-suspending by using the crosslinking buffer solution, then adding a double-end functional group crosslinking agent solution of 0.002AmL into the solution, uniformly shaking and mixing, and carrying out crosslinking reaction for 2 hours at the constant temperature of 37 ℃.
S4. Seal
And after the crosslinking reaction is finished, adding D mL of sealing buffer solution into the crosslinked magnetic beads for cleaning, adding D mL of sealing buffer solution after the cleaning is finished for resuspension, sealing for 18-28h at the constant temperature of 37 ℃, cleaning by using a preservation solution after the sealing is finished, and adding D mL of preservation buffer solution for preservation after the cleaning is finished to obtain the antibody-coupled tosyl magnetic beads.
Compared with the prior art, the invention can achieve the following technical effects:
according to the invention, the antibody to be modified is coupled on the magnetic bead by using the double-end functional group cross-linking agent and the inert protein, the coupling of the inert protein can reduce the physical adsorption of the antibody on the surface of the magnetic bead, and for the physically adsorbed antibody, the bifunctional cross-linking agent can cross-link the antibody with the covalently connected antibody and fix the antibody on the magnetic bead, so that the effect of improving the stability of the magnetic bead is finally achieved.
The preparation method of the high-stability magnetic bead provided by the invention is a method for obtaining the magnetic bead containing the antibody based on coupling of the double-end functional group cross-linking agent and the inert protein, and the physically adsorbed protein and the covalently connected protein are cross-linked, so that the antibody is connected and fixed on the magnetic bead as much as possible, and the stability of the magnetic bead is improved. Compared with the magnetic beads obtained by the conventional process, the method provided by the invention can obviously improve the stability of the magnetic beads, reduce the falling of the antibody, and is simple to operate and time-saving.
Drawings
FIG. 1 is a schematic diagram illustrating the poor stability of tosyl magnetic beads in the prior art.
Detailed Description
The technical solutions in the embodiments will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, wherein like reference numerals represent like elements in the drawings. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the embodiments of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the embodiments of the invention. As used in the description of embodiments of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Example 1
The embodiment of the invention provides a preparation method of high-stability magnetic beads, which is applicable to tosyl magnetic beads of all coupled proteins, and takes tosyl magnetic beads modified with iPTH antibodies as an example, and the specific operation is as follows:
s1, cleaning of magnetic beads
The stock solution of magnetic beads includes magnetic beads and a storage solution, and the amount of the magnetic beads needs to be determined during coupling. In this embodiment, the mass of the tosyl magnetic bead to be coupled is 20mg, and the concentration of the magnetic bead stock solution is 100mg/mL, the volume of the measured magnetic bead stock solution is 0.2mL;
accurately measuring tosyl magnetic bead concentrated solution with the volume of 0.2mL, putting the tosyl magnetic bead concentrated solution into a container, adding a first coating buffer solution with the volume of 1mL, shaking and uniformly mixing, carrying out magnetic adsorption for a period of time, discarding the supernatant, repeating the process, and washing for 3 times to obtain the washed magnetic beads.
S2. Coupling of magnetic beads
And (2) resuspending the washed magnetic beads with a first coating buffer solution, adding 0.4mg of iPTH antibody and 0.4mg of inert protein, adding 0.3mL of a second coating solution to enable the total volume of the final solution to be 1mL, rapidly shaking and uniformly mixing, and carrying out coupling reaction for 20 hours while uniformly mixing in a 37 ℃ incubator.
The inert protein used in this example was BSA.
S3, crosslinking of magnetic beads
Weighing a certain mass of the double-end functional group cross-linking agent DSS, and dissolving the DSS by using DMSO to obtain a double-end functional group cross-linking agent solution with the concentration of 10 mg/mL.
And (3) washing the coupled magnetic bead solution for 3 times by using a crosslinking buffer solution, then re-suspending by using the crosslinking buffer solution, then adding 0.04mL of a double-end functional group crosslinking agent solution into the solution, uniformly shaking and mixing, and carrying out crosslinking reaction for 2 hours at the constant temperature of 37 ℃.
S4. Seal
And after the crosslinking reaction is finished, adding 1mL of sealing buffer solution into the crosslinked magnetic beads for washing for 3 times, adding 1mL of sealing buffer solution after washing for resuspending, sealing for 18-28h at the constant temperature of 37 ℃, washing for 3 times by using preservation solution after sealing is finished, and adding 1mL of preservation buffer solution for preservation after washing is finished to obtain the tosyl magnetic beads coupled with the iPTH antibody.
In the embodiment of the invention, the used reagent comprises the following specific components:
first coating buffer: comprising 100mM boric acid, pH9.5;
second coating buffer: comprising 100mM boric acid, 3M ammonium sulphate, pH9.5.
Crosslinking buffer solution: comprises 100mM TEA,100mM NaCl, pH8.0;
blocking buffer: contains 1 mM tris,0.5% BSA, pH7.4;
preservation buffer solution: contains 1 mM PBS,5% sucrose, 0.5% BSA, pH7.0.
Comparative example 1
Preparing iPTH tosyl magnetic beads by a conventional process:
s1, cleaning of magnetic beads
Accurately measuring tosyl magnetic bead concentrated solution with the volume of 0.2mL, putting the tosyl magnetic bead concentrated solution into a container, adding a first coating buffer solution with the volume of 1mL, shaking and uniformly mixing, carrying out magnetic adsorption for a period of time, discarding the supernatant, repeating the process, and washing for 3 times to obtain the washed magnetic beads.
S2. Coupling of magnetic beads
And (3) resuspending the washed magnetic beads with a first coating buffer solution, adding 0.2mg of iPTH antibody, adding 0.3mL of a second coating solution to make the total volume of the final solution be 1mL, rapidly shaking and uniformly mixing, and carrying out coupling reaction for 20 hours while uniformly mixing in a 37 ℃ incubator.
S3. Seal
And after the coupling reaction is finished, adding 1mL of blocking buffer solution into the magnetic beads for washing for 3 times, adding 1mL of blocking buffer solution after the washing is finished for resuspending, blocking for 18-28h at the constant temperature of 37 ℃, washing for 3 times by using preservation solution after the blocking is finished, and adding 1mL of preservation buffer solution after the washing is finished for preservation to obtain the tosyl magnetic beads coupled with the iPTH antibody.
Comparative example 2
Adding only inert protein and adding no bifunctional cross-linking agent to prepare iPTH tosyl magnetic beads:
s1, cleaning of magnetic beads
Accurately measuring tosyl magnetic bead concentrated solution with the volume of 0.2mL, putting the tosyl magnetic bead concentrated solution into a container, adding a first coating buffer solution with the volume of 1mL, shaking and uniformly mixing, carrying out magnetic adsorption for a period of time, discarding the supernatant, repeating the process, and washing for 3 times to obtain the washed magnetic beads.
S2. Coupling of magnetic beads
Resuspending the washed magnetic beads with a first coating buffer solution, adding 0.4mg of iPTH antibody and 0.4mg of inert protein, adding 0.3mL of a second coating solution to make the total volume of the final solution 1mL, rapidly shaking and uniformly mixing, and carrying out coupling reaction for 20h while uniformly mixing in a 37 ℃ incubator.
S3. Seal
And after the coupling reaction is finished, adding 1mL of sealing buffer solution into the magnetic beads for cleaning for 3 times, adding 1mL of sealing buffer solution after the cleaning is finished for resuspension, sealing for 18-28h at the constant temperature of 37 ℃, cleaning for 3 times by using preservation solution after the sealing is finished, and adding 1mL of preservation buffer solution after the cleaning is finished for preservation to obtain the tosyl magnetic beads coupled with the iPTH antibody.
A plurality of samples of iPTH tosyl magnetic beads obtained in example 1 and comparative examples 1-2 were selected and tested for their light values after acceleration at 37 ℃ for 7 days and after standing at 2-8 ℃ for one year, respectively, and the test results are shown in tables 1-6.
TABLE 1 light value of each sample before and after acceleration at 37 ℃ of iPTH tosyl magnetic beads obtained in comparative example 1
Sample(s) | Initial value | The seventh day | Amplitude reduction |
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 light values of samples of iPTH tosyl magnetic beads obtained in comparative example 1 after standing at 2-8 ℃ for one year
TABLE 3 light values of the samples before and after acceleration at 37 ℃ of iPTH obtained in comparative example 2
Sample(s) | Initial value | The seventh day | Amplitude reduction |
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 light values of samples of iPTH tosyl magnetic beads obtained in comparative example 2 after standing at 2-8 ℃ for one year
Sample(s) | Initial value at 4 ℃ | Standing at 4 deg.C for one year | Amplitude reduction |
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 light values of the iPTH tosyl magnetic beads obtained in example 1 before and after acceleration at 37 ℃ for each sample
TABLE 6 light values of samples of iPTH magnetic beads obtained in example 1 after standing at 2-8 ℃ for one year
Sample(s) | Initial value at 4 ℃ | Standing at 4 deg.C for one year | Amplitude reduction |
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% |
As can be seen from the results in tables 1-6, the decrease in optical value of the magnetic beads obtained in comparative example 1 by the conventional coupling process was 20-30% before and after acceleration at 37 ℃, and the decrease in optical value of the magnetic beads after being placed at 4 ℃ for one year was also 20-30%. The light value of the magnetic beads after the inert protein is added is reduced by 10-20% before and after the magnetic beads are accelerated at 37 ℃, and the light value of the magnetic beads after the magnetic beads are placed for one year at 4 ℃ is reduced by 10-20%. According to the scheme of the invention, after the magnetic beads are added with the inert protein coupling and the cross-linking agent for cross-linking, the optical values of samples of the obtained magnetic beads are within 10% before and after acceleration at 37 ℃ and after the magnetic beads are placed at 4 ℃ for one year. Obviously, the magnetic beads obtained by the method have obviously improved stability.
The results show that the method provided by the invention can obviously improve the stability of the tosyl magnetic beads coupled with the iPTH antibody.
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to 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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CN118033110A (en) * | 2024-02-21 | 2024-05-14 | 河北医陆生物科技有限公司 | Preparation method of antigen-coupled amino magnetic beads |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105842456A (en) * | 2015-01-14 | 2016-08-10 | 北京康诺生物科技有限公司 | Oriented immunomagnetic beads of lactoferrin and preparation method and application of oriented immunomagnetic beads |
CN111983221A (en) * | 2020-08-19 | 2020-11-24 | 深圳市卓润生物科技有限公司 | Surface modified magnetic bead and preparation method and application thereof |
CN114544939A (en) * | 2022-01-26 | 2022-05-27 | 天津鸿宇泰生物科技有限公司 | Streptavidin magnetic bead marking method |
CN115015556A (en) * | 2022-05-26 | 2022-09-06 | 宁波海尔施智造有限公司 | Liver type fatty acid binding protein (L-FABP) detection kit and preparation method thereof |
WO2022198925A1 (en) * | 2021-03-22 | 2022-09-29 | 深圳市亚辉龙生物科技股份有限公司 | Nano-magnetic bead coating, and preparation method therefor, detection reagent thereof and detection kit thereof |
CN115166230A (en) * | 2022-06-30 | 2022-10-11 | 郑州凌思生物科技有限责任公司 | Method for improving efficiency of magnetic particle coupled protein by using dendrimer |
-
2022
- 2022-12-30 CN CN202211723851.XA patent/CN115856286A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105842456A (en) * | 2015-01-14 | 2016-08-10 | 北京康诺生物科技有限公司 | Oriented immunomagnetic beads of lactoferrin and preparation method and application of oriented immunomagnetic beads |
CN111983221A (en) * | 2020-08-19 | 2020-11-24 | 深圳市卓润生物科技有限公司 | Surface modified magnetic bead and preparation method and application thereof |
WO2022198925A1 (en) * | 2021-03-22 | 2022-09-29 | 深圳市亚辉龙生物科技股份有限公司 | Nano-magnetic bead coating, and preparation method therefor, detection reagent thereof and detection kit thereof |
CN114544939A (en) * | 2022-01-26 | 2022-05-27 | 天津鸿宇泰生物科技有限公司 | Streptavidin magnetic bead marking method |
CN115015556A (en) * | 2022-05-26 | 2022-09-06 | 宁波海尔施智造有限公司 | Liver type fatty acid binding protein (L-FABP) detection kit and preparation method thereof |
CN115166230A (en) * | 2022-06-30 | 2022-10-11 | 郑州凌思生物科技有限责任公司 | Method for improving efficiency of magnetic particle coupled protein by using dendrimer |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN118033110A (en) * | 2024-02-21 | 2024-05-14 | 河北医陆生物科技有限公司 | Preparation method of antigen-coupled amino magnetic beads |
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