CN112763703B - Immunomagnetic bead and preparation method and application thereof - Google Patents
Immunomagnetic bead and preparation method and application thereof Download PDFInfo
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- G—PHYSICS
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- 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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
Abstract
The invention relates to an immunomagnetic bead, a preparation method and application thereof, and belongs to the technical field of immunodetection. The invention provides a method for preparing immunomagnetic beads, which is characterized in that an antigen introduced with a pyridine disulfide group is covalently coupled to the sulfhydryl magnetic beads through covalent reaction of the sulfhydryl group and the pyridine disulfide group, so that the immunomagnetic beads are obtained. Furthermore, the method can react under mild activation and coupling conditions to obtain the immunomagnetic beads, thereby effectively ensuring the bioactivity of the antigen and being more beneficial to improving the efficiency and sensitivity of the immunomagnetic beads for immune reaction.
Description
Technical Field
The invention relates to an immunomagnetic bead, a preparation method and application thereof, and belongs to the technical field of immunodetection.
Background
The magnetic beads, also called magnetic particles, are spherical composite materials with diameters up to nanometer or micrometer level, and the cores are made of superparamagnetic materials such as ferroferric oxide or ferric oxide, so that the magnetic beads can move towards the magnetic field direction under the action of an externally applied magnetic field to achieve the purpose of separating target objects. The outer shell of the magnetic beads is generally a polymer material such as polystyrene or dextran which can be activated to generate amino (-NH 2), carboxyl (-COOH) or hydroxyl (-OH) functional groups, and antigen, antibody and the like can be combined on the surfaces of the magnetic beads through the coupling reaction among the groups, so that the magnetic beads become carriers of substances such as antigen, antibody and the like. The antigen/antibody on the magnetic beads can be combined with the specific antibody/antigen to form immune complex, has high specificity of immune reaction, can be specifically directed to target substances and can be separated from other substances under the action of magnetic force, so that the magnetic beads are widely applied to the fields of immunodetection, protein separation and purification, cell screening and the like.
At present, the immunomagnetic beads widely used in immunodetection mainly comprise amino magnetic beads and carboxyl magnetic beads. Wherein, the carboxyl magnetic beads are coupled with substances such as antigen, antibody and the like mainly by using ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) as a cross-linking agent. Because EDC is very unstable in aqueous solution, when the applied activation buffer is an inorganic phase, the problems of low coupling efficiency and large batch-to-batch difference are easy to occur, and when the EDC is subjected to organic reaction, the biological activity of antigens and antibodies is reduced due to the organic phase, and 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 adopting glutaraldehyde as a coupling agent, and the coupling of the amino magnetic beads and the amino groups of the protein can be realized because the two ends of the molecule of glutaraldehyde are respectively provided with an aldehyde group which can be subjected to covalent reaction with the amino groups (-NH 2). However, glutaraldehyde coupling reaction is nondirectional, so that coupling reaction of magnetic beads, antigens and antibodies is easy to occur, the magnetic beads are seriously adsorbed in a nonspecific manner, the magnetic beads are easy to aggregate in the modification process, the detection is inaccurate, the batch-to-batch difference is large, and the like, and in addition, the problem of low coupling efficiency is also caused.
Therefore, it is urgently needed to find a method for preparing immunomagnetic beads, which has high coupling efficiency, small batch-to-batch difference, strong specificity of the immunomagnetic beads prepared and difficult aggregation in the magnetic bead modification process, so as to realize accurate detection of antigen concentration.
Disclosure of Invention
In order to solve the defects of low coupling efficiency, large batch-to-batch variability and serious nonspecific 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 an antigen introduced with a pyridine disulfide group;
the preparation method of the immunomagnetic beads comprises the following steps: and (3) reacting the antigen introduced with the pyridine disulfide group with a sulfhydryl magnetic bead to obtain the immunomagnetic bead.
In one embodiment of the present invention, the preparation method of the thiol magnetic beads comprises the following steps:
a magnetic bead activation step: reacting the amino magnetic beads with an activator to obtain amino magnetic beads introduced with pyridine disulfide groups;
a reduction reaction step: and (3) reacting the amino magnetic beads introduced with the pyridine disulfide groups with a reducing agent to obtain the sulfhydryl magnetic beads.
In one embodiment of the invention, the activator used in the antigen activation step and/or the bead activation step is an organic compound containing one pyridyldisulfide group, preferably N-hydroxysuccinimide ester of 3- (2-pyridyldisulfide) propionic acid.
The activator is dissolved or diluted with a conventional solvent, for example DMSO or DMF, to a concentration of 5-50mM.
The amino magnetic beads or the sulfhydryl magnetic beads can be washed at least 1 time before or after the reaction and then resuspended, the antigen can be dissolved and diluted by adopting a buffer before the reaction, the buffer can be sodium phosphate, sodium bicarbonate, sodium citrate, disodium hydrogen phosphate or sodium dihydrogen phosphate buffer, especially 10-30mM sodium phosphate buffer, 100-200mM chloride, 0.5-2mM EDTA and the like can be added into the buffer, and the pH of the buffer 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 is used; in other embodiments, a magnetic bead separation wash containing 20mM sodium phosphate, 150mM NaCl, pH 7.5 is used. The suspension is re-suspended to the concentration of 15-30 mg/mL.
In one embodiment of the 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 amino group-containing antigen.
In one embodiment of the present invention, the amino group-containing antigen is one or more of a triiodothyronine antigen, thyroxine antigen, S-adenosylhomocysteine antigen or calcitonin antigen.
In one embodiment of the invention, the antigen is one or more of a triiodothyronine antigen or 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: the reaction is carried out for 15-60min at 20-30 ℃ and the rotating speed of 50-70 rpm.
The antigen activation buffer liquefaction comprises 10-30mM buffer salt, 100-200mM chloride salt, and the pH value 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 activating step, the concentration of the antigen in the antigen activating buffer is 0.5 to 5mg/mL, and the concentration of the activator in the reaction solution is 5 to 100mM.
In one embodiment of the present invention, in the immunomagnetic bead preparation step, the ratio of antigen to thiol magnetic bead into which a pyridine disulfide group is introduced is: 1mg of sulfhydryl magnetic beads corresponds to 10-80nmol of antigen with pyridine disulfide group introduced.
Specifically, in the magnetic bead activation step, amino magnetic beads are taken, washed with a magnetic bead activation buffer, resuspended with the magnetic bead activation buffer, and then mixed with an active agent to react.
Wherein, the reaction conditions are as follows: the reaction is carried out for 15-60min at 20-30 ℃ and the rotating speed of 50-70 rpm. The cleaning times are 1-3 times.
The magnetic bead activation buffer solution comprises 10-30mM buffer salt, 100-200mM chloride salt, 0.5-2mM EDTA, and pH of 6.0-8.0.
The buffer salt is selected from one or more of 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 activator in the reaction solution is 5 to 100mM.
Specifically, in the reduction reaction step, amino magnetic beads introduced with pyridine disulfide groups are taken and washed by a magnetic bead coupling solution, resuspended by the magnetic bead coupling solution, and mixed with an active agent to react.
Wherein, the reaction conditions are as follows: the reaction is carried out for 15-60min at 20-30 ℃ and the rotating speed of 50-70 rpm. The cleaning times are 1-3 times.
The magnetic bead coupling solution comprises 10-30mM buffer salt, 100-200mM chloride salt, 0.5-2mM EDTA, and pH of 6.0-8.0.
The buffer salt is selected from one or more of sodium phosphate, sodium bicarbonate, sodium citrate, disodium hydrogen phosphate or sodium dihydrogen phosphate; the chloride salt is NaCl.
In one embodiment of the invention, in the reduction reaction step, the concentration of the amino magnetic beads introduced with the pyridine disulfide groups in the magnetic bead coupling solution is 5-30 mg/mL, and the concentration of the activator in the reaction solution is 5-100 mM.
Specifically, the method further comprises the steps of taking sulfhydryl magnetic beads, cleaning the sulfhydryl magnetic beads by using a magnetic separation cleaning solution, and re-suspending the sulfhydryl magnetic beads by using the magnetic separation cleaning solution.
Wherein, the reaction conditions are as follows: the reaction is carried out for 18 to 24 hours at the temperature of 20 to 30 ℃ and the rotating speed of 50 to 70 rpm. The cleaning times are 1-3 times.
The magnetic separation cleaning solution comprises 10-30mM buffer salt, 100-200mM chloride salt and pH of 6.0-8.0.
The buffer salt is selected from one or more of sodium phosphate, sodium bicarbonate, sodium citrate, disodium hydrogen phosphate or sodium dihydrogen phosphate; the chloride salt is NaCl.
The suspension is re-suspended to the concentration of 15-30 mg/mL.
The invention also provides an immunomagnetic bead which is prepared by the method.
The invention also provides a detection kit which contains the immunomagnetic beads.
The invention also provides application of the immunomagnetic beads or the detection kit in detecting the concentration of target substances capable of being specifically combined with antigens in the immunomagnetic beads or antigens in the immunomagnetic beads contained in the detection kit.
In one embodiment of the invention, the antigen is an amino group-containing antigen.
In one embodiment of the present invention, the amino group-containing antigen is one or more of a triiodothyronine antigen, thyroxine antigen, S-adenosylhomocysteine antigen or calcitonin antigen.
In one embodiment of the invention, the antigen is one or more of a triiodothyronine antigen or 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, provided by the invention, 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 is adopted to react with the sulfhydryl magnetic beads, and the antigen introduced with the pyridine disulfide group is covalently coupled to the sulfhydryl magnetic beads, so that the immunomagnetic beads are obtained.
2. According to the method for preparing the immunomagnetic beads, both antigen activation and immunomagnetic bead preparation can be performed under mild reaction conditions, so that the bioactivity of the antigen is effectively ensured, and the efficiency, accuracy and sensitivity of immune reaction of the immunomagnetic beads are improved.
3. In the method for preparing the immunomagnetic beads, in the antigen activation step, an activator which is an organic matter containing pyridine disulfide bond is adopted as the activator, preferably N-hydroxysuccinimide ester (SPDP) of 3- (2-pyridine disulfide) propionic acid is adopted as the activator to introduce pyridine disulfide group on antigen, and a spacer arm of the SPDP is
Coupling of SPDP increases the magnetic beads and conjugates compared to zero length coupling agentsThe distance between the magnetic beads and the conjugate reduces the steric hindrance effect, is more beneficial to the specific recognition of the conjugate, the antigen and the antibody, can effectively avoid the nonspecific adsorption of the magnetic beads, and the aggregation of the magnetic bead modification process, thereby improving the efficiency, the accuracy and the sensitivity of the immune magnetic beads for immune reaction.
4. In the method for preparing the immunomagnetic beads, organic matters containing pyridine disulfide bonds are adopted as an activating agent, preferably N-hydroxysuccinimide ester (SPDP) of 3- (2-pyridine disulfide) propionic acid is adopted as the activating agent in the step of activating the magnetic beads, pyridine disulfide groups are introduced into the 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 beneficial to the specific recognition of the conjugate and the antigen and antibody, effectively avoid the nonspecific adsorption of the magnetic beads, and the aggregation in the magnetic bead modification process, thereby improving the immune reaction efficiency and sensitivity of the immune magnetic beads.
5. The invention provides an immunomagnetic bead which is obtained by covalent reaction between sulfhydryl groups on the magnetic bead and pyridyl disulfide groups on an antigen; the immune magnetic beads prepared by the method have the advantages of strong specificity, small batch-to-batch difference, high coupling efficiency, strong specificity, high accuracy and high sensitivity when immunoreaction occurs. In addition, the immune magnetic beads can simultaneously couple the magnetic beads with thyroxine and triiodothyronine antigens, so that the immune reaction efficiency is improved.
6. The immunomagnetic beads provided by the invention can be simultaneously applied to the determination of thyroxine (TT 4), total triiodothyronine (TT 3), free thyroxine (FT 4) and free triiodothyronine (FT 3) concentration, and meanwhile, the immunomagnetic beads solve the interference problem existing in the detection of alpha-functional hormone by using a biotin-avidin system on the premise of ensuring the detection performance to meet the requirements, and improve the detection accuracy. And compared with the common Tianshenjiawong hormone detection kit on the market, the preparation process of the immunomagnetic beads is simpler.
Drawings
Fig. 1: the activation reaction formula of the amino magnetic beads.
Fig. 2: the synthesis reaction formula of the sulfhydryl magnetic beads.
Fig. 3: activation reaction formula of triiodothyronine (T3) antigen.
Fig. 4: activation reaction formula of thyroxine (T4) antigen.
Fig. 5: synthetic reaction formula of immunomagnetic beads.
Fig. 6: the different detection kits detect the determination result of the serum sample of the total triiodothyronine (TT 3).
Fig. 7: different detection kits detect the determination results of the total thyroxine (T4) serum samples.
Fig. 8: the different detection kits detect the determination results of the serum sample of free triiodothyronine (FT 3).
Fig. 9: the different detection kits detect the determination results of the free thyroxine (FT 4) serum samples.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1: immunomagnetic beads and preparation thereof
The embodiment provides an immunomagnetic bead, and the preparation method thereof comprises the following steps:
(1) A magnetic bead activation step:
1mL of amino magnetic beads (MB-NH) with a concentration of 100mg/mL was taken 2 PurchasingFrom ThermoFisher company), washed 2 times with a bead activation buffer containing 20mM sodium phosphate, 150mM NaCl, 1mM EDTA, pH 7.5, and resuspended to a concentration of 20mg/mL with this bead activation buffer to give bead suspension A; dissolving an activator 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) with dimethyl sulfoxide (DMSO) to a concentration of 30mM, adding the mixture into the 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; the reaction system A is reacted for 30 minutes at room temperature (24 ℃) in a shaking table (60 rpm) to activate, so as to obtain amino magnetic beads introduced with pyridine disulfide groups; after the reaction system A is clarified, sucking the liquid in the reaction system A by using a liquid-transferring gun, separating the amino magnetic beads introduced with pyridine disulfide groups from the reaction system A, washing the amino magnetic beads introduced with pyridine disulfide groups obtained by separation for 2-3 times by using a magnetic bead coupling solution containing 20mM sodium phosphate, 150mM NaCl and 1mM EDTA and having the pH of 7.5, and re-suspending the amino magnetic beads introduced with pyridine disulfide groups obtained by separation by using the magnetic bead coupling solution until the concentration is 20mg/mL to obtain a magnetic bead suspension B, wherein the magnetic bead suspension B is stored at 4 ℃ for standby (the activation reaction formula of the amino magnetic beads is shown in figure 1).
(2) Reduction reaction step
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 rpm) at room temperature for 30 minutes, and removing disulfide bonds introduced by an activator SPDP to obtain mercapto-modified mercapto magnetic beads; after the reaction system B is clarified, sucking out the liquid in the reaction system B by using a liquid-transferring gun, separating the sulfhydryl magnetic beads from the reaction system B, washing the separated sulfhydryl magnetic beads for 2-3 times by using a magnetic separation washing liquid containing 20mM sodium phosphate and 150mM NaCl with the pH of 7.5, removing redundant reducing agent, and resuspending the separated sulfhydryl magnetic beads by using the magnetic separation washing liquid until the concentration is 20mg/mL to obtain a magnetic bead suspension C (the synthetic reaction formula of the sulfhydryl magnetic beads is shown in figure 2).
(3) Antigen activation step
Respectively dissolving triiodothyronine antigen (purchased from SIGMA company) and thyroxine antigen (purchased from SIGMA company) in an antigen activation buffer containing 20mM sodium bicarbonate, 150mM NaCl and pH of 9.0 until the concentrations of the triiodothyronine antigen and thyroxine antigen in the antigen activation buffer are 1mg/mL respectively to obtain a triiodothyronine antigen solution and a thyroxine antigen solution; dissolving an activator 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) with dimethyl sulfoxide (DMSO) to a concentration of 30mM, and then respectively adding the mixture into the triiodothyronine antigen solution and the thyroxine antigen solution 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) carrying out reaction on the reaction system C for 30 minutes at room temperature to activate, so as to respectively obtain the triiodothyronine antigen introduced with the pyridine disulfide group and the thyroxine antigen introduced with the pyridine disulfide group (the activation reaction formula of the triiodothyronine 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
Respectively taking the triiodothyronine antigen introduced with the pyridine disulfide group and prepared in the step (3), the thyroxine antigen introduced with the pyridine disulfide group and prepared in the step (3) and the magnetic bead suspension C prepared in the step (2), and controlling the mass ratio of the triiodothyronine antigen, the thyroxine antigen and the sulfhydryl magnetic beads to be 0.65 mug/mg and 1.55 mug/mg respectively when mixing to obtain a reaction system D; reacting the reaction system D on a shaking table (60 rpm) at room temperature for 20 hours to obtain immunomagnetic beads; after the reaction system D is clarified, sucking out the liquid in the reaction system D by using a liquid-transfering gun to separate the immunomagnetic beads from the reaction system D, firstly performing magnetic separation on the immunomagnetic beads obtained by separation (placing a coating tube containing immunomagnetic bead suspension on a magnetic separator for standing, sucking out the liquid in the coating tube after the liquid and the magnetic beads are separated, the process is called magnetic separation), then 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 by using the magnetic bead diluent until the concentration is 10mg/mL, and obtaining immunomagnetic bead suspension, wherein the immunomagnetic bead suspension is stored at 4 ℃ (the synthetic reaction formula of the immunomagnetic beads is shown in figure 5).
Example 2: total triiodothyronine (TT 3) detection kit and preparation thereof
The embodiment provides a total triiodothyronine detection kit, which comprises a general alpha-work immunomagnetic bead working solution, an alkaline phosphatase marked triiodothyronine antibody working solution, a total triiodothyronine calibrator, a test cleaning solution and a substrate solution, and the preparation method comprises the following steps:
2.1 preparation of general A-work immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
The immunomagnetic bead suspension is diluted to a concentration of 0.2mg/mL by a magnetic bead dilution containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4, and the general A-work immunomagnetic bead working solution is obtained.
2.2 preparation of alkaline phosphatase-labeled triiodothyronine antibody working solution
0.1mg of the triiodothyronine antibody was activated (the activation method can be referred to in the literature "Uto I, ishimatsu T, hirayama H, et al, determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation [ J ]. J.Immunol. Methods,1991,138 (1): 87-94)") and then 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 give an alkaline phosphatase-labeled triiodothyronine antibody conjugate.
The modified alkaline phosphatase-labeled triiodothyronine antibody conjugate was buffered with an enzyme-labeled buffer containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-phenylnaphthalenesulfonic-1-acid salt, pH 6.0 at a volume ratio of 1:3000 to obtain the enzyme-labeled antibody working solution.
2.3 preparation of Total triiodothyronine calibrator
The triiodothyronine antigen was diluted to concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0nmol/L with a calibrator dilution containing 50mM HEPES, 150mM NaCl, 1g/L BSA, pH 7.4, and the diluted triiodothyronine antigen and the calibrator dilutions were used as a total triiodothyronine assay work calibrator.
2.4 reagent test
The method for detecting the coupling efficiency of the general A-work immunomagnetic beads comprises the following steps: the reaction system D after 20 hours of reaction is subjected to magnetic separation, supernatant is reserved, and the supernatant is diluted by different times by using a calibrator diluent containing 50mM HEPES, 150mM NaCl, 1g/L BSA and pH of 7.4, and concentration test is carried out by using a Rogowski 411 instrument and a matched triiodothyronine and thyroxine detection kit.
The method for detecting the accuracy of the general A-work immunomagnetic beads comprises the following steps: absorbing 50 mu L of total triiodothyronine working calibrator or serum sample in the 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 magnetic separation was washed, 100. Mu.L of a substrate solution (APS-5) was added thereto, and after incubation at 37℃for 20S, the luminescence value was read.
2.5 detection results
Coupling efficiency was measured by EDC coupling method (EDC coupling method reference "Zhang Erli, EDC/NHS method immunomagnetic beads and effect verification, chemical engineering management, 2016 (30): pages 104-105") as a control, and the measurement results are shown in Table 1:
according to the coupling effect, the lower the concentration of the antigen to be labeled is in the supernatant, the more the antigen substances are coupled to the surface of the immunomagnetic beads, and as can be seen from Table 1, the lower the content of the antigen in the supernatant when the immunomagnetic beads are prepared by the method of this example, compared with the EDC coupling method, which means that the coupling efficiency of the immunomagnetic beads prepared by the method of this example is higher.
The results of the accuracy tests are shown in Table 2 and FIG. 6 (unit: nmol/L):
the results in Table 2 and FIG. 6 show that the linear equation fitting the detection result of the total triiodothyronine concentration in 60 serum and the concentration of the reference system is y=1.0137x+0.1604, and the correlation coefficient is R, by adopting the general alpha-immunomagnetic beads of the embodiment as the components of the total triiodothyronine detection kit 2 0.9733, accurate detection resultThe method is good in degree, and can be applied to determination of total triiodothyronine in serum.
TABLE 1 coupled supernatant antigen concentration assay results
TABLE 2 determination results of the various detection kits for detecting serum samples of total triiodothyronine (TT 3)
Note that: the alignment system was a triiodothyronine detection kit purchased from roche company.
Example 3: total thyroxine (TT 4) detection kit and preparation thereof
The embodiment provides a total thyroxine detection kit, which comprises a general alpha-immune magnetic bead working solution, an alkaline phosphatase labeled thyroxine antibody working solution, a total thyroxine calibrator, a test cleaning fluid and a substrate solution, wherein the preparation method comprises the following steps:
3.1 preparation of general A-work immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
The immunomagnetic bead suspension is diluted to a concentration of 0.2mg/mL by a magnetic bead dilution containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4, and the general A-work immunomagnetic bead working solution is obtained.
3.2 preparation of working solution for alkaline phosphatase-labeled thyroxine antibody
0.1mg of thyroxine antibody was activated (for activation, see "Uto I, ishimatsu T, hirayama H, et al determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation [ J ]. J.Immunol. Methods,1991,138 (1): 87-94)") and then 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 give an alkaline phosphatase-labeled thyroxine antibody conjugate.
The modified alkaline phosphatase-labeled thyroxine antibody conjugate was buffered with an enzyme-labeled buffer containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-phenylnaphthalenesulfonic-1-acid salt, pH 6.0 at a volume ratio of 1:2000, diluting to obtain the enzyme-labeled antibody working solution.
3.3 preparation of Total thyroxine calibrator
Thyroxine antigen was diluted to a concentration of 15, 25, 50, 75, 100, 150, 200, 250, 320nmol/L with a calibrator dilution containing 50mM HEPES, 150mM NaCl, 1g/L BSA, pH 7.4, and the diluted thyroxine antigen and calibrator dilutions were used as thyroxine assay work calibrator.
3.4 reagent test
The method for detecting the accuracy of the general A-work immunomagnetic beads comprises the following steps: as in 2.4 of example 2.
3.5 detection results
The results of the accuracy tests are shown in Table 3 and FIG. 7 (unit: nmol/L):
the results in Table 3 and FIG. 7 show that, by using the general A-work immunomagnetic beads of the present example as a thyroxine detection kit component, the linear equation fitting the thyroxine concentration detection results in 60 serum samples to the concentration of the reference system is y=1.0137x+3.4919, and the correlation coefficient is R 2 The detection result accuracy is better in the detection range of the kit (0.9602), and the kit can be applied to the determination of thyroxine in serum.
TABLE 3 determination of the total thyroxine (T4) serum samples by different detection kits
Note that: the alignment system was a thyroxine detection kit purchased from roche company.
Example 4: kit for detecting free triiodothyronine (FT 3) and preparation thereof
The embodiment provides a kit for detecting free triiodothyronine, which comprises a general alpha-work immunomagnetic bead working solution, an alkaline phosphatase marked free triiodothyronine antibody working solution, a free triiodothyronine calibrator, a test cleaning solution and a substrate solution, and the preparation method comprises the following steps:
4.1 preparation of general A-work immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
The immunomagnetic bead suspension is diluted to a concentration of 0.2mg/mL by a magnetic bead dilution containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4, and the general A-work immunomagnetic bead working solution is obtained.
4.2 preparation of alkaline phosphatase-labeled triiodothyronine antibody working solution
After 0.1mg of the triiodothyronine antibody was activated (for activation, see "Uto I, ishimatsu T, hirayama H, et al determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation [ J ]. J. Immunol. Methods,1991,138 (1): 87-94)"), it was 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 give an alkaline phosphatase-labeled triiodothyronine antibody conjugate.
The modified alkaline phosphatase-labeled triiodothyronine antibody conjugate was buffered with an enzyme-labeled buffer containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-phenylnaphthalenesulfonic-1-acid salt, pH 6.0 at a volume ratio of 1:4000 to obtain the enzyme-labeled antibody working solution.
4.3 preparation of free Triiodothyronine calibrator
The triiodothyronine antigen was diluted to concentrations of 2.0, 4.0, 6.0, 8.0, 10, 20, 30, 50, 60pmol/L with a calibrator dilution containing 50mM HEPES, 150mM NaCl, 1g/L BSA, pH 7.4, and the diluted triiodothyronine antigen and the calibrator dilution were used as free triiodothyronine assay work calibrators.
4.4 reagent test
The method for detecting the accuracy of the general A-work immunomagnetic beads comprises the following steps: as in 2.4 of example 2.
4.5 detection 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 the linear equation fitting the detection result of the concentration of free triiodothyronine in 60 serum and the concentration of the reference system is y= 1.0449x-0.187, and the correlation coefficient is R, by adopting the general alpha-immunomagnetic beads of the embodiment as the components of the free triiodothyronine detection kit 2 The detection result of the comparative manufacturer is better in consistency, namely 0.9859, and the method can be applied to the determination of free triiodothyronine in serum.
TABLE 4 determination results of the detection of serum samples of free triiodothyronine (FT 3) by different detection kits
| Sequence number | Comparison system | The kit | Sequence 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 that: the comparison system was a free triiodothyronine detection kit purchased from roche company.
Example 5: free thyroxine (FT 4) detection kit and preparation thereof
The embodiment provides a free thyroxine detection kit, which comprises a general alpha-immune magnetic bead working solution, an alkaline phosphatase labeled free thyroxine antibody working solution, a free thyroxine calibrator, a test cleaning fluid and a substrate solution, wherein the preparation method comprises the following steps:
5.1 preparation of general A-work immunomagnetic bead working solution
The immunomagnetic bead suspension was prepared as in example 1.
The immunomagnetic bead suspension is diluted to a concentration of 0.2mg/mL by a magnetic bead dilution containing 50mM Tris, 150mM NaCl, 1g/L BSA and pH 7.4, and the general A-work immunomagnetic bead working solution is obtained.
5.2 preparation of working solution for alkaline phosphatase-labeled thyroxine antibody
0.1mg of thyroxine antibody was activated (for activation, see "Uto I, ishimatsu T, hirayama H, et al determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation [ J ]. J.Immunol. Methods,1991,138 (1): 87-94)") and then 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 give an alkaline phosphatase-labeled thyroxine antibody conjugate.
The modified alkaline phosphatase-labeled thyroxine antibody conjugate was buffered with an enzyme-labeled buffer containing 50mM MES, 150mM NaCl, 1g/L BSA, 1mg/mL 8-phenylnaphthalenesulfonic-1-acid salt, pH 6.0 at a volume ratio of 1:3000 to obtain the enzyme-labeled antibody working solution.
5.3 preparation of free thyroxine calibrator
Thyroxine antigen was diluted to 5, 10, 15, 20, 30, 40, 50, 77, 85pmol/L with calibrator dilutions containing 50mM HEPES, 150mM NaCl, 1g/L BSA, pH 7.4, and the diluted thyroxine antigen and calibrator dilutions were used as free thyroxine assay working calibrator.
5.4 reagent test
The method for detecting the accuracy of the general A-work immunomagnetic beads comprises the following steps: as in 2.4 of example 2.
5.5 detection 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 the use of the general A-work immunomagnetic beads of the present example as a component of a free thyroxine detection kit resulted in the detection of free thyroxine concentration in 60 serum samples and the referenceThe linear equation fitted to the system concentration is y= 0.9823x-0.6971, and the correlation coefficient is R 2 The accuracy of the detection result is good, and the method can be applied to the determination of free triiodothyronine in serum.
TABLE 5 determination of free thyroxine (FT 4) serum samples by different detection kits
| Sequence number | Comparison system | The kit | Sequence 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 that: the alignment system was a free thyroxine detection kit purchased from roche company.
In summary, the general alpha-fetid immunomagnetic beads provided in examples 2-5 can accurately detect the concentration of thyroxine (TT 4), total triiodothyronine (TT 3), free thyroxine (FT 4) and free triiodothyronine (FT 3) in serum, and the preparation method of the general alpha-fetid immunomagnetic beads provided in examples 1-4 can simultaneously couple the magnetic beads with thyroxine and triiodothyronine antigens by using only one activator, so that the immune reaction efficiency is improved, the implementation process is simple and controllable, and the operability and repeatability of the preparation process are ensured.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (4)
1. A method of preparing an immunomagnetic bead comprising the steps of:
a magnetic bead activation step: reacting the amino magnetic beads with 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester to obtain amino magnetic beads introduced with pyridyldithio;
a reduction reaction step: amino magnetic beads introduced with pyridine disulfide groups react with dithiothreitol to obtain sulfhydryl magnetic beads;
antigen activation step: respectively reacting the triiodothyronine antigen and thyroxine antigen with 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester to obtain a triiodothyronine antigen introduced with a pyridyldithio group and a thyroxine antigen introduced with a pyridyldithio group;
the preparation method of the immunomagnetic beads comprises the following steps: reacting the triiodothyronine antigen introduced with the pyridine disulfide group, the thyroxine antigen introduced with the pyridine disulfide group with sulfhydryl magnetic beads to obtain immunomagnetic beads;
in the antigen activation step, an antigen is dissolved in an antigen activation buffer solution and then mixed with an active agent to react under the following reaction conditions: reacting for 15-60min at 20-30 ℃ and rotating speed of 50-70 rpm; the concentration of the antigen in the antigen activation buffer solution is 0.5-5 mg/mL, and the concentration of the activator in the reaction solution is 5-100 mM;
in the preparation step of the immunomagnetic beads, amino magnetic beads are taken and washed by a magnetic bead activation buffer solution, resuspended by the magnetic bead activation buffer solution, and then mixed with an active agent for reaction under the following reaction conditions: reacting for 15-60min at 20-30 ℃ and rotating speed of 50-70 rpm; the addition ratio of the antigen introduced with the pyridine disulfide group and the sulfhydryl magnetic beads is as follows: 1mg of sulfhydryl magnetic beads corresponds to 10 to 80nmol of antigen introduced with pyridine disulfide groups; the concentration of the amino magnetic beads in the magnetic bead activation buffer solution is 5-30 mg/mL, and the concentration of the activating agent in the reaction solution is 5-100 mM;
in the reduction reaction step, amino magnetic beads introduced with pyridine disulfide groups are taken and washed by magnetic bead coupling liquid, and are resuspended by the magnetic bead coupling liquid, and are mixed with an active agent for reaction under the following reaction conditions: reacting for 15-60min at 20-30 ℃ and rotating speed of 50-70 rpm; the concentration of the amino magnetic beads introduced with the pyridine disulfide groups in the magnetic bead coupling solution is 5-30 mg/mL, and the concentration of the activating agent in the reaction solution is 5-100 mM.
2. An immunomagnetic bead prepared by the method of claim 1.
3. A test kit comprising the immunomagnetic bead of claim 2.
4. Use of an immunomagnetic bead according to claim 2 or a detection kit according to claim 3 for detecting the concentration of a substance that specifically binds to an antigen in the immunomagnetic bead or an antigen in an immunomagnetic bead contained in the detection kit, characterized in that the antigen is one or more of a triiodothyronine antigen or a thyroxine antigen and the target substance that specifically binds to an antigen is one or more of thyroxine, total triiodothyronine, free thyroxine or free triiodothyronine.
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