CN115980352A - A kind of HCV antigen coating pretreatment agent, antigen coating method and detection kit - Google Patents

Abstract

The invention is suitable for the technical field of immunodetection, and provides an HCV antigen coating pretreatment agent, an antigen coating method and a detection kit, which comprise a nonionic surfactant, a nonionic detergent, an antigen coating buffer solution and purified water; wherein the antigen coating buffer solution is phosphate buffer solution with the concentration of 0.1M and the pH value of 8.0; the invention provides an HCV antigen coating pretreatment agent, an antigen coating method and a detection kit, wherein the antigen pretreatment agent contains Tween, triton and tetraethylenepentamine, and compared with the prior art, the antigen coating pretreatment agent and the antigen coating preparation method provided by the invention can obviously improve the antigen coating efficiency, thereby effectively improving the sensitivity of the detection reagent, obtaining higher signal-to-noise ratio and being beneficial to reducing detection errors.

Description

HCV antigen coating pretreatment agent, antigen coating method and detection kit

Technical Field

The invention relates to the technical field of immune detection, in particular to an HCV antigen coating pretreatment agent, an antigen coating method and a detection kit.

Background Art

Hepatitis C is a systemic infectious disease caused by the hepatitis C virus (HCV) and mainly transmitted through the blood, with liver damage as the main feature. It is estimated that about 170 million people are infected with HCV worldwide, and about 70% of them become chronic. Among them, about 20% to 30% develop cirrhosis in 10 to 30 years, and 1% to 5% of them can further develop hepatocellular carcinoma. Hepatitis C infection has become a serious global public health problem.

At present, the commonly used methods for detecting HCV-specific antibodies include enzyme-linked immunosorbent assay, chemiluminescence and colloidal gold rapid test. The most commonly used method for detecting hepatitis C virus is immunoassay, which is based on the specific recognition of antigen-antibody.

Chemiluminescent immunoassay technology is currently recognized as an advanced immunodiagnostic technology in the world and is widely used in the diagnosis of tumor markers, infectious diseases, endocrine function, hormones, etc.

Chemiluminescent immunoassay includes two parts: immune reaction and chemiluminescence. Chemiluminescence refers to the formation of an excited intermediate by the catalysis of a catalyst or oxidation of an oxidant. When this excited intermediate returns to a stable ground state, it will release photons of equal energy level, resulting in luminescence. The chemiluminescent immunoassay process is to first label the luminescent substance or enzyme on the antigen or antibody. After the antigen and antibody specifically bind, an oxidant or luminescent substrate is added. After oxidation or reaction with the substrate, luminescence occurs. Based on the principle that the concentration of the analyte in the chemical detection system is linearly quantitatively related to the chemiluminescence intensity of the system under certain conditions, the chemiluminescence intensity of the system is detected by the instrument to determine the content of the analyte. According to the different antigen or antibody coating methods, chemiluminescence is divided into microplate type and microparticle type. The microparticle type is divided into magnetic microparticle type and non-magnetic microparticle type. Among them, the magnetic microparticle type is the most advanced chemiluminescence technology.

Under certain conditions, the functional groups on the surface of the magnetic microspheres react with the groups (amino/carboxyl, etc.) on proteins such as antigens or antibodies to form covalent bonds or non-covalent bonds, thereby coupling the target protein to the magnetic microspheres. Due to the particularity of the HCV antigen structure - small molecules and hydrophobicity, the current common antigen coating efficiency is generally not high, which leads to problems such as low sensitivity of the HCV detection kit, low signal-to-noise ratio, and high production cost.

Summary of the invention

The purpose of the present invention is to provide an HCV antigen coating pretreatment agent, an antigen coating method and a detection kit to solve the above technical problems.

To achieve the above object, the present invention provides the following technical solution: an HCV antigen coating pretreatment agent, comprising a nonionic surfactant, a nonionic detergent, an antigen coating buffer, and purified water.

Preferably, the nonionic surfactant includes Tween, Triton and tetraethylenepentamine.

Preferably, the type of Tween is one of Tween-20, Tween-60 and Tween-80, and the type of Triton is Triton-100.

Preferably, in the HCV antigen coating pretreatment agent, the mass percentage content of Tween is 0.01%-10%, the mass percentage content of Triton is 0.001%-10%, and the mass percentage content of tetraethylenepentamine is 0.0005%-1%.

Preferably, the antigen coating buffer is one of phosphate buffer, carbonate buffer and borate buffer.

A HCV antigen coating method comprises the following steps:

Step (1), firstly performing HCV antigen pretreatment with an HCV antigen pretreatment agent;

Step (2), binding the pretreated HCV antigen to a solid phase carrier.

Preferably, the HCV antigen pretreatment time in step (1) is 0.5-5 hours.

Preferably, the HCV antigen-coated solid phase carrier is one of carboxyl magnetic beads, amino magnetic beads and toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent. The HCV marker antigen reagent is one of alkaline phosphatase, horseradish peroxidase and acridinium ester.

The present invention has at least the following beneficial effects:

The present invention provides an HCV antigen coating pretreatment agent, an antigen coating method and a detection kit. The antigen pretreatment agent contains Tween, Triton and tetraethylenepentamine. Compared with the prior art, the antigen coating pretreatment agent and the antigen coating preparation method provided by the present invention can significantly improve the antigen coating efficiency, thereby effectively improving the sensitivity of the detection reagent, obtaining a higher signal-to-noise ratio, and being conducive to reducing the detection error.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

The antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0.

Among them, the non-ionic surfactants are Tween-20, Triton-100 and tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-20 is 0.1%, the mass percentage of Triton-100 is 0.01%, and the mass percentage of tetraethylenepentamine is 0.001%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 2

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

Among them, the antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0;

Among them, the non-ionic surfactants are Tween-20, Triton-100 and tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-20 is 0.5%, the mass percentage of Triton-100 is 0.1%, and the mass percentage of tetraethylenepentamine is 0.005%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 3

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

Among them, the antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0;

Among them, the non-ionic surfactants are Tween-20, Triton-100 and tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-20 is 1%, the mass percentage of Triton-100 is 0.05%, and the mass percentage of tetraethylenepentamine is 0.01%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 4

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

Among them, the antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0;

Among them, the non-ionic surfactants are Tween-20, Triton-100 and tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-20 is 3%, the mass percentage of Triton-100 is 1%, and the mass percentage of tetraethylenepentamine is 0.05%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 5

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

Among them, the antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0;

Among them, the non-ionic surfactants are Tween-20, Triton-100 and tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-20 is 5%, the mass percentage of Triton-100 is 2%, and the mass percentage of tetraethylenepentamine is 0.1%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 6

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

Among them, the antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0;

Among them, the non-ionic surfactants are Tween-20, Triton-100 and tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-20 is 8%, the mass percentage of Triton-100 is 5%, and the mass percentage of tetraethylenepentamine is 0.5%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 7

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

Among them, the antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0;

Among them, the non-ionic surfactants are Tween-20, Triton-100 and tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-20 is 10%, the mass percentage of Triton-100 is 6%, and the mass percentage of tetraethylenepentamine is 1%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 8

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

The antigen coating buffer is a carbonate buffer with a concentration of 0.1 M and a pH of 8.0.

Among them, the non-ionic surfactants are Tween-20 and Triton-100; the non-ionic detergent is tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-60 is 0.01%, the mass percentage of Triton-100 is 0.0001%, and the mass percentage of tetraethylenepentamine is 0.0005%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 0.5 h;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 9

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

The antigen coating buffer is a carbonate buffer with a concentration of 0.1 M and a pH of 8.0.

Among them, the non-ionic surfactants are Tween-20 and Triton-100; the non-ionic detergent is tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-60 is 0.01%, the mass percentage of Triton-100 is 0.0001%, and the mass percentage of tetraethylenepentamine is 0.005%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 5 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is carboxyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

Example 10

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

The antigen coating buffer is a borate buffer with a concentration of 0.1 M and a pH of 8.0.

Among them, the non-ionic surfactants are Tween-20 and Triton-100; the non-ionic detergent is tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-80 is 5%, the mass percentage of Triton-100 is 0.01%, and the mass percentage of tetraethylenepentamine is 1%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 5 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is carboxyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV labeled antigen reagent and an HCV coated antigen reagent, wherein the HCV labeled antigen reagent is horseradish peroxidase.

Embodiment 11

This embodiment provides an antigen coating pretreatment agent, which includes a non-ionic surfactant, a non-ionic detergent, an antigen coating buffer, and purified water.

The antigen coating buffer is a borate buffer with a concentration of 0.1 M and a pH of 8.0.

Among them, the non-ionic surfactants are Tween-20 and Triton-100; the non-ionic detergent is tetraethylenepentamine.

In the HCV antigen coating pretreatment agent, the mass percentage of Tween-80 is 5%, the mass percentage of Triton-100 is 10%, and the mass percentage of tetraethylenepentamine is 1%.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time being 1 hour;

Step (2), binding the pretreated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is amino magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is an acridinium ester.

Comparative Example 1

This embodiment provides an antigen coating pretreatment agent, including a non-ionic detergent, an antigen coating buffer, and purified water.

The antigen coating buffer is a phosphate buffer with a concentration of 0.1 M and a pH of 8.0.

A HCV antigen coating method comprises the following steps:

Step (1), firstly pre-treating HCV antigen with an HCV antigen pre-treating agent, the pre-treatment time is 2 hours;

Step (2), binding the pre-treated HCV antigen to a solid phase carrier, wherein the solid phase carrier coated with the HCV antigen is toluene yellow acyl magnetic beads.

An HCV coated antigen reagent is prepared by the above method.

A HCV detection kit comprises an HCV marker antigen reagent and an HCV coated antigen reagent, wherein the HCV marker antigen reagent is alkaline phosphatase.

The parameters of Examples 1-11 and Comparative Example 1 are shown in Table 1 below:

Table 1 Parameter comparison between Examples 1-11 and Comparative Example 1

Comparative Example 1 and Examples 1-11 were subjected to parallel comparative tests. During the experiment, each set of reagents simultaneously tested negative quality control and positive quality control (quality control samples were all substances that met domestic standards), and compared the sensitivity and signal-to-noise ratio. The following is a specific description of the method for preparing a reagent for detecting HCV antibodies. The specific test scheme is as follows:

Preparation of HCV antigen-coated methyl benzoyl magnetic beads:

(1) Take HCV antigen, add the above antigen pretreatment agent, and incubate at 37°C for 2 h;

(2) Take toluene yellow acyl magnetic beads (Toysl) and wash once with phosphate buffer;

(3) separation by magnetic separator and removal of supernatant;

(4) adding the antigen pretreated in (1) to the washed magnetic beads;

(5) After mixing, add the catalyst and react at 37°C for 24 h;

(6) Add 1% BSA and block at 37°C for 6 h;

(7) Wash 4 times with cleaning solution and store at 2-8℃.

(8) When in use, dilute the magnetic beads obtained in step (7) to 0.1 mg/mL with TRIS buffer.

Preparation of HCV antigen-coated carboxyl magnetic beads:

(1) Take HCV antigen, add the above antigen pretreatment agent, and incubate at 37°C for 5 h;

(2) Take the carboxyl magnetic beads and wash them three times with phosphate buffer;

(3) separation by magnetic separator and removal of supernatant;

(4) adding the antigen pretreated in (1) to the washed magnetic beads;

(5) After mixing, add activation reagent NHS and react at room temperature for 3 h;

(6) Add 1% BSA and block at room temperature for 2 h;

(7) Wash 4 times with cleaning solution and store at 2-8℃.

(8) When in use, dilute the magnetic beads obtained in step (7) to 0.1 mg/mL with TRIS buffer.

Preparation of HCV antigen-coated amino magnetic beads:

(1) Take HCV antigen, add the above antigen pretreatment agent, and incubate at 37°C for 1 h;

(2) Take the amino magnetic beads and wash them three times with phosphate buffer;

(3) separation by magnetic separator and removal of supernatant;

(4) adding the antigen pretreated in (1) to the washed magnetic beads;

(5) After mixing, add activation reagent glutaraldehyde and react at room temperature for 2 h;

(6) Add 2% BSA and block at room temperature for 1 h;

(7) Wash 4 times with cleaning solution and store at 2-8℃.

(8) When in use, dilute the magnetic beads obtained in step (7) to 0.1 mg/mL with TRIS buffer.

Preparation of HCV antigen labeled alkaline phosphatase:

(1) Take 10 mg of alkaline phosphatase (ALP) and add phosphate buffer to dilute ALP to 10 mg/mL;

(2) Add Trut's reagent (2 mg/mL) and mix well, and react at room temperature for 30 min;

(3) Ultrafiltration tube to remove unreacted Trut's reagent;

(4) Collect the ALP after the reaction is completed.

(5) Take 10 mg of antigen, dilute it to 10 mg/mL with phosphate buffer, add cross-linking agent SMCC, and mix carefully;

(6) Reaction at room temperature for 30 mins;

(7) removing unreacted cross-linking agent SMCC by ultrafiltration tube;

(8) Collect the antigen after the reaction is completed.

(9) mixing the product obtained in step (4) and step (8) in a volume ratio of 1:1;

(10) Reaction at room temperature for 1 h;

(11) Add 50% glycerol for storage.

(12) When used, dilute the labeled antigen obtained in step (11) to 0.1 ug/mL with TRIS buffer;

Preparation of HCV antigen labeled with horseradish peroxidase:

(1) Take 10 mg of horseradish peroxidase (HRP) and dilute it to 10 mg/mL in phosphate buffer;

(2) Add NaIO4 solution, mix well, and react at 4°C for 30 mins;

(3) Add ethylene glycol aqueous solution, mix well, and let stand for 30 minutes;

(4) Add antigen, put into dialysis bag, and dialyze overnight at 4°C;

(5) Add NaBH4 solution and let stand at 4°C for 2 h;

(6) Add an equal volume of saturated ammonium sulfate and let stand at 4°C for 1 h;

(7) Centrifuge, remove the supernatant, and resuspend the precipitate in ammonium sulfate;

(8) The solution in (7) was placed in a dialysis bag and dialyzed overnight;

(9) Centrifuge and take the supernatant as the enzyme conjugate.

(10) Add 50% glycerol for storage.

(11) When used, dilute the labeled antigen obtained in step (10) with TRIS buffer to 0.1 ug/mL

Preparation of HCV antigen labeled acridinium ester:

(1) Take 10 mg of acridinium ester (AE) and add DMSO to dissolve it to 5 mg/mL;

(2) Take the antigen, add phosphate buffer, and mix well;

(3) Add acridinium ester (AE) and mix well, and react at room temperature in the dark for 30 minutes;

(4) removing unreacted acridinium ester (AE) by ultrafiltration tube;

(5) The product is the AE marker;

(6) When used, dilute the labeled antigen obtained in step (5) to 0.1 ug/mL with TRIS buffer.

Testing procedures:

Take 50uL of the analyte and 50uL of HCV-coated magnetic bead antigen reagent, mix them evenly, react at 37℃ for 20mins, wash 4 times, then add 50uL of alkaline phosphatase-labeled antigen, react at 37℃ for 20mins, wash 4 times, then add substrate, perform luminescent reaction, and record the corresponding luminescent value of each analyte.

Test results

The test results are shown in Table 2, Table 3 and Table 4.

Table 2 Negative/positive quality control results of each embodiment

Table 3 Signal-to-noise ratio of positive quality control in each example

Table 4 Clinical sample test results

in:

P/N: RLU of the sample to be tested/RLU of the negative sample;

RLU1/P/N1: the test result of the present invention;

RLU2/P/N2: is the test result of a well-known domestic marketed product.

A total of 108 negative clinical samples were tested in this experiment. All the test results of the present invention were negative, with a specificity of 100% and a compliance rate of 100%. However, among the 108 negative samples tested by a well-known domestic marketed product, 3 false positives were detected, with a false positive probability of 2.8% and a specificity/compliance rate of 97.2%.

The experimental results show that the use of the antigen coating pretreatment agent to coat solid phase carriers such as magnetic beads significantly increases the coating efficiency, and the reagent sensitivity and signal-to-noise ratio are significantly improved.

In the prior art, the coating efficiency of antigens and magnetic beads is very low, the activity of the antigens is impaired after coating, and a large amount of antigens needs to be wasted, increasing the production cost. The present invention optimizes the antigen coating method through a large number of experiments, and adds an antigen pretreatment stage before the antigen and magnetic beads are coated. Adding this process is beneficial to the extension of the antigen protein structure, better exposing the functional groups on the antigen protein: such as -NH2/-COOH, etc., which is more conducive to the chemical reaction between the antigen and the corresponding groups of the magnetic beads, while maintaining the stability of the secondary structure of the antigen.

The current existing antigen coating processes (such as the public documents CN105954510A/CN202010469073) do not involve an antigen pretreatment process, but directly mix the antigen with the magnetic beads for reaction. The coating efficiency of the antigen coating preparation method provided by the present invention is more than 10 times higher than that of the prior art, while maintaining the antigen activity well, thereby increasing the sensitivity and detection rate of the detection reagent.

The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. An HCV antigen coating pretreatment agent, characterized in that: comprises nonionic surfactant, nonionic detergent, antigen coating buffer solution and purified water.

2. An HCV antigen coated pretreatment agent according to claim 1, wherein: the nonionic surfactant comprises tween, triton and tetraethylenepentamine.

3. An HCV antigen coated pretreatment agent according to claim 2, wherein: the type of the Tween is one of Tween-20, tween-60 and Tween-80, and the type of the triton is triton-100.

4. An HCV antigen coated pretreatment agent according to claim 2, wherein: in the HCV antigen coating pretreatment agent, the mass percent content of Tween is 0.01-10%, the mass percent content of Triton is 0.001-10%, and the mass percent content of tetraethylenepentamine is 0.0005-1%.

5. An HCV antigen coated pretreatment agent according to claim 1, wherein: the antigen coating buffer is one of phosphate buffer, carbonate buffer and borate buffer.

6. A method of coating an HCV antigen, comprising the steps of:

firstly, carrying out HCV antigen pretreatment by using an HCV antigen pretreatment agent;

and (2) binding the pretreated HCV antigen on a solid phase carrier.

7. An HCV antigen coating method as claimed in claim 6, wherein: the HCV antigen pretreatment in the step (1) lasts for 0.5-5h.

8. An HCV antigen coating method as claimed in claim 6, wherein: the solid phase carrier coated by the HCV antigen is one of carboxyl magnetic beads, amino magnetic beads and toluoyl magnetic beads.

9. An HCV-coated antigen reagent, comprising: prepared by the method of claim 6.

10. An HCV detection kit characterized in that: comprising an HCV labeled antigen reagent which is one of alkaline phosphatase, horseradish peroxidase and acridinium esters, and the HCV-coated antigen reagent obtained in claim 9.