CN112763703A - Immunomagnetic bead and preparation method and application thereof - Google Patents
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Abstract
The invention relates to an immunomagnetic bead, a preparation method and application thereof, and belongs to the technical field of immunoassay. The invention provides a method for preparing immunomagnetic beads, which is characterized in that covalent reaction is carried out between sulfydryl and pyridyl disulfide groups, and an antigen introduced with a pyridyl disulfide group is covalently coupled to the sulfydryl magnetic beads to obtain the immunomagnetic beads. Furthermore, the method can react under mild activation and coupling conditions to obtain the immunomagnetic beads, so that the bioactivity of the antigen is effectively ensured, and the efficiency and the sensitivity of the immunomagnetic beads for immunoreaction are improved.
Description
Technical Field
The invention relates to an immunomagnetic bead, a preparation method and application thereof, and belongs to the technical field of immunoassay.
Background
Magnetic beads, also called magnetic particles, are spherical composite materials with a diameter of nanometer or micrometer level, and because the core is made of superparamagnetic materials such as ferroferric oxide or ferric oxide, the magnetic beads can move towards the direction of a magnetic field under the action of an external magnetic field to achieve the purpose of separating a target object. The shell of the magnetic beads is generally a polymer material such as polystyrene or dextran that can be activated to generate functional groups such as amino group (-NH2), carboxyl group (-COOH), or hydroxyl group (-OH), and antigens, antibodies, etc. can be bound to the surface of the magnetic beads by coupling reaction between the groups to become carriers for the substances such as antigens, antibodies, etc. The antigen/antibody on the magnetic bead can be combined with a specific antibody/antigen to form an immune complex, has high specificity of immune reaction, can be specifically directed to a target substance, and can be separated from other substances under the action of magnetic force, so that the magnetic bead is widely applied to the fields of immunoassay, protein separation and purification, cell screening and the like.
At present, the immunomagnetic beads widely used in immunoassay mainly include amino magnetic beads and carboxyl magnetic beads. The carboxyl magnetic beads are mainly coupled with substances such as antigens and antibodies by using ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) as a cross-linking agent. Since EDC is extremely unstable in aqueous solution, when the activation buffer applied to EDC is an inorganic phase, the problems of low coupling efficiency and large batch-to-batch variation are easily caused, and when the organic phase reacts, the biological activity of antigens and antibodies is reduced by the organic phase, the problems of low coupling efficiency and inaccurate detection result are also caused. The amino magnetic beads are usually connected with antigens and antibodies by using glutaraldehyde as a coupling agent, and because two ends of each molecule of the glutaraldehyde have an aldehyde group capable of performing covalent reaction with amino (-NH2), the coupling of the amino magnetic beads and protein amino can be realized. However, the coupling reaction of glutaraldehyde is non-directional, and the coupling reaction of magnetic beads, antigens and antibodies is easy to occur, so that the non-specific adsorption of the magnetic beads is serious, the magnetic bead modification process is easy to aggregate, the detection is inaccurate, the difference between batches is large, and the like, and in addition, the problem of low coupling efficiency exists.
Therefore, a method for preparing immunomagnetic beads, which has high coupling efficiency, small batch difference, strong specificity of the prepared immunomagnetic beads and difficult aggregation in the magnetic bead modification process, is urgently needed to be found so as to realize accurate detection of antigen concentration.
Disclosure of Invention
In order to overcome the defects of low coupling efficiency, large batch-to-batch difference, serious non-specific adsorption and easy aggregation of the prepared immunomagnetic beads in the method for preparing the immunomagnetic beads in the prior art, the invention provides a method for preparing the immunomagnetic beads, which comprises the following steps:
antigen activation step: reacting the antigen with an activator to obtain the antigen introduced with a pyridine disulfide group;
the preparation method of the immunomagnetic beads comprises the following steps: and reacting the antigen introduced with the pyridine disulfide group with the thiol magnetic beads to obtain the immunomagnetic beads.
In an embodiment of the present invention, the method for preparing thiol magnetic beads includes the following steps:
magnetic bead activation step: reacting the amino magnetic beads with an activating agent to obtain amino magnetic beads with a pyridine disulfide group introduced;
a reduction reaction step: and (3) reacting the amino magnetic beads introduced with the pyridine disulfide groups with a reducing agent to obtain the thiol magnetic beads.
In one embodiment of the present invention, the activating agent used in the antigen activation step and/or the magnetic bead activation step is an organic substance having one pyridine disulfide bond group, preferably N-hydroxysuccinimide 3- (2-pyridyldithio) propionate.
The activator is dissolved or diluted with a conventional solvent, such as DMSO or DMF, to a concentration of 5-50 mM.
The amino magnetic beads or the thiol magnetic beads can be washed at least 1 time by using a buffer solution before or after the reaction and then resuspended, the antigen can also be dissolved by using a buffer solution before the reaction and then diluted, the buffer solution can adopt a sodium phosphate buffer solution, a sodium bicarbonate buffer solution, a sodium citrate buffer solution, a disodium hydrogen phosphate buffer solution or a sodium dihydrogen phosphate buffer solution, particularly a 10-30mM sodium phosphate buffer solution, 100-200mM chloride salt, 0.5-2mM EDTA and the like can also be added into the buffer solution, and the pH of the buffer solution can be adjusted to 6.0-8.0, preferably 7.5. For example, in certain embodiments a bead activation buffer containing 20mM sodium phosphate, 150mM NaCl, 1mM EDTA, pH 7.5; in other embodiments, the wash solution is separated using magnetic beads with pH 7.5 containing 20mM sodium phosphate, 150mM NaCl. Resuspending to a concentration of 15-30 mg/mL.
In one embodiment of the present invention, the reducing agent is one or more of dithiothreitol, tris (2-carboxyethyl) phosphine, or mercaptoethanol.
In one embodiment of the invention, the antigen is an antigen containing an amino group.
In one embodiment of the present invention, the antigen containing an amino group is one or more of a triiodothyronine antigen, a thyroxine antigen, a S-adenosylhomocysteine antigen, or a calcitonin antigen.
In one embodiment of the invention, the antigen is one or more of a triiodothyronine antigen or a thyroxine antigen.
Specifically, in the antigen activation step, an antigen is dissolved in an antigen activation buffer, and then mixed with an active agent to react.
Wherein the reaction conditions are as follows: reacting at 20-30 deg.C and 50-70rpm for 15-60 min.
The antigen activation buffer solution includes 10-30mM buffer salt and 100-200mM chloride salt, and the pH is 8.0-10.0.
The buffer salt is selected from one or more of sodium bicarbonate, sodium phosphate, sodium citrate, disodium hydrogen phosphate or sodium dihydrogen phosphate; the chloride salt is NaCl.
In one embodiment of the present invention, in the antigen activation step, the concentration of the antigen in the antigen activation buffer is 0.5 to 5mg/mL, and the concentration of the activating agent in the reaction solution is 5 to 100 mM.
In one embodiment of the present invention, in the step of preparing immunomagnetic beads, the ratio of the antigen introduced with pyridinedithio group to the thiol magnetic beads is: 1mg of thiol magnetic beads corresponds to 10-80nmol of antigen introduced with pyridine disulfide groups.
Specifically, in the magnetic bead activation step, the amino magnetic beads are taken and washed by magnetic bead activation buffer, and are resuspended by the magnetic bead activation buffer, and then are mixed with the active agent for reaction.
Wherein the reaction conditions are as follows: reacting at 20-30 deg.C and 50-70rpm for 15-60 min. The number of washing times is 1-3.
The magnetic bead activation buffer comprises 10-30mM buffer salt, 100-200mM chloride salt and 0.5-2mM EDTA, and the pH value is 6.0-8.0.
The buffer salt is one or more selected from sodium phosphate, sodium bicarbonate, sodium citrate, disodium hydrogen phosphate or sodium dihydrogen phosphate; the chloride salt is NaCl.
In one embodiment of the present invention, in the magnetic bead activation step, the concentration of the amino magnetic beads in the magnetic bead activation buffer is 5 to 30mg/mL, and the concentration of the activating agent in the reaction solution is 5 to 100 mM.
Specifically, in the reduction reaction step, the amino magnetic beads with the introduced pyridinedithio groups are washed by the magnetic bead coupling solution, resuspended by the magnetic bead coupling solution, and mixed with the active agent for reaction.
Wherein the reaction conditions are as follows: reacting at 20-30 deg.C and 50-70rpm for 15-60 min. The number of washing times is 1-3.
The magnetic bead coupling solution comprises 10-30mM buffer salt, 100-200mM chloride salt and 0.5-2mM EDTA, and the pH value is 6.0-8.0.
The buffer salt is one or more selected from sodium phosphate, sodium bicarbonate, sodium citrate, disodium hydrogen phosphate or sodium dihydrogen phosphate; the chloride salt is NaCl.
In one embodiment of the present invention, in the reduction reaction step, the concentration of the pyridinedithio group-introduced amino magnetic beads in the magnetic bead coupling solution is 5 to 30mg/mL, and the concentration of the activating agent in the reaction solution is 5 to 100 mM.
Specifically, the method further comprises the steps of taking the thiol magnetic beads, washing with a magnetic separation washing solution, and resuspending with the magnetic separation washing solution before the step of preparing the immunomagnetic beads.
Wherein the reaction conditions are as follows: reacting for 18-24 hours at the temperature of 20-30 ℃ and the rotating speed of 50-70 rpm. The number of washing times is 1-3.
The magnetic separation cleaning solution comprises 10-30mM buffer salt and 100-200mM chloride salt, and has a pH of 6.0-8.0.
The buffer salt is one or more selected from sodium phosphate, sodium bicarbonate, sodium citrate, disodium hydrogen phosphate or sodium dihydrogen phosphate; the chloride salt is NaCl.
Resuspending to a concentration of 15-30 mg/mL.
The invention also provides an immunomagnetic bead which is prepared by using the method.
The invention also provides a detection kit, which contains the immunomagnetic beads.
The invention also provides the application of the immunomagnetic beads or the detection kit in detecting the concentration of the target substance which can be specifically combined with the antigen in the immunomagnetic beads or the antigen in the immunomagnetic beads contained in the detection kit.
In one embodiment of the invention, the antigen is an antigen containing an amino group.
In one embodiment of the present invention, the antigen containing an amino group is one or more of a triiodothyronine antigen, a thyroxine antigen, a S-adenosylhomocysteine antigen, or a calcitonin antigen.
In one embodiment of the invention, the antigen is one or more of a triiodothyronine antigen or a thyroxine antigen.
In one embodiment of the present invention, the target substance capable of specifically binding to an antigen is one or more of thyroxine, total triiodothyronine, free thyroxine, or free triiodothyronine.
The technical scheme of the invention has the following advantages:
1. according to the method for preparing the immunomagnetic beads, the antigen introduced with the pyridine disulfide group is obtained through the reaction of the antigen and the activating agent, then the antigen introduced with the pyridine disulfide group reacts with the mercapto magnetic beads, and the antigen introduced with the pyridine disulfide group is covalently coupled to the mercapto magnetic beads to obtain the immunomagnetic beads.
2. According to the method for preparing the immunomagnetic beads, the antigen activation and the immunomagnetic beads can be carried out under mild reaction conditions, so that the bioactivity of the antigen is effectively ensured, and the efficiency, accuracy and sensitivity of the immunomagnetic beads in immunoreaction are improved.
3. In the method for preparing immunomagnetic beads provided by the invention, in the antigen activation step, the adopted activator is an organic matter containing a pyridine disulfide bond as an activator, preferably 3- (2-pyridine disulfide group) N-hydroxysuccinimide ester propionate (SPDP) is used as an activator to introduce a pyridine disulfide group into an antigen, and the spacer arm of the SPDP is
Compared with a zero-length coupling agent, the coupling of the SPDP can increase the distance between the magnetic beads and the conjugate, reduce the steric hindrance effect, be more favorable for the specific recognition of the conjugate, an antigen and an antibody, effectively avoid the nonspecific adsorption of the magnetic beads, and improve the aggregation of the magnetic bead modification process, thereby improving the efficiency, the accuracy and the sensitivity of the immune reaction of the immunomagnetic beads.
4. In the method for preparing the immunomagnetic beads, an organic matter containing a pyridine disulfide bond is used as an activator in a bead activation step, preferably 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) is used as the activator to introduce a pyridyldithio group into amino magnetic beads, and a spacer arm of the SPDP is
Compared with a zero-length coupling agent, the coupling of the SPDP can increase the distance between the magnetic beads and the conjugate, reduce the steric hindrance effect, be more favorable for the specific recognition of the conjugate, an antigen and an antibody, effectively avoid the nonspecific adsorption of the magnetic beads, and improve the aggregation of the magnetic bead modification process, thereby improving the efficiency and the sensitivity of the immune reaction of the immunomagnetic beads.
5. The invention provides an immunomagnetic bead, which is obtained by covalent reaction between a sulfydryl on the magnetic bead and a pyridyl disulfide group on an antigen; the immunomagnetic beads prepared by the method have the advantages of strong specificity, small batch difference, high coupling efficiency, strong specificity, high accuracy and high sensitivity when immunoreaction occurs. In addition, the immunomagnetic beads can simultaneously couple the magnetic beads with two antigens, namely thyroxine and triiodothyronine, so that the efficiency of immunoreaction is improved.
6. The immunomagnetic bead provided by the invention can be simultaneously applied to the determination of the concentrations of thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4) and free triiodothyronine (FT3), and meanwhile, on the premise of ensuring that the detection performance meets the requirement, the immunomagnetic bead solves the problem of interference existing in the detection of the alpha hormone by using a biotin-avidin system, and improves the detection accuracy. And compared with the common Tianshen Jiagong hormone detection kit on the market, the preparation process of the immunomagnetic beads is simpler.
Drawings
FIG. 1: and (3) an activation reaction formula of the amino magnetic beads.
FIG. 2: the synthetic reaction formula of the thiol magnetic beads.
FIG. 3: activation of triiodothyronine (T3) antigen.
FIG. 4: thyroxine (T4) antigen activation reaction.
FIG. 5: synthetic reaction formula of immunomagnetic beads.
FIG. 6: different detection kits detect the determination result of the serum sample of the total triiodothyronine (TT 3).
FIG. 7: different test kits tested the results of the total thyroxine (T4) serum sample.
FIG. 8: different detection kits detect the determination result of the serum sample of the free triiodothyronine (FT 3).
FIG. 9: different test kits tested the results of the determination of the serum sample of free thyroxine (FT 4).
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1: immunomagnetic beads and preparation thereof
The embodiment provides an immunomagnetic bead, and a preparation method thereof comprises the following steps:
(1) magnetic bead activation step:
1mL of amino magnetic beads (MB-NH) with a concentration of 100mg/mL was taken2Purchased from thermo fisher corporation), washed 2 times with a magnetic bead activation buffer containing 20mM sodium phosphate, 150mM NaCl, 1mM EDTA and having a pH of 7.5, and resuspended with this magnetic bead activation buffer to a concentration of 20mg/mL, resulting in a magnetic bead suspension a; dissolving an activator N-hydroxysuccinimide ester (SPDP) of 3- (2-pyridine dithio) propionic acid into dimethyl sulfoxide (DMSO) until the concentration is 30mM, adding the solution into a magnetic bead suspension until the final concentration of the SPDP in the magnetic bead suspension is 3mM, and fully and uniformly mixing to obtain a reaction system A; reacting the reaction system A in a shaking table (60 revolutions per minute) at room temperature (24 ℃) for 30 minutes for activation to obtain amino magnetic beads with introduced pyridine disulfide groups; adsorbing the reaction system A by using a magnet, sucking liquid in the reaction system A by using a liquid transfer gun after the reaction system A is clarified to separate amino magnetic beads with pyridine disulfide groups introduced from the reaction system A, washing the separated amino magnetic beads with the pyridine disulfide groups introduced by using a magnetic bead coupling solution containing 20mM sodium phosphate, 150mM NaCl and 1mM EDTA and having the pH of 7.5 for 2-3 times, and resuspending the separated amino magnetic beads with the pyridine disulfide groups introduced by using the magnetic bead coupling solution to obtain a magnetic bead suspension B with the concentration of 20mg/mL, wherein the magnetic bead suspension B is stored at 4 ℃ for later use (the ammonia suspension B is stored for later use) (the ammonia suspension B is stored at 4℃)The activation reaction of the base magnetic beads is shown in FIG. 1).
(2) Step of reduction reaction
Adding a reducing agent Dithiothreitol (DTT) into the magnetic bead suspension B until the final concentration of the DTT in the magnetic bead suspension B is 20mM, and fully and uniformly mixing to obtain a reaction system B; reacting the reaction system B in a shaking table (60 revolutions per minute) at room temperature for 30 minutes, and removing a disulfide bond introduced by an activating agent SPDP to obtain thiol-modified thiol magnetic beads; adsorbing the reaction system B by using a magnet, after the reaction system B is clarified, sucking out liquid in the reaction system B by using a pipette gun, separating the thiol magnetic beads from the reaction system B, cleaning the separated thiol magnetic beads for 2-3 times by using a magnetic separation cleaning solution containing 20mM sodium phosphate, 150mM NaCl and having a pH value of 7.5, removing redundant reducing agents, and re-suspending the separated thiol magnetic beads to a concentration of 20mg/mL by using the magnetic separation cleaning solution to obtain a magnetic bead suspension C (a synthetic reaction formula of the thiol magnetic beads is shown in figure 2).
(3) Antigen activation step
Dissolving triiodothyronine antigen (purchased from SIGMA company) and thyroxine antigen (purchased from SIGMA company) in an antigen activation buffer solution containing 20mM sodium bicarbonate and 150mM NaCl and having a pH value of 9.0, respectively, until the concentrations of the triiodothyronine antigen and the thyroxine antigen in the antigen activation buffer solution are 1mg/mL, respectively, to obtain a triiodothyronine antigen solution and a thyroxine antigen solution; dissolving an activator N-hydroxysuccinimide ester (SPDP) 3- (2-pyridyldithio) propionate into dimethyl sulfoxide (DMSO) until the concentration is 30mM, adding the solution into triiodothyronine antigen solution and thyroxine antigen solution respectively until the final concentrations of the SPDP in the triiodothyronine antigen solution and the thyroxine antigen solution are 20mM respectively, and fully and uniformly mixing to obtain a reaction system C; and (3) reacting the reaction system C at room temperature for 30 minutes to activate, so as to respectively obtain triiodothyronine antigen introduced with the pyridine disulfide group and thyroxine antigen introduced with the pyridine disulfide group (the activation reaction formula of the triiodothyronine and the antigen is shown in figure 3, and the activation reaction formula of the thyroxine antigen is shown in figure 4).
(4) Preparation of immunomagnetic beads
Mixing the triiodothyronine antigen introduced with the pyridinedithio group prepared in the step (3), the thyroxine antigen introduced with the pyridinedithio group prepared in the step (3) and the magnetic bead suspension C prepared in the step (2) respectively, and controlling the mass ratios of the triiodothyronine antigen, the thyroxine antigen and the mercapto magnetic bead to be 0.65 mu g/mg and 1.55 mu g/mg respectively when mixing to obtain a reaction system D; reacting the reaction system D for 20 hours at room temperature in a shaking table (60 revolutions per minute) to obtain immunomagnetic beads; adsorbing the reaction system D by using a magnet, after the reaction system D is clarified, sucking out liquid in the reaction system D by using a pipette gun to separate the immunomagnetic beads from the reaction system D, performing magnetic separation on the immunomagnetic beads obtained by separation (placing a coating tube filled with immunomagnetic bead suspension on a magnetic separator for standing, after the liquid is separated from the immunomagnetic beads, sucking out the liquid in the coating tube, wherein the process is called magnetic separation), washing the immunomagnetic beads obtained by separation for 2-3 times by using a magnetic bead diluent containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH of 7.4, finally diluting the immunomagnetic beads obtained by separation to a concentration of 10mg/mL by using the magnetic bead diluent to obtain an immunomagnetic bead suspension, and storing the immunomagnetic beads at 4 ℃ (the immunomagnetic bead synthesis reaction formula is shown in figure 5).
Example 2: total triiodothyronine (TT3) detection kit and preparation method thereof
The embodiment provides a total triiodothyronine detection kit, which comprises a general A-functional immunomagnetic bead working solution, an alkaline phosphatase labeled triiodothyronine antibody working solution, a total triiodothyronine calibrator, a test cleaning solution and a substrate solution, wherein the preparation method comprises the following steps:
2.1 preparation of Universal A-Gong immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
Diluting the immunomagnetic bead suspension with a magnetic bead diluent containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4 to a concentration of 0.2mg/mL to obtain the universal A-functional immunomagnetic bead working solution.
2.2 preparation of alkaline phosphatase-labeled triiodothyronine antibody working solution
After activating 0.1mg of triiodothyronine antibody (see "Uto I, Ishimatsu T, Hirayama H, et al. determination of origin Tamm-Horsfall protein by ELISA using a maleimid method for enzyme-antibody coupling [ J ]. J. immunological methods,1991,138(1): 87-94."), the antibody is mixed with 0.2mg of alkaline phosphatase, reacted in PBS buffer (pH 7.2) at room temperature for 1 hour, and after completion of the reaction, purified by an affinity column to obtain an alkaline phosphatase-labeled triiodothyronine antibody conjugate.
The modified alkaline phosphatase-labeled triiodothyronine antibody conjugate was treated with an enzyme-labeled buffer solution containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-anilinonaphthalenesulfon-1-carboxylate, pH 6.0 in a volume ratio of 1: 3000, diluting to obtain an enzyme-labeled antibody working solution.
2.3 preparation of Total Triiodothyronine calibrator
Triiodothyronine antigen was diluted to concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, and 12.0nmol/L with a calibrator diluent containing 50mM HEPES, 150mM NaCl, 1g/L BSA, and PH 7.4, and the diluted triiodothyronine antigen and this calibrator diluent were used as total triiodothyronine assay working calibrators.
2.4 reagent test
The detection method of the coupling efficiency of the general A-function immunomagnetic beads comprises the following steps: and (3) carrying out magnetic separation on the reaction system D after 20 hours of reaction, reserving the supernatant, diluting the supernatant by different times by using a calibrator diluent containing 50mM HEPES, 150mM NaCl, 1g/L BSA and having a pH of 7.4, and carrying out concentration test by adopting a Roche e411 instrument and a matched triiodothyronine and thyroxine detection kit.
The detection method of the accuracy of the general A function immunomagnetic beads comprises the following steps: sucking 50 mu L of total triiodothyronine working calibrator or serum sample into a reaction hole 1, adding 50 mu L of enzyme-labeled antibody working solution, reacting for 5 minutes at 37 ℃, then adding 50 mu L of magnetic bead working solution, and reacting for 10 minutes at 37 ℃; the cells were washed by magnetic separation, 100. mu.L of substrate solution (APS-5) was added, and the cells were incubated at 37 ℃ for 20 seconds to read the luminescence.
2.5 test results
The detection of coupling efficiency is carried out by using EDC coupling method (EDC coupling method reference "Zhang Erli, EDC/NHS method for preparing immunomagnetic beads and verifying its effect. chemical management, 2016 (30): page 104-:
according to the coupling effect, the lower the concentration of the antigen to be labeled in the supernatant is, the more the antigen substances coupled to the surface of the immunomagnetic beads are, and as can be seen from table 1, compared with the EDC coupling method, when the immunomagnetic beads are prepared by using the method of the present embodiment, the amount of the antigen in the supernatant is lower, which indicates that the coupling efficiency of the immunomagnetic beads prepared by using the method of the present embodiment is higher.
The results of the accuracy measurements are shown in Table 2 and FIG. 6 (unit: nmol/L):
the results in table 2 and fig. 6 show that, when the universal afoa beads of this example are used as components of a total triiodothyronine assay kit, the linear equation fitting the assay results of the total triiodothyronine concentration in 60 cases of serum with the reference system concentration is y ═ 1.0137x +0.1604, and the correlation coefficient is R20.9733, the detection result has good accuracy, and the method can be applied to the determination of the total triiodothyronine in serum.
TABLE 1 detection of antigen concentration in the coupled supernatants
TABLE 2 determination results of serum samples of total triiodothyronine (TT3) with different detection kits
Note: the comparison system is a triiodothyronine detection kit purchased from Roche.
Example 3: total thyroxine (TT4) detection kit and preparation thereof
The embodiment provides a total thyroxine detection kit, which comprises general A functional immunomagnetic bead working solution, alkaline phosphatase labeled thyroxine antibody working solution, a total thyroxine calibrator, a test cleaning solution and a substrate solution, wherein the preparation method comprises the following steps:
3.1 preparation of Universal A-Gong Immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
Diluting the immunomagnetic bead suspension with a magnetic bead diluent containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4 to a concentration of 0.2mg/mL to obtain the universal A-functional immunomagnetic bead working solution.
3.2 preparation of alkaline phosphatase-labeled thyroxine antibody working solution
After activating 0.1mg of thyroxine antibody (see "Uto I, Ishimatsu T, Hirayama H, et al. determination of origin Tamm-Horsfall protein by ELISA using a matrix method for enzyme-antibody coupling [ J ]. J. immunological. methods,1991,138(1): 87-94."), the thyroxine antibody conjugate is mixed with 0.2mg of alkaline phosphatase, reacted in PBS buffer (pH 7.2) at room temperature for 1 hour, and purified by an affinity column after the reaction is completed, thereby obtaining an alkaline phosphatase-labeled thyroxine antibody conjugate.
The modified alkaline phosphatase-labeled thyroxine antibody conjugate was treated with an enzyme-labeled buffer containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-anilinonaphthalenesulfon-1-carboxylate, pH 6.0, in a volume ratio of 1: 2000, diluting to obtain the enzyme-labeled antibody working solution.
3.3 preparation of Total thyroxine calibrator
Thyroxine antigens were diluted to concentrations of 15, 25, 50, 75, 100, 150, 200, 250, and 320nmol/L with a calibrator diluent containing 50mM HEPES, 150mM NaCl, 1g/L BSA, and pH 7.4, and the diluted thyroxine antigens and this calibrator diluent were used as a thyroxine detection working calibrator.
3.4 reagent test
The detection method of the accuracy of the general A function immunomagnetic beads comprises the following steps: the same as 2.4 in embodiment 2.
3.5 test results
The results of the accuracy measurements are shown in Table 3 and FIG. 7 (unit: nmol/L):
the results in table 3 and fig. 7 show that, when the universal afar immunomagnetic beads of the present example are used as components of a thyroxine detection kit, the linear equation of the detection result of the concentration of thyroxine in 60 cases and the concentration of the reference system is y ═ 1.0137x +3.4919, and the correlation coefficient is R20.9602 shows that the detection result has high accuracy in the detection range, and can be applied to the determination of thyroxine in serum.
TABLE 3 determination of serum samples for total thyroxine (T4) with different detection kits
Note: the comparison system was a thyroxine detection kit purchased from roche.
Example 4: free triiodothyronine (FT3) detection kit and preparation thereof
The embodiment provides a free triiodothyronine detection kit, which comprises universal A-immunomagnetic bead working solution, alkaline phosphatase labeled free triiodothyronine antibody working solution, a free triiodothyronine calibrator, a test cleaning solution and a substrate solution, wherein the preparation method comprises the following steps:
4.1 preparation of Universal A-Gong immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
Diluting the immunomagnetic bead suspension with a magnetic bead diluent containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4 to a concentration of 0.2mg/mL to obtain the universal A-functional immunomagnetic bead working solution.
4.2 preparation of alkaline phosphatase-labeled triiodothyronine antibody working solution
After activating 0.1mg triiodothyronine antibody (the activation method can be referred to in the literature "Uto I, Ishimatsu T, Hirayama H, et al.determination of origin Tamm-Horsfall protein by ELISA using a maleimid method for enzyme-antibody coupling [ J ]. J.immunological methods,1991,138(1): 87-94."), the mixture is mixed with 0.2mg alkaline phosphatase, and the mixture is reacted in PBS buffer (pH 7.2) at room temperature for 1 hour, and after the reaction is completed, the mixture is purified by an affinity column to obtain an alkaline phosphatase-labeled triiodothyronine antibody conjugate.
The modified alkaline phosphatase-labeled triiodothyronine antibody conjugate was treated with an enzyme-labeled buffer solution containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-anilinonaphthalenesulfon-1-carboxylate, pH 6.0 in a volume ratio of 1: 4000 to obtain the enzyme-labeled antibody working solution.
4.3 preparation of free Triiodothyronine calibrator
Triiodothyronine antigen was diluted to concentrations of 2.0, 4.0, 6.0, 8.0, 10, 20, 30, 50, and 60pmol/L with a calibrator diluent containing 50mM HEPES, 150mM NaCl, 1g/L BSA, and pH 7.4, and the diluted triiodothyronine antigen and this calibrator diluent were used as free triiodothyronine assay working calibrators.
4.4 reagent test
The detection method of the accuracy of the general A function immunomagnetic beads comprises the following steps: the same as 2.4 in embodiment 2.
4.5 test results
The results of the accuracy measurements are shown in Table 4 and FIG. 8 (unit: pmol/L):
the results in Table 4 and FIG. 8 show that, using the universal functional A immunomagnetic beads of this example as the components of the kit for detecting free triiodothyronine, the linear equation of the concentration of free triiodothyronine in 60 cases of sera and the concentration of the reference system is y-1.0449 x-0.187, and the correlation coefficient is R20.9859, the consistency with the test result of the comparison factory is better, and the kit can be applied to the determination of the free triiodothyronine in the serum.
TABLE 4 determination results of serum samples for free triiodothyronine (FT3) with different detection kits
| Serial number | Comparison system | The kit | Serial number | Comparison system | The kit |
| 1 | 0.56 | 0.53 | 21 | 4.44 | 4.75 |
| 2 | 0.83 | 0.96 | 22 | 4.68 | 4.15 |
| 3 | 1.05 | 1.13 | 23 | 4.82 | 4.43 |
| 4 | 1.27 | 1.38 | 24 | 5.24 | 5.31 |
| 5 | 1.52 | 1.63 | 25 | 5.48 | 4.55 |
| 6 | 1.97 | 2.19 | 26 | 5.92 | 5.52 |
| 7 | 2.04 | 2.19 | 27 | 6.01 | 5.41 |
| 8 | 2.57 | 2.49 | 28 | 6.18 | 6.43 |
| 9 | 2.78 | 2.58 | 29 | 6.33 | 6.86 |
| 10 | 2.93 | 2.37 | 30 | 6.49 | 6.08 |
| 11 | 3.12 | 3.47 | 31 | 6.77 | 6.93 |
| 12 | 3.26 | 2.69 | 32 | 7.08 | 7.71 |
| 13 | 3.48 | 3.73 | 33 | 9.03 | 8.63 |
| 14 | 3.52 | 3.51 | 34 | 10.01 | 12.45 |
| 15 | 3.74 | 3.59 | 35 | 12.46 | 14.78 |
| 16 | 3.85 | 3.57 | 36 | 17.29 | 14.13 |
| 17 | 3.99 | 3.52 | 37 | 21.91 | 23.98 |
| 18 | 4.13 | 4.09 | 38 | 26.08 | 28.87 |
| 19 | 4.18 | 3.88 | 39 | 34.08 | 32.13 |
| 20 | 4.37 | 5.25 | 40 | 40.84 | 44.27 |
Note: the comparison system is a free triiodothyronine detection kit purchased from Roche.
Example 5: free thyroxine (FT4) detection kit and preparation thereof
The embodiment provides a free thyroxine detection kit, which comprises general A functional immunomagnetic bead working solution, alkaline phosphatase labeled free thyroxine antibody working solution, a free thyroxine calibrator, a test cleaning solution and a substrate solution, wherein the preparation method comprises the following steps:
5.1 preparation of Universal A-functional immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
Diluting the immunomagnetic bead suspension with a magnetic bead diluent containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4 to a concentration of 0.2mg/mL to obtain the universal A-functional immunomagnetic bead working solution.
5.2 preparation of alkaline phosphatase-labeled thyroxine antibody working solution
After activating 0.1mg of thyroxine antibody (see "Uto I, Ishimatsu T, Hirayama H, et al. determination of origin Tamm-Horsfall protein by ELISA using a matrix method for enzyme-antibody coupling [ J ]. J. immunological. methods,1991,138(1): 87-94."), the thyroxine antibody conjugate is mixed with 0.2mg of alkaline phosphatase, reacted in PBS buffer (pH 7.2) at room temperature for 1 hour, and purified by an affinity column after the reaction is completed, thereby obtaining an alkaline phosphatase-labeled thyroxine antibody conjugate.
The modified alkaline phosphatase-labeled thyroxine antibody conjugate was treated with an enzyme-labeled buffer containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-anilinonaphthalenesulfon-1-carboxylate, pH 6.0, in a volume ratio of 1: 3000, diluting to obtain an enzyme-labeled antibody working solution.
5.3 preparation of free thyroxine calibrator
Thyroxine antigen was diluted to concentrations of 5, 10, 15, 20, 30, 40, 50, 77, 85pmol/L with a calibrator diluent containing 50mM HEPES, 150mM NaCl, 1g/L BSA, pH 7.4, and the diluted thyroxine antigen and this calibrator diluent were used as free thyroxine detection working calibrators.
5.4 reagent test
The detection method of the accuracy of the general A function immunomagnetic beads comprises the following steps: the same as 2.4 in embodiment 2.
5.5 test results
The results of the accuracy measurements are shown in Table 5 and FIG. 9 (unit: pmol/L):
the results in table 5 and fig. 9 show that, when the universal afar immunomagnetic beads of the present example are used as components of a kit for detecting free thyroxine, the linear equation of the detection result of the concentration of free thyroxine in 60 cases of serum and the concentration of the reference system is y ═ 0.9823 x-0.6971, and the correlation coefficient is R2The detection result has better accuracy when the content is 0.982, and the method can be applied to the determination of the free triiodothyronine in the serum.
TABLE 5 determination of serum samples for free thyroxine (FT4) with different test kits
| Serial number | Comparison system | The kit | Serial number | Comparison system | The kit |
| 1 | 4.05 | 3.12 | 21 | 19.31 | 20.37 |
| 2 | 5.21 | 4.87 | 22 | 19.89 | 17.48 |
| 3 | 9.64 | 8.89 | 23 | 20 | 17.19 |
| 4 | 9.97 | 8.12 | 24 | 20.45 | 20.21 |
| 5 | 10.05 | 9.78 | 25 | 21.69 | 20.78 |
| 6 | 11.27 | 13.47 | 26 | 22.53 | 23.86 |
| 7 | 11.42 | 8.96 | 27 | 22.57 | 25.05 |
| 8 | 12.87 | 14.26 | 28 | 24.88 | 24.96 |
| 9 | 13.39 | 11.72 | 29 | 25.08 | 22.26 |
| 10 | 14.9 | 12.47 | 30 | 27.71 | 22.36 |
| 11 | 14.98 | 14.72 | 31 | 28.34 | 27.96 |
| 12 | 15.07 | 13.96 | 32 | 29.09 | 25.94 |
| 13 | 15.19 | 17.77 | 33 | 31.78 | 33.22 |
| 14 | 15.24 | 12.53 | 34 | 33.51 | 29.93 |
| 15 | 15.97 | 12.86 | 35 | 39.55 | 39.89 |
| 16 | 16.63 | 19.45 | 36 | 41.15 | 44.85 |
| 17 | 17.11 | 16.98 | 37 | 47.84 | 54.49 |
| 18 | 17.95 | 17.04 | 38 | 58.96 | 52.46 |
| 19 | 18.13 | 14.48 | 39 | 63.69 | 60.19 |
| 20 | 18.76 | 22.12 | 40 | 76.78 | 72.54 |
Note: the comparison system was a free thyroxine detection kit purchased from roche.
In summary, the universal afte immunomagnetic beads provided in embodiments 2 to 5 can accurately detect the concentrations of thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), and free triiodothyronine (FT3) in serum, and the preparation method of the universal afte immunomagnetic beads provided in embodiments 1 to 4 can simultaneously couple the beads with two antigens, i.e., thyroxine and triiodothyronine, using only one activator, thereby improving the efficiency of immunoreaction, and the implementation process is simple and controllable, and ensures the operability and repeatability of the preparation process.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for preparing immunomagnetic beads is characterized by comprising the following steps:
antigen activation step: reacting the antigen with an activator to obtain the antigen introduced with a pyridine disulfide group;
the preparation method of the immunomagnetic beads comprises the following steps: and reacting the antigen introduced with the pyridine disulfide group with the thiol magnetic beads to obtain the immunomagnetic beads.
2. The method of claim 1, wherein the thiol magnetic bead is prepared by a method comprising the steps of:
magnetic bead activation step: reacting the amino magnetic beads with an activating agent to obtain amino magnetic beads with a pyridine disulfide group introduced;
a reduction reaction step: and (3) reacting the amino magnetic beads introduced with the pyridine disulfide groups with a reducing agent to obtain the thiol magnetic beads.
3. The method according to claim 1 or 2, wherein the activator used in the antigen activation step and/or the magnetic bead activation step is an organic substance containing a pyridine disulfide group, preferably N-hydroxysuccinimide 3- (2-pyridyldithio) propionate.
4. The method of claim 2, wherein the reducing agent is one or more of dithiothreitol, tris (2-carboxyethyl) phosphine, or mercaptoethanol.
5. The method of any one of claims 1 to 4, wherein the antigen is an amino-containing antigen; preferably, the antigen containing amino group is one or more of triiodothyronine antigen, thyroxine antigen, S-adenosylhomocysteine antigen or calcitonin antigen.
6. The method according to any one of claims 1 to 5, wherein in the antigen activation step, the antigen is dissolved in an antigen activation buffer and then mixed with an active agent to react;
preferably, the concentration of the antigen in the antigen activation buffer solution is 0.5-5 mg/mL; the concentration of the activating agent in the reaction liquid is 5-100 mM.
7. The method of any one of claims 1 to 6, wherein in the step of preparing immunomagnetic beads, the antigen with the introduced pyridinedithio group and the thiol magnetic beads are added in a ratio of: 1mg of thiol magnetic beads corresponds to 10-80nmol of antigen introduced with pyridine disulfide groups.
8. An immunomagnetic bead prepared by the method of any one of claims 1 to 7.
9. A test kit comprising the immunomagnetic bead according to claim 8.
10. Use of the immunomagnetic beads according to claim 8 or the detection kit according to claim 9 for detecting the concentration of a substance that specifically binds to an antigen in the immunomagnetic beads or to an antigen in the immunomagnetic beads contained in the detection kit.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113533745A (en) * | 2021-07-21 | 2021-10-22 | 苏州立禾生物医学工程有限公司 | Homocysteine detection kit and detection method thereof |
| CN117110601A (en) * | 2023-08-11 | 2023-11-24 | 江苏奥雅生物科技有限公司 | Immune magnetic bead preservation solution, preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6150181A (en) * | 1995-06-29 | 2000-11-21 | Universite Pierre Et Marie Curie | Magnetic nanoparticles coupled to annexine, and utilization thereof |
| CN103168238A (en) * | 2010-07-27 | 2013-06-19 | 微球实验公司 | Covalent immobilization of molecules comprising an amino group |
| CN106226513A (en) * | 2016-08-02 | 2016-12-14 | 北京乐普医疗科技有限责任公司 | A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof |
| CN106468632A (en) * | 2015-08-16 | 2017-03-01 | 复旦大学 | A kind of magnetic Nano material and its production and use |
| CN108333361A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | Bridging has magnetic microsphere compound formulation of antibody and the preparation method and application thereof |
| CN109444437A (en) * | 2018-12-19 | 2019-03-08 | 广东菲鹏生物有限公司 | Miao's Le Shi pipe hormone determination kit based on restoring method label attached luminescent indicator |
| CN111308066A (en) * | 2020-03-10 | 2020-06-19 | 北京国科融智生物技术有限公司 | Novel method for enriching and detecting biomedical and environmental samples by magnetic particles |
| US20200209226A1 (en) * | 2017-01-20 | 2020-07-02 | Shenzhen New Industries Biomedical Engineering Co. | Labeled complex, preparation method thereof, kit containing the same, application of kit and detection system comprising kit |
-
2020
- 2020-12-31 CN CN202011635675.5A patent/CN112763703B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6150181A (en) * | 1995-06-29 | 2000-11-21 | Universite Pierre Et Marie Curie | Magnetic nanoparticles coupled to annexine, and utilization thereof |
| CN103168238A (en) * | 2010-07-27 | 2013-06-19 | 微球实验公司 | Covalent immobilization of molecules comprising an amino group |
| CN106468632A (en) * | 2015-08-16 | 2017-03-01 | 复旦大学 | A kind of magnetic Nano material and its production and use |
| CN106226513A (en) * | 2016-08-02 | 2016-12-14 | 北京乐普医疗科技有限责任公司 | A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof |
| CN108333361A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | Bridging has magnetic microsphere compound formulation of antibody and the preparation method and application thereof |
| US20200209226A1 (en) * | 2017-01-20 | 2020-07-02 | Shenzhen New Industries Biomedical Engineering Co. | Labeled complex, preparation method thereof, kit containing the same, application of kit and detection system comprising kit |
| CN109444437A (en) * | 2018-12-19 | 2019-03-08 | 广东菲鹏生物有限公司 | Miao's Le Shi pipe hormone determination kit based on restoring method label attached luminescent indicator |
| CN111308066A (en) * | 2020-03-10 | 2020-06-19 | 北京国科融智生物技术有限公司 | Novel method for enriching and detecting biomedical and environmental samples by magnetic particles |
Non-Patent Citations (1)
| Title |
|---|
| 曾戎: "《多糖基高分子 药物轭合物的设计、合成、表征和评价》", 31 May 2011 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113533745A (en) * | 2021-07-21 | 2021-10-22 | 苏州立禾生物医学工程有限公司 | Homocysteine detection kit and detection method thereof |
| CN117110601A (en) * | 2023-08-11 | 2023-11-24 | 江苏奥雅生物科技有限公司 | Immune magnetic bead preservation solution, preparation method and application thereof |
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