CN113311154A - Coupling method, diluent and application of immunomagnetic particles - Google Patents
Coupling method, diluent and application of immunomagnetic particles Download PDFInfo
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- CN113311154A CN113311154A CN202110596633.3A CN202110596633A CN113311154A CN 113311154 A CN113311154 A CN 113311154A CN 202110596633 A CN202110596633 A CN 202110596633A CN 113311154 A CN113311154 A CN 113311154A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Abstract
The invention provides a coupling method, diluent and application of immunomagnetic particles. According to the antibody-magnetic bead coupling process, the carboxyl magnetic beads are activated under the acidic pH condition, and then the activated carboxyl magnetic beads are coupled with the antibody under the condition close to neutrality, so that the biological activity of the antibody is maintained under the mild reaction condition, the aggregation of the activated carboxyl magnetic beads is reduced, and the efficiency of coupling the carboxyl magnetic beads with the antibody is improved. The blocking liquid provided by the invention enables carboxyl sites of unconjugated antibodies on magnetic beads to be combined by BSA, amino acid or/and small molecular peptide and PEG through ionic bonds or covalent bonds, and simultaneously enables the antibodies conjugated on the carboxyl magnetic beads to keep correct and stable forms, thereby improving the detection performance and enhancing the sensitivity. The immune magnetic particle diluent provided by the invention can ensure that the prepared immune magnetic particles, such as CA125 antibody-carboxyl magnetic beads, can still keep good dispersibility and stability after being diluted by the diluent and placed at 4 ℃ for 12 months or at 37 ℃ for 7 days.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a coupling method, dispersion liquid and application of immunomagnetic particles.
Background
The magnetic particle has wide application in chemiluminescence immunoassay, common surface active groups of the magnetic particle have carboxyl, amino and the like, and the magnetic particle can be coupled with an antibody or amino or carboxyl on the antigen through intermediate activation under certain conditions and is used for detecting the antigen or the antibody. The common way of coupling carboxyl magnetic beads with antibodies is a one-step method and a two-step method: the one-step method is to mix the magnetic beads and the antibody and then add the cross-linking agent for reaction. The one-step method has high requirements on the antibody and needs to tolerate a high concentration of the cross-linking agent, but if the amount of the cross-linking agent is too low, the coupling efficiency is affected, so that the cross-linking mode is generally rarely adopted.
The two-step method is to activate the magnetic beads and then add the antibody for coupling. Carboxyl magnetic beads are usually activated by selecting MES buffer solution with acidic pH (pH 4.0-6.5), and then coupling reaction is carried out by adding a certain amount of antibody under the same conditions, for example, the activation and coupling reaction are carried out under the same acidic conditions in the patent application with the application number of CN105319356A and the invention patent with the application number of CN103926398B, but the coupled antibody-magnetic beads are usually easy to aggregate under the low pH condition. The carboxyl magnetic beads are also selected to carry out the second coupling reaction in a phosphate buffer solution with neutral to alkaline pH (pH 7.0-8.0), for example, the coupling mode is adopted in the invention patent with the patent number of CN105478087B and the invention patent application with the application number of CN108503710A, but the coupling efficiency is often low under the condition of phosphate with high pH. Alternatively, two coupling steps were selected in phosphate buffer solution with pH7.0, such as the coupling method disclosed in patent CN102305858B, but the high pH condition combined with phosphate usually results in low EDC activation efficiency, and it is difficult to obtain the desired coupling effect.
The immunomagnetic particles with high coupling efficiency are difficult to obtain by the existing method, or even if the immunomagnetic particles with high coupling efficiency are obtained, the particles are often poor in dispersibility, aggregation is easy to occur as long as the particles are placed for a long time, and obvious agglomerate-shaped aggregates can be seen under a microscope, so that the chemiluminescence reagent prepared by the immunomagnetic particles is poor in precision, sensitivity and stability, and the performance of the reagent is greatly influenced.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a coupling method, dispersion liquid and application of immunomagnetic particles, and solve the problems of low coupling efficiency and poor dispersibility of immunomagnetic particles, poor chemiluminescence test performance prepared by using the immunomagnetic particles and the like in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method of coupling immunomagnetic particles, comprising the steps of:
(1) and (3) activation: activating clean magnetic beads by using a cross-linking agent in a buffer system a to obtain activated magnetic beads;
(2) coupling: adding an antibody into the activated magnetic beads in the buffer system b, uniformly mixing and incubating to obtain an antibody-magnetic bead conjugate;
(3) and (3) sealing: adding a sealing solution into the antibody-magnetic bead conjugate obtained in the step (2) for sealing to obtain an antibody-magnetic bead coupled immunomagnetic particle final product;
the pH value of the buffer system a is 4-6, and the pH value of the buffer system b is 6.5-7.3. The antibody-magnetic bead coupling process adopts an improved two-step method, firstly carboxyl magnetic beads are activated under an acidic pH condition, then the activated carboxyl magnetic beads are coupled with antibodies under a condition close to neutrality, and are sealed by optimized sealing liquid, the mild reaction condition not only keeps the bioactivity of the antibodies, but also effectively reduces the aggregation of the activated carboxyl magnetic beads, effectively improves the coupling efficiency of the carboxyl magnetic beads coupled with the antibodies, and compared with the existing coupling process, the background value is lower, the sensitivity is higher, the magnetic bead dispersibility and the stability are better, and the reagent performance is improved.
In the step (1), the concentration of the cross-linking agent is 10-100 mg/ml, the mass ratio of the cross-linking agent to the magnetic beads is 1: 100-100: 1, and the activation time is 10-60 min.
Preferably, the concentration of the cross-linking agent is 10mg/ml, the mass ratio of the cross-linking agent to the magnetic beads is 1: 1, and the activation time is 30 min.
The concentration of the buffer system a is 10-500 mM, and the concentration of the buffer system b is 10-500 mM.
Preferably, the pH value of the buffer system a is 5, the pH value of the buffer system b is 6.8, the concentration of the buffer system a is 100mM, and the concentration of the buffer system b is 100 mM. The carboxyl magnetic beads are activated under the pH condition that the pH value of acid is 5, and then the activated carboxyl magnetic beads are coupled with the antibody under the pH value of 6.8, so that the mild reaction condition not only keeps the biological activity of the antibody, but also effectively reduces the aggregation of the activated carboxyl magnetic beads, and effectively improves the coupling efficiency of the carboxyl magnetic beads coupled with the antibody.
Preferably, the buffer system a is MES buffer solution, and the buffer system b is Good's buffer solution; the Good's buffer is selected from one of MES, PIPES, MOPSO, MOPS, HEPES, HEPPSO and EPPS buffers; more preferably, the Good's buffer is selected from MOPS buffers; good's buffer solution does not participate in and interfere with biochemical reaction, and the efficiency of coupling the carboxyl magnetic beads with the antibody can be effectively improved.
In the step (2), the mass ratio of the antibody to the magnetic beads is 1: 50-1: 200, and the incubation time is 3-24 h;
preferably, the mass ratio of the antibody to the magnetic beads is 1: 50, and the incubation time is 6 h.
In the step (2), the components of the confining liquid comprise 0.01-0.5M Tris with pH of 7.0-9.0, 50-300 mM NaCl, 0.01-10% BSA (w/v), 0.01-1% Triton X-100(v/v), 0.1-10% PEG (v/v), 0.1-5% amino acid or/and small molecule peptide (w/v), and 0.01-0.09% sodium azide (w/v), and the confining time is 1-24 h. The confining liquid enables carboxyl sites of unconjugated antibodies on magnetic beads to be combined by BSA, amino acid or small molecular peptide and PEG through ionic bonds or covalent bonds, and enables the antibodies conjugated on the carboxyl magnetic beads to keep correct and stable forms, so that interference of nonspecific factors during detection can be further reduced, background signals are reduced, detection performance is improved, and sensitivity is enhanced.
Preferably, the components of the blocking solution comprise 0.1M Tris, 150mM NaCl, 0.5% BSA (w/v), 0.5% Triton X-100(v/v), 0.1-10% PEG (v/v), 0.1-5% amino acid or/and small molecule peptide (w/v), 0.05% sodium azide (w/v) with pH 7.5.
Preferably, the PEG comprises PEG200-10000, preferably PEG600-6000, most preferably PEG4000, the PEG concentration being 2% (v/v). PEG can be physically adsorbed to the immunomagnetic particles to effectively block the surfaces of the magnetic particles, but when the molecular weight of the PEG is too low, blocking of macromolecular blocking agents (such as BSA, casein and the like) to the immunomagnetic particles through chemical bonds can be prevented, and when the molecular weight is too high, the chain density of a PEG layer on the surfaces of the immunomagnetic particles is reduced, and further, nonspecific substances penetrate through the PEG layer to weaken the blocking effect. The PEG4000 is used for sealing the immune magnetic particles, on one hand, the molecular weight is proper, so that biological micromolecules (such as amino acids, micromolecule peptides and the like) and macromolecular sealing agents can be allowed to seal the surfaces of the magnetic particles in a chemical bond mode, on the other hand, the PEG4000 is covered on the surfaces of the immune magnetic particles to maintain the correct conformation of the coupling antibody on the surfaces of the magnetic particles, and the stability and the detection specificity of the immune magnetic particles are improved.
Preferably, the amino acid comprises at least one of alanine, glycine, lysine or arginine.
Preferably, the small molecule peptide comprises at least one of diglycine, dypeptide, carnosine or tripeptide.
More preferably, the concentration of the amino acid or/and the small molecule peptide is 2% (w/v).
The magnetic beads comprise carboxyl magnetic beads, the crosslinker comprises EDC crosslinker, and the antibody comprises CA125 antibody.
The invention also provides the immunomagnetic particles obtained by the coupling method.
The invention also provides application of the immunomagnetic particles obtained by the coupling method in preparation of a kit detection reagent or a kit.
The present invention also provides a dilution of immunomagnetic particles obtained by the coupling method according to any one of claims 1 to 7.
The components of the diluent comprise: 0.01-0.5M Tris, 50-300 mM NaCl, 0.05-5% BSA (w/v), 0.01-10% PVP (w/v), 0.1-10% trehalose (w/v), 0.01-1% Tween20(v/v), 0.01-0.09% sodium azide and 0.01-0.1% ProClin300 with the pH value of 7.0-9.0. After the immune magnetic particles prepared by the invention, such as CA125 antibody-magnetic beads, are diluted by the diluent, the magnetic beads have good dispersibility and stability, and can still maintain good detection precision without aggregation after being placed for 12 months for a long time. Specifically, the use method of the diluent comprises the following steps: prepared immune magnetic particles (such as CA125 antibody-magnetic beads) are used as raw materials, and the diluted solution is diluted by 1: 10(v/v) and then can be used for detecting the sugar antigen 125 determination kit.
Preferably, the components of the diluent comprise: 0.1M Tris, 200mM NaCl, 1% BSA (w/v), 5% PVP (w/v), 2% trehalose (w/v), 0.05% Tween20(v/v), 0.05% sodium azide, 0.05% ProClin300, pH 7.5. The diluent component PVP has a good dispersing effect and can weaken the precipitation caused by mutual aggregation of magnetic particles, BSA and trehalose both have the effect of stabilizing biological macromolecules and can keep good biological activity of a coupling antibody on the immune magnetic particles, and the combined use of the components is beneficial to keeping good dispersibility and stability of the immune magnetic particles after being placed for a long time.
The invention has the following beneficial effects:
(1) the antibody-magnetic bead coupling process adopts an improved two-step method, namely, carboxyl magnetic beads are activated under the condition of low pH, then antibody coupling is carried out in a mild buffer system close to neutrality, and preferred confining liquid is used for confining, the mild reaction condition not only keeps the bioactivity of the antibody, but also effectively reduces aggregation after the activation of the carboxyl beads, improves the coupling efficiency of the magnetic bead coupling antibody, and compared with the existing coupling process, the background value is lower, the sensitivity is higher, the dispersibility and the stability of the magnetic beads are better, and the performance of a reagent is improved.
(2) According to the invention, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is used as a cross-linking agent, carboxyl magnetic beads are activated under an acidic pH condition, and then the activated carboxyl magnetic beads are coupled with CA125 antibodies in Good's buffer solution (not participating in and interfering biochemical reaction) with nearly neutral pH (pH6.5-7.3), so that the mild reaction condition not only maintains the biological activity of the CA125 antibodies, but also effectively reduces aggregation after activation of the carboxyl magnetic beads and improves the coupling efficiency of the carboxyl magnetic bead coupled antibodies.
(3) The blocking liquid provided by the invention enables carboxyl sites of unconjugated antibodies on magnetic beads to be combined by BSA, amino acid or/and small molecular peptide and PEG through ionic bonds or covalent bonds, and enables the antibodies conjugated on the carboxyl magnetic beads to keep a correct and stable form, so that the interference of nonspecific factors during detection can be further reduced, background signals are reduced, the detection performance is improved, and the sensitivity is enhanced.
(4) The immune magnetic particle diluent provided by the invention can keep good dispersibility and stability for a long time after the prepared immune magnetic particles are diluted by the diluent, and for example, the CA125 antibody-carboxyl magnetic beads prepared by the invention can still keep good dispersibility and stability after being diluted by the diluent and placed at 4 ℃ for 12 months or at 37 ℃ for 7 days.
Drawings
FIG. 1: the effect of different dilutions on reagent stability was analyzed.
FIG. 2: the influence of different diluents on the dispersibility of the immunomagnetic particles.
Detailed Description
In order to better explain the invention, the following further explains the content of the invention in conjunction with specific embodiments and the accompanying drawings.
Example 1: preparation of immunomagnetic particles
The first step is as follows: activation of carboxyl magnetic beads
(1) Washing 10mg magnetic beads with 1ml 100mM MES (pH5.0), incubating for 2min, adsorbing with magnetic plate, and removing supernatant;
(2) adding 100mM MES (pH5.0) for resuspension;
(3) 100mM MES (pH5.0) was taken to dissolve EDC to 10mg/ml, and an appropriate amount of diluted EDC was added to the magnetic beads of step (3), EDC: the mass ratio of the magnetic beads is 1: 1, vortex and mix evenly, and incubate for 30min at room temperature;
(4) after incubation, adsorbing by a magnetic plate, and removing supernatant;
(5 washing 1 time with 100mM MES (pH5.0), adsorbing with magnetic plate, and discarding the supernatant;
(6) adding 100mM Good's buffer solution (pH6.8) to obtain activated carboxyl magnetic beads;
the second step is that: antibody-magnetic bead coupling
The CA125 antibody was diluted with 100mM Good's buffer (pH6.8), and the carboxyl magnetic beads activated in step one were added to the diluted antibody, antibody: the mass ratio of the magnetic beads is 1: 50, the mixture is evenly mixed by vortex and incubated for 6 hours at room temperature.
The third step: sealing and washing
(1) Taking the antibody-magnetic bead conjugate in the second step, adsorbing by a magnetic plate, removing the supernatant, adding 1ml of a blocking solution containing 0.1M Tris (pH7.5), 150mM NaCl, 0.5% BSA (w/v), 0.5% Triton X-100(v/v), 2% PEG4000, 2% force peptide and 0.05% sodium azide (w/v) and blocking for 16 hours;
(2) adsorbing by a magnetic plate, and discarding the supernatant;
(3) washing with TBST (0.1M Tris, 150mM NaCl, 0.05% Tween20(v/v), pH7.4) for 3 times, adsorbing with magnetic plate, and discarding the supernatant;
(4) and (3) preserving the coupled magnetic beads by using 1ml of storage buffer solution to prepare an antibody-magnetic bead coupled immune magnetic particle final product: CA125 antibody-magnetic beads.
Example 2: preparation of immune magnetic particle diluent
The immune magnetic particle diluent comprises the following components: 0.1M Tris (pH7.5), 200mM NaCl, 1% BSA (w/v), 5% PVP (w/v), 2% trehalose (w/v), 0.05% Tween20(v/v), 0.05% sodium azide, 0.05% ProClin 300.
Diluting CA125 antibody-magnetic beads with the diluent at a ratio of 1: 10(v/v) to obtain solid phase reagents, detecting background values on a full-automatic chemiluminescence immunoassay analyzer, and recording RLU values.
Comparative example 1
The antibody and the activated carboxyl magnetic beads were coupled under the condition of 100mM MES (pH5.0), and the rest of the procedure was the same as in example 1.
Comparative example 2
The antibody and the activated carboxyl magnetic beads were coupled in a 100mM phosphate buffer solution (pH6.8), and the procedure was the same as in example 1.
Comparative example 3
The blocking solution used was composed of 1ml of the same composition containing 0.1M Tris (pH7.5), 150mM NaCl, 0.5% BSA (w/v), 2% PEG4000(w/v), 0.5% Triton X-100(v/v), 0.05% sodium azide (w/v), and the rest was the same as in example 1.
Comparative example 4
The blocking solution used was composed of 1ml of the same composition containing 0.1M Tris (pH7.5), 150mM NaCl, 0.5% BSA (w/v), 2% propeptide (w/v), 0.05% Tween20(v/v), 0.05% sodium azide (w/v), and the rest of the procedure was as in example 1.
Comparative example 5
The CA125 antibody-magnetic beads were diluted 1: 10(v/v) using a common immunomagnetic particle diluent, and the rest of the procedure was the same as in example 2.
The formula of the common immune magnetic particle diluent is as follows: 0.1M PBS (pH7.5), 2% BSA (w/v), 0.05% Tween20(v/v), 0.05% sodium azide (w/v), 0.05% ProClin300 (v/v).
Test results
(1) Effect of different immunomagnetic particle coupling protocols on background values
The CA125 antibody-carboxyl magnetic beads prepared in the example 1 and the comparative examples 1 to 4 are diluted by the immune magnetic particle diluent according to the ratio of 1: 10(v/v) to prepare a solid phase reagent, the background values are respectively detected on a full-automatic chemiluminescence immunoassay analyzer, the RLU values are recorded, and the related test results are shown in Table 1.
Table 1: analysis of influence of different immunomagnetic particle coupling schemes on background value
As can be seen from Table 1, the background value of the CA125 antibody-carboxyl magnetic beads obtained by coupling in example 1 is the lowest, the signal-to-noise ratio is the highest, and the repeatability is the best compared with that of comparative examples 1-4. The improved two-step antibody-magnetic bead coupling process is shown, namely, the carboxyl magnetic beads are activated in the acidic MES buffer solution, then the activated carboxyl magnetic beads are coupled with the antibodies in the Good's buffer solution which is nearly neutral, and the antibodies are sealed by the preferable sealing solution, so that the moderate reaction conditions not only keep the biological activity of the antibodies, but also effectively reduce the aggregation of the activated carboxyl magnetic beads and improve the efficiency of coupling the carboxyl magnetic beads with the antibodies. The Good's buffer solution does not participate in and interfere with biochemical reaction, and compared with a phosphate buffer solution with the same pH value, the efficiency of coupling the carboxyl magnetic beads with the antibody can be effectively improved.
In addition, the blocking solution provided by the invention enables the carboxyl sites of the unconjugated antibodies on the magnetic beads to be combined by BSA, amino acid or small molecular peptide and PEG through ionic bonds or covalent bonds, and enables the antibodies conjugated on the carboxyl magnetic beads to keep a correct and stable form.
(2) Effect of different immunomagnetic particle dilutions on reagent stability and precision the RLU values of the bur controls of the same batch were determined on a full-automatic chemiluminescence immunoassay analyzer using the solid phase reagents prepared in example 2 and comparative example 5 at different time periods, respectively, and the results of the related tests are shown in table 2 and fig. 1.
Table 2: effect of different dilutions on reagent stability and precision
As can be seen from Table 2 and FIG. 1 (A: effect of dilution of antibody-magnetic beads on reagent stability at 4 ℃ C.; B: effect of dilution of antibody-magnetic beads on reagent stability at 37 ℃ C.), the solid-phase reagents prepared in example 2 and comparative example 5, after standing at 4 ℃ for 1 year, the quality control of 3 levels of berle is respectively reduced by 12.15%, 10.4%, 8.95% and 33.4%, 26.4%, 21.4%, after being placed at 37 ℃ for 7 days, the 3 levels of bere quality control decreased by 13.6%, 11.3%, 10.3%, and 35.4%, 29.9%, and 24.4%, respectively, indicating that the stability of example 2 was superior to that of comparative example 5, and after the solid-phase reagent prepared in example 2 was left at 4 ℃ for 12 months or at 37 ℃ for 7 days, the precision thereof was not lowered, but the precision of the solid phase reagent prepared in comparative example 5 was gradually deteriorated as the standing time was increased, mainly due to aggregation of magnetic beads caused by the decrease in the dispersibility of magnetic beads (see fig. 2). In FIG. 2, A is the result of leaving the solid-phase reagent prepared with the ordinary diluent at 4 ℃ for 12 months, B is the result of leaving the solid-phase reagent prepared with the ordinary diluent at 37 ℃ for 7 days, D is the result of leaving the solid-phase reagent prepared with the antibody-magnetic bead diluent of the present invention at 0 months, E is the result of leaving the solid-phase reagent prepared with the diluent of the present invention at 4 ℃ for 12 months, and F is the result of leaving the solid-phase reagent prepared with the diluent of the present invention at 37 ℃ for 7 days.
The results show that the immune magnetic particle diluent provided by the invention can keep good dispersity and stability of the immune magnetic particles. The diluent component PVP has a good dispersing effect and can weaken the precipitation caused by mutual aggregation of magnetic particles, BSA and trehalose both have the effect of stabilizing biological macromolecules and can keep good biological activity of a coupling antibody on the immune magnetic particles, and the combined use of the components is beneficial to keeping good dispersibility and stability of the immune magnetic particles after being placed for a long time.
Claims (10)
1. A coupling method of immunomagnetic particles is characterized by comprising the following steps:
(1) and (3) activation: activating clean magnetic beads by using a cross-linking agent in a buffer system a to obtain activated magnetic beads;
(2) coupling: adding an antibody into the activated magnetic beads in the buffer system b, uniformly mixing and incubating to obtain an antibody-magnetic bead conjugate;
(3) and (3) sealing: adding a sealing solution into the antibody-magnetic bead conjugate obtained in the step (2) for sealing to obtain an antibody-magnetic bead coupled immunomagnetic particle final product;
the pH value of the buffer system a is 4-6, and the pH value of the buffer system b is 6.5-7.3.
2. The coupling method of immunomagnetic particles according to claim 1, wherein in the step (1), the concentration of the crosslinking agent is 10-100 mg/ml, and the mass ratio of the crosslinking agent to the magnetic beads is 1: 100-100: 1, the activation time is 10-60 min;
preferably, the concentration of the cross-linking agent is 10mg/ml, and the mass ratio of the cross-linking agent to the magnetic beads is 1: 1, and the activation time is 30 min.
3. The method for coupling immunomagnetic particles according to claim 1, wherein the buffer system a has a pH of 5, and the buffer system b has a pH of 6.8;
preferably, the buffer system a is MES buffer solution, and the buffer system b is Good's buffer solution; the Good's buffer is selected from MES, PIPES, MOPSO, MOPS, HEPES, HEPPSO or EPPS buffer.
4. The method for coupling immunomagnetic particles according to claim 1, wherein in the step (2), the mass ratio of the antibody to the magnetic beads is 1: 50-1: 200, wherein the incubation time is 3-24 h;
preferably, the mass ratio of the antibody to the magnetic beads is 1: 50, and the incubation time is 6 h.
5. The coupling method of immunomagnetic particles according to claim 1, wherein in the step (2), the components of the blocking solution comprise 0.01-0.5M Tris with pH of 7.0-9.0, 50-300 mM NaCl, 0.01-10% BSA (w/v), 0.01-1% Triton X-100(v/v), 0.1-10% PEG (v/v), 0.1-5% amino acid or/and small molecule peptide (w/v), 0.01-0.09% sodium azide (w/v), and the blocking time is 1-24 h;
preferably, the components of the blocking solution comprise 0.1M Tris with the pH of 7.5, 150mM NaCl, 0.5% BSA (w/v), 0.5% Triton X-100(v/v), 0.1-10% PEG (v/v), 0.1-5% amino acid or/and small molecule peptide (w/v), 0.05% sodium azide (w/v), and the blocking time is 1-24 h;
preferably, the PEG comprises PEG200-10000, the PEG concentration being 2% (v/v);
preferably, the amino acid comprises at least one of alanine, glycine, lysine, or arginine;
preferably, the small molecule peptide comprises at least one of diglycine, dypeptide, carnosine or tripeptide;
more preferably, the concentration of the amino acid or/and the small molecule peptide is 5% (w/v).
6. The method of any one of claims 1 to 5, wherein the magnetic beads comprise carboxyl magnetic beads, the cross-linking agent comprises EDC cross-linking agent, and the antibody comprises CA125 antibody.
7. Immunomagnetic particles obtainable by the coupling process according to any one of claims 1 to 6.
8. Use of immunomagnetic particles obtained by the coupling method according to any one of claims 1 to 6 for the preparation of a kit or a kit detection reagent.
9. A dilution solution of immunomagnetic particles, wherein the dilution solution is used for diluting immunomagnetic particles obtained by the coupling method according to any one of claims 1 to 6.
10. The dilution of immunomagnetic particles according to claim 9, wherein the components of the dilution comprise: 0.01-0.5M Tris, 50-300 mM NaCl, 0.05-5% BSA (w/v), 0.01-10% PVP (w/v), 0.1-10% trehalose (w/v), 0.01-1% Tween20(v/v), 0.01-0.09% sodium azide and 0.01-0.1% ProClin300 with the pH value of 7.0-9.0;
preferably, the components of the diluent comprise: 0.1M Tris pH7.5, 200mM NaCl, 1% BSA (w/v), 5% PVP (w/v), 2% trehalose (w/v), 0.05% Tween20(v/v), 0.05% sodium azide, 0.05% ProClin 300.
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