阅读说明：本技术 一种睾酮的磁颗粒酶促化学发光免疫检测试剂 (Magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone ) 是由 汪紫晶 于琴 丁军发 谢钧 于 2021-08-04 设计创作，主要内容包括：本发明免疫分析技术领域,具体涉及一种睾酮的磁颗粒酶促化学发光免疫检测试剂。本发明公开了一种睾酮的磁颗粒酶促化学发光免疫检测试剂,其组成成分：0.01-0.05％的包被有睾酮抗原的超顺磁性微粒、碱性磷酸酶标记睾酮抗体、睾酮释放试剂、碱性磷酸酶发光底物以及磁珠清洗液；所述睾酮释放试剂为双氢睾酮、达那唑、肝素钠、8-苯胺-1-萘磺酸(ANS)、8-苯胺-1-萘磺酸盐、三氯乙酸钠、2-HydroxymethyleneEthisterone(CAS:2787-02-2)、炔孕酮(ethisterone,CAS:434-03-7)、2-Hydroxymethyl-1,2-dehydroethisterone(CAS:60142-08-7)中的一种或多种组合。(The invention belongs to the technical field of immunoassay, and particularly relates to a magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone. The invention discloses a magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone, which comprises the following components: 0.01-0.05% of superparamagnetic particles coated with testosterone antigens, an alkaline phosphatase marked testosterone antibody, a testosterone release reagent, an alkaline phosphatase luminescent substrate and a magnetic bead cleaning solution; the testosterone releasing reagent is one or more of dihydrotestosterone, danazol, heparin sodium, 8-aniline-1-naphthalene sulfonic Acid (ANS), 8-aniline-1-naphthalene sulfonate, sodium trichloroacetate, 2-hydroxymethyene Ethisterone (CAS:2787-02-2), ethisterone (CAS: 434-03-7) and 2-hydroxymethyne-1, 2-dehydroethisterone (CAS: 60142-08-7).)

1. A magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone, which is characterized in that: the components are as follows: 0.01-0.05% of superparamagnetic particles coated with testosterone antigens, an alkaline phosphatase marked testosterone antibody, a testosterone release reagent, an alkaline phosphatase luminescent substrate and a magnetic bead cleaning solution;

the testosterone releasing reagent is one or more of dihydrotestosterone, danazol, heparin sodium, 8-aniline-1-naphthalene sulfonic Acid (ANS), 8-aniline-1-naphthalene sulfonate, sodium trichloroacetate, 2-hydroxymethyene Ethisterone (CAS:2787-02-2), ethisterone (CAS: 434-03-7) and 2-hydroxymethyne-1, 2-dehydroethisterone (CAS: 60142-08-7).

2. The magnetic particle enzymatic chemiluminescent immunoassay reagent for testosterone according to claim 1, wherein: the concentration of each component in the testosterone releasing agent is as follows: the concentration of dihydrotestosterone is 0.01-0.05mg/mL, the concentration of danazol is 0.05-0.5mg/mL, the concentration of heparin sodium is 2-50mg/mL, the concentration of 8-aniline-1-naphthalene sulfonic Acid (ANS) or 8-aniline-1-naphthalene sulfonate is 1-5mg/mL, the concentration of sodium trichloroacetate is 1-25mg/mL, the concentration of ethisterone is 0.05-0.25mg/mL, the concentration of 2-hydroxymethyethlesterone (CAS:2787-02-2) is 0.01-0.5mg/mL, the concentration of 2-Hydroxymethyl-1,2-dehydroethisterone is 0.01-0.60 mg/mL;

the preparation method of the testosterone releasing agent comprises the following steps,

s1: taking a compound with testosterone releasing effect according to the component ratio, dissolving with DMSO or DMF or pure water to obtain a mother solution with the concentration of 500mg/mL,

s2: the testosterone releasing agent is prepared by diluting the buffer solution to the corresponding concentration.

3. The magnetic particle enzymatic chemiluminescent immunoassay reagent for testosterone according to claim 2, wherein: in the S2, the buffer solution is one of a PBS buffer solution, a quinoline ethanesulfonic acid buffer solution (MES), a Tris-HCL buffer solution, a citrate buffer solution, a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO) buffer solution, a glycine-hydrochloric acid buffer solution, an acetic acid-sodium acetate buffer solution, a 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution and a TRIS buffer solution.

4. The magnetic particle enzymatic chemiluminescent immunoassay reagent for testosterone according to claim 1, wherein: the preparation method of the superparamagnetic particles coated with the testosterone antigens comprises the following steps,

a1: putting 1mL of commercial carboxyl magnetic beads (10mg/mL) into a 2.0mL centrifuge tube, placing the centrifuge tube on a magnetic separator for magnetic separation, and absorbing the supernatant;

a2: adding 1mL of labeling buffer solution (0.1M MES, pH5.0) into the centrifuge tube, resuspending the magnetic beads, placing the centrifuge tube on a magnetic separator for magnetic separation, and sucking the supernatant;

a3: adding 1mL of labeling buffer solution into the centrifuge tube, and uniformly shaking the labeling buffer solution to resuspend the magnetic beads;

a4: adding 100ul of testosterone antigen (testosterone-BSA conjugate) into the resuspended magnetic beads, and mixing at room temperature for 30 min;

a5: adding 100ul of coupling agent EDC (10mg/mL, prepared by labeled buffer solution) into the mixture of the magnetic beads and the testosterone antigen, shaking and uniformly mixing, and rotationally mixing at room temperature for 3 h;

a6: after the reaction, the centrifuge tube was placed on a magnetic separator for magnetic separation, after the supernatant was removed, 1ml of a magnetic bead washing buffer (25mM Tris-HCl,150mM NaCl, 0.05% Tween 20, pH7.2) was added, and after shaking and mixing, the centrifuge tube was placed on a magnetic separator for magnetic separation, and then the supernatant was removed. The magnetic bead cleaning is repeated for 3 times;

a7: after the final washing step, 500ul of a bead preservation buffer (50mM Tris,150mM NaCl, 1% BSA, 0.05% PC300, pH7.2) was added, and the beads were resuspended and stored under refrigeration.

5. The magnetic particle enzymatic chemiluminescent immunoassay reagent for testosterone according to claim 1, wherein: the preparation method of the alkaline phosphatase marked testosterone antibody comprises the following steps,

b1: dissolving a proper amount of alkaline phosphatase in 0.8mL of pure water, adding 0.2mL of prepared 0.1M NaIO4 solution into the alkaline phosphatase, and reacting for 20min at room temperature in a dark place;

b2: adding the buffer solution into a 10KD ultrafiltration centrifugal tube, carrying out ultrafiltration centrifugation at 4 ℃, and carrying out buffer solution replacement by using 0.2M carbonate buffer solution with pH 9.5;

b3: adding a certain amount of testosterone antibody into the alkaline phosphatase after buffer solution replacement, and reacting for 3 hours at room temperature in a dark place;

b4: adding 0.1mL of prepared 4mg/mL sodium borohydride solution, uniformly quenching, and standing for 2 hours at 4 ℃;

b5: after the reaction is finished, a 50kD ultrafiltration centrifugal tube is used for ultrafiltration and centrifugation, and 50mM PBS buffer solution with pH7.4 is used for buffer solution replacement, so that the alkaline phosphatase labeled testosterone antibody is prepared.

6. The magnetic particle enzymatic chemiluminescent immunoassay reagent for testosterone according to claim 1, wherein: the alkaline phosphatase luminescent substrate contains 50mM Tris, 0.5mM APS-5 and 5mM MgCl₂、2.5mM ZnCl₂0.1% TritonX100, 0.05% Proclin300, and the pH value of the alkaline phosphatase luminescent substrate is 9.5 +/-0.2.

Technical Field

The invention belongs to the technical field of immunoassay, and particularly relates to a magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone.

Background

Testosterone, also known as testosterone, testosterone or testosterone, is a steroid estrogen that is secreted by the male testes or the female ovaries, and the adrenal glands also secrete small amounts of testosterone. Testosterone can promote the generation of epididymis, vas deferens and seminal vesicles, promote the growth and development of external genital organs and secondary sexual characteristics, and influence the generation of sperms, which have important health effects in both men and women. For men, elevated testosterone levels are common in adrenal cortical hyperplasia, adrenal cortical tumor, testicular tumor, male sexual precocity, and other conditions. The decreased testosterone level is commonly seen in primary male hypofunction such as cryptorchidism, azorchidism, mesenchymal cell hypoplasia and congenital seminal tubule hypoplasia, hyperprolactinemia, hypercortisolism and the like. In women, abnormalities in testosterone levels are common in idiopathic hirsutism, polycystic ovary syndrome, pineal tumors, ovarian masculinizing tumors, and the like. Accurate detection of testosterone levels in the body is helpful in the auxiliary diagnosis and treatment of these diseases.

In serum, testosterone is mainly bound to sex hormone binding protein, albumin and cortisol binding protein, and only 2% of testosterone is in a free state. Immunoassay of serum total testosterone requires release of testosterone from sex hormone binding protein, albumin, and cortisol binding protein to facilitate detection of antibody recognition. Therefore, the formulation composition of the testosterone releasing reagent in the detection reagent is related to the release effect of testosterone, and the sensitivity and specificity of the serum total testosterone immunoassay reagent are directly influenced.

Disclosure of Invention

In view of the problems raised by the above background art, the present invention is directed to: aims to provide a magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

a magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone comprises the following components: 0.01-0.05% of superparamagnetic particles coated with testosterone antigens, an alkaline phosphatase marked testosterone antibody, a testosterone release reagent, an alkaline phosphatase luminescent substrate and a magnetic bead cleaning solution;

the testosterone releasing reagent is one or more of dihydrotestosterone, danazol, heparin sodium, 8-aniline-1-naphthalene sulfonic Acid (ANS), 8-aniline-1-naphthalene sulfonate, sodium trichloroacetate, 2-hydroxymethyene Ethisterone (CAS:2787-02-2), ethisterone (CAS: 434-03-7) and 2-hydroxymethyne-1, 2-dehydroethisterone (CAS: 60142-08-7).

As a preferred embodiment of the present invention, the concentration of each component in the testosterone releasing agent is: the concentration of dihydrotestosterone is 0.01-0.05mg/mL, the concentration of danazol is 0.05-0.5mg/mL, the concentration of heparin sodium is 2-50mg/mL, the concentration of 8-aniline-1-naphthalene sulfonic Acid (ANS) or 8-aniline-1-naphthalene sulfonate is 1-5mg/mL, the concentration of sodium trichloroacetate is 1-25mg/mL, the concentration of ethisterone is 0.05-0.25mg/mL, the concentration of 2-hydroxymethyethlesterone (CAS:2787-02-2) is 0.01-0.5mg/mL, the concentration of 2-Hydroxymethyl-1,2-dehydroethisterone is 0.01-0.60 mg/mL;

the preparation method of the testosterone releasing agent comprises the following steps,

s1: taking the compound with testosterone releasing effect according to the component ratio, dissolving with DMSO or DMF or pure water, and making into mother liquor with concentration of 500 mg/mL.

S2: the testosterone releasing agent is prepared by diluting the buffer solution to the corresponding concentration.

3. The magnetic particle enzymatic chemiluminescent immunoassay reagent for testosterone according to claim 2, wherein: in the S2, the buffer solution is one of a PBS buffer solution, a quinoline ethanesulfonic acid buffer solution (MES), a Tris-HCL buffer solution, a citrate buffer solution, a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO) buffer solution, a glycine-hydrochloric acid buffer solution, an acetic acid-sodium acetate buffer solution, a 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution and a TRIS buffer solution.

In a preferred embodiment of the present invention, the superparamagnetic particles coated with testosterone antigens are prepared by,

a1: putting 1mL of commercial carboxyl magnetic beads (10mg/mL) into a 2.0mL centrifuge tube, placing the centrifuge tube on a magnetic separator for magnetic separation, and absorbing the supernatant;

a2: adding 1mL of labeling buffer solution (0.1MMES, pH5.0) into the centrifuge tube, resuspending the magnetic beads, placing the centrifuge tube on a magnetic separator for magnetic separation, and sucking the supernatant;

a3: adding 1mL of labeling buffer solution into the centrifuge tube, and uniformly shaking the labeling buffer solution to resuspend the magnetic beads;

a4: adding 100ul of testosterone antigen (testosterone-BSA conjugate) into the resuspended magnetic beads, and mixing at room temperature for 30 min;

a5: adding 100ul of coupling agent EDC (10mg/mL, prepared by labeled buffer solution) into the mixture of the magnetic beads and the testosterone antigen, shaking and uniformly mixing, and rotationally mixing at room temperature for 3 h;

a6: after the reaction, the centrifuge tube was placed on a magnetic separator for magnetic separation, and after the supernatant was removed, 1ml of a magnetic bead washing buffer (25mM Tris-HCl,150mM NaCl, 0.05% Tween 20, pH7.2) was added, followed by shaking and mixing, and then placed on a magnetic separator for magnetic separation, and then the supernatant was removed. The magnetic bead cleaning is repeated for 3 times;

a7: after the final washing step, 500ul of magnetic bead preservation buffer (50mM Tris,150mM NaCl, 1% BSA, 0.05% PC300, pH7.2) was added, and the beads were resuspended and then stored under refrigeration.

As a preferable proposal of the invention, the preparation method of the alkaline phosphatase labeled testosterone antibody comprises the following steps,

b1: dissolving a proper amount of alkaline phosphatase in 0.8mL of pure water, adding 0.2mL of prepared 0.1M NaIO4 solution into the alkaline phosphatase, and reacting for 20min at room temperature in a dark place;

b2: adding the buffer solution into a 10KD ultrafiltration centrifugal tube, carrying out ultrafiltration centrifugation at 4 ℃, and carrying out buffer solution replacement by using 0.2M carbonate buffer solution with pH 9.5;

b3: adding a certain amount of testosterone antibody into the alkaline phosphatase after buffer solution replacement, and reacting for 3 hours at room temperature in a dark place;

b4: adding 0.1mL of prepared 4mg/mL sodium borohydride solution, uniformly quenching, and standing for 2 hours at 4 ℃;

b5: after the reaction is finished, a 50kD ultrafiltration centrifugal tube is used for ultrafiltration and centrifugation, and the buffer solution is replaced by a PBS buffer solution of 50mMPH7.4 to prepare the alkaline phosphatase labeled testosterone antibody.

In a preferred embodiment of the present invention, the substrate for alkaline phosphatase luminescence comprises 50mM Tris, 0.5mM PS-5, 5mM MgCl₂、2.5mMZnCl₂0.1% TritonX100, 0.05% Proclin300, and the pH value of the alkaline phosphatase luminescent substrate is 9.5 +/-0.2.

By adopting the technical scheme of the invention,

compared with the prior art, the releasing agent in the detection reagent is used as a component of the testosterone enzymatic chemiluminescence immunoassay reagent, and can rapidly and effectively dissociate testosterone from a combined egg, so that the sensitivity and specificity of the testosterone immunoassay reagent can be obviously improved.

Drawings

The invention is further illustrated by the non-limiting examples given in the accompanying drawings;

FIG. 1 is a graph showing the results of measurement of a reference example of an testosterone free releasing reagent;

FIG. 2 is a graph showing the results of detection in example 1;

FIG. 3 is a graph showing the results of the detection in example 2;

FIG. 4 is a graph showing the results of example 3;

FIG. 5 is a graph showing the results of detection in example 4;

FIG. 6 is a graph showing the results of example 5.

Detailed Description

In order that those skilled in the art can better understand the present invention, the following technical solutions are further described with reference to the accompanying drawings and examples.

A magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone comprises the following components: 0.01-0.05% of superparamagnetic particles coated with testosterone antigens, an alkaline phosphatase marked testosterone antibody, a testosterone release reagent, an alkaline phosphatase luminescent substrate and a magnetic bead cleaning solution;

the testosterone releasing agent is one or more of dihydrotestosterone, danazol, heparin sodium, 8-aniline-1-naphthalene sulfonic Acid (ANS), 8-aniline-1-naphthalene sulfonate, sodium trichloroacetate, 2-hydroxymethyene Ethisterone (CAS:2787-02-2), ethisterone (CAS: 434-03-7), and 2-hydroxymethyne-1, 2-dehydroethisterone (CAS:60142-08-7)

Wherein, the preparation method of the superparamagnetic particles coated with the testosterone antigen comprises the following steps,

a1: putting 1mL of commercial carboxyl magnetic beads (10mg/mL) into a 2.0mL centrifuge tube, placing the centrifuge tube on a magnetic separator for magnetic separation, and absorbing the supernatant;

a2: adding 1mL of labeling buffer solution (0.1MMES, pH5.0) into the centrifuge tube, resuspending the magnetic beads, placing the centrifuge tube on a magnetic separator for magnetic separation, and sucking the supernatant;

a3: adding 1mL of labeling buffer solution into the centrifuge tube, and uniformly shaking the labeling buffer solution to resuspend the magnetic beads;

a4: adding 100ul of testosterone antigen (testosterone-BSA conjugate) into the resuspended magnetic beads, and mixing at room temperature for 30 min;

a5: adding 100ul of coupling agent EDC (10mg/mL, prepared by labeled buffer solution) into the mixture of the magnetic beads and the testosterone antigen, shaking and uniformly mixing, and rotationally mixing at room temperature for 3 h;

a6: after the reaction, the centrifuge tube was placed on a magnetic separator for magnetic separation, and after the supernatant was removed, 1ml of a magnetic bead washing buffer (25mM Tris-HCl,150mM NaCl, 0.05% Tween 20, pH7.2) was added, followed by shaking and mixing, and then placed on a magnetic separator for magnetic separation, and then the supernatant was removed. The magnetic bead cleaning is repeated for 3 times;

a7: after the final washing step, 500ul of magnetic bead preservation buffer (50mM Tris,150mM NaCl, 1% BSA, 0.05% PC300, pH7.2) was added, and the beads were resuspended and then stored under refrigeration.

The preparation method of the alkaline phosphatase marked testosterone antibody comprises the following steps,

b1: dissolving a proper amount of alkaline phosphatase in 0.8mL of pure water, adding 0.2mL of prepared 0.1M NaIO4 solution into the alkaline phosphatase, and reacting for 20min at room temperature in a dark place;

b2: adding the buffer solution into a 10KD ultrafiltration centrifugal tube, carrying out ultrafiltration centrifugation at 4 ℃, and carrying out buffer solution replacement by using 0.2M carbonate buffer solution with pH 9.5;

b3: adding a certain amount of testosterone antibody into the alkaline phosphatase after buffer solution replacement, and reacting for 3 hours at room temperature in a dark place;

b4: adding 0.1mL of prepared 4mg/mL sodium borohydride solution, uniformly quenching, and standing for 2 hours at 4 ℃;

b5: after the reaction is finished, a 50kD ultrafiltration centrifugal tube is used for ultrafiltration and centrifugation, and the buffer solution is replaced by a PBS buffer solution of 50mMPH7.4 to prepare the alkaline phosphatase labeled testosterone antibody.

The alkaline phosphatase luminescent substrate contains 50mM Tris, 0.5mM PS-5, 5mM MgCl₂、2.5mMZnCl₂0.1% TritonX100, 0.05% Proclin300, and the pH value of the alkaline phosphatase luminescent substrate is 9.5 +/-0.2.

Example 1: a magnetic particle enzymatic chemiluminescence immunoassay reagent for testosterone comprises the following components: 0.01-0.05% of superparamagnetic particles coated with testosterone antigens, an alkaline phosphatase marked testosterone antibody, a testosterone release reagent, an alkaline phosphatase luminescent substrate and a magnetic bead cleaning solution,

wherein the superparamagnetic particles coated with testosterone antigen, the alkaline phosphatase labeled testosterone antibody and the alkaline phosphatase luminescent substrate are prepared according to the method;

the testosterone releasing agent contains 0.02mg/mL dihydrotestosterone, 0.1mg/mL danazol, and 50mMPBS buffer solution, and has a pH value of 7.0.

Example 2: the difference from example 1 is a testosterone releasing agent comprising 3.0mg/mL sodium trichloroacetate, 0.1mg/mL ethisterone, 100mM Tris buffer, pH 7.4.

Example 3: the difference from example 1 was that a testosterone releasing agent comprising 4.0mg/mL heparin sodium, 0.03mg/mL dihydrotestosterone, 2mg/mL 8-aniline-1-naphthalenesulfonic Acid (ANS), and 60mM Tris-HCl buffer was adjusted to pH 7.2.

Example 4: the difference from example 1 is that a testosterone releasing agent comprising 3.0mg/mL sodium trichloroacetate, 0.1mg/mL 2-hydroxymethylene Ethisterone (CAS:2787-02-2), 0.02mg/mL dihydrotestosterone, and 50mM MES buffer was adjusted to pH 6.0.

Example 5: the difference from example 1 is that the testosterone releasing agent contains 0.05mg/mL dihydrotestosterone, 0.1mg/mL 2-hydroxymethylene Ethisterone (CAS:2787-02-2), 2mg/mL 8-aniline-1-naphthalenesulfonic Acid (ANS), and 50mM HEPES buffer, and the pH is adjusted to 8.0.

Specificity: collecting 100 clinical samples, using a Roche testosterone kit to perform assignment, testing the samples by using the detection reagents obtained in the 5 different embodiments, and comparing the test results with the Roche reagent test results, wherein the test results are shown in a figure 1-a figure 6;

FIG. 1 is a graph showing the results of a reference example of an testosterone free releasing reagent, FIGS. 2 to 6 are graphs showing the results of examples 1 to 5, respectively,

the experimental results of fig. 1 show that the linear equation is y-0.8952 x +0.4372 and the correlation coefficient is R²＝0.7812；

The experimental result of fig. 2 shows that the equation of the straight line is y 1.0358x +0.0112, and the correlation coefficient is R²＝0.9743；

The experimental results of fig. 3 show that the linear equation is y, 1.0031x +0.0063 and the correlation coefficient is R²＝0.9759；

The experimental results of fig. 4 show that the linear equation is y-0.9749 x +0.0700 and the correlation coefficient is R²＝0.9783；

The experimental results of fig. 5 show that the linear equation is y ═ 1.0001x +0.0319, and the correlation coefficient is R²＝0.9766；

The experimental results of fig. 6 show that the linear equation is y 1.0453x +0.0146 and the correlation coefficient is R²＝0.9803；

By comparing fig. 1 with fig. 2 to fig. 6, we find that the detection reagent can rapidly and effectively dissociate testosterone from the combined eggs, so that the sensitivity and specificity of the testosterone immunoassay reagent can be obviously improved.

The foregoing embodiments are merely illustrative of the principles of the present invention and its efficacy, and are not to be construed as limiting the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.