WO2022198925A1 - Nano-magnetic bead coating, and preparation method therefor, detection reagent thereof and detection kit thereof - Google Patents

Abstract

Disclosed is a nano-magnetic bead coating, and a preparation method therefor, a detection reagent thereof and a detection kit thereof. The method for preparing the nano-magnetic bead coating comprises: reducing a disulfide bond in a hinge region of an antibody to prepare an antibody fragment having a mercapto group; and mixing and reacting the antibody fragment having the mercapto group, a bifunctional crosslinking agent and a nano-magnetic bead to prepare the nano-magnetic bead coating.

Description

Nanomagnetic bead coating and preparation method thereof, detection reagent and detection kit

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of the Chinese patent application filed on March 22, 2021 with the application number 202110303552X and the invention titled "Nano Magnetic Bead Coating and Its Preparation Method, Detection Reagent and Detection Kit", The entire contents of which are incorporated herein by reference.

technical field

The invention relates to the technical field of immunodetection, in particular to a nano-magnetic bead coating and a preparation method thereof, a detection reagent and a detection kit.

Background technique

Immunomagnetic beads ((Immunomagnetic bead, IMB, magnetic beads for short)) have a core-shell structure, generally including core metal particles (Fe ₂ O ₃ , Fe ₃ O ₄ , etc.), polymer materials ( Such as polystyrene, polyvinyl chloride, etc.) and the outermost functional ligands (such as _-NH2 , -COOH, -OH, -CHO). Immunomagnetic beads have small particle size and large specific surface area, which can capture more analytes, and can directly perform enzymatic color development, fluorescence or isotope display on their surface. Therefore, they are more and more widely used in immunodetection.

In the immunoassay, the nano-magnetic bead coating formed by coating the antibody on the surface of the immunomagnetic bead is an important component of the immunological reagent. Another antibody labeled with tracers such as acridine ester, ruthenium terpyridine, adamantane, luminol, horseradish peroxidase or alkaline phosphatase binds to form an immune complex to achieve antigen detection. However, in practice, it is found that the specificity and sensitivity of the nano-magnetic bead coating formed by coating the antibody on the surface of the immunomagnetic beads need to be improved in detection.

SUMMARY OF THE INVENTION

According to some embodiments, a preparation method of a nano-magnetic bead coating, a nano-magnetic bead coating, a detection reagent and a detection kit are provided.

A preparation method of a nano-magnetic bead coating, comprising:

reducing the disulfide bond of the hinge region of the antibody to prepare an antibody fragment having a sulfhydryl group; and

The antibody fragment with sulfhydryl group, the bifunctional cross-linking agent and the nano-magnetic beads are mixed and reacted to prepare a nano-magnetic bead coating.

Different from the traditional connection between the amino group on the antibody and the nano-magnetic beads to form a covalent bond, the above-mentioned preparation method of the nano-magnetic bead coating is to form other covalent bonds between the nano-magnetic beads and the sulfhydryl group of the antibody fragment except for the coordination bond. valence bond to form a nano-magnetic bead coating, which enables the antibody fragment to achieve directional coupling with the nano-magnetic beads, so that no Fc end of the prepared nano-magnetic bead coating faces outwards, which can improve the prepared nano-magnetic beads. The effective antibody amount of the bead coating is beneficial to improve the sensitivity and specificity of detection.

A nano-magnetic bead coating is prepared by the above-mentioned preparation method of the nano-magnetic bead coating.

A detection reagent, comprising the above-mentioned nano-magnetic bead coating.

A detection kit, comprising a first reagent and a second reagent, the first reagent contains a labeled antibody that can specifically bind to an antigen, the second reagent contains the above-mentioned nano-magnetic bead coating, and the The nano-magnetic bead coating can also specifically bind to the antigen, and the site where the nano-magnetic bead coating binds to the antigen is the same as the binding site of the labeled antibody to the antigen. The location is different.

The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, and to implement according to the content of the description, the preferred embodiments of the present invention are described below in detail with the accompanying drawings.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application or in the traditional technology, the following briefly introduces the accompanying drawings that are used in the description of the embodiments or the traditional technology. Obviously, the drawings in the following description are only the For some embodiments of the application, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

1 is a schematic diagram of the reaction for preparing half-antibody fragments in Example 1;

FIG. 2 is a flow chart of the preparation of the nano-magnetic bead coating in Example 1. FIG.

Detailed ways

In order to facilitate an understanding of the present invention, the present invention will be described more fully below, which may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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 invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

Definition of Terms

Antibodies: Antibodies refer to immunoglobulins produced by the immune system of the body under the stimulation of antigens, which are produced by the proliferation and differentiation of B lymphocytes or memory cells into plasma cells that can specifically bind to the corresponding antigens. A typical antibody molecule has a symmetrical structure of four polypeptide chains, including two identical heavy chains (H chains) with relatively large relative molecular weights; two identical light chains (L chains) with relatively small relative molecular weights. ). The chains are linked by disulfide bonds and non-covalent bonds to form a monomer molecule composed of four polypeptide chains. There are two types of light chains, κ and λ, and five types of heavy chains: μ, δ, γ, ε, and α. The whole antibody molecule can be divided into two parts: constant region and variable region. In a given species, the constant regions of different antibody molecules all have the same or nearly the same amino acid sequence. The variable regions are located at the ends of the two arms of "Y". In the variable region, a small number of amino acid residues have particularly strong changes, and the residue composition and arrangement of these amino acids are more prone to variation regions, which are called hypervariable regions. The hypervariable region is located on the molecular surface and consists of 17 amino acid residues at most, and as few as 2 to 3. The amino acid sequence of the hypervariable region determines the antigen-binding specificity of the antibody. The two antigen-binding sites on an antibody molecule are the same, located at the ends of the two arms, called antigen-binding fragments (Fab fragments). The handle of the "Y" is called the crystalline fragment (Fc fragment), and the sugar is bound to the Fc fragment. Structurally, in this text, unless otherwise specified, when referring to an antibody, it refers to a whole antibody, that is, an antibody structure including 4 chains and an Fc segment. In addition, functionally, the "antibody" in the present invention refers to an antibody specific to a target antigen, that is, an antibody having a specific binding ability to an antigen.

Antibody fragments include half-antibody fragments and Fab' fragments. Half-antibody fragments: Whole antibodies are split into symmetrical fragments with thiol groups each by a disulfide reducing agent, each half-antibody fragment contains a complete light chain and a complete heavy chain and an Fc fragment. Fab' fragment: It is a monovalent antigen-binding fragment with a thiol group. Each Fab' fragment is based on the half-antibody fragment and omits the Fc fragment.

Antigen: a class of substances that can induce an immune response in the immune system and can specifically bind to the product of the immune response (antibody or effector cells).

In the field of immunodetection technology, the sensitivity and specificity of detection are generally improved by screening antibodies with strong affinity and good specificity for antigens to prepare nano-magnetic beads coating. However, in the research process of this application, it is found that in the traditional method of coupling antibodies to the surface of immunomagnetic beads to prepare nanomagnetic beads coating, the primary ammonia of the antibody is generally selected as the coupling target, however, The specific coupling position of the immunomagnetic beads and the antibody is random, some of the immunomagnetic beads are coupled to the amino group of the Fc end of the antibody, while the other part of the immunomagnetic beads is coupled to the amino group of the Fab end. Moreover, when the immunomagnetic beads are coupled to the Fab end of the antibody, the Fc end of the antibody is located on the outside of the immunomagnetic beads, and the antigen-binding site is not easily exposed, thereby reducing the amount of effective antibody that actually immunoreacts with the antigen, thereby making the Low specificity and sensitivity when applied to detection.

Therefore, an embodiment provides a preparation method of a nano-magnetic bead coating, which enables the directional coupling of antibody fragments by reacting sulfhydryl groups of antibody fragments with nano-magnetic beads to form covalent bonds other than coordination bonds On the magnetic nanobeads, the Fab end is fully exposed, and the effective antibody amount is ensured, so that when the nanomagnetic bead coating is used for immunodetection, the specificity is good and the sensitivity is high. Specifically, the preparation method comprises the following steps:

Step 1: Use a reducing agent to reduce the disulfide bond of the hinge region of the antibody to prepare an antibody fragment having a sulfhydryl group.

The disulfide bonds between the heavy chains of the hinge region of an antibody are more easily reduced than the disulfide bonds between the heavy and light chains of the antigen-binding region of the antibody. Thus, the disulfide bonds of the hinge region of an antibody can be reduced to form half-antibody fragments. Alternatively, all the disulfide bonds in the hinge region of the antibody are reduced to prepare antibody fragments having thiol groups.

Chitinase 3-like 1 (CHI3L1), also known as human chondrocyte protein 39 (YKL-30), is a member of the chitinase family, and its expression level is increased in multiple malignant tumors. High, including gastric cancer, liver cancer, endometrial cancer, ovarian cancer, lung cancer, squamous cell skin cancer, etc. CHI3L1 can play a regulatory role in the occurrence and development of tumors by promoting tumor angiogenesis, increasing adhesion, promoting tumor invasion and metastasis, and activating signaling pathways such as transforming growth factor-β/mitogen-activated protein kinase/extracellular signal-regulated kinase. At the same time, serum chitin-3-like protein 1 is involved in pathological processes such as acute and chronic inflammation and extracellular matrix remodeling. It has a wide range of clinical application prospects. The identification of liver diseases has attracted more and more attention of researchers. detection meaning.

Thus, in one of the embodiments, the antibody fragment is from the CHI3L1 antibody. It is understood that, in other embodiments, the antibody fragments can also be derived from other antibodies, such as procalcitonin antibodies, cardiac troponin I antibodies, and the like.

Optionally, the reducing agent for reducing the disulfide bond of the hinge region of the antibody is mercaptoethylamine (2-MEA), and the mass ratio of the antibody to the reducing agent is 1:(1-50). Further, the mass ratio of the antibody to the reducing agent is 1:(1-10). It can be understood that, in other embodiments, the reducing agent may also be other disulfide bond reducing agents, such as 2-mercaptoethanol, dithiothreitol, and the like.

In the present embodiment, the antibody fragment is a half-antibody fragment, and therefore, all the disulfide bonds in the hinge region of the antibody may be reduced. Of course, in other embodiments, antibody fragments can also be Fab' fragments. In this case, a corresponding step of stripping the Fc fragment from the antibody or antibody fragment is required. For example, before reducing the disulfide bond with a reducing agent, the antibody is digested with a protease to excise the Fc fragment. It can be understood that, in another embodiment, the disulfide bond in the hinge region of the antibody can also be partially reduced, and at this time, the bifunctional cross-linking agent forms a covalent bond with the reduced disulfide bond.

Of course, after the step of mixing and reacting the reducing agent for reducing the disulfide bond of the hinge region of the antibody with the antibody, the step of desalting or ultrafiltration to remove impurities is also included on the reaction product, so as to obtain a high-purity half-antibody fragment.

Step 2: The antibody fragment with sulfhydryl group, the bifunctional cross-linking agent and the nano-magnetic beads are mixed and reacted to prepare the nano-magnetic bead coating.

Specifically, the magnetic nanobeads are amino nanomagnetic beads or carboxyl nanomagnetic beads.

The bifunctional cross-linking agent not only has groups that can react with groups on the magnetic nanobeads to form covalent bonds other than coordination bonds, but also has groups that can react with groups of antibody fragments to form covalent bonds other than coordination bonds. bond group. Optionally, the bifunctional cross-linking agent is selected from at least one of maleimide-based bi-functional cross-linking agents, iodoacetic acid-based bi-functional cross-linking agents, and acetylated thiol-based bi-functional cross-linking agents.

In one embodiment, the bifunctional crosslinking agent is a maleimide-based bifunctional crosslinking agent. For example, the nano-magnetic beads are amino nano-magnetic beads, and the maleimide-based bifunctional cross-linking agent is selected from 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide Ester (abbreviated as SMCC), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt (abbreviated as Sulfo-SMCC) and maleimide- (PEG) at least one of n-succinimide esters, wherein n is an integer between 2 and 24. Further, n is an integer between 2 and 12. For another example, in one embodiment, the magnetic nanobeads are carboxyl nanomagnetic beads, and the maleimide bifunctional crosslinking agent is selected from 1-(2-aminoethyl)maleimide hydrochloride and At least one of N-ε-maleimidohexanoic acid hydrazide.

In one embodiment, the bifunctional crosslinking agent is an iodoacetic acid bifunctional crosslinking agent. Optionally, the iodoacetic acid bifunctional crosslinking agent is selected from iodoacetic acid N-hydroxysuccinimide ester (abbreviated as SIAB) and succinimidyl ester (4-iodoacetic acid) aminobenzoate (abbreviated as Sulfo-SIAB). ) at least one of them.

In one embodiment, the bifunctional crosslinking agent is an acetylated thiol-based bifunctional crosslinking agent. Optionally, the acetylated thiol-based bifunctional cross-linking agent is selected from at least one of N-succinic acid S-acetoacetate and N-succinic acid-(PEG)m-S-acetoacetate, wherein, m is an integer between 2 and 24.

It can be understood that, in other embodiments, the bifunctional cross-linking agent is not limited to the above, and can also be other substances that can simultaneously link the group of the nanomagnetic beads and the sulfhydryl group of the antibody fragment in the form of covalent bond formation.

Optionally, the mass ratio of the antibody fragment with sulfhydryl group to the bifunctional cross-linking agent and the magnetic nanobead is 1:(0.5-10):(5-100). Further, the mass ratio of the antibody fragment having a thiol group to the bifunctional cross-linking agent and the magnetic nanobead is 1:(1-5):(10-50).

Optionally, after mixing the nano-magnetic beads with the bifunctional cross-linking agent, the antibody fragments with thiol groups are added.

Of course, after the mixing reaction is completed, the step of purification and removal of impurities is also included.

The preparation method of the above-mentioned nano-magnetic bead coating is simple and convenient, and is beneficial to industrial production. In addition, the nano-magnetic bead coating prepared by the above-mentioned preparation method of the nano-magnetic bead coating is through the reaction between the sulfhydryl group of the antibody fragment and the nano-magnetic bead to form a covalent bond other than the coordination bond, so that the antibody fragment is directionally coupled The nano-magnetic beads are coupled to the nano-magnetic beads to ensure the effective amount of antibodies, so that when the nano-magnetic beads coating is used for immunodetection, the detection specificity is good and the sensitivity is high.

In addition, an embodiment also provides a nano-magnetic bead coating, the nano-magnetic bead coating being prepared by the above-mentioned preparation method of the nano-magnetic bead coating. The nano-magnetic bead coating includes nano-magnetic beads and antibody fragments, the antibody fragments are half antibody fragments or Fab' fragments, and the sulfhydryl groups of the nano-magnetic beads and the antibody fragments are connected by forming covalent bonds other than coordination bonds. When the nano-magnetic bead coating is used for immunodetection, the detection specificity is good and the sensitivity is high.

In addition, according to an embodiment, there is also provided a detection reagent, the above-mentioned nanomagnetic bead coating.

Specifically, the above detection reagent also includes a buffer. Optionally, the buffer is selected from at least one of phosphate buffer, carbonate buffer and borate buffer.

Since the above-mentioned detection reagent includes the above-mentioned nano-magnetic bead coating, it has high sensitivity and specificity.

In addition, an embodiment also provides a detection kit, the detection kit includes a first reagent and a second reagent, the first reagent contains a marker-labeled antibody that can specifically bind to an antigen, and the second reagent contains the above-mentioned nanometer Magnetic bead coating, the nano-magnetic bead coating can also specifically bind to antigen, and the site where the nano-magnetic bead coating binds to the antigen is the same as the site where the labeled antibody binds to the antigen. point different. That is, the detection kit adopts the principle of double-antibody sandwich method for antigen detection.

Optionally, in the first reagent, the label is selected from acridine ester, ruthenium terpyridine, adamantane, luminol, derivatives of luminol, isoluminol, derivatives of isoluminol, One of root peroxidase and alkaline phosphatase. It can be understood that, in other embodiments, the marker is not limited to the above, and can also be other substances that can be used in immunodetection.

The above-mentioned detection kit comprises the above-mentioned nano-magnetic bead coating, which has high sensitivity and specificity when used to detect antigens.

The following describes in detail with reference to specific embodiments. The following examples do not include other components except inevitable impurities unless otherwise specified. Unless otherwise specified, the reagents and instruments used in the examples are routinely selected in the art. The experimental methods for which specific conditions are not indicated in the examples are realized according to conventional conditions, such as conditions described in literatures, books or methods recommended by manufacturers. The following CHI3L1 antibody was purchased from Hangzhou Puwang Biotechnology Co., Ltd. "M" used herein to represent concentration is an abbreviation for "mol/L" and "mM" is an abbreviation for "mmol/L".

Example 1

The magnetic nanobead coating of this embodiment is formed by using maleimide-(PEG) _4- succinimidyl ester to activate the magnetic nanobeads and react with half-antibody fragments. Specifically, please refer to FIG. 1 and FIG. 2, the preparation of the nano-magnetic bead coating in this embodiment includes but is not limited to the following steps:

(1) Dilute the CHI3L1 antibody with 0.02M PBS buffer to 0.2 mg/mL to prepare an antibody solution.

(2) Cytoethylamine (2-MEA) was added to the antibody solution in step (1) to make the final concentration 10 mM, and after gentle mixing, the reaction was carried out at room temperature (25° C.) for 60 min. PBS was used to balance the desalting column, and the product after the reaction was desalted to remove impurities to obtain a purified half-antibody fragment solution.

(3) Take 10 mg of amino magnetic nanoparticles, wash with 0.02M PBS, and resuspend to prepare a nanomagnetic bead solution with a concentration of 10 mg/mL. Then, maleimide-(PEG) ₄ was added to the nano-magnetic bead solution according to the mass ratio of the nano-magnetic beads to maleimide-(PEG) ₄ -succinimide ester of 1:0.05. -Succinimidyl ester, reacted at room temperature (25°C) for 30 min, then magnetically separated, discarded the supernatant, and prepared activated nanomagnetic beads.

(4) After washing the activated magnetic nanobeads obtained in step (3) with PBS, mix them with the purified half-antibody fragment solution obtained in step (2), so that the mass ratio of antibody fragments to magnetic nanobeads is The ratio was 1:50, and the reaction was carried out at room temperature (25 °C) for 3 h.

(5) After the reaction in step (4), use 0.1% (m/v) BSA and 1 mM NEM (N-ethylmaleimide, NEM is a monofunctional cross-linking with maleimide) The function is to quench the residual small amount of sulfhydryl groups on the antibody fragment, and there is no need to remove it after the reaction) to wash the magnetic beads once, and mix and react at room temperature (25°C) for 3h.

(6) After the reaction in step (5), replace the magnetic beads with a PBS solution containing 0.1% (m/v) BSA, resuspend at a concentration of 10 mg/mL, and store at 4°C.

Example 2

The magnetic nanobead coating of this embodiment is formed by activating the magnetic nanobeads with carbodiimide and 1-(2-aminoethyl)maleimide hydrochloride and reacting with the half-antibody fragments. Specifically, the preparation of the nano-magnetic bead coating of this embodiment includes but is not limited to the following steps:

(1) Dilute the CHI3L1 antibody with 0.02M PBS buffer to 0.2 mg/mL to prepare an antibody solution.

(2) Cytoethylamine (2-MEA) was added to the antibody solution in step (1) to make the final concentration 10 mM, and after mixing, react at room temperature (25° C.) for 60 min. PBS was used to balance the desalting column, and the product after the reaction was desalted to remove impurities to obtain a purified half-antibody fragment solution.

(3) Take 10 mg of carboxyl magnetic nanoparticles, wash with 0.02M MES buffer, and resuspend to prepare a nanomagnetic bead solution with a concentration of 10 mg/mL. Then, according to the mass ratio of nanomagnetic beads, carbodiimide and 1-(2-aminoethyl)maleimide hydrochloride to 1:0.05:0.05, add carbon dioxide to the nanomagnetic bead solution Imine and 1-(2-aminoethyl)maleimide hydrochloride were reacted at room temperature (25° C.) for 30 min, then magnetically separated, and the supernatant was discarded to obtain activated magnetic nanobeads.

(4) After washing the activated magnetic nanobeads obtained in step (3) with PBS, mix them with the purified half-antibody fragment solution obtained in step (2), so that the mass ratio of antibody fragments to magnetic nanobeads is The ratio was 1:50, and the reaction was carried out at room temperature (25 °C) for 3 h.

(5) After the reaction in step (4), wash the magnetic beads once with a PBS solution containing 0.1% (m/v) BSA and 1 mM NEM, and mix and react at room temperature (25°C) for 3 hours.

(6) After the reaction in step (5), replace the magnetic beads with a PBS solution containing 0.1% (m/v) BSA, resuspend at a concentration of 10 mg/mL, and store at 4°C.

Comparative Example 1

The nanomagnetic bead coating of this comparative example is a complex formed by linking the direct CHI3L1 antibody to the surface of the nanomagnetic beads through carbodiimide.

The preparation of the nano-magnetic bead coating of this comparative example includes but is not limited to the following steps:

(1) Take 10 mg of carboxyl magnetic nanoparticles, wash with 0.02M MES buffer, and resuspend to prepare a nanomagnetic bead solution with a concentration of 10 mg/mL. Then, according to the ratio of the mass ratio of nano-magnetic beads to carbodiimide of 1:0.05, carbodiimide was added to the nano-magnetic bead solution, and the reaction was carried out at room temperature (25° C.) for 30 min, followed by magnetic separation, and the supernatant was discarded. , followed by adding MES buffer for washing and resuspending to prepare a solution of activated nanomagnetic beads with a concentration of 10 mg/mL.

(2) Add 0.2 mg of CHI3L1 antibody to the activated nano-magnetic beads solution prepared in step (1), mix well, and react at room temperature (25°C) for 3 hours.

(3) After the reaction in step (2), wash the magnetic beads with a PBS solution containing 0.1% (m/v) BSA, and resuspend the beads with a PBS solution containing 0.1% (m/v) BSA to a concentration of 10 mg/ mL, stored at 4°C.

test

(1) Dilute the nano-magnetic bead coatings obtained in each example and comparative example 1 to 0.15 mg/mL, and then test the sensitivity of the nano-magnetic bead coatings in each embodiment and comparative example 1 for detecting CHI3L1 and accuracy, in which: the sensitivity is characterized by the detection signal value. Under the same sample concentration, the higher the signal value, the higher the sensitivity; The higher the accuracy, the higher the accuracy and the better the specificity. The results are shown in Tables 1 and 2.

Table 1

project	Example 1	Example 2	Comparative Example 1
specificity	98.2%	95.4%	94.2%

Table 2

It can be seen from Table 1 and Table 2 that the sensitivity and specificity of Example 1 and Example 2 are significantly better than those of Comparative Example 1, indicating that the use of a disulfide bond reducing agent to reduce the disulfide bond in the hinge region of the antibody to obtain antibody fragments and The nano-magnetic bead coating formed after the nano-magnetic beads is connected has higher sensitivity and better specificity than the magnetic bead coating formed by directly connecting the antibody to the nano-magnetic beads.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (17)

1. A preparation method of a nano-magnetic bead coating, comprising:

   using a reducing agent to reduce the disulfide bond of the hinge region of the antibody to prepare an antibody fragment having a sulfhydryl group; and

   The antibody fragment with sulfhydryl group, the bifunctional cross-linking agent and the nano-magnetic beads are mixed and reacted to prepare a nano-magnetic bead coating.
2. The method according to claim 1, wherein the bifunctional crosslinking agent is selected from the group consisting of maleimide bifunctional crosslinking agent, iodoacetic acid bifunctional crosslinking agent and acetylated thiol bifunctional crosslinking agent at least one of the combination agents.
3. The method according to claim 2, wherein the maleimide bifunctional crosslinking agent is selected from 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid Succinimide ester, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt and maleimide-(PEG)n-succinimide At least one of imide esters, wherein n is an integer between 2 and 24.
4. The method according to claim 2, wherein the maleimide bifunctional crosslinking agent is selected from 1-(2-aminoethyl)maleimide hydrochloride and N-ε- At least one of maleimide caproic acid hydrazide.
5. The method according to claim 2, wherein the iodoacetic acid bifunctional cross-linking agent is selected from the group consisting of iodoacetic acid N-hydroxysuccinimide ester and succinimide ester (4-iodoacetic acid) aminobenzyl at least one of the acid esters.
6. The method according to claim 2, wherein the acetylated thiol-based bifunctional cross-linking agent is selected from N-succinic acid S-acetoacetate and N-succinic acid-(PEG)m-S-acetyl At least one of acetates, wherein m is an integer between 2 and 24.
7. The method according to claim 1, wherein the reducing agent is mercaptoethylamine, and the mass ratio of the antibody to the reducing agent is 1:(1-50).
8. The method according to claim 1, wherein the mass ratio of the antibody fragment with thiol group to the bifunctional crosslinking agent and the magnetic nanobead is 1:(0.5-10):(5-100 ).
9. The method according to claim 1, wherein after reducing the disulfide bond of the hinge region of the antibody, the method further comprises: removing impurities from the reduced product.
10. The method of claim 1, wherein the antibody fragment is an antibody fragment of a monoclonal antibody.
11. The method according to any one of claims 1 to 10, wherein the antibody is a CHI3L1 antibody.
12. The method according to any one of claims 1 to 10, wherein the magnetic nanobeads are amino magnetic nanobeads or carboxyl nanomagnetic beads.
13. A nano-magnetic bead coating is prepared by the method of any one of claims 1-12.
14. The magnetic nanobead coating according to claim 13, characterized in that it comprises magnetic nanobeads and antibody fragments, the antibody fragments are half-antibody fragments or Fab' fragments, and the magnetic nanobeads and the antibody fragments The sulfhydryl groups are linked by forming covalent bonds other than coordinative bonds.
15. A detection reagent, comprising the nanomagnetic bead coating of claim 14 .
16. The detection reagent according to claim 15, further comprising a buffer selected from at least one of phosphate buffer, carbonate buffer and borate buffer.
17. A detection kit, comprising a first reagent and a second reagent, the first reagent contains an antibody labeled with a label that can specifically bind to an antigen, and the second reagent contains the nano-magnetic as claimed in claim 13 A bead coating, the nanomagnetic bead coating can also specifically bind to the antigen, and the site where the nanomagnetic bead coating binds to the antigen is associated with the labeled antibody. The site of binding to the antigen is different.

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