WO2024138959A1

WO2024138959A1 - Sialidase neu1 chemiluminescent detection kit and method for detecting sialidase neu1

Info

Publication number: WO2024138959A1
Application number: PCT/CN2023/088733
Authority: WO
Inventors: 马芳; 姚凯成
Original assignee: 成都思瑞多医疗科技有限公司
Priority date: 2022-12-29
Filing date: 2023-04-17
Publication date: 2024-07-04
Also published as: CN116047072A

Abstract

A sialidase NEU1 chemiluminescent detection kit and a method for detecting a sialidase NEU1. The kit comprises a first reagent and a second reagent. The first reagent contains immunomagnetic particles, wherein the immunomagnetic particles are first magnetic particles or second magnetic particles; the first magnetic particles are streptavidin magnetic particles having surfaces connected to a coating, and the coating is a first antibody marked by a biotin ester; and the second magnetic particles are carboxyl magnetic particles having surfaces connected to a second antibody. The second reagent contains a third antibody marked by a luminescent marker. The first antibody, the second antibody and the third antibody are all antibodies capable of specifically binding to NEU1. The kit has high sensitivity, high accuracy, high repeatability, a wide detection range and high stability, and can quickly detect the content of the NEU1 in a sample to be tested, especially a seminal plasma sample.

Description

Sialidase NEU1 chemiluminescence detection kit and method for detecting sialidase NEU1

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure claims priority to a Chinese patent application with application number CN202211715911.3 filed with the Chinese Patent Office on December 29, 2022, and entitled “Sialidase NEU1 Chemiluminescent Detection Kit and Method for Detecting Sialidase NEU1”, the entire contents of which are incorporated by reference into the present disclosure.

Technical Field

The present disclosure relates to the technical field of immunoassay, and in particular to a chemiluminescent detection kit for sialidase NEU1 and a method for detecting sialidase NEU1.

Background technique

Sialic acid (SA), also known as neuraminic acid, is a general term for a class of nonacarbonic monosaccharides commonly found at the end of glycoprotein or glycolipid molecules in organisms. Sialic acid exists on the surface of sperm membranes and plays an important role in spermatogenesis, epididymal sperm maturation, and sperm-egg binding.

Sialidases, also known as neuraminidase (NEU), are a family of enzymes that break down sialic acid on the cell surface. Sialidases mainly include four subtypes of proteins: NEU1, NEU2, NEU3, and NEU4. Among them, NEU1 and NEU3 exist on the surface of sperm membranes, and NEU1 and NEU3 can affect sperm reproductive ability. During the process of in vitro capacitation of sialic acid in humans, sialic acid on the sperm membrane falls off from the cell membrane of sperm in the form of a single sugar molecule. At the same time, sialidases (NEU1 or NEU3) on the sperm membrane fall off due to the fluidity of the sperm membrane and participate in shearing to cause single sialic acid molecules to fall off. Therefore, the detection of NEU1 or NEU3 in seminal plasma samples can be used to assess sperm quality to determine whether men are infertile.

Therefore, there is an urgent need for a kit for detecting sialidase NEU1 in seminal plasma samples that has high sensitivity, high accuracy, high repeatability, wide detection range and high stability.

Summary of the invention

The present disclosure provides a chemiluminescent detection kit for sialidase NEU1, characterized by comprising: a first reagent and a second reagent;

The first reagent contains immunomagnetic particles, which are first magnetic particles and/or second magnetic particles; the first magnetic particles are streptavidin magnetic particles with a coating connected to the surface, and the coating is a first antibody labeled with biotin ester; the second magnetic particles are carboxyl magnetic particles with a second antibody connected to the surface;

The second reagent contains a third antibody labeled with a luminescent marker;

Wherein, the first antibody, the second antibody and the third antibody are all antibodies that can specifically bind to NEU1.

Optionally, the luminescent marker is selected from any one of acridinium ester, terpyridine ruthenium, luminol and AMPPD.

Optionally, the luminescent marker is selected from acridinium esters.

Optionally, in the first magnetic particles, the mass ratio of the streptavidin magnetic particles to the coating is 100:(0.2-2); in the coating, the mass ratio of the first antibody to the biotin ester is 20:(1-2);

And/or, in the second magnetic particles, the mass ratio of the carboxyl magnetic particles to the second antibody is 100:(0.2-2).

Optionally, the immunomagnetic particles are first magnetic particles.

Optionally, the mass concentration of the immunomagnetic particles in the first reagent is 0.2-1 mg/mL.

Optionally, in the third antibody labeled with the luminescent marker, the mass ratio of the third antibody to the luminescent marker is 20:(1-5).

Optionally, the mass concentration of the third antibody labeled with the luminescent marker in the second reagent is 0.05-0.5 μg/mL.

Optionally, the first reagent also contains a protein protective agent.

Optionally, the mass ratio of the protein protective agent to the immunomagnetic particles is 1:(0.4-0.8).

Optionally, the mass concentration of the protein protective agent in the first reagent is 0.5-1.0 mg/mL.

Optionally, the immunomagnetic particles are first magnetic particles; the preparation method of the first reagent comprises: mixing the streptavidin magnetic particles with a first diluent, and obtaining a first precipitate after magnetic liquid separation; mixing the first precipitate with a second diluent, and then adding the coating material to mix for 30-60 minutes, and obtaining a second precipitate after magnetic liquid separation; mixing the second precipitate with a third diluent, and then adding a first blocking solution to mix for 30-60 minutes, and obtaining a third precipitate after magnetic liquid separation; mixing the third precipitate with a fourth diluent;

Wherein, the first diluent, the second diluent, the third diluent and the fourth diluent each independently include 5-20mM PBS buffer, 0.1-1wt% bovine serum albumin, 0.01-0.05wt% Triton X-100 and 0.05-0.1wt% of the protein protective agent; the first blocking solution includes 0.1-0.5wt% biotin buffer.

Optionally, the method for preparing the coating comprises: dissolving the biotin ester in dimethyl sulfoxide, adding the first antibody and mixing for 30-60 minutes; then adding the first stop solution and mixing for 10-30 minutes, and dialyzing in PBS buffer for 12-24 hours; wherein the first stop solution comprises 0.5-1M Tris buffer.

Optionally, the immunomagnetic particles are second magnetic particles; the preparation method of the first reagent comprises: mixing the carboxyl magnetic particles with a fifth diluent, and obtaining a fourth precipitate after magnetic liquid separation; mixing the fourth precipitate with a sixth diluent, adding a seventh diluent to mix, and then adding the second antibody to mix for 2-4 hours, and obtaining a fifth precipitate after magnetic liquid separation; mixing the fifth precipitate with an eighth diluent, and then adding a second blocking solution to mix for 30-45 minutes, and obtaining a sixth precipitate after magnetic liquid separation; mixing the sixth precipitate with a ninth diluent;

Wherein, the fifth dilution liquid, the sixth dilution liquid and the eighth dilution liquid each independently include 0.05-0.2M morpholineethanesulfonic acid buffer; the seventh dilution liquid includes 15-35mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride buffer; the second blocking liquid includes 20-30mM aminoacetic acid buffer; the ninth dilution liquid includes 5-20mM PBS buffer, 0.1-1wt% bovine serum albumin, 0.01-0.05wt% Triton X-100 and 0.05-0.1wt% of the protein protective agent.

Optionally, the preparation method of the second reagent comprises: firstly mixing the solution containing the luminescent marker and the third antibody for 30-60 minutes under light-proof conditions; then adding the second stop solution and mixing for 10-30 minutes under light-proof conditions; after purification, dissolving the purified substance in the tenth dilution solution;

Wherein, the second stop solution includes 0.1-1wt% lysine buffer; the tenth dilution solution includes 5-20mM PBS buffer, 0.1-1wt% casein and 0.01-0.05wt% Triton X-100.

The present disclosure also provides a chemiluminescent detection reagent for sialidase NEU1, comprising: a first reagent and a second reagent;

The second reagent contains a third antibody labeled with a luminescent marker;

Optionally, the luminescent marker is selected from acridinium esters.

Optionally, the immunomagnetic particles are first magnetic particles.

Optionally, the first reagent also contains a protein protective agent.

The present disclosure also provides a method for detecting sialidase NEU1, wherein the method for detecting sialidase NEU1 adopts the sialidase NEU1 chemiluminescent detection kit according to any one of claims 1 to 9; the method for detecting sialidase NEU1 comprises: mixing a sample to be tested, the first reagent and the second reagent, performing a first incubation, and then performing detection using a chemiluminescent immunoassay analyzer.

Optionally, the method for detecting sialidase NEU1 further comprises: after the first incubation, adding pre-excitation solution and excitation solution for second incubation, and then detecting using the chemiluminescence immunoassay analyzer; wherein the pre-excitation solution comprises 0.5-1.0wt% peroxide; and the excitation solution comprises 0.2-0.5M sodium hydroxide.

Optionally, the temperatures of the first incubation and the second incubation are independently 36.5-37.5° C., the time of the first incubation is 8-12 min, and the time of the second incubation is 1-5 min.

The present disclosure also provides a method for diagnosing fertility of a male subject, comprising:

A) contacting the sialidase NEU1 chemiluminescent detection kit or the sialidase NEU1 chemiluminescent detection reagent described above with a sample from the subject under conditions sufficient for a binding reaction to occur; and

B) Detection of immune complexes produced by the binding reaction.

Optionally, the sample is seminal plasma.

Detailed ways

In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be described clearly and completely below. If no specific conditions are specified in the embodiments, the conventional conditions or the manufacturer's The reagents and instruments used were all commercially available without manufacturer indication.

In one embodiment of the present disclosure, a sialidase detection kit is provided, comprising a first reagent and a second reagent. The first reagent contains immunomagnetic particles, which are first magnetic particles and/or second magnetic particles; the first magnetic particles are streptavidin magnetic particles with a coating connected to the surface, and the coating is a first antibody labeled with biotin ester; the second magnetic particles are carboxyl magnetic particles with a second antibody connected to the surface. The second reagent contains a third antibody labeled with a luminescent marker.

Among them, the first antibody, the second antibody and the third antibody are all antibodies that can specifically bind to NEU1.

It should be noted that, in the present disclosure, streptavidin magnetic particles refer to magnetic particles containing streptavidin; and carboxyl magnetic particles refer to magnetic particles containing carboxyl groups.

The chemiluminescent detection kit for sialidase NEU1 provided by the present disclosure adopts the double antibody sandwich detection principle, and the immunomagnetic particles (containing the first antibody or the second antibody) and the third antibody labeled with a luminescent marker in the kit can bind to NEU1. Using the chemiluminescent detection kit for sialidase NEU1 provided by the present disclosure and with the help of magnetic particle chemiluminescent technology, the content of NEU1 in the sample to be tested can be quickly detected, and the kit has high sensitivity, high accuracy, high repeatability, wide detection range and high stability.

In the present disclosure, the luminescent marker is selected from any one of acridinium ester, terpyridine ruthenium, luminol and AMPPD. The luminescent marker has a larger luminescence intensity and is easier to be detected by a chemiluminescent immunoassay instrument.

Optionally, the luminescent marker is selected from acridinium ester. Without being bound by theory, acridinium ester has a high quantum yield, high luminescence efficiency and high intensity; the luminescence system of acridinium ester is simple and does not require a catalyst, thereby improving the signal-to-noise ratio; the molecular weight of acridinium ester is small, and the conformation of the third antibody is minimally affected, and the third antibody labeled with the luminescent marker has good stability; therefore, when the luminescent marker is selected from acridinium, the kit is more sensitive and more stable.

In an optional embodiment of the present disclosure, the immunomagnetic particles are the first magnetic particles. Without being bound by theory, the immunomagnetic particles are the first magnetic particles, and the biotin ester in the coating can specifically bind to streptavidin to achieve signal amplification.

In the present disclosure, in the first magnetic particle, the mass ratio of streptavidin magnetic particle to coating is 100:(0.2-2); in the coating, the mass ratio of the first antibody to biotin ester is 20:(1-2). Without being bound by theory, under the above ratio conditions, the sensitivity, accuracy, repeatability, detection range and stability of the kit are further improved.

As an example, the mass ratio of streptavidin magnetic particles to the coating can be, for example, 100:(0.4-1.8), 100:(0.6-1.6) or 100:(0.8-1.4), such as 100:0.2, 100:0.5, 100:1, 100:1.5 or 100:2, etc., or an interval value between any two of the above endpoints. As an example, the mass ratio of the first antibody to the biotin ester can be, for example, 100:(0.4-1.8), 100:(0.6-1.6) or 100:(0.8-1.4), such as 100:0.2, 100:0.5, 100:1, 100:1.5 or 100:2, etc., or an interval value between any two of the above endpoints. The ratio can be, for example, 20:(1.1-1.9), 20:(1.3-1.7) or 20:(1.5-1.6), such as 20:1.2, 20:1.4, 20:1.6, 20:1.8 or 20:2.0, etc., or an interval value between any two of the above endpoint values.

In the second magnetic particles, the mass ratio of the second antibody to the carboxyl magnetic particles is 100:(0.2-2). Without being bound by theory, the above ratio conditions are conducive to further improving the sensitivity, accuracy, repeatability, detection range and stability of the kit.

As an example, the mass ratio of the second antibody to the carboxyl magnetic particles can be, for example, 100:(0.4-1.8), 100:(0.6-1.6) or 100:(0.8-1.4), such as 100:0.2, 100:0.5, 100:1, 100:1.5 or 100:2, etc., or an interval value between any two of the above endpoint values.

Optionally, the mass concentration of the immunomagnetic particles in the first reagent is 0.2-1.0 mg/mL, which is beneficial to further improve the sensitivity, accuracy, repeatability, detection range and stability of the kit.

As an example, the mass concentration of the immunomagnetic particles in the first reagent can be, for example, 0.3-0.9 mg/mL, 0.4-0.8 mg/mL or 0.5-0.7 mg/mL, such as 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL or 1.0 mg/mL, etc., or an interval value between any two of the above endpoint values.

In some optional embodiments of the present disclosure, the first reagent further contains a protein protecting agent.

It should be noted that, in the present disclosure, the protein protecting agent refers to: a protein protecting agent used to protect the antibody (first antibody or second antibody, third antibody) in the first reagent and the second reagent to maintain the biological activity of the first antibody (or second antibody) and the third antibody.

Optionally, the first reagent also contains a protein protective agent, which is beneficial to improving the stability of the kit.

As an example, the protein protective agent can be the IVD-specific protein protective agent of Zhengzhou Saitukang Biotechnology Co., Ltd.

Optionally, the mass ratio of the protein protective agent to the immunomagnetic microparticles is 1:(0.4-0.8). Without being bound by theory, the mass ratio of the protein protective agent to the immunomagnetic microparticles within the above range is beneficial to further improve the stability of the kit.

As an example, the mass ratio of the protein protective agent to the immunomagnetic particles can be 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, etc., or an interval value between any two of the above endpoints.

Optionally, the mass concentration of the protein protective agent in the first reagent is 0.5-1.0 mg/mL. Without being bound by theory, the mass concentration of the protein protective agent in the first reagent is within the above range, which is conducive to further improving the stability of the kit.

As an example, the mass concentration of the protein protectant in the first reagent can be, for example, 0.55-0.85 mg/mL, 0.6-0.8 mg/mL or 0.65-0.75 mg/mL, such as 0.5 mg/mL, 0.8 mg/mL and 1.0 mg/mL, etc., or an interval value between any two of the above endpoint values.

Optionally, when the immunomagnetic particles are the first magnetic particles, the preparation method of the first reagent includes: mixing the streptavidin magnetic particles with the first diluent, and obtaining a first precipitate after magnetic liquid separation; mixing the first precipitate with the second diluent, then adding the coating material and mixing for 30-60 minutes, and obtaining a second precipitate after magnetic liquid separation; mixing the second precipitate with the third diluent, and then adding the first blocking liquid and mixing for 30-60 minutes, and obtaining a third precipitate after magnetic liquid separation; mixing the third precipitate with the fourth diluent. Wherein, the first diluent, the second diluent, the third diluent and the fourth diluent each independently include 5-20mM PBS buffer, 0.1-1wt% bovine serum albumin, 0.01-0.05wt% Triton X-100 and 0.05-0.1wt% protein protective agent; the first blocking liquid includes 0.1-0.5wt% biotin buffer.

The above technical solution can quickly prepare the first reagent, and the preparation method is simple and easy to carry out large-scale production.

Optionally, the preparation method of the coating includes: dissolving biotin ester in dimethyl sulfoxide, adding the first antibody and mixing for 30-60 minutes; then adding the first stop solution and mixing for 10-30 minutes, and dialyzing in PBS buffer for 12-24 hours; wherein the first stop solution includes 0.5-1M Tris buffer.

The above technical solution can quickly prepare the coating, and the preparation method is simple and easy to carry out large-scale production.

When the immunomagnetic particles are the second magnetic particles, the preparation method of the first reagent includes: mixing the carboxyl magnetic particles with the fifth diluent, and obtaining a fourth precipitate after magnetic liquid separation; mixing the fourth precipitate with the sixth diluent, adding the seventh diluent to mix, and then adding the second antibody to mix for 2-4 hours, and obtaining a fifth precipitate after magnetic liquid separation; mixing the fifth precipitate with the eighth diluent, and then adding the second blocking liquid to mix for 30-45 minutes, and obtaining a sixth precipitate after magnetic liquid separation; mixing the sixth precipitate with the ninth diluent. Among them, the fifth dilution liquid, the sixth dilution liquid and the eighth dilution liquid each independently include 0.05-0.2M morpholineethanesulfonic acid buffer; the seventh dilution liquid includes 15-35mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride buffer; the second blocking liquid includes 20-30mM aminoacetic acid buffer; the ninth dilution liquid includes 5-20mM PBS buffer, 0.1-1wt% bovine serum albumin, 0.01-0.05wt% Triton X-100 and 0.05-0.1wt% protein protective agent.

The above technical solution uses the EDC/NHS principle to covalently link the carboxyl group of the carboxyl magnetic microparticles and the amino group on the second antibody, which can quickly prepare the first reagent, and the preparation method is simple and easy to mass produce.

It should be noted that in the present disclosure, morpholineethanesulfonic acid refers to a substance with a CAS number of 4432-31-9, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride refers to a substance with a CAS number of 25952-53-8, and aminoacetic acid refers to a substance with a CAS number of 56-40-6.

In some optional embodiments of the present disclosure, the immunomagnetic particles are the first magnetic particles; in the first magnetic particles, the biotin ester in the coating can specifically bind to streptavidin to achieve signal amplification.

In the present disclosure, in the third antibody labeled with a luminescent marker, the mass ratio of the third antibody to the luminescent marker is 20:(1-5). Without being bound by theory, the above ratio conditions are conducive to further improving the sensitivity, accuracy, repeatability, detection range and stability of the kit.

It should be noted that the third antibody labeled with a fluorescent marker refers to a whole substance containing a fluorescent marker and the third antibody.

As an example, the mass ratio of the third antibody to the luminescent marker can be, for example, 20:(1.5-4.5), 20:(2-4) or 20:(2.5-3), such as 20:1, 20:2.5, or 20:5, etc., or an interval value between any two of the above endpoint values.

Optionally, the mass concentration of the third antibody labeled with a luminescent marker in the second reagent is 0.05-0.5 μg/mL, which is beneficial to further improve the sensitivity, accuracy, repeatability, detection range and stability of the kit.

As an example, the mass concentration of the third antibody labeled with a luminescent marker in the second reagent can be, for example, 0.1-0.04 μg/mL, 0.15-0.35 μg/mL or 0.2-0.3 μg/mL, such as 0.05 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.25 μg/mL, 0.35 μg/mL and 0.5 μg/mL, etc., or an interval value between any two of the above endpoint values.

The preparation method of the second reagent includes: first mixing the solution containing the luminescent marker and the third antibody under light-proof conditions for 30-60 minutes; then adding the second stop solution and mixing under light-proof conditions for 10-30 minutes; after purification, dissolving the purified substance in the tenth dilution solution. The second stop solution includes 0.1-1wt% lysine buffer; the tenth dilution solution includes 5-20mM PBS buffer, 0.1-1wt% casein and 0.01-0.05wt% Triton X-100.

The above technical solution can quickly prepare the second reagent, and the preparation method is simple and easy to carry out large-scale production.

The sialidase detection kit also includes a pre-excitation solution and an excitation solution used in chemiluminescent immunoassay detection. The pre-excitation solution includes 0.5-1.0 wt% peroxide; the excitation solution includes 0.2-0.5 M sodium hydroxide.

The present disclosure also provides a method for detecting sialidase NEU1, and the method for detecting sialidase NEU1 adopts the above-mentioned sialidase NEU1 chemiluminescent detection kit; the method for detecting sialidase NEU1 comprises: mixing a sample to be tested, a first reagent and a second reagent, performing a first incubation, and then detecting using a chemiluminescent immunoassay analyzer.

The method for detecting sialidase NEU1 provided in the present disclosure uses the sialidase NEU1 chemiluminescent detection kit provided in the present disclosure, and can quickly, efficiently and accurately measure the content of NEU1 in a sample to be tested (especially a seminal plasma sample).

Optionally, the method for detecting sialidase NEU1 further comprises: after the first incubation, adding pre-excitation solution and excitation solution for second incubation, and then detecting using a chemiluminescence immunoassay analyzer; wherein the pre-excitation solution comprises 0.5-1.0wt% peroxide; and the excitation solution comprises 0.2-0.5M sodium hydroxide.

In some optional embodiments, the temperatures of the first incubation and the second incubation are independently 36.5-37.5° C., the time of the first incubation is 8-12 min, and the time of the second incubation is 1-5 min.

As an example, the temperature of the first incubation and the second incubation are both 37°C.

An embodiment of the present disclosure also provides a method for diagnosing fertility of a male subject, comprising:

A) contacting the sialidase NEU1 chemiluminescent detection kit or sialidase NEU1 chemiluminescent detection reagent with the sample from the subject under conditions sufficient for a binding reaction to occur to perform a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

Optionally, the sample is seminal plasma.

The present disclosure provides a chemiluminescent detection kit for sialidase NEU1 and a method for detecting sialidase NEU1, which can be used to detect NEU1 in seminal plasma samples and have high sensitivity, high accuracy, high repeatability, wide detection range and high stability.

Example

Example 1

This embodiment provides a chemiluminescent detection kit for sialidase NEU1. The chemiluminescent detection kit for sialidase NEU1 consists of a first reagent and a second reagent, and is prepared by the following method:

(1) Preparation of biotin ester-labeled NEU1 antibody 1

NEU1 antibody 1 (purchased from Chengdu Besty Biotechnology Co., Ltd., model B9) was dialyzed against 20mM PBS buffer for 20h and then recovered. 2mg of biotin ester was dissolved in 220μL dimethyl sulfoxide to obtain a biotin ester solution. 1mg of the dialyzed NEU1 antibody 1 was added with 15μL of the biotin ester solution and mixed for 45min; 30μL of the first stop solution was added and mixed for 20min; then dialyzed against 20mM PBS buffer for 20h to recover the biotin ester-labeled NEU1 antibody 1.

Wherein, the first stop solution is 1.0 M Tris buffer.

(2) Preparation of the first reagent

10 mg of streptavidin magnetic particles were added to 10 mL of the first diluent and mixed, and then placed in a magnetic separator for magnetic liquid separation, and the supernatant was removed to obtain a first precipitate; after washing the first precipitate twice, 10 mL of the second diluent was used to resuspend the first precipitate.

Add 0.1 mg of the biotin ester-labeled NEU1 antibody 1 obtained in step (1) to the solution of the resuspended first precipitate, mix and react for 45 minutes; place in a magnetic separator for magnetic liquid separation, remove the supernatant, and obtain a second precipitate. Use 10 mL of the third diluent to resuspend the second precipitate, then add 1 mL of the first blocking solution, mix The reaction was continued for 45 minutes; the mixture was placed in a magnetic separator for magnetic liquid separation, and the supernatant was removed to obtain a third precipitate. The third precipitate was washed twice with the first washing solution, and then the third precipitate was resuspended with 30 mL of the fourth diluent to obtain a first reagent.

The first diluent, the second diluent, the third diluent and the fourth diluent are all composed of 10 mM PBS buffer, 0.5 wt% bovine serum albumin, 0.02 wt% Triton X-100 and 0.05 wt% protein protectant (IVD-specific protein protectant of Zhengzhou Saitukang Biotechnology Co., Ltd.). The first blocking solution is 0.2 wt% biotin buffer.

(3) Preparation of the second reagent

NEU1 antibody 2 (purchased from Chengdu Besty Biotechnology Co., Ltd., model G3) was dialyzed against 20 mM PBS buffer for 20 h and then recovered.

To the dialyzed NEU1 antibody 2, add 30 μL of 10 mM acridinium ester solution, mix and react for 45 minutes in the dark; add 0.2 mL of the second stop solution, mix and react for 20 minutes in the dark; then purify through a desalting column, dissolve the purified substance in the tenth dilution solution, dilute to a mass concentration of 0.1 μg/mL of acridinium-labeled NEU1 antibody 2, and obtain a second reagent.

Among them, the second stop solution is 1.0wt% lysine buffer; the tenth dilution solution is composed of 10mM PBS buffer, 0.5wt% bovine serum albumin and 0.02wt% Triton X-100.

Example 2

This embodiment provides a chemiluminescent detection kit for sialidase NEU1, which consists of a first reagent and a second reagent. The difference between this embodiment and embodiment 1 is that this embodiment does not include step (1), and step (2) of this embodiment is different from that of embodiment 1.

Step (2) of this embodiment is as follows:

NEU1 antibody 1 (purchased from Chengdu Besty Biotechnology Co., Ltd., model B9) was dialyzed for 20 h with 20 mM PBS buffer and then recovered. Then 10 mg of carboxyl magnetic particles were added to 10 mL of the fifth dilution, mixed, placed in a magnetic separator for magnetic liquid separation, and the supernatant was removed to obtain a fourth precipitate; after washing the fourth precipitate twice, the fourth precipitate was resuspended with 10 mL of the sixth dilution.

Add 1.5 mL of the seventh dilution to the solution of the resuspended fourth precipitate, mix well; then add 100 μg of the dialyzed NEU1 antibody 1, mix well for 3 hours; place in a magnetic separator for magnetic liquid separation, remove the supernatant, obtain the fifth precipitate, and wash the fifth precipitate twice. Use 5 mL of the eighth dilution to resuspend the fifth precipitate, then add 5 mL of the second blocking solution, mix well for 40 minutes; place in a magnetic separator for magnetic liquid separation, remove The supernatant was used to obtain the sixth precipitate, which was washed twice and then resuspended in 30 mL of the ninth dilution solution to obtain the first reagent.

Among them, the fifth dilution liquid, the sixth dilution liquid and the eighth dilution liquid are all 0.1M morpholineethanesulfonic acid buffer; the seventh dilution liquid is 25mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride buffer; the second blocking liquid is 25mM aminoacetic acid buffer; the ninth dilution liquid is composed of 10mM PBS buffer, 0.5wt% bovine serum albumin, 0.02wt% Triton X-100 and 0.05wt% protein protectant (IVD-specific protein protectant of Zhengzhou Saitukang Biotechnology Co., Ltd.).

Example 3

The present embodiment provides a sialidase NEU1 chemiluminescent detection kit, which consists of a first reagent and a second reagent. The difference between the present embodiment and embodiment 1 is that the first dilution liquid, the second dilution liquid, the third dilution liquid and the fourth dilution liquid are all composed of 10 mM PBS buffer, 0.5 wt% bovine serum albumin and 0.02 wt% Triton X-100.

Example 4

The present embodiment provides a sialidase NEU1 chemiluminescent detection kit, which consists of a first reagent and a second reagent. The difference between the present embodiment and embodiment 2 is that the ninth dilution solution consists of 10 mM PBS buffer, 0.5 wt% bovine serum albumin and 0.02 wt% Triton X-100.

Experimental Example 1

The sensitivity of the sialidase NEU1 chemiluminescent detection kit provided in Example 1 was tested. The sensitivity test results are shown in Table 1.

The luminescence value in the sensitivity detection process is determined by a chemiluminescence immunoassay, and the steps for determining the luminescence value are as follows: (1) diluting the seminal plasma 5 times with 20 mM PBS buffer to prepare a semen sample; (2) taking 30 μL of the semen sample, adding 50 μL of the first reagent and 100 μL of the second reagent, mixing, and incubating at 37°C for 10 minutes to form an immune complex; (3) using a fully automatic chemiluminescence immunoassay to clean the immune complex; (4) adding 100 μL of pre-excitation solution and 100 μL of excitation solution to the cleaned immune complex, mixing, and incubating at 37°C for 2 minutes, and using a fully automatic chemiluminescence immunoassay to detect the luminescence signal value; wherein the pre-excitation solution contains 0.7 wt% peroxide, and the excitation solution contains 0.3 M sodium hydroxide.

The sensitivity detection method is as follows: Repeat the measurement of zero-concentration calibrator A (0ng/mL) 20 times, obtain the luminescence values of the 20 measurements, calculate the mean value (M) and standard deviation (SD), and obtain the luminescence value corresponding to (M+2SD). Based on the mean of the luminescence values of the 20 measurements of zero-concentration calibrator A and the mean of the luminescence values of the 3 measurements of the adjacent calibrator B (1ng/mL), a linear equation is obtained, and the luminescence value corresponding to (M+2SD) is substituted into the linear equation. The corresponding concentration is calculated and is the limit of blank (LOB).

Table 1

It can be seen that the LOB of the sialidase detection kit of Example 1 of the present disclosure is 0.0318 ng/mL, which is much smaller than the current industry requirement for the sensitivity of the kit (0.2 ng/mL), indicating that the sialidase detection kit provided in Example 1 has high sensitivity.

Experimental Example 2

The accuracy of the sialidase NEU1 chemiluminescent detection kit provided in Example 1 was tested. The accuracy test results are shown in Table 2.

The luminescence value in the accuracy detection process is measured by a chemiluminescence immunoassay analyzer, and the steps for measuring the luminescence value are the same as those in Experimental Example 1.

The recovery rate is calculated according to the following formula:

Where:

R is the recovery rate; C is the detection concentration of the recovered sample, in ng/mL; Vo is the volume of the added sample to be tested, in mL; V is the volume of the added sample, in mL; Co is the detection concentration of the added sample to be tested, in ng/mL; Cs is the detection concentration of the added sample, which is 1000ng/mL.

Table 2

In Table 2, S1, S2 and S3 are samples of low, medium and high concentrations, respectively. “Before adding” refers to the sample to be tested without adding the high-value sample; “After adding 10% high-value sample” refers to adding the high-value sample to the low, medium and high concentration samples, and the volume of the added high-value sample is 10% of the total volume of the sample to be tested.

As can be seen from Table 2, the recovery rate of the sialidase NEU1 chemiluminescent detection kit of Example 1 of the present disclosure is 96-101%, which is much higher than the current industry requirements for the accuracy of the kit (recovery rate: 85% to 115%), indicating that the sialidase detection kit provided in Example 1 has high accuracy.

Experimental Example 3

The linearity detection was performed on the sialidase NEU1 chemiluminescence detection kit provided in Example 1. The linearity detection results are shown in Table 3.

The luminescence value in the linear detection process is measured by a chemiluminescence immunoassay analyzer, and the steps for measuring the luminescence value are the same as those in Experimental Example 1.

The linear detection method is as follows: dilute the high-value sample close to the upper limit of the detection range into 9 concentrations according to a certain ratio, among which the lowest concentration sample should be close to the lower limit of the detection range; measure the luminescence values of the 9 diluted concentrations, repeat the measurement 3 times for each, and use the least squares method to fit the average value of the measured concentration to the theoretical concentration or dilution ratio, and calculate the linear correlation coefficient (r).

table 3

In Table 3, L1 to L9 are 9 samples to be tested with concentrations from high to low.

As can be seen from Table 3, the linear correlation coefficient r of the sialidase detection kit of Example 1 of the present disclosure is 1.00, which meets the current industry requirements for the linearity of the kit (r≥0.99), indicating that the sialidase detection kit provided in Example 1 has a wide detection range.

Experimental Example 4

The chemiluminescent detection kit for sialidase NEU1 provided in Example 1 was tested for repeatability. The test results for repeatability are shown in Table 4.

The luminescence value in the repeatability detection process was measured using a chemiluminescence immunoassay analyzer, and the steps for measuring the luminescence value were the same as those in Experimental Example 1.

The repeatability test method is as follows: the luminescence value of a sample is measured 10 times, and the coefficient of variation (CV) is calculated according to the following formula:

Where:

CV is the coefficient of variation, M is the mean of 10 luminescence values, and SD is the standard deviation of 10 luminescence values.

Table 4

As can be seen from Table 4, the coefficient of variation CV of the sialidase detection kit of Example 1 of the present disclosure is 0.9-2.7%, which is much lower than the current industry requirement for the coefficient of variation CV of the kit (CV≤10%), indicating that the sialidase detection kit provided in Example 1 has high repeatability.

Experimental Example 5

The stability of the sialidase NEU1 chemiluminescent detection kits provided in Example 1 and Example 3 was tested respectively. The results of the stability test are shown in Table 5.

The luminescence value during the stability test was measured using a chemiluminescence immunoassay analyzer, and the steps for measuring the luminescence value were the same as those in Experimental Example 1.

table 5

It can be seen from Table 5 that the sialidase NEU1 chemiluminescent detection kits provided in Examples 1 and 3 of the present disclosure both have good thermal stability, and the thermal stability of the sialidase NEU1 chemiluminescent detection kit provided in Example 1 is higher than the thermal stability of the sialidase NEU1 chemiluminescent detection kit provided in Example 3.

The sialidase NEU1 chemiluminescent detection kit provided by the present disclosure has high sensitivity, high accuracy, high repeatability, wide detection range and high stability, and can quickly detect the content of NEU1 in semen samples.

The embodiments described above are part of the embodiments of the present disclosure, rather than all of the embodiments. The detailed description of the embodiments of the present disclosure is not intended to limit the scope of the present disclosure claimed for protection, but merely represents selected embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure.

Industrial Applicability

The sialidase NEU1 chemiluminescent detection kit provided by the present disclosure has high sensitivity, high accuracy, high repeatability, wide detection range and high stability, and can quickly detect the content of NEU1 in a sample to be tested (especially a seminal plasma sample), and therefore has excellent industrial practical performance.

Claims

A chemiluminescent detection kit for sialidase NEU1, characterized by comprising: a first reagent and a second reagent;

The first reagent contains immunomagnetic particles, which are first magnetic particles and/or second magnetic particles; the first magnetic particles are streptavidin magnetic particles with a coating connected to the surface, and the coating is a first antibody labeled with biotin ester; the second magnetic particles are carboxyl magnetic particles with a second antibody connected to the surface;

The second reagent contains a third antibody labeled with a luminescent marker;

Wherein, the first antibody, the second antibody and the third antibody are all antibodies that can specifically bind to NEU1.
The chemiluminescent detection kit for sialidase NEU1 according to claim 1, characterized in that the luminescent marker is selected from any one of acridinium ester, terpyridine ruthenium, luminol and AMPPD;

Optionally, the luminescent marker is selected from acridinium esters.
The chemiluminescent detection kit for sialidase NEU1 according to claim 1 or 2, characterized in that in the first magnetic particles, the mass ratio of the streptavidin magnetic particles to the coating is 100:(0.2-2); in the coating, the mass ratio of the first antibody to the biotin ester is 20:(1-2);

And/or, in the second magnetic particles, the mass ratio of the carboxyl magnetic particles to the second antibody is 100:(0.2-2).
The sialidase NEU1 chemiluminescent detection kit according to claim 3, characterized in that the immunomagnetic particles are first magnetic particles;

Optionally, the mass concentration of the immunomagnetic particles in the first reagent is 0.2-1 mg/mL.
The sialidase NEU1 chemiluminescent detection kit according to any one of claims 1 to 4, characterized in that, in the third antibody labeled with the luminescent marker, the mass ratio of the third antibody to the luminescent marker is 20:(1-5);

Optionally, the mass concentration of the third antibody labeled with the luminescent marker in the second reagent is 0.05-0.5 μg/mL.
The sialidase NEU1 chemiluminescent detection kit according to any one of claims 3 to 5, characterized in that the first reagent further contains a protein protective agent;

Optionally, the mass ratio of the protein protective agent to the immunomagnetic particles is 1:(0.4-0.8);

Optionally, the mass concentration of the protein protective agent in the first reagent is 0.5-1.0 mg/mL.
The chemiluminescent detection kit for sialidase NEU1 according to claim 6, characterized in that the immunomagnetic particles are first magnetic particles; and the preparation method of the first reagent comprises: Mixing with the first diluent, and obtaining a first precipitate after magnetic liquid separation; mixing the first precipitate with the second diluent, and then adding the coating material to mix for 30-60 minutes, and obtaining a second precipitate after magnetic liquid separation; mixing the second precipitate with the third diluent, and then adding the first blocking solution to mix for 30-60 minutes, and obtaining a third precipitate after magnetic liquid separation; mixing the third precipitate with the fourth diluent;

Wherein, the first diluent, the second diluent, the third diluent and the fourth diluent each independently comprise 5-20 mM PBS buffer, 0.1-1 wt% bovine serum albumin, 0.01-0.05 wt% Triton X-100 and 0.05-0.1 wt% of the protein protective agent; the first blocking solution comprises 0.1-0.5 wt% biotin buffer;

Optionally, the method for preparing the coating comprises: dissolving the biotin ester in dimethyl sulfoxide, adding the first antibody and mixing for 30-60 minutes; then adding the first stop solution and mixing for 10-30 minutes, and dialyzing in PBS buffer for 12-24 hours; wherein the first stop solution comprises 0.5-1M Tris buffer.
The chemiluminescent detection kit for sialidase NEU1 according to claim 6, characterized in that the immunomagnetic particles are second magnetic particles; the preparation method of the first reagent comprises: mixing the carboxyl magnetic particles with a fifth diluent, and obtaining a fourth precipitate after magnetic liquid separation; mixing the fourth precipitate with a sixth diluent, adding a seventh diluent to mix, and then adding the second antibody to mix for 2-4 hours, and obtaining a fifth precipitate after magnetic liquid separation; mixing the fifth precipitate with an eighth diluent, and then adding a second blocking solution to mix for 30-45 minutes, and obtaining a sixth precipitate after magnetic liquid separation; mixing the sixth precipitate with a ninth diluent;

Wherein, the fifth dilution liquid, the sixth dilution liquid and the eighth dilution liquid each independently include 0.05-0.2M morpholineethanesulfonic acid buffer; the seventh dilution liquid includes 15-35mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride buffer; the second blocking liquid includes 20-30mM aminoacetic acid buffer; the ninth dilution liquid includes 5-20mM PBS buffer, 0.1-1wt% bovine serum albumin, 0.01-0.05wt% Triton X-100 and 0.05-0.1wt% of the protein protective agent.
The chemiluminescent detection kit for sialidase NEU1 according to claim 1, characterized in that the preparation method of the second reagent comprises: first mixing the solution containing the luminescent marker and the third antibody under light-proof conditions for 30-60 minutes; then adding the second stop solution and mixing for 10-30 minutes under light-proof conditions; after purification, dissolving the purified substance in the tenth dilution solution;

Wherein, the second stop solution includes 0.1-1wt% lysine buffer; the tenth dilution solution includes 5-20mM PBS buffer, 0.1-1wt% casein and 0.01-0.05wt% Triton X-100.
A chemiluminescent detection reagent for sialidase NEU1, characterized by comprising: a first reagent and a second reagent;

The first reagent contains immunomagnetic particles, which are first magnetic particles and/or second magnetic particles; the first magnetic particles are streptavidin magnetic particles with a coating connected to the surface, and the coating is a first antibody labeled with biotin ester; the second magnetic particles are carboxyl magnetic particles with a second antibody connected to the surface;

The second reagent contains a third antibody labeled with a luminescent marker;

Wherein, the first antibody, the second antibody and the third antibody are all antibodies that can specifically bind to NEU1.
The chemiluminescent detection reagent for sialidase NEU1 according to claim 10, characterized in that the luminescent marker is selected from any one of acridinium ester, terpyridine ruthenium, luminol and AMPPD;

Optionally, the luminescent marker is selected from acridinium esters.
The sialidase NEU1 chemiluminescent detection reagent according to claim 10 or 11, characterized in that in the first magnetic particles, the mass ratio of the streptavidin magnetic particles to the coating is 100:(0.2-2); in the coating, the mass ratio of the first antibody to the biotin ester is 20:(1-2);

And/or, in the second magnetic particles, the mass ratio of the carboxyl magnetic particles to the second antibody is 100:(0.2-2).
The sialidase NEU1 chemiluminescent detection reagent according to claim 12, characterized in that the immunomagnetic particles are first magnetic particles;

Optionally, the mass concentration of the immunomagnetic particles in the first reagent is 0.2-1 mg/mL;

Optionally, in the third antibody labeled with the luminescent marker, the mass ratio of the third antibody to the luminescent marker is 20:(1-5);

Optionally, the mass concentration of the third antibody labeled with the luminescent marker in the second reagent is 0.05-0.5 μg/mL;

Optionally, the first reagent further contains a protein protective agent;

Optionally, the mass ratio of the protein protective agent to the immunomagnetic particles is 1:(0.4-0.8);

Optionally, the mass concentration of the protein protective agent in the first reagent is 0.5-1.0 mg/mL.
A method for detecting sialidase NEU1, characterized in that the method for detecting sialidase NEU1 adopts the sialidase NEU1 chemiluminescent detection kit according to any one of claims 1 to 9; the method for detecting sialidase NEU1 comprises: mixing a sample to be tested, the first reagent and the second reagent and performing a first incubation, and then detecting using a chemiluminescent immunoassay analyzer;

Optionally, the method for detecting sialidase NEU1 further comprises: after the first incubation, adding a pre-excitation solution and an excitation solution for a second incubation, and then using the chemiluminescence immunoassay analyzer for detection; wherein the pre-excitation solution comprises 0.5-1.0wt% peroxide; the excitation solution comprises 0.2-0.5M sodium hydroxide;

Optionally, the temperatures of the first incubation and the second incubation are independently 36.5-37.5° C., the time of the first incubation is 8-12 min, and the time of the second incubation is 1-5 min.
A method for diagnosing fertility in a male subject, comprising:

A) contacting the sialidase NEU1 chemiluminescent detection kit according to any one of claims 1 to 9 or the sialidase NEU1 chemiluminescent detection reagent according to any one of claims 10 to 13 with a sample from the subject under conditions sufficient for a binding reaction to occur; and

B) detecting immune complexes produced by the binding reaction;

Optionally, the sample is seminal plasma.