CN110954691B

CN110954691B - Sample pretreatment reagent and application thereof

Info

Publication number: CN110954691B
Application number: CN201911304158.7A
Authority: CN
Inventors: 刘春龙; 付彦凯; 张舟; 盛长忠; 周泽奇; 粟艳
Original assignee: Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2021-02-26
Anticipated expiration: 2039-12-17
Also published as: CN110954691A

Abstract

The invention provides a sample pretreatment reagent and application thereof, wherein the sample pretreatment reagent comprises streptavidin magnetic particles and/or anti-free biotin antibodies. In the invention, in the process of detecting the fungal antigen, in order to avoid the influence of the biotin in the sample to be detected on the result, the sample to be detected is firstly pretreated by using a sample pretreatment reagent, and after the biotin in the sample to be detected is removed by adsorption, the antigen detection is carried out, so that the interference of the biotin in the sample to be detected on the result is eliminated, and the detection accuracy is obviously improved.

Description

Sample pretreatment reagent and application thereof

Technical Field

The invention belongs to the technical field of in-vitro diagnosis, relates to a sample pretreatment reagent and application thereof, and particularly relates to a sample pretreatment reagent for removing biotin interference, a constructed chemiluminescence kit and application thereof.

Background

Magnetic particles refer to superparamagnetic materials with particle sizes ranging from nanometer to micrometer, and are often used as solid phase carriers for immunoreaction. The immune magnetic particle formed by coating the antibody (or antigen) on the surface of the magnetic particle in a covalent or non-covalent mode can participate in immune reaction, and the phase separation of immune complexes is realized by means of superparamagnetism of the magnetic particle, so that simple, convenient and quick in-vitro diagnosis is realized.

Streptavidin is a tetrameric protein, has strong affinity with small-molecule biotin, can form a streptavidin-biotin complex in a short time, and is difficult to dissociate once formed. Thus, streptavidin and biotin often participate as a bridge structure in the immune response. For example, an antibody labeled with streptavidin and a biotin-labeled horseradish peroxidase can form an antibody-streptavidin-biotin-horseradish peroxidase complex in a short time.

The streptavidin magnetic particle refers to a magnetic particle with streptavidin covalently coated on the surface, and can be used for grabbing antibody (or antigen) molecules marked with biotin to form a magnetic particle-streptavidin-biotin-antibody (or antigen) immune magnetic particle compound, and further participate in subsequent immune reactions. The streptavidin-based immunological technology is convenient to operate and has been widely and deeply applied to the in vitro diagnosis industry.

Biotin, also known as vitamin B7, is a common substance found in humans, and normally, low levels of biotin in patient samples (e.g., serum, cerebrospinal fluid) do not interfere with in vitro diagnostic techniques based on streptavidin magnetic particles. With the improvement of living standard, more and more people begin to take dietary supplements such as compound vitamins, and the biotin level in the human body is too high due to the large-dose taking of the dietary supplements, so that the in-vitro diagnosis technology based on the streptavidin magnetic particles can be interfered, and misdiagnosis is caused. Biotin interference is a problem that has been revealed in recent years, and no good solution has been found in academia and industry.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the sample pretreatment reagent and the application thereof, the sample pretreatment reagent is combined with biotin in a sample in advance, and the problem of biotin interference in immunoassay is solved, so that the prepared kit has the advantages of good accuracy, high detection speed, high sensitivity and high automation degree, and is favorable for providing a powerful basis for rapid clinical diagnosis of fungal infection diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a sample pretreatment reagent comprising streptavidin magnetic particles and/or anti-free biotin antibodies.

In the invention, in the process of detecting the fungal antigen, in order to avoid the influence of the biotin in the sample to be detected on the result, the sample to be detected is firstly pretreated by using a sample pretreatment reagent, and after the biotin in the sample to be detected is removed by adsorption, the antigen detection is carried out, so that the interference of the biotin in the sample to be detected on the result is eliminated, and the detection accuracy is obviously improved.

The invention adopts streptavidin magnetic particles to carry out sample pretreatment, adsorbs free biotin in the sample, realizes phase separation by virtue of superparamagnetism of the magnetic particles, and uses the sample without the biotin for next detection.

In the invention, the anti-free biotin antibody is adopted to carry out sample pretreatment, the free biotin in the sample is neutralized, but the free biotin is not combined with the biotin covalently coupled on the antibody (or antigen), and the reagent does not need to be separated after being used and can be directly used for the next detection.

Preferably, the sample pretreatment reagent further comprises PBS and/or Proclin-300.

Preferably, the concentration of the PBS buffer solution is 0.01-0.05M, and the concentration of the Proclin-300 as a preservative is 0.1% -0.5%.

In a second aspect, the present invention provides a chemiluminescent detection kit comprising a sample pretreatment reagent according to the first aspect.

Preferably, the kit further comprises a capture antibody and a signal antibody.

Preferably, the capture antibody and the signal antibody are monoclonal antibodies against fungal antigens.

Preferably, the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 1, and the light chain variable region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 2;

SEQ ID NO:1：

MTLTCFCARTISKTSTRLTGLEYIGMISGATVDLKMYGMDLWGPGTLVTVSSVQCQSVEEVSGFSLSSYITNYYYVRQAPGKDMNWVTYVTPGTPLSGGNTGETGLRWLLLVAVLKGYANWTSPTTEDTANGRF；

SEQ ID NO:2：

MDTRAPTQLLGATFAIVMTQSEACAGYKYTGLLLLNRLANCQATEVVVKWLPGTWYQFTLTISDGSVPVCDQDATLASGVPSRFKGSGATYYTPSSVGGTIDSQKPGSSGTQVGDTVPPKLIAFGLIYQSVYSN。

in the invention, the monoclonal antibody is a rabbit-derived monoclonal antibody against candida mannan, has good specificity and stability, has a dissociation constant smaller than that of a mouse-derived monoclonal antibody, has strong affinity with candida mannan, can be rapidly combined with the candida mannan, and is favorable for shortening the detection time.

Preferably, the heavy chain variable region of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 3, and the light chain variable region of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 4;

SEQ ID NO:3：

MEVSGFSLSSYITAPGKSGRWLVDLKMGRLTGLETGLYGMDLDTANGWGPGTLVTVSSLLVAVLKGVQCQSVEEYIGMISGANTGYANWTSPTTERFDMNWVTYVTPGTPLTLNYYYVRQTCFCARTISKTSTT；

SEQ ID NO:4：

MLLPSEACAGDQDAATYYGATLIVMGTQLWFSNNVPLTLLPKLLIYARLANCQAVIAFGGGTEVVVKTWYQFTLTISDGSVPVCYKYTGVYDSQKPSGSGTQVGSSTGSTLASGVDTQSDTRAPTPPSRFKGIQ。

in the invention, the monoclonal antibody is a rabbit-derived monoclonal antibody against cryptococcus capsular polysaccharide, has good specificity and stability, has a dissociation constant smaller than that of a mouse-derived monoclonal antibody, has strong affinity with cryptococcus capsular polysaccharide, can be rapidly combined with cryptococcus capsular polysaccharide, and is favorable for shortening the detection time.

Preferably, the capture antibody is modified with biotin.

Preferably, the concentration of the capture antibody is 0.5 to 3. mu.g/mL, and may be, for example, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, 2. mu.g/mL, 3.5. mu.g/mL, or 3. mu.g/mL.

Preferably, the signal antibody is modified with a chemiluminescent group.

Preferably, the chemiluminescent group comprises any one or a combination of at least two of acridine ester, acridine sulfonyl, isoluminol, luminol, horseradish peroxidase, (adamantane) -1, 2-dioxyethane, or alkaline phosphatase, preferably acridine ester.

Preferably, the concentration of the signal antibody is 0.5-3. mu.g/mL, and may be, for example, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, 2. mu.g/mL, 3.5. mu.g/mL, or 3. mu.g/mL.

Preferably, the kit further comprises a solid support.

Preferably, the solid phase carrier comprises any one or combination of at least two of magnetic particles, an enzyme label plate, microspheres, an affinity membrane or a liquid phase chip, and the solid phase carrier is preferably magnetic particles.

Preferably, the surface of the solid phase carrier is modified with streptavidin.

Preferably, the concentration of the magnetic fine particles is 100 to 1000. mu.g/mL, and may be, for example, 100. mu.g/mL, 200. mu.g/mL, 300. mu.g/mL, 400. mu.g/mL, 500. mu.g/mL, 600. mu.g/mL, 700. mu.g/mL, 800. mu.g/mL, 900. mu.g/mL, or 1000. mu.g/mL.

Preferably, the kit further comprises a calibrator, wherein the calibrator is a fungal antigen.

Preferably, the fungal antigen comprises candida mannopolysaccharides and/or cryptococcus capsular polysaccharides.

Preferably, the concentration of the calibrator is 0.2-25.0 ng/mL, such as 0.2ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, 12ng/mL, 13ng/mL, 14ng/mL, 15ng/mL, 16ng/mL, 17ng/mL, 18ng/mL, 19ng/mL, 20ng/mL, 21ng/mL, 22ng/mL, 23ng/mL, 24ng/mL or 25ng/mL, preferably 0.2-1.0 ng/mL (Candida mannosan) or 5.0-25.0 ng/mL (Candida cryptococci capsular polysaccharide), respectively.

In a third aspect, the present invention provides a method for detecting a fungal antigen, wherein the method employs the kit of the second aspect.

Preferably, the detection method comprises the following steps:

(1) pretreating a sample to be detected by adopting a sample pretreatment reagent to remove biotin in the sample to be detected;

(2) adding a biotin-labeled capture antibody, a streptavidin-coated solid phase carrier and a signal antibody, mixing, incubating and removing supernatant.

In the invention, in the process of detecting the fungal antigen, in order to avoid the influence of the biotin in the sample to be detected on the result, the sample to be detected is firstly pretreated by using a sample pretreatment reagent, the biotin in the sample to be detected is adsorbed and removed, and then the biotin-labeled capture antibody, the streptavidin-coated solid phase carrier and the signal antibody are added to complete the binding reaction of the antigen and the antibody, so that the interference of the biotin in the sample to be detected on the result is eliminated.

The method provided by the invention obviously reduces the interference of biotin in the sample to be detected on the result, and the influence on the detection concentration is less than 10%.

Preferably, the pretreatment time in step (1) is 1-10 min, such as 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10 min.

Preferably, the mixing incubation time in the step (2) is 1-20 min, for example, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20 min.

Preferably, the detection method further comprises the step of chemiluminescent detection after antigen binding.

Preferably, the chemiluminescent detection comprises adding a substrate and measuring the intensity of the chemiluminescence; and calculating to obtain the concentration of the antigen in the sample to be detected according to the standard curve.

In a fourth aspect, the present invention provides a sample pretreatment reagent according to the first aspect and/or a kit according to the second aspect for use in preparing a fungal infection disease detection reagent.

Compared with the prior art, the invention has the following beneficial effects:

(1) in the process of detecting the fungal antigen, the sample pretreatment reagent is firstly incubated with the sample to be detected to remove free biotin in the sample to be detected, and then antigen detection is carried out, so that the interference of the biotin in the sample to be detected on the result is eliminated, and the detection accuracy is obviously improved;

(2) the kit adopts the rabbit-derived monoclonal antibody as a capture antibody and a signal antibody, has good specificity and stability and strong affinity with a fungal antigen, can be quickly combined with the antigen, and is favorable for shortening the detection time to within 15 min;

(3) the kit has strong specificity, high sensitivity and detection limit of 0.05ng/mL, achieves the clinical coincidence rate of 94 percent, is superior to the existing kits on the market, and has wide application prospect in the early rapid clinical diagnosis of fungal infection diseases.

Detailed Description

To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

EXAMPLE 1 preparation of the kit

(1) Preparation of Biotin-labeled Capture antibody

Dissolving long-chain activated biotin in dimethyl sulfoxide according to the concentration of 1 mg/mL; dissolving an anti-candida mannan monoclonal antibody (heavy chain is SEQ ID NO:1, light chain is SEQ ID NO:2) to be coupled and purified according to the concentration of 1mg/mL in a sodium bicarbonate solution with the pH value of 9.0 of 0.1 mol/L; mixing the activated biotin solution with an antibody solution to be coupled according to a ratio of 1:8, and incubating for 4 hours at room temperature; the solution was dialyzed at 4 ℃ for 24h against 0.05mol/L PBS buffer pH 7.2, with 4 changes to remove unbound free biotin, yielding a biotin-labeled capture antibody.

(2) Preparation of streptavidin-labeled magnetic microparticles

Resuspending the magnetic particles with 2-morpholine ethanesulfonic acid buffer (MES) to make the concentration of the magnetic particles appropriate, adding streptavidin into the magnetic particle solution, and suspending at 37 ℃ for 60min, wherein the mass ratio of the magnetic particles to the streptavidin is 25: 1; adding a freshly prepared carbodiimide aqueous solution, suspending at 37 ℃ for 6h, wherein the volume ratio of a 2-morpholine ethanesulfonic acid buffer solution (MES) to the carbodiimide aqueous solution is 15:1, and carrying out magnetic separation after the incubation is finished; blocking with 2% bovine serum albumin blocking solution, mixing, incubating at 37 ℃ for 1h, magnetic separation, removing supernatant, resuspending streptavidin-coupled magnetic microparticles in 1% BSA-containing Tris-HCl buffer (0.01mol/L, pH 7-7.5), and standing at 4 ℃ for further use.

(3) Preparation of acridinium ester labeled signal antibody

Adding equal volumes of 12.8mg/mL sodium periodate solution and 1% glycol solution into the acridine ester solution in sequence, and reacting at 2-8 ℃ in a dark place for 1 h; adding Mn monoclonal antibody into the mixed solution according to the molar ratio of the acridinium ester solution to the antibody of (0.5-2) to 1, and dialyzing overnight at 4 ℃ in 0.05M carbonate buffer solution; taking out the mixed solution, adding 5mg/mL sodium borohydride solution into the mixed solution according to the proportion of adding 20 mu L sodium borohydride solution into each milligram of antibody, and reacting for 2 hours at the temperature of 2-8 ℃ in a dark place; adding an equal volume of saturated ammonium sulfate solution, and reacting for 1h at the temperature of 2-8 ℃ in a dark place; centrifuging at 8000rpm at 2-8 deg.C for 30min, discarding supernatant, dissolving precipitate with appropriate amount of 0.01M phosphate buffer, and dialyzing at 2-8 deg.C overnight; the acridinium ester-labeled signal antibody was diluted with 0.05M 2- (N-morpholino) ethanesulfonic acid buffer (pH 6.0).

Example 2 one-step assay of Candida mannan

In this example, candida mannan was detected by a one-step method, which comprises the following steps:

(1) incubating 10 μ L of mannan at a concentration of 1ng/mL with 150 μ L of streptavidin-labeled magnetic microparticles (biotin loading of 5000pmol/mg) at a concentration of 1mg/mL for 10min at 37 deg.C, magnetically separating for 1min, and retaining the supernatant;

(2) adding 150 μ L of biotin-labeled capture antibody with concentration of 0.3 μ g/mL, 150 μ L of streptavidin-labeled magnetic particles with concentration of 0.3mg/mL and 150 μ L of acridinium ester-labeled signal antibody with concentration of 0.3 μ g/mL into the supernatant, fully mixing, incubating at 37 ℃ for 10min, magnetically separating for 1min, removing supernatant, and washing with washing solution for 5 times;

(3) adding 200 mu L of excitation liquid, fully mixing, and measuring the chemiluminescence intensity of the solution by adopting a full-automatic chemiluminescence apparatus Lumiray series.

EXAMPLE 3 preparation of the kit

(1) Preparation of Biotin-labeled Capture antibody

Dissolving long-chain activated biotin in dimethyl sulfoxide according to the concentration of 1 mg/mL; dissolving an anti-cryptococcus capsular polysaccharide monoclonal antibody (heavy chain is SEQ ID NO:3, and light chain is SEQ ID NO:4) to be coupled and purified in a sodium bicarbonate solution with the pH value of 9.0 of 0.1mol/L according to the concentration of 1 mg/mL; mixing the activated biotin solution with an antibody solution to be coupled according to a ratio of 1:8, and incubating for 4 hours at room temperature; the solution was dialyzed at 4 ℃ for 24h against 0.05mol/L PBS buffer pH 7.2, with 4 changes to remove unbound free biotin, yielding a biotin-labeled capture antibody.

(2) Preparation of acridinium ester labeled signal antibody

Adding equal volumes of 12.8mg/mL sodium periodate solution and 1% glycol solution into the acridine ester solution in sequence, and reacting at 2-8 ℃ in a dark place for 1 h; adding GXM monoclonal antibody into the mixed solution according to the molar ratio of the acridinium ester solution to the antibody of (0.5-2) to 1, and dialyzing overnight at 4 ℃ in 0.05M carbonate buffer solution; taking out the mixed solution, adding 5mg/mL sodium borohydride solution into the mixed solution according to the proportion of adding 20 mu L sodium borohydride solution into each milligram of antibody, and reacting for 2 hours at the temperature of 2-8 ℃ in a dark place; adding an equal volume of saturated ammonium sulfate solution, and reacting for 1h at the temperature of 2-8 ℃ in a dark place; centrifuging at 8000rpm at 2-8 deg.C for 30min, discarding supernatant, dissolving precipitate with appropriate amount of 0.01M phosphate buffer, and dialyzing at 2-8 deg.C overnight; the acridinium ester-labeled signal antibody was diluted with 0.05M 2- (N-morpholino) ethanesulfonic acid buffer (pH 6.0).

Example 4 one-step assay for Cryptococcus capsular polysaccharide

In this example, the detection of cryptococcus capsular polysaccharide is performed by a one-step method, which includes the following steps:

(1) incubating 10 μ L of capsular polysaccharide at a concentration of 10ng/mL with 150 μ L of anti-free biotin antibody at a concentration of 1 μ g/mL for 10min at 37 ℃;

(2) adding 150 μ L of biotin-labeled capture antibody with concentration of 0.3 μ g/mL, 150 μ L of streptavidin-labeled magnetic particles with concentration of 0.3mg/mL and 150 μ L of acridinium ester-labeled signal antibody with concentration of 0.3 μ g/mL into the mixed solution, fully mixing, incubating at 37 ℃ for 10min, magnetically separating for 1min, removing supernatant, and washing with washing solution for 5 times;

Example 5 specificity test

Compared with mouse-derived monoclonal antibodies, the rabbit-derived monoclonal antibodies have the advantages of multiple recognition sites, strong affinity, good specificity and the like, the test data of the kit and the commercially available kit are shown in table 1, and the specificity of the kit is superior to that of the existing candida mannan detection kit in the market.

TABLE 1 clinical coincidence rates of different Candida mannan assay kits

Detection kit manufacturer	DNK (invention)	BioRad
			Methodology of	Chemical luminescence method	ELISA method
Clinical compliance rate	94％	76％

Example 6 sensitivity test

After the cryptococcus capsular polysaccharide is subjected to gradient dilution, sensitivity detection is carried out. As a result, the signal value is increased along with the increase of the concentration of the capsular polysaccharide; the limit of detection (LOD) reached 0.05ng/mL, as compared to a commercially available kit, as shown in Table 2.

TABLE 2 sensitivity of Cryptococcus different capsular polysaccharide kits

Brand	DNK (invention)	IMMY	IMMY
				Methodology of	Chemical luminescence method	Colloidal gold method	ELISA
Sensitivity index	LOD	C5	LOD
				Antigen concentration ng/mL	0.05	1.00	1.70

Example 7 anti-Biotin interference Capacity test

The existing chemiluminescence diagnostic kit mostly adopts one-step operation in order to shorten the reaction time, and biotin in a sample can interfere the combination of the biotin-labeled antibody and avidin magnetic beads to influence the accuracy of the result because the biotin-labeled antibody reacts with the sample and the avidin magnetic beads simultaneously.

In the invention, in the process of detecting the fungal antigen, in order to avoid the influence of biotin in a sample to be detected on the result, the scheme of embodiment 2 or 4 is adopted to realize the combination of the antigen and the antibody in the sample to be detected, wherein, in embodiment 2, the sample to be detected is pretreated by adopting a streptavidin coated solid phase carrier, and after the biotin in the sample to be detected is removed by adsorption, the biotin labeled capture antibody, the streptavidin coated solid phase carrier and the signal antibody are added to complete the combination reaction of the antigen and the antibody; in the embodiment 4, firstly, a sample to be detected is pretreated by an anti-free biotin antibody, biotin in the sample to be detected is removed through adsorption, and then a biotin-labeled capture antibody, a streptavidin-coated solid phase carrier and a signal antibody are added to complete the binding reaction of an antigen and an antibody; the interference of the biotin in the sample to be detected on the result is completely eliminated. The results are shown in Table 3.

TABLE 3 comparison of anti-Biotin interference Capacity of the examples

In conclusion, in the process of detecting the fungal antigen, the sample pretreatment reagent is incubated with the sample to be detected firstly to remove free biotin in the sample to be detected, and then the antigen detection is carried out, so that the interference of the biotin in the sample to be detected on the result is eliminated, and the detection accuracy is obviously improved; the kit constructed by the method adopts the rabbit-derived monoclonal antibody as a capture antibody and a signal antibody, has good specificity and stability and strong affinity with a fungal antigen, can be quickly combined with the antigen, and is favorable for shortening the detection time to within 15 min; the kit has strong specificity, high sensitivity and detection limit of 0.05ng/mL, achieves the clinical coincidence rate of 94 percent, is superior to the existing kits on the market, and has wide application prospect in the early rapid clinical diagnosis of fungal infection diseases.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

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Claims

1. A chemiluminescent detection kit, characterized in that the kit comprises a sample pretreatment reagent, wherein the sample pretreatment reagent comprises streptavidin magnetic particles and/or anti-free biotin antibodies;

the kit further comprises a capture antibody and a signal antibody;

the capture antibody and the signal antibody are monoclonal antibodies against fungal antigens;

the heavy chain variable region of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 1, and the light chain variable region of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 2;

or the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID NO. 3, and the light chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID NO. 4.

2. The kit of claim 1, wherein the sample pretreatment reagents further comprise PBS and/or Proclin-300.

3. The kit of claim 2, wherein the capture antibody is modified with biotin;

the concentration of the capture antibody is 0.5-3 mug/mL;

the signal antibody is modified with a chemiluminescent group;

the chemiluminescent group comprises any one or the combination of at least two of acridine ester, acridine sulfonyl, isoluminol, luminol, horseradish peroxidase, (adamantane) -1, 2-dioxyethane or alkaline phosphatase;

the concentration of the signal antibody is 0.5-3 mug/mL.

4. The kit of claim 3, wherein the chemiluminescent group is an acridinium ester.

5. The kit of claim 3 or 4, further comprising a solid support;

the solid phase carrier comprises any one or the combination of at least two of magnetic particles, an enzyme label plate, microspheres, an affinity membrane or a liquid phase chip.

6. The kit of claim 5, wherein the solid support is a magnetic microparticle;

the surface of the solid phase carrier is modified with streptavidin;

the concentration of the magnetic particles is 100-1000 mug/mL.

7. The kit of claim 3 or 6, further comprising a calibrator which is a fungal antigen;

the fungal antigen comprises candida mannosan and/or cryptococcus capsular polysaccharide;

the concentration of the calibrator is 0.2-25.0 ng/mL.

8. A method for the detection of fungal antigens for non-diagnostic purposes, wherein the method employs a kit according to any one of claims 1 to 7.

9. The detection method according to claim 8, characterized in that it comprises the steps of:

10. The detection method according to claim 9, wherein the pretreatment time in the step (1) is 1-10 min;

and (3) the mixed incubation time in the step (2) is 1-20 min.

11. The detection method according to any one of claims 8 to 10, further comprising a step of chemiluminescence detection after antigen binding;

the chemiluminescence detection comprises adding a substrate and measuring chemiluminescence intensity; and calculating to obtain the concentration of the antigen in the sample to be detected according to the standard curve.

12. Use of the kit according to any one of claims 1 to 7 for the preparation of a fungal infection disease detection reagent.