Magnetic bead coating, preparation method and application thereof, and detection kit
阅读说明：本技术 磁珠包被物及其制备方法和应用、检测试剂盒 (Magnetic bead coating, preparation method and application thereof, and detection kit ) 是由 高晨燕 王光亮 祝亮 何凡 王晓炜 于 2019-09-30 设计创作，主要内容包括：本发明涉及一种磁珠包被物及其制备方法和应用、检测试剂盒。该磁珠包被物包括：蛋白质、聚乙二醇修饰剂和纳米磁珠,聚乙二醇修饰剂与蛋白质共价连接,纳米磁珠与蛋白质连接。上述磁珠包被物的稳定性较好。(The invention relates to a magnetic bead coating material, a preparation method and application thereof, and a detection kit. The magnetic bead coating comprises: the protein-based nano magnetic bead modifying agent comprises protein, a polyethylene glycol modifying agent and a nano magnetic bead, wherein the polyethylene glycol modifying agent is covalently connected with the protein, and the nano magnetic bead is connected with the protein. The stability of the magnetic bead coating is good.)
1. A magnetic bead coating, comprising: the protein-based nano magnetic bead protein comprises a protein, a polyethylene glycol modifier and a nano magnetic bead, wherein the polyethylene glycol modifier is covalently connected with the protein, and the nano magnetic bead is connected with the protein.
2. The coating for magnetic beads of claim 1, wherein the polyethylene glycol modifier has a structural formulaThe polyethylene glycol modifier forms an-NH-CO-structure with the protein to link the polyethylene glycol modifier to the protein;
further, the amino group of the polyethylene glycol modifier and the carboxyl group of the protein form the-NH-CO-structure.
3. The coating for magnetic beads according to any one of claims 1 to 2, wherein the polyethylene glycol modifier has a weight average molecular weight of 200 or more;
and/or n is greater than or equal to 4;
and/or, the protein is an antigen or an antibody.
4. The coating for magnetic beads according to any one of claims 1 to 2, wherein the nanobead has a reactive group, and the reactive group reacts with an amino group of the protein to link the nanobead to the protein;
further, the active group is a carboxyl group, and the active group and the amino group of the protein form an-NH-CO-structure so that the nano magnetic bead is connected with the protein.
5. A preparation method of a magnetic bead coating material is characterized by comprising the following steps:
coupling a polyethylene glycol modifier with a protein to ensure that the polyethylene glycol modifier is covalently connected with the protein to obtain a modified protein; and
coupling reaction is carried out on the modified protein and the nano magnetic beads, so that the modified protein is connected with the nano magnetic beads, and a magnetic bead coating object is obtained.
6. The method of claim 5, wherein the polyethylene glycol modifier has a formula of
7. The method of claim 6, wherein the step of mixing and reacting the polyethylene glycol modifier with the protein to form an-NH-CO-structure with the protein to covalently link the polyethylene glycol modifier to the protein comprises: reacting a cross-linking agent with a carboxyl group of the protein to form an intermediate; reacting the intermediate with an amino group of the polyethylene glycol modifier to form the-NH-CO-structure, thereby covalently linking the polyethylene glycol modifier with the protein to obtain the modified protein.
8. The method of any one of claims 5 to 7, wherein the step of coupling the modified protein to the nanobead comprises: the nano magnetic beads are provided with active groups, and the active groups react with amino groups of the modified proteins to connect the nano magnetic beads with the modified proteins to obtain the magnetic bead coating materials.
9. Use of the coating of magnetic beads according to any one of claims 1 to 4 or the coating of magnetic beads prepared by the method of any one of claims 5 to 8 in the preparation of a detection reagent, a detection kit or a detection device.
10. A detection kit comprising the coating material for magnetic beads according to any one of claims 1 to 4 or the coating material for magnetic beads prepared by the method for preparing the coating material for magnetic beads according to any one of claims 5 to 8.
Technical Field
The invention relates to immunological detection, in particular to a magnetic bead coating, a preparation method and application thereof, and a detection kit.
Background
The coating of magnetic beads is obtained by binding an antigen or an antibody to the surface of the magnetic beads. The magnetic bead coating can be used as a component of an immunoassay reagent to be applied to immunoassay and immunodiagnosis, such as immunoassay of a chemiluminescent platform and immunodiagnosis. Generally, a sample to be detected and an antigen or an antibody on the surface of a magnetic bead coating are subjected to an immunoreaction to capture an object to be detected in the sample to be detected, and then the captured object to be detected is subjected to quantitative or qualitative detection by using a chemiluminescence technology. The magnetic bead coating is used as an important component of the immunoassay reagent, and has great influence on the performance of the immunoassay reagent. However, the stability of the existing magnetic bead coating is relatively poor, and the actual requirement cannot be met.
Disclosure of Invention
Therefore, it is necessary to provide a coating material for magnetic beads with good stability.
In addition, a preparation method and application of the magnetic bead coating material and a detection kit are also provided.
A magnetic bead coating, comprising: the protein-based nano magnetic bead protein comprises a protein, a polyethylene glycol modifier and a nano magnetic bead, wherein the polyethylene glycol modifier is covalently connected with the protein, and the nano magnetic bead is connected with the protein.
The magnetic bead coating material is characterized in that a polyethylene glycol modifier is covalently connected with protein, so that the protein is connected with a PEG arm, the space structure rigidity of the protein is increased, the stability of the protein is improved, and the magnetic bead coating material with higher stability can be obtained after the protein connected with the polyethylene glycol modifier is connected with nano magnetic beads.
In one embodiment, the polyethylene glycol modifier has a structural formula
further, the amino group of the polyethylene glycol modifier and the carboxyl group of the protein form the-NH-CO-structure.
In one embodiment, the polyethylene glycol modifier has a weight average molecular weight of 200 or more;
and/or n is greater than or equal to 4;
and/or, the protein is an antigen or an antibody.
In one embodiment, the nano magnetic beads have a reactive group, and the reactive group reacts with an amino group of the protein to connect the nano magnetic beads with the protein;
further, the active group is a carboxyl group, and the active group and the amino group of the protein form an-NH-CO-structure so that the nano magnetic bead is connected with the protein.
A preparation method of a magnetic bead coating comprises the following steps:
coupling a polyethylene glycol modifier with a protein to ensure that the polyethylene glycol modifier is covalently connected with the protein to obtain a modified protein; and
coupling reaction is carried out on the modified protein and the nano magnetic beads, so that the modified protein is connected with the nano magnetic beads, and a magnetic bead coating object is obtained.
In one embodiment, the polyethylene glycol modifier has a structural formula
In one embodiment, the step of mixing and reacting the polyethylene glycol modifier with the protein to form an-NH-CO-structure with the protein to covalently attach the polyethylene glycol modifier to the protein comprises: reacting a cross-linking agent with a carboxyl group of the protein to form an intermediate; reacting the intermediate with an amino group of the polyethylene glycol modifier to form the-NH-CO-structure, thereby covalently linking the polyethylene glycol modifier with the protein to obtain the modified protein.
In one embodiment, the step of coupling the modified protein to the nanobead comprises: the nano magnetic beads are provided with active groups, and the active groups react with amino groups of the modified proteins to connect the nano magnetic beads with the modified proteins to obtain the magnetic bead coating materials.
The magnetic bead coating material or the magnetic bead coating material prepared by the preparation method is applied to the preparation of a detection reagent, a detection kit or a detection device.
A detection kit comprises the magnetic bead coating or the magnetic bead coating prepared by the preparation method of the magnetic bead coating.
Drawings
Fig. 1 is a schematic flow chart of a method for preparing a magnetic bead coating material according to an embodiment.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, 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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The magnetic bead coating material of one embodiment has high stability, and can be used for preparing a detection reagent, a detection kit or a detection device. Specifically, the magnetic bead coating comprises: the protein-based nano magnetic bead modifying agent comprises protein, a polyethylene glycol modifying agent and a nano magnetic bead, wherein the polyethylene glycol modifying agent is covalently connected with the protein, and the nano magnetic bead is connected with the protein.
The magnetic bead coating material is characterized in that a polyethylene glycol modifier is covalently connected with protein, so that the protein is connected with a PEG arm, the space structure rigidity of the protein is increased, the stability of the protein is improved, and the magnetic bead coating material with higher stability can be obtained after the protein connected with the polyethylene glycol modifier is connected with nano magnetic beads.
The protein can be combined with the substance to be detected, so that the substance to be detected in the test sample can be qualitatively or quantitatively detected. The test sample may be blood, for example. The test sample is not limited to blood, and may be other test samples, such as saliva or gastric juice.
In one embodiment, the protein is an antigen or an antibody. It should be noted that, in the process of coating antigen and antibody, the antigen structure is complex, and the coating difficulty is greater than that of antibody.
In one embodiment, the protein is an EB NA IgG antigen (DNA virus nuclear antigen IgG of lymphotropic cells of the herpesviridae family) or a Toxo IgM antigen (toxoplasma virus IgM antigen). The protein is not limited to the antigen specified above, and may be another antigen.
In one embodiment, the protein is a troponin i (ctni) antibody. The protein is not limited to the antibody described above, and may be another antibody, for example, a cancer antigen 15-3(CA15-3) antibody.
Polyethylene glycol modifiers can affect the spatial structure of proteins, causing changes in various biochemical properties of the protein, including: the chemical stability is increased, the resistance of proteolytic enzyme is improved, the water solubility of protein is improved, the nonspecific adsorption generated by the hydrophobic effect of the protein is reduced in the immune reaction process, the toxic and side effects of the protein are reduced, the half-life period of the protein is prolonged, and the stability of the protein is improved. And the polyethylene glycol modifier does not block the immunological binding site of the protein in space, so that the immunological activity of the protein is not affected basically by modifying the protein with the polyethylene glycol modifier.
In one embodiment, the polyethylene glycol modifier has the structural formula
In one embodiment, the weight average molecular weight of the polyethylene glycol modifier is 200-1200. The arrangement ensures that the polyethylene glycol modifier can not block the immunological binding site of the protein in space, and the modification of the polyethylene glycol modifier has little influence on the immunological activity of the protein. Modification with polyethylene glycol modifiers having too large a molecular weight tends to decrease the biological activity of the protein, while modification with polyethylene glycol modifiers having too small a molecular weight tends to have a weak effect on maintaining the stability of the modified protein.
In one embodiment, n is greater than or equal to 4. Further, n is 4-24. It should be noted that the higher the polymerization degree of the polyethylene glycol modifier, the higher the molecular weight, and modification with the polyethylene glycol modifier having an excessively high molecular weight is likely to decrease the biological activity of the protein.
The nano magnetic beads are provided with active groups, and the active groups of the nano magnetic beads can be coupled with proteins, so that the coupled proteins and the substances to be detected can be combined to realize the high-efficiency separation and detection of the substances to be detected by using the action of an external magnetic field. Further, the active groups of the nanobead react with the amino groups of the protein to link the nanobead to the protein. Furthermore, the active group of the nano magnetic bead is carboxyl. At this time, the carboxyl group of the nanobead and the amino group of the protein form an-NH-CO-structure to link the nanobead and the protein. Specifically, the binding site of the protein is a primary amine group. The active group of the nanobead is not limited to a carboxyl group, and may be another active group, for example, a tosyl group.
The magnetic bead coating material is characterized in that a polyethylene glycol modifier is covalently connected with protein, so that the protein is connected with a PEG arm, the space structure rigidity of the protein is increased, the stability of the protein is improved, and the magnetic bead coating material with higher stability can be obtained after the protein connected with the polyethylene glycol modifier is connected with nano magnetic beads.
Furthermore, in the magnetic bead coating material, the structural formula of the polyethylene glycol modifier is shown in the specification
The nanobead is not limited to be directly linked to the modified protein via an active group, and may be indirectly linked to the modified protein. For example: the nano magnetic beads are connected with the modified protein by adopting a biotin-avidin system. That is, avidin is linked to the nanobead, and then linked to the biotinylated modified protein. Because the affinity between the biotinylation modified protein and the nano magnetic bead connected with the avidin is very high, the nano magnetic bead can be simply, conveniently, quickly and effectively and tightly combined with the modified protein. The biotinylated modified protein is a modified protein to which biotin is linked. The indirect linkage is not limited to the biotin-avidin system, and may be another intermediate linkage system.
As shown in fig. 1, the method for preparing a magnetic bead coating according to the above embodiment can prepare a magnetic bead coating having high stability, and can be used for preparing a detection reagent, a detection kit, or a detection device. Specifically, the preparation method comprises the following steps of S110 to S120:
s110, coupling reaction is carried out on the polyethylene glycol modifier and the protein, so that the polyethylene glycol modifier is covalently connected with the protein, and the modified protein is obtained.
In one embodiment, S110 includes: and mixing and reacting the cross-linking agent, the protein and the polyethylene glycol modifier to obtain the modified protein. Further, S110 includes: mixing and reacting a polyethylene glycol modifier, a protein and a cross-linking agent to enable the polyethylene glycol modifier and the protein to form an-NH-CO-structure so as to enable the polyethylene glycol modifier and the protein to be covalently connected, thereby obtaining the modified protein. Wherein the structural formula of the polyethylene glycol modifier is shown in the specification
In one embodiment, the crosslinking agent is a crosslinking agent capable of activating carboxyl groups. Further, the crosslinking agent is carbodiimide. Carbodiimide is mainly used to activate carboxyl groups and promote formation of amide bonds. The crosslinking agent is not limited to carbodiimide, and may be other crosslinking agents capable of activating carboxyl groups, and may be provided as needed. Further, the cross-linking agent can activate both the carboxyl group of the protein and the carboxyl group of the polyethylene glycol modifier.
In one embodiment, when the cross-linking agent activates the carboxyl group of the protein, S110 comprises the following reaction processes S111 to S112:
s111, reacting the cross-linking agent with the protein to obtain a first intermediate.
In one embodiment, the crosslinking agent is a carbodiimide. The cross-linking agent reacts with the carboxyl groups of the protein to give a first intermediate. Specifically, the reaction formula of the cross-linking agent with the protein is as follows:
and S112, reacting the first intermediate with a polyethylene glycol modifier so that the polyethylene glycol modifier is covalently connected with the protein to obtain the modified protein.
Specifically, the structural formula of the polyethylene glycol modifier is shown in the specificationThe first intermediate reacts with the amino group of the polyethylene glycol modifier so that the polyethylene glycol modifier and the protein form an-NH-CO-structure to obtain the modified protein. The reaction of the first intermediate with the polyethylene glycol modifier is as follows:
in one embodiment, when the crosslinking agent activates the carboxyl group of the polyethylene glycol modifier, S110 comprises the following reaction processes S111 'to S112':
and S111', reacting the cross-linking agent with a polyethylene glycol modifier to obtain a first intermediate.
In one embodiment, the crosslinking agent is a carbodiimide. The structural formula of the polyethylene glycol modifier isThe first intermediate is obtained by the reaction of the cross-linking agent and the carboxyl of the polyethylene glycol modifier. Specifically, the reaction formula of the reaction of the cross-linking agent and the polyethylene glycol modifier is as follows:
s112', the first intermediate and the protein react to covalently link the polyethylene glycol modifier to the protein, resulting in a modified protein.
Specifically, the first intermediate reacts with the amino group of the protein to allow the polyethylene glycol modifier to form an-NH-CO-structure with the protein, thereby obtaining the modified protein. More specifically, the reaction of the first intermediate with the protein is represented by the following equation:
in the process of mixing and reacting the crosslinking agent, the protein, and the polyethylene glycol modifier, the crosslinking agent can simultaneously activate the carboxyl group of the protein and the carboxyl group of the polyethylene glycol modifier. In this case, S110 can obtain two modified proteins; in one of the modified proteins, carboxyl of the protein reacts with amino of a polyethylene glycol modifier to form an-NH-CO-structure; in another modified protein, the amino group of the protein reacts with the carboxyl group of the polyethylene glycol modifier to form an-NH-CO-structure.
In one embodiment, the mass ratio of the carbodiimide to the protein to the polyethylene glycol modifier is (0.016-0.024): (1-2): (2-4). The reaction pH is 5.0-7.0. The reaction temperature is 20-30 ℃. The reaction time is 10 min-20 min. Further, carbodiimide, protein and polyethylene glycol modifier are dissolved in buffer solution respectively, and then mixed and reacted. The carbodiimide may be mixed with the protein and then mixed with the polyethylene glycol modifier and reacted, or the carbodiimide may be mixed with the protein and reacted, or the carbodiimide may be mixed with the polyethylene glycol modifier and reacted, and then mixed with the protein and reacted. Further, the buffer was MES buffer (2-morpholino-ethanesulfonic acid, 2- (N-morpholino) ethanesulfonic acid). Specifically, the concentration of the MES buffer solution is 2.13 mg/mL-5.33 mg/mL, and the pH is 5.0-7.0. More specifically, the MES buffer concentration was 3.20mg/mL and the pH was 6.0. Wherein the protein concentration in the MES buffer solution containing the protein was 1.67mg/mL, the carbodiimide concentration in the MES buffer solution containing the carbodiimide was 1mg/mL, and the polyethylene glycol modifier concentration in the MES buffer solution containing the polyethylene glycol modifier was 10 mg/mL.
In one embodiment, after the step of mixing and reacting the cross-linking agent, the protein and the polyethylene glycol modifier to obtain the modified protein, the method further comprises the following steps: and mixing the cross-linking agent, the protein and the polyethylene glycol modifier, and reacting to obtain a reactant for purification. The arrangement can remove free polyethylene glycol modifier, obtain modified protein with the purity of more than 99 percent, and is more favorable for the subsequent combination with nanometer magnetic beads. Further, the purification mode is ultrafiltration. Furthermore, the molecular weight cut-off of the ultrafiltration is 20 KD-40 KD. Specifically, in the ultrafiltration process, the buffer solution for changing the solution is PBS buffer solution. More specifically, the concentration of the PBS buffer solution is 15 mmol/L-25 mmol/L, and the pH value is 7.4-8.4. In one specific example, the concentration of PBS buffer is 15mmol/L and the pH is 7.4. Wherein, the reactant obtained by mixing and reacting the cross-linking agent, the protein and the polyethylene glycol modifier is ultrafiltered for a plurality of times. In one specific example, the number of ultrafiltration is 3.
And S120, coupling reaction of the modified protein and the nano magnetic beads so as to connect the modified protein and the nano magnetic beads to obtain a magnetic bead coating.
In one embodiment, the active group of the nano magnetic bead reacts with the amino group of the modified protein to connect the nano magnetic bead with the modified protein, thereby obtaining the magnetic bead coating. Further, the active group of the nano magnetic bead is carboxyl, the carboxyl of the nano magnetic bead reacts with the amino of the modified protein to form an-NH-CO-structure, so that the nano magnetic bead is connected with the modified protein, and the magnetic bead coating is obtained.
Specifically, the nanobead has a carboxyl group, and S120 includes S121 to S122:
and S121, reacting the nano magnetic beads with a cross-linking agent to obtain a second intermediate.
In one embodiment, the mass ratio of the nano magnetic beads to the cross-linking agent is 1 (0.1-0.2). The reaction pH is 5.0-7.0. The reaction temperature is 20-30 ℃. The reaction time is 25 min-35 min.
In one embodiment, the crosslinking agent comprises a carbodiimide. Carbodiimide is mainly used to activate carboxyl groups and promote formation of amide bonds. Further, the crosslinking agent also includes N-hydroxysuccinimide. N-hydroxysuccinimide is mainly used for activating carbonyl groups during amide bond formation, protecting synthesized amino acid and improving yield. Further, the preparation process of the cross-linking agent comprises the following steps: and respectively dissolving carbodiimide and N-hydroxysuccinimide in dimethyl sulfoxide, and mixing to obtain the cross-linking agent. Specifically, the volume ratio of carbodiimide to N-hydroxysuccinimide in the crosslinking agent is 1: 1. More specifically, the carbodiimide concentration in the dimethylsulfoxide solution with the carbodiimide dissolved therein was 10 mg/mL. The concentration of N-hydroxysuccinimide in the dimethyl sulfoxide solution in which N-hydroxysuccinimide was dissolved was 10 mg/mL.
When the cross-linking agent comprises carbodiimide and N-hydroxysuccinimide and the nano magnetic bead has carboxyl, the cross-linking agent reacts with the carboxyl of the nano magnetic bead to obtain a second intermediate. Specifically, the reaction formula of the cross-linking agent and the nano magnetic beads is as follows:
in one embodiment, before the step of mixing and reacting the nanobead with the cross-linking agent, the step of washing the nanobead is further included. With this arrangement, the residual preservation solution on the surface of the nano magnetic beads can be removed. Specifically, the cleaning solution is MES buffer. Furthermore, the concentration of the MES buffer solution is 2.13 mg/mL-5.13 mg/mL, and the pH value is 5.0-7.0. Further, the MES buffer concentration was 3.20mg/mL and the pH was 6.0. Specifically, washing is performed a plurality of times. More specifically, the number of washing times was 3.
In one embodiment, after the step of mixing and reacting the nanobead with the cross-linking agent, the method further comprises the following steps: and mixing the nano magnetic beads with a cross-linking agent, and washing a reactant obtained by reaction. This arrangement enables the removal of the free crosslinking agent and the production of a second intermediate having a higher purity. Further, the washing solution is MES buffer. Furthermore, the concentration of the MES buffer solution is 2.13 mg/mL-5.13 mg/mL, the pH is 5.0-7.0, and the temperature is 2-8 ℃. Specifically, MES buffer was at a concentration of 3.20mg/mL, pH 6.0, and temperature 4 ℃. More specifically, the number of washing is 1.
And S122, reacting the second intermediate with the modified protein to enable carboxyl of the nano magnetic beads to react with amino of the modified protein to form an-NH-CO-structure, so as to obtain the magnetic bead coating.
Further, the amino group of the modified protein is a primary amino group. Specifically, the reaction of the second intermediate with the modified protein is represented by the following formula:
in one embodiment, the mass ratio of the second intermediate to the modified protein is (25-50): 1. the reaction pH is 5.0-7.0. The reaction temperature is 20-30 ℃. The reaction time is 2.5 h-3.5 h.
Further, the step of mixing and reacting the second intermediate with the modified protein comprises: after resuspending the second intermediate with MES buffer, modified protein was added and mixed and reacted. Furthermore, the pH value of the MES buffer solution is 5-7, the temperature is 2-8 ℃, and the concentration is 2.13-5.33 mg/mL. Specifically, MES buffer was at pH 6.0, temperature 4 ℃ and concentration 3.20 mg/mL.
In one embodiment, the step of mixing and reacting the second intermediate with the modified protein further comprises the steps of: and (3) carrying out sealing treatment on a reactant obtained by mixing and reacting the second intermediate with the modified protein. The arrangement can reduce the active sites on the surface of the nano magnetic beads which are not combined with the modified protein, reduce the non-specific adsorption in the application process of the product and improve the sensitivity and accuracy of the product. Further, the step of blocking the reactant obtained by mixing and reacting the second intermediate with the modified protein comprises: and mixing and reacting the second intermediate and the modified protein to obtain a reactant, mixing and reacting the reactant with the sealing liquid at the reaction temperature of 20-30 ℃ for 2-4 h to obtain the reactant subjected to sealing treatment. Further, the blocking solution was PBS buffer (phosphate buffer) containing 5% by mass of BSA (bovine serum albumin). Specifically, the concentration of PBS buffer was 150mmol/L, and the pH was 7.4.
In one embodiment, the step of blocking the reactant obtained by mixing and reacting the second intermediate with the modified protein further comprises the following steps: and carrying out magnetic separation on the reaction product after the sealing treatment. By the arrangement, the reactant after the sealing treatment can be quickly separated from the sealing liquid, and the magnetic bead coating substance can be obtained.
In one embodiment, after the step of magnetically separating the reactants after the blocking treatment, the method further comprises the following steps: and (4) carrying out constant volume on the magnetic bead coating obtained after the magnetic separation treatment. Further, the solvent used in the volume fixing process was PBS buffer (phosphate buffer) containing 5% by mass of BSA (bovine serum albumin). Further, the concentration of PBS buffer was 15mmol/L and the pH was 7.4. Specifically, the PBS buffer solution containing the magnetic bead coating material of 5 mg/mL-15 mg/mL is obtained after constant volume.
The preparation method of the magnetic bead coating has at least the following advantages:
(1) according to the preparation method of the magnetic bead coating, the polyethylene glycol modifier is covalently connected with the protein, so that the protein is connected with the PEG arm, the space structure rigidity of the protein is increased, the stability of the protein is improved, and the magnetic bead coating with high stability can be obtained after the protein connected with the polyethylene glycol modifier is connected with the nano magnetic beads.
(2) In the preparation method of the magnetic bead coating, carbodiimide is used as a cross-linking agent in the modification process of the protein and the coupling process of the modified protein and the nano magnetic beads, so that the coupling efficiency is facilitated.
(3) The magnetic bead coating prepared by the preparation method of the magnetic bead coating has the advantages of enhanced hydrophilicity, lower non-specific adsorption and smaller toxic and side effects.
(4) The magnetic bead coating prepared by the preparation method of the magnetic bead coating has longer half-life period and higher stability.
The detection kit of an embodiment includes the magnetic bead coating material of the embodiment or the magnetic bead coating material prepared by the method for preparing the magnetic bead coating material.
In one embodiment, the detection kit further comprises a second antibody labeled with a luminescent substance. The second antibody marked by the luminescent substance can be connected with a conjugate formed by combining the object to be detected and the coating material of the magnetic beads, so that the object to be detected can be detected through a luminescent signal. Further, the second antibody was a mouse anti-human IgG. The second antibody is not limited to mouse anti-human IgG, and may be another second antibody, for example, mouse anti-human IgM. Further, the luminescent material is an acridine substituent. Specifically, the luminescent substance is an acridinium ester. The light-emitting substance is not limited to an acridinium ester, and may be other acridine substitutes, for example, acridine sulfonamide. The detection kit is not limited to the one including the luminescent substance-labeled secondary antibody, and may include a luminescent substance-labeled antigen. When the luminescent substance is used for labeling the antigen, the antigen labeled by the luminescent substance can be connected with a conjugate formed by combining the substance to be detected and the magnetic bead coating substance, so that the substance to be detected can be detected through a luminescent signal.
The detection kit at least has the following advantages:
(1) the magnetic bead coating used by the detection kit has high chemical stability and long half-life period, so that the detection kit has long service life and is beneficial to saving the use cost.
(2) The non-specific adsorption of the magnetic bead coating used by the detection kit is low, and the accuracy of the detection result is high.
The following are specific examples:
unless otherwise specified, in the following examples: EB NA IgG antigen was purchased from Fenpeng Bio Inc; ToxoIgM antigen available from MICROBIX under the trade designation EL-18-08; acridinium ester-labeled mouse anti-human IgG antibodies were purchased from fenpeng bio-ltd; polyethylene glycol modifiers were purchased from Thermo Fisher corporation; nanobead (with carboxyl groups on the surface) was purchased from Merck; carbodiimide (EDC) available from Thermo corporation; n-hydroxysuccinimide (NHS) from Thermo; the luminous liquid is purchased from Shenzhen Shenhuilong Biotech limited, and comprises pre-excitation liquid and excitation liquid, wherein the product number of the pre-excitation liquid is C89999, and the product number of the excitation liquid is C89968; purchased from Shenzhen, Yahuilong Biotech, Inc.; molecular weight cut-off ultrafiltration tubes were purchased from Millipore corporation; the ultramicro ultraviolet spectrophotometer is purchased from Thermo corporation, and is Nanodrop lite; the chemiluminescence determinator is purchased from Shenzhen Shenhuilong Biotech, Inc. with model number iFlash 3000.
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