阅读说明：本技术 磁珠、试剂盒、制备方法及用途 (Magnetic bead, kit, preparation method and application ) 是由 林定彪 李玲 于 2021-08-19 设计创作，主要内容包括：为解决现有的用于测定抗甲状腺球蛋白抗体的试剂盒,在超声混匀的仪器上使用时,精密度较差导致临床样本测定异常的技术问题,本发明实施例提供一种磁珠、试剂盒、制备方法及用途,包括：包被有活化官能团的磁珠；以及TG抗原,通过与所述活化官能团反应包被在磁珠上。本发实施例通过TG抗原包被在包被有活化官能团的磁珠上,使在超声混匀的仪器上测定抗甲状腺球蛋白抗体时,精密度和准确度较现有的测定抗甲状腺球蛋白抗体的试剂更优。(In order to solve the technical problem that when the existing kit for measuring the antithyroid globulin antibody is used on an ultrasonic mixing instrument, the precision is poor, so that the clinical sample measurement is abnormal, the embodiment of the invention provides a magnetic bead, a kit, a preparation method and an application, wherein the preparation method comprises the following steps: magnetic beads coated with an activating functional group; and a TG antigen coated on the magnetic bead by reacting with the activated functional group. According to the embodiment, the TG antigen is coated on the magnetic beads coated with the activated functional groups, so that when the antithyroid globulin antibody is measured on an ultrasonic uniform mixing instrument, the precision and the accuracy are better than those of the existing reagent for measuring the antithyroid globulin antibody.)

1. A magnetic bead, comprising:

magnetic beads coated with an activating functional group; and

TG antigen, through with the activation functional group coating on the magnetic bead.

2. The magnetic bead of claim 1, wherein the magnetic bead coated with the activating functional group is a streptavidin magnetic bead, an epoxy magnetic bead, a carboxyl magnetic bead, or a tosyl magnetic bead.

3. The magnetic bead of claim 2, wherein the magnetic bead coated with the activating functional group is a tosyl magnetic bead.

4. The magnetic bead of claim 2, wherein 1mg of the TG antigen coated on the magnetic bead having the activating functional group has a coating mass of 20 ug.

5. A method for preparing a magnetic bead as defined in claim 3 or 4, comprising:

mixing the magnetic beads with a boric acid buffer solution to prepare a mixed solution;

after the mixed solution is subjected to vortex and ultrasonic treatment, removing supernatant through magnetic adsorption to obtain a treated first magnetic bead;

mixing the treated first magnetic beads with a boric acid buffer solution, vortexing, incubating and uniformly mixing, and removing supernatant through magnetic adsorption to obtain second magnetic beads;

mixing the boric acid buffer solution and the TG antigen, adding the mixture into a second magnetic bead, and performing vortex, rolling and uniform mixing to obtain a uniform mixing solution;

mixing the uniformly mixed solution with a potassium phosphate aqueous solution, performing vortex incubation and rolling incubation, and removing the supernatant to obtain a third magnetic bead;

and rolling and uniformly mixing the third magnetic beads and Tris-HCl buffer solution containing bovine serum albumin, and carrying out magnetic adsorption to remove supernatant so as to obtain the TG antigen-coated tosyl magnetic beads.

6. The production method according to claim 5, wherein the boric acid concentration in the boric acid buffer is 0.1 mol/L; the concentration of potassium phosphate in the aqueous potassium phosphate solution was 3 mol/L.

7. The method according to claim 5, wherein the Tris-HCl buffer containing bovine serum albumin is a Tris-HCl buffer containing 0.5% by mass of BSA.

8. Use of the magnetic bead according to any one of claims 1 to 4 for the determination of anti-thyroglobulin antibodies.

9. A kit for assaying an anti-thyroglobulin antibody comprising the magnetic bead according to any one of claims 1 to 3.

10. The kit for measuring an anti-thyroglobulin antibody according to claim 6 further comprising an enzyme working solution.

Technical Field

The invention relates to a magnetic bead, a kit, a preparation method and application.

Background

Thyroglobulin (TG) is a water-soluble glycoprotein with a molecular weight of 660000 daltons, produced by the thyroid gland, and is the major component of the thyroid follicular cavity. TG consists of four peptide chains that are folded tightly together, have a spatial configuration, and are the predominant form of presence in the thyroid gland. The primary function of TG is to store and synthesize thyroid hormone, which is a mechanism in which TG protein contains 140 tyrosine residues, 40 of which (L-tyrosine) are iodinated under the action of Thyroid Peroxidase (TPO) for the synthesis of thyroxine (T4) and triiodothyronine (T3).

Although Anti-T and Anti-TPO are commonly detected simultaneously in Hashimoto thyroiditis, mucoedema, and toxic diffuse goiter disease, up to 1% of thyroid hypofunction diseases are only associated with Anti-TG. The determination of Anti-TG in the patient can determine hypothyroidism and disseminated goiter caused by autoimmune thyroiditis and is also helpful for distinguishing Graves from virus nodular goiter. In addition, the Anti-TG determination can also be used as an auxiliary check of serum TG determination, and the correctness of the TG determination on thyroid tumor diagnosis is improved. When an existing antithyroid globulin antibody determination kit (chemiluminescence method) is used for an instrument for uniformly mixing reagents by ultrasonic waves, antigens coated on magnetic beads fall off along with the increase of ultrasonic frequency, so that the precision and accuracy of the reagents are poor, abnormal measurement values appear in clinical sample detection, and clinical diagnosis and treatment are affected.

However, when the existing kit for anti-thyroglobulin antibodies is used on an ultrasonic mixing instrument, the precision is poor, and the clinical sample measurement is abnormal.

Disclosure of Invention

In order to solve the technical problem that when the existing kit for measuring the antithyroid globulin antibody is used on an ultrasonic mixing instrument, the precision is poor, so that the clinical sample measurement is abnormal, the embodiment of the invention provides a magnetic bead, a kit, a preparation method and an application.

The embodiment of the invention is realized by the following technical scheme:

in a first aspect, an embodiment of the present invention provides a magnetic bead, including:

magnetic beads coated with an activating functional group; and

TG antigen, through with the activation functional group coating on the magnetic bead.

Further, the magnetic beads coated with the activated functional groups are streptavidin magnetic beads, epoxy magnetic beads, carboxyl magnetic beads or tosyl magnetic beads.

Further, the magnetic beads coated with the activated functional groups are tosyl magnetic beads.

Further, the coating mass of TG antigen corresponding to 1mg of magnetic beads coated with an activating functional group was 20 ug.

In a second aspect, an embodiment of the present invention provides a method for preparing a magnetic bead, including:

mixing the magnetic beads with a boric acid buffer solution to prepare a mixed solution;

after the mixed solution is subjected to vortex and ultrasonic treatment, removing supernatant through magnetic adsorption to obtain a treated first magnetic bead;

mixing the treated first magnetic beads with a boric acid buffer solution, vortexing, incubating and uniformly mixing, and removing supernatant through magnetic adsorption to obtain second magnetic beads;

mixing the boric acid buffer solution and the TG antigen, adding the mixture into a second magnetic bead, and performing vortex, rolling and uniform mixing to obtain a uniform mixing solution;

mixing the uniformly mixed solution with a potassium phosphate aqueous solution, performing vortex incubation and rolling incubation, and removing the supernatant to obtain a third magnetic bead;

and rolling and uniformly mixing the third magnetic beads and Tris-HCl buffer solution containing bovine serum albumin, and carrying out magnetic adsorption to remove supernatant so as to obtain the TG antigen-coated tosyl magnetic beads.

Further, the concentration of boric acid in the boric acid buffer solution is 0.1 mol/L; the concentration of potassium phosphate in the aqueous potassium phosphate solution was 3 mol/L.

Further, the Tris-HCl buffer solution containing bovine serum albumin is a Tris-HCl buffer solution containing BSA with the mass fraction of 0.5%.

In a third aspect, the embodiments of the present invention provide a use of a magnetic bead for determining an anti-thyroglobulin antibody.

In a fourth aspect, the embodiments of the present invention provide a kit for determining an anti-thyroglobulin antibody, including the magnetic bead.

Further, the kit also comprises an enzyme working solution.

Compared with the prior art, the embodiment of the invention has the following advantages and beneficial effects:

according to the magnetic bead, the kit, the preparation method and the application provided by the embodiment of the invention, the TG antigen is coated on the magnetic bead coated with the activated functional group, so that when the antithyroid globulin antibody is measured on an ultrasonic uniformly-mixed instrument, the precision and the accuracy are better than those of the conventional reagent for measuring the antithyroid globulin antibody.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that: it is not necessary to employ these specific details to practice the present invention. In other instances, well-known structures, circuits, materials, or methods have not been described in detail so as not to obscure the present invention.

Throughout the specification, reference to "one embodiment," "an embodiment," "one example," or "an example" means: the particular features, structures, or characteristics described in connection with the embodiment or example are included in at least one embodiment of the invention. Thus, the appearances of the phrases "one embodiment," "an embodiment," "one example" or "an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable combination and/or sub-combination in one or more embodiments or examples. Further, those of ordinary skill in the art will appreciate that the illustrations provided herein are for illustrative purposes and are not necessarily drawn to scale. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In the description of the present invention, the terms "front", "rear", "left", "right", "upper", "lower", "vertical", "horizontal", "upper", "lower", "inner", "outer", etc., indicate an orientation or positional relationship, merely for convenience of description and simplicity of description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore should not be construed as limiting the scope of the present invention.

Examples

In order to solve the technical problem that when an existing kit for measuring an antithyroid globulin antibody is used on an ultrasonic mixing instrument, the precision is poor, so that the clinical sample measurement is abnormal, in a first aspect, an embodiment of the present invention provides a magnetic bead, including: magnetic beads coated with an activating functional group; and a TG antigen coated on the magnetic bead by reacting with the activated functional group.

The TG antigen is coated on the magnetic beads coated with the activated functional groups, so that the precision and accuracy of the reagent for measuring the antithyroid globulin antibody are better than those of the existing reagent for measuring the antithyroid globulin antibody when the antithyroid globulin antibody is measured on an ultrasonic uniformly-mixed instrument.

The working principle is as follows: when the magnetic beads are mixed with Anti-TG antibody in a solution, the TG antigen and the Anti-TG antibody are subjected to specific reaction and combination, so that the Anti-TG antibody in the solution is fixed on the magnetic beads, then ALP-labeled mouse Anti-human IgG antibody is added to be combined with the Anti-TG antibody to form a TG antigen-Anti-TG antibody-ALP-labeled mouse Anti-human IgG antibody compound, then a luminescent substrate is added, the enzyme conjugate catalyzes the luminescent substrate to emit photons, an instrument is used for measuring the photons, and the number of the photons is in direct proportion to the concentration of the Anti-TG in a sample. The concentration of Anti-TG in the sample can be calculated through a calibration curve of concentration-Relative Luminescence (RLU).

Further, the magnetic beads coated with the activated functional groups are streptavidin magnetic beads, epoxy magnetic beads, carboxyl magnetic beads or tosyl magnetic beads.

Further, the magnetic beads coated with the activated functional groups are tosyl magnetic beads.

Further, the coating mass of TG antigen corresponding to 1mg of magnetic beads coated with an activating functional group was 20 ug.

In a second aspect, an embodiment of the present invention provides a method for preparing a magnetic bead, including:

mixing the magnetic beads with a boric acid buffer solution to prepare a mixed solution;

after the mixed solution is subjected to vortex and ultrasonic treatment, removing supernatant through magnetic adsorption to obtain a treated first magnetic bead;

mixing the treated first magnetic beads with a boric acid buffer solution, vortexing, incubating and uniformly mixing, and removing supernatant through magnetic adsorption to obtain second magnetic beads;

mixing the boric acid buffer solution and the TG antigen, adding the mixture into a second magnetic bead, and performing vortex, rolling and uniform mixing to obtain a uniform mixing solution;

mixing the uniformly mixed solution with a potassium phosphate aqueous solution, performing vortex incubation and rolling incubation, and removing the supernatant to obtain a third magnetic bead;

and rolling and uniformly mixing the third magnetic beads and Tris-HCl buffer solution containing bovine serum albumin, and carrying out magnetic adsorption to remove supernatant so as to obtain the TG antigen-coated tosyl magnetic beads.

Further, the concentration of boric acid in the boric acid buffer solution is 0.1 mol/L; the concentration of potassium phosphate in the aqueous potassium phosphate solution was 3 mol/L.

Further, the Tris-HCl buffer solution containing bovine serum albumin is a Tris-HCl buffer solution containing BSA with the mass fraction of 0.5%.

In a third aspect, the embodiments of the present invention provide a use of a magnetic bead for determining an anti-thyroglobulin antibody.

In a fourth aspect, the embodiments of the present invention provide a kit for determining an anti-thyroglobulin antibody, including the magnetic bead.

Further, the kit also comprises an enzyme working solution.

Example 1

A kit for measuring an anti-thyroglobulin antibody comprises streptavidin magnetic beads coated by TG antigen and enzyme working solution.

The streptavidin magnetic bead coated by the TG antigen is as follows:

A. 0.25mg of TG antigen was dialyzed against 5L of 0.02M PBS (pH7.4) buffer at 2-8 ℃ overnight.

B. And taking out the dialyzed TG antigen in a proper container, accurately measuring and recording the volume, and diluting the TG antigen to 0.2mg/mL for later use.

C. 1.0mg of BNHS was weighed out and dissolved in 217. mu.L of DMSO to prepare a BNHS solution of 10 mmol/L.

D. According to the mass ratio of BNHS to TG antigen of 20: and 1, adding the BNHS solution into the TG antigen diluent obtained in the step B, fully and uniformly mixing, and reacting at room temperature for 30 min. (i.e., 1.0mg of antibody added to 10mmol/L BNHS solution 13.3. mu.L; 0.25mg added to prepared 10mmol/L BNHS solution 3.3. mu.L)

E. The reaction was terminated with 1mol/L Tris buffer, and 1mol/L Tris buffer was added to the reaction mixture. Adding Tris with a volume one percent of the final volume of the reaction solution, fully and uniformly mixing, and reacting at room temperature for 10 min.

F. The reaction solution after the termination was taken out and put into a dialysis bag, and dialyzed overnight at 2 to 8 ℃ with 5L of 0.02M PBS (pH7.4) buffer solution.

G. And taking out the dialyzed biotin antibody, accurately measuring the volume, adding glycerol with the same volume, labeling, recording the concentration, and storing at-20 ℃ for later use.

H. And coating streptavidin magnetic beads with biotin-labeled biotin antibodies, wherein the coating amount is 20 mu g/mg, and obtaining the streptavidin magnetic beads coated with TG antigens. Diluted with Buffer of 1% BSA for use.

Example 2

A kit for measuring an antithyroid globulin antibody comprises TG antigen-coated epoxy magnetic beads and an enzyme working solution.

The preparation method of the TG antigen-coated epoxy magnetic bead comprises the following steps:

A. 6mg of epoxy-based magnetic beads are put into a clean tube, and 0.3mL of ultrapure water is added to ensure that the concentration of the epoxy-based magnetic beads is 20 mg/mL;

B. vortex for 15S, and place in water bath for ultrasound for 5min twice;

C. placing on a magnetic frame for adsorption for 1min, and removing the supernatant;

D. repeat step B, C;

E. antigen dosage: adding TG antigen with a mass of 120ug (TG antigen concentration of 1mg/mL, i.e. adding 0.12mL antigen);

volume of f.0.1m PBS: 0.18mL of 0.1M PBS was added;

G. mixing the TG antigen of the E and the PBS solution of the F, fully and uniformly mixing, and adding the mixture into the solution obtained by the D for vortex for 5 s;

H. adding 0.2mL of 3M ammonium sulfate solution into the solution obtained in the step G, uniformly mixing the solution for 5s in a vortex mode, and carrying out rolling reaction for 3h at the temperature of 37 ℃;

I. after the reaction is finished, placing the mixture in a magnetic rack for adsorption for 1min, and removing the supernatant;

j.0.3mL100mM glycine solution (pH 11.3) is vortexed for 5s, placed in a magnetic frame for adsorption for 1min, and the supernatant is removed;

swirling K.0.3mL200mM glycine solution (pH 2.8) for 5s, placing in a magnetic frame for adsorption for 1min, and removing supernatant;

l. adding 0.3mL of Tris-HCl buffer solution containing 0.5% BSA, carrying out rolling reaction at room temperature for 10min, placing the mixture in a magnetic frame for adsorption for 1min, and removing the supernatant;

m, adding 0.3mL of Tris-HCl buffer solution containing 0.5% BSA, carrying out rolling reaction at 37 ℃ for 24 hours, placing the solution on a magnetic frame for adsorption for 1min, and removing supernatant; obtaining epoxy magnetic beads coated with TG antigen; the TG antigen-coated epoxy magnetic beads were diluted with 1% BSA buffer for use.

Example 3

A kit for measuring an antithyroid globulin antibody comprises TG antigen-coated carboxyl magnetic beads and an enzyme working solution.

The preparation method of the TG antigen coated carboxyl magnetic bead comprises the following steps:

A. 5mg of carboxyl magnetic beads are put into a clean tube, and 1mL of 15mM MES buffer solution with the pH value of 6.0 is added;

B. swirling for 15S, and placing in a rolling mixing transportation instrument to mix for 10 min;

C. repeating the step B;

D. accurately weighing 10mgEDC, adding 1mL of precooled water, fully dissolving and uniformly mixing;

E. adding 0.08mL of the solution obtained in step D into 0.92mL of 15mM MES buffer solution (pH 6.0), and mixing well;

F. adding the solution obtained by the step E into the solution obtained by the step C to enable the final concentration of the carboxyl magnetic beads to be 5mg/mL, carrying out vortex for 10s, and rolling and uniformly mixing for 30 min;

G. placing in a magnetic frame for adsorption for 1min, and removing the supernatant;

H. antigen dosage: adding 100uL of TG antigen with the concentration of 1mg/mL, namely the antigen mass is 0.1 mg;

I. adding 150uL15mM MES buffer solution (pH 6.0) to dilute TG antigen to 0.4mg/mL, and mixing well;

J. adding the solution obtained in the step I into the solution obtained in the step G, performing vortex for 10s, performing rolling incubation for 3h, placing the solution in a magnetic frame for adsorption for 1min, and removing the supernatant;

p, adding 1mL of Tris-HCl buffer solution containing 0.5% BSA into the solution obtained by J to enable the concentration of the carboxyl magnetic beads to be 5mg/mL, carrying out rolling reaction for 10min, placing the solution in a magnetic frame for adsorption for 1min, and removing the supernatant;

K. repeating P twice;

l, adding 1mL of Tris-HCl buffer solution containing 0.5% BSA into the solution obtained by the K, enabling the concentration of magnetic beads to be 5mg/mL, carrying out rolling reaction at 37 ℃ for 24 hours, placing the solution on a magnetic rack for adsorption for 1min, and removing the supernatant;

obtaining carboxyl magnetic beads coated by TG antigen; TG antigen-coated carboxyl magnetic beads were diluted with 1% BSA buffer for use.

Example 4

A kit for measuring an anti-thyroglobulin antibody comprises a TG antigen-coated tosyl magnetic bead and an enzyme working solution.

A kit for measuring an anti-thyroglobulin antibody comprises a TG antigen-coated tosyl magnetic bead and an enzyme working solution.

A. 6mg of tosyl magnetic beads were put into a clean tube, and 1.2mL of 0.1M boric acid buffer (pH 9.5) was added thereto so that the concentration of the magnetic beads became 5 mg/mL;

B. swirling for 30s, and placing in a water bath for ultrasonic treatment for 5min twice;

C. placing on a magnetic frame for adsorption for 3min, and removing the supernatant;

D. adding 1.2ml of 0.1M boric acid buffer solution with the pH value of 9.5, vortexing for 30S, and rolling, incubating and uniformly mixing for 5 min;

E. placing on a magnetic frame for adsorption for 3min, and removing the supernatant;

F. e is repeated;

G. antigen dosage: the amount of TG antigen added at a concentration of 1mg/mL is 120ug, and 80uL of 0.1M boric acid buffer (pH 9.5) is required;

I. mixing the antigen of G with boric acid buffer solution, mixing well, adding into the solution obtained from F, vortexing for 5s, and rolling for 5 min;

J. adding 66.6uL of 3M potassium phosphate aqueous solution, quickly vortexing for 5s and standing at 37 ℃ for 24h in a rolling way;

K. adding 1.2mL of Tris-HCl buffer solution containing 0.5% BSA into the solution obtained by J, and rolling and mixing for 10 min;

l, placing the supernatant on a magnetic frame for adsorption, and removing the supernatant;

m. K, L repeat twice;

adding 1.2mL of Tris-HCl buffer solution containing 0.5% BSA into the solution obtained by M, and performing rolling incubation at 37 ℃ for 24 hours;

o, placing the supernatant on a magnetic frame for adsorption, and removing the supernatant; obtaining tosyl magnetic beads coated by TG antigen; the TG antigen-coated tosyl magnetic beads were diluted with 1% BSA buffer for use.

Comparative experiment

Experiment one: and (3) performing a kit sensitivity comparison experiment under the condition that four kinds of magnetic beads such as streptavidin magnetic beads, epoxy magnetic beads, carboxyl magnetic beads and tosyl magnetic beads are coated with the same amount of TG antigen.

Experimental materials: BNHS, DMSO, glycerol, 0.02M PBS (pH7.4) Buffer, 1mol/LTris Buffer, TG antigen, EDC, 0.1M boric acid Buffer, 3mol/L ammonium sulfate solution, 3mol/L potassium phosphate aqueous solution, 100mmol/L glycine solution pH11.3 and 200mmol/L glycine solution pH2.8, Tris-HCl Buffer containing 0.5% BSA, 15mmol/LMES Buffer pH6.0, ultrapure water, Buffer containing 1% BSA, ALP-labeled mouse Anti-human IgG antibody, naked magnetic beads, Anti-TG calibrator Cal-1, Cal-6 (concentration of Cal-1 to Cal-6 is 0, 1, 5, 20, 40, 80IU/mL), substrate solution, washing solution, Wwinter Bioluminescence immunoassay analyzer 2000 LA.

The method of example 1, example 2, example 3 and example 4 was adopted, the four different binding modes of magnetic beads and the enzyme working solution were combined to form a kit, Anti-TG calibrators cal-1 to cal-6 were measured, the measurement curves and sensitivities of the different magnetic beads were observed, and samples S1-S40 were measured and compared with Rogowski values.

The reaction mode is as follows: adding 50 mu L of calibrator and 50 mu L of magnetic beads, incubating for 10min at 37 ℃, adding cleaning solution, cleaning and separating for three times in a magnetic field, adding 100 mu L of enzyme working solution, incubating for 10min at 37 ℃, adding cleaning solution, cleaning and separating for three times in the magnetic field, adding substrate solution, incubating for 5min at 37 ℃, and detecting on a full-automatic chemiluminescence determinator. The results of the experiment are shown in table 1.

TABLE 1

And (4) experimental conclusion: from the experimental result, the kit consisting of the four magnetic beads can have better reactivity with an Anti-TG calibrator, the four magnetic beads coated with the same coating amount of TG antigen can be seen from the sensitivity, the signal of the streptavidin magnetic bead is about half lower than that of the other three magnetic beads, and the maximum coating amount of the magnetic bead is 10ug/mg, so that even if the coating amount is 20ug of TG antigen coated on 1mg of magnetic beads, the actual TG antigen coated on the streptavidin magnetic beads is 10ug of TG antigen coated on 1mg of magnetic beads, and the kit curves of the four magnetic beads can meet the use requirement.

Experiment two: precision verification experiment of four magnetic bead kits with different connection modes.

Referring to a method related to precision verification experiments of a human antithyroid antibody industry standard (YY/T1594-; a kit prepared from four magnetic beads is used for continuously testing the Cal-4 level 50 test, the signal of the 50 test of the reagent is observed, and the falling condition of the TG antigen on each magnetic bead is judged according to the trend of the signal.

Experimental materials: BNHS, DMSO, glycerol, 0.02M PBS (pH7.4) Buffer, 1mol/LTris Buffer, TG antigen, EDC, 0.1M boric acid Buffer, 3mol/L ammonium sulfate solution, 3mol/L potassium phosphate aqueous solution, 100mmol/L glycine solution pH11.3 and 200mmol/L glycine solution pH2.8, Tris-HCl Buffer containing 0.5% BSA, 15mmol/LMES Buffer pH6.0, ultrapure water, Buffer containing 1% BSA, ALP-labeled mouse Anti-human IgG antibody, naked magnetic beads, Anti-TG calibrator Cal-1, Cal-6 (concentration of Cal-1 to Cal-6 is 0, 1, 5, 20, 40, 80IU/mL), substrate solution, washing solution, Wwinter Bioluminescence immunoassay analyzer 2000 LA.

Reaction mode refer to experiment one. The results of the experiments are shown in tables 2 and 3.

TABLE 2

TABLE 3

And (4) experimental conclusion: table 2 shows that the precision of the four magnetic beads is tosyl magnetic bead ═ epoxy magnetic bead ═ carboxyl magnetic bead > streptavidin magnetic bead; from the results in table 3, as the test proceeds, signals of streptavidin magnetic beads and carboxyl magnetic beads are significantly reduced, while signals of tosyl magnetic beads and epoxy magnetic beads are not significantly reduced, which indicates that the shedding amount of the antigen on the magnetic beads is streptavidin magnetic beads > carboxyl magnetic beads > tosyl magnetic beads ═ epoxy magnetic beads.

Experiment three: and preparing a kit by four magnetic beads, and testing the condition of a clinical sample.

Experimental materials: BNHS, DMSO, glycerol, 0.02M PBS (pH7.4) Buffer, 1mol/LTris Buffer, TG antigen, EDC, 0.1M boric acid Buffer, 3mol/L ammonium sulfate solution, 3mol/L potassium phosphate aqueous solution, 100mmol/L glycine solution, pH11.3 and 200mmol/L glycine solution, pH2.8, Tris-HCl Buffer containing 0.5% BSA, 15mmol/LMES Buffer, pH6.0, ultrapure water, Buffer containing 1% BSA, ALP-labeled mouse Anti-human IgG antibody, naked magnetic beads, Anti-TG calibrator Cal-1, Cal-6 (concentration of Cal-1 to Cal-6 is 0, 1, 5, 20, 40, 80IU/mL), sample S1-S40, substrate solution, washing solution, Wavente Bioluminescence immunoassay analyzer LA 2000.

Reaction mode refer to experiment one. The experimental results are shown in table 4 below.

TABLE 4

And (4) experimental conclusion: the experimental results show that, after the clinical samples are detected by four kinds of magnetic beads, the more the streptavidin magnetic bead detection clinical samples are detected, the lower the detected concentration trend, the abnormal samples are detected by both epoxy magnetic beads and carboxyl magnetic beads, and the measured values are higher, because the same antigen and antibody are used, the higher the measured values are caused by the nonspecific adsorption of the magnetic beads, and only the measured values of the tosyl magnetic beads are closest to the roche measured values and have no abnormal samples, the least nonspecific adsorption of the tosyl magnetic beads is indicated.

Experiment four: and (3) performing a tosyl magnetic bead thermal acceleration experiment to observe the stability of the kit.

The experimental method comprises the following steps: the prepared tosyl magnetic beads are divided into two parts in equal volume and then put into a kit, one part is stored at 2-8 ℃, and the other part is stored at 37 ℃ in a constant temperature incubator. And (3) matching the two magnetic beads with an enzyme working solution to determine an Anti-TG calibrator. And (4) observing the luminescent signals of the calibrator on the third day and the sixth day respectively, calculating the signal retention rate, and verifying the stability of the kit.

The results are shown in Table 5.

TABLE 5

And (4) experimental conclusion: the signal retention rate of the tosyl magnetic beads is still higher after the heat treatment for 3 days and 6 days at 37 ℃, which indicates that the stability of the reagent is better.

In conclusion, four experiments can be carried out to obtain: the Anti-TG determination kit is shown to have the advantages that when the tosyl magnetic beads are used, the precision and the clinical sample test are obviously improved compared with other commonly used magnetic beads, the clinical compliance rate is higher, the accuracy is higher, and the market advantage is achieved.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.