阅读说明：本技术 1,3-β-D葡聚糖衍生物、试剂盒及制备方法及测定1,3-β-D葡聚糖含量的方法 (1, 3-beta-D glucan derivative, kit, preparation method and method for determining content of 1, 3-beta-D glucan ) 是由 鲁衡 高燕 孙金超 石云齐 于 2020-11-25 设计创作，主要内容包括：1,3-β-D葡聚糖衍生物、试剂盒及制备方法及测定1,3-β-D葡聚糖含量的方法。本发明提供一种结合了磁微粒分离技术、生物素-亲和素放大技术和化学发光分析技术的1,3-β-D葡聚糖磁微粒化学发光免疫检测试剂盒。同时用1,3-β-D葡聚糖抗原衍生物制备了生物素化1,3-β-D葡聚糖抗原衍生物和酶标1,3-β-D葡聚糖抗原衍生物,这样就构建了2个体系(1、生物素化1,3-β-D葡聚糖抗原衍生物+抗体酶标试剂+定标液；2、酶标1,3-β-D葡聚糖抗原衍生物+生物素化1,3-β-D葡聚糖鼠单抗+定标液)可以进行样本中1,3-β-D葡聚糖的测试,这样不同企业在开发试剂盒时就会有更灵活的选取,并且只需要通过制备1,3-β-D葡聚糖抗原衍生物这个中间体就能实现。(1, 3-beta-D glucan derivative, kit and preparation method thereof, and method for determining content of 1, 3-beta-D glucan. The invention provides a 1, 3-beta-D glucan magnetic particle chemiluminescence immunoassay kit combining a magnetic particle separation technology, a biotin-avidin amplification technology and a chemiluminescence analysis technology. Meanwhile, the 1, 3-beta-D glucan antigen derivative and the enzyme-labeled 1, 3-beta-D glucan antigen derivative are prepared by using the 1, 3-beta-D glucan antigen derivative, so that 2 systems (1, biotinylation 1, 3-beta-D glucan antigen derivative, an antibody enzyme-labeled reagent and a calibration solution; 2, enzyme-labeled 1, 3-beta-D glucan antigen derivative, biotinylation 1, 3-beta-D glucan mouse monoclonal antibody and the calibration solution) are constructed, the 1, 3-beta-D glucan in a sample can be tested, and different enterprises can select the kit more flexibly during development and can realize the test only by preparing the intermediate of the 1, 3-beta-D glucan antigen derivative.)

1. A1, 3-beta-D glucan antigen derivative and a preparation method thereof are characterized in that the 1, 3-beta-D glucan antigen derivative is prepared by the following steps:

p1: respectively dissolving 1, 3-beta-D glucan and succinic anhydride by using DMF buffer solution to obtain 1, 3-beta-D glucan-DMF solution and succinic anhydride-DMF solution;

p2, adding the 1, 3-beta-D glucan-DMF solution into the succinic anhydride-DMF solution, and reacting for 6h at 65 ℃; after the reaction is finished, tributylamine and isobutyl chloroformate are added, and the mixture reacts for 30min at room temperature to obtain a DMF solution of 1, 3-beta-D glucan-succinic anhydride; adding a lysine solution, and reacting at room temperature for 3 hours;

p3: dialyzing the reaction mixture with water overnight, adjusting pH to precipitate, standing the precipitate at room temperature for 1h, standing at 4 deg.C for 3h, centrifuging at 4 deg.C for 10-1For 5min, use Na₂CO₃Dissolving the precipitate with buffer solution to obtain 1, 3-beta-D glucan antigen derivative.

2. The 1,3- β -D glucan antigen derivative according to claim 1, wherein said 1,3- β -D glucan antigen derivative is produced by said method.

3. The kit for measuring the content of the 1, 3-beta-D glucan is characterized by comprising a biotinylation 1, 3-beta-D glucan antigen derivative, an antibody enzyme labeling reagent, an enzyme labeling 1, 3-beta-D glucan antigen derivative, a biotinylation 1, 3-beta-D glucan mouse monoclonal antibody, a calibration solution and a quality control product.

4. The kit for measuring the content of 1, 3-beta-D glucan according to claim 3 and the preparation method thereof, wherein the preparation method of the kit is as follows:

(1) the biotinylated 1, 3-beta-D glucan antigen derivative was prepared as follows:

calculated from the 1,3- β -D glucan antigen derivative molecular weight (605) and the biotin-NHS molecular weight (587), in a molar ratio of 1: weighing biotin-NHS at the feeding amount of 20, dissolving the biotin-NHS in DMSO, adding the dissolved biotin-NHS into the 1, 3-beta-D glucan antigen derivative in the claim 2, fully mixing the mixture uniformly, and reacting the mixture at room temperature for 2 hours; dialyzing the reaction product with a dialysis bag; diluting the dialysis product to working solution concentration by using buffer solution;

(2) the preparation process of the antibody enzyme labeling reagent comprises the following steps:

respectively activating the 1, 3-beta-D glucan mouse monoclonal antibody and alkaline phosphatase, mixing the two, and reacting for 18 hours at the temperature of 2-8 ℃; purifying the reactant by a chromatographic column, collecting a peak I and a peak II, mixing, and preparing the mixture into working solution by using a buffer solution;

(3) the preparation process of the enzyme-labeled 1, 3-beta-D glucan antigen derivative comprises the following steps:

separately activating the 1,3- β -D glucan antigen derivative of claim 2 and alkaline phosphatase, and mixing the activated glucan antigen derivative and alkaline phosphatase at 2-8 ℃ for 18 hours; purifying the reactant by a chromatographic column, collecting a peak I and a peak II, mixing, and preparing the mixture into working solution by using a buffer solution;

(4) the preparation process of the biotinylation 1, 3-beta-D glucan mouse monoclonal antibody comprises the following steps:

calculated from the molecular weight of 1,3- β -D glucan murine mab (150000) and the molecular weight of biotin-NHS (587) in a molar ratio of 1: weighing biotin-NHS at the feeding amount of 20, dissolving the biotin-NHS with DMSO, adding the dissolved biotin-NHS into 1, 3-beta-D dextran mouse monoclonal antibody, fully and uniformly mixing, reacting at room temperature for 2 hours, dialyzing a reaction product with a dialysis bag, and diluting the dialyzed product with a buffer solution to the concentration of a working solution.

(5) Subpackaging the biotin 1, 3-beta-D glucan antigen derivative, an antibody enzyme labeling reagent, an enzyme labeled 1, 3-beta-D glucan antigen derivative, a biotinylation 1, 3-beta-D glucan murine monoclonal antibody, a calibration solution and a quality control product.

5. The kit for measuring the content of 1,3- β -D glucan according to claim 4 and the preparation method thereof, wherein the 1,3- β -D glucan murine monoclonal antibody in the step (2) is dialyzed with 0.1 × PBS and then activated with 2IT to obtain an activated antibody; and (3) dialyzing the 1, 3-beta-D glucan antigen derivative in the step (3) by using 0.1-fold PBS, and then activating by using 2IT to obtain the activated antigen.

6. The kit for measuring the content of 1,3- β -D glucan according to claim 4, wherein the alkaline phosphatase in the steps (2) and (3) is activated by SMCC to obtain activated alkaline phosphatase.

7. A method for measuring the content of 1, 3-beta-D glucan is characterized by comprising the following steps:

p1: taking 15 ul of a sample, a calibration solution or a quality control product, adding 30ul of the biotinylated 1, 3-beta-D glucan antigen derivative as described in claims 3-6, adding 30ul of the antibody enzyme-labeled reagent as described in claims 3-6, and incubating for 15 min;

p2: adding 30 μ l of magnetic beads into the P1 step, and incubating for 5 min;

p3 magnetic separation for 2min, removing supernatant;

p4 washing 3 times, 300 mul washing liquid each time;

p5-add 200. mu.l of substrate, found.

8. A method for measuring the content of 1, 3-beta-D glucan is characterized by comprising the following steps:

p1, taking 15 μ l of sample, calibration solution and quality control product, adding 30 μ l of the enzyme-labeled 1, 3-beta-D glucan antigen derivative as described in claims 3-6, adding 30 μ l of the biotinylated 1, 3-beta-D glucan murine monoclonal antibody as described in claims 3-6, and incubating for 15 min;

p2: clearing to P1;

p4, washing for 3 times, adding 30 mu l of magnetic beads in each step, and incubating for 5 min;

p3 magnetic separation for 2min, and removing 300 μ l of lotion;

p5-add 200. mu.l of substrate, found.

9. The method for measuring the content of 1,3- β -D glucan according to claim 7 or 8, wherein the magnetic beads are streptavidin magnetic beads.

Technical Field

The invention belongs to the field of biochemical detection, and particularly relates to a 1, 3-beta-D glucan derivative and a preparation method thereof, as well as a kit for determining the content of 1, 3-beta-D glucan by adopting a magnetic particle chemiluminescence method and a preparation method thereof.

Background

1, 3-beta-D glucan is a polysaccharide, widely present in fungal cell walls other than Zygomycetes, accounts for more than 50% of the components of fungal cell walls, and is a unique component on fungal cell walls. After fungi enter human blood or deep tissues, the 1, 3-beta-D glucan can be released from cell walls through phagocytosis, digestion and other treatments of phagocytes, so that the content of blood and other body fluids is increased; on the other hand, when superficial fungal infection occurs, 1, 3-beta-D glucan is not released, so that the content of 1, 3-beta-D glucan in body fluid is not increased, and whether the organism is infected with invasive fungi or not can be known through detecting 1, 3-beta-D glucan in blood or non-blood samples.

The main detection method of 1, 3-beta-D glucan at present is a limulus reagent detection method, namely a commonly known G test method, and the principle is as follows: during the experiment, a limulus reagent is added into a specimen, wherein a factor G is activated by 1, 3-beta-D glucan to become an activated factor G, and the activated factor G enables prothrombin to become coagulase, so that the coagulogen becomes coagulin, the specimen is just like turbidity, the more the coagulation is, the worse the light transmittance is, and then the content of the 1, 3-beta-D glucan in the specimen can be measured by measuring the light transmittance of the specimen. However, the limulus reagent method is not highly specific and has relatively severe reaction conditions, such as the laboratory requires clean and dust-free flow, the optimum pH value of the reaction is 6-8, and the reaction result is greatly influenced by the temperature and the reaction time. Therefore, in view of the above disadvantages, the immunoassay method is widely used in clinical laboratories, that is, a method for determining the presence or absence of a test substance by specific binding of an antigen-antibody.

The immunoassay method has the advantages of convenient and rapid detection, low requirements on detection environment and reaction conditions, strong specificity and higher sensitivity than a limulus reagent method. At present, the immunoassay of 1, 3-beta-D glucan is mainly an elisa method, namely the content of a substance to be detected in a sample is judged by a color development method, but because the coating amount of an elisa reaction antigen antibody is limited by a 96-well plate, and the antigen antibody reaction is on an approximately two-dimensional plane, the reaction speed is slow, and the complete reaction time is long. In addition, the final signal is based on the calculation of the color depth, which has a certain limit. Therefore, the elisa method has somewhat poor sensitivity and linearity when testing low and high concentration samples. False negative detection results are likely to occur in clinical tests for low-value samples, and inaccurate detection results are likely to occur for high-value samples.

The magnetic particle chemiluminescence method can reduce the influence of nonspecific adsorption of other components in a sample by cleaning the magnetic bead-antibody-antigen complex, and effectively improve the sensitivity. Secondly, the magnetic particles are in a nanometer level, the specific surface area is large, the reaction speed is high, and the reaction time is short. Thirdly, since the luminescence is the number of detected photons, the detection of low-concentration and high-concentration samples is not comparable in color depth, so that the detection range can be greatly expanded.

From the above comparison, it is apparent that detection of 1, 3-. beta. -D-glucan by magnetic particle chemiluminescence is a suitable method. However, since 1,3- β -D-glucan itself does not contain an amino group that can be used for coupling biotin and alkaline phosphatase, it is necessary to prepare a derivative by coupling it with an amino group-containing compound, and then prepare a kit by coating and labeling. In view of the fact that domestic chemiluminescence mostly adopts a luminous system (enzymatic chemiluminescence) of alkaline phosphatase (ALP) or horseradish peroxidase (HRP), some solid-phase companies adopt a biotin-streptomycin avidin magnetic particle and FITC-anti-FITC magnetic bead system, and some companies adopt a system directly coated by magnetic beads. Under the condition, the groups on the 1, 3-beta-D glucan derivative can be coupled with biotin, FITC or magnetic beads and the like for solid-phase coupling of a magnetic particle system, and can be coupled with enzyme (ALP or HRP) and the like for luminescent labeling, so that the flexibility of establishing a reaction system is greatly improved. Therefore, the invention of the 1, 3-beta-D glucan derivative which can be used for solid phase coupling and luminescent labeling is key for developing a 1, 3-beta-D glucan kit.

Disclosure of Invention

In order to solve the problems existing in the background technology and overcome the defects that the existing 1, 3-beta-D glucan kit can not be used for both solid phase coupling and luminescent labeled 1, 3-beta-D glucan derivatives, the technical scheme has the greatest characteristic of providing the 1, 3-beta-D glucan antigen derivative and the preparation method thereof, the 1, 3-beta-D glucan antigen can be flexibly connected with biotin or enzyme indirectly according to the needs by preparing the derivative, the coupling problem which is the key problem of developing the project is solved, and a magnetic particle enzymatic chemiluminescence system of the 1, 3-beta-D glucan can be conveniently established.

The technical solution of the invention is as follows:

a method for preparing 1, 3-beta-D glucan antigen derivatives comprises the following steps:

p1: respectively dissolving 1, 3-beta-D glucan and succinic anhydride by using DMF buffer solution to obtain 1, 3-beta-D glucan-DMF solution and succinic anhydride-DMF solution;

p2, adding the 1, 3-beta-D glucan-DMF solution into the succinic anhydride-DMF solution, and reacting for 6h at 65 ℃; after the reaction is finished, tributylamine and isobutyl chloroformate are added, and the mixture reacts for 30min at room temperature to obtain a DMF solution of 1, 3-beta-D glucan-succinic anhydride; adding a lysine solution, and reacting at room temperature for 3 hours;

p3: dialyzing the reaction mixture with water overnight, adjusting pH to precipitate, standing the precipitate at room temperature for 1h, standing at 4 deg.C for 3h, centrifuging at 4 deg.C for 10-15min, and adding Na₂CO₃Dissolving the precipitate with buffer solution to obtain 1, 3-beta-D glucan antigen derivative.

A1, 3-beta-D glucan antigen derivative is prepared by the method.

The kit comprises a biotinylation 1, 3-beta-D glucan antigen derivative, an antibody enzyme labeling reagent, an enzyme labeling 1, 3-beta-D glucan antigen derivative, a biotinylation 1, 3-beta-D glucan mouse monoclonal antibody, a calibration solution and a quality control product. The preparation method of the kit comprises the following steps:

(1) the biotinylated 1, 3-beta-D glucan antigen derivative was prepared as follows:

calculated from the 1,3- β -D glucan antigen derivative molecular weight (605) and the biotin-NHS molecular weight (587), in a molar ratio of 1: weighing biotin-NHS at the feeding amount of 20, dissolving the biotin-NHS in DMSO, adding the dissolved biotin-NHS into the 1, 3-beta-D glucan antigen derivative, fully and uniformly mixing, and reacting at room temperature for 2 hours; dialyzing the reaction product with a dialysis bag; the dialysis product was diluted with buffer to working solution concentration.

(2) The preparation process of the antibody enzyme labeling reagent comprises the following steps:

respectively activating the 1, 3-beta-D glucan mouse monoclonal antibody and alkaline phosphatase, mixing the two, and reacting for 18 hours at the temperature of 2-8 ℃; purifying the reactant by a chromatographic column, collecting a peak I and a peak II, mixing, and preparing the mixture into working solution by using a buffer solution; further dialyzing the 1, 3-beta-D glucan mouse monoclonal antibody by using 0.1-fold PBS (phosphate buffer solution), and then activating by using 2IT (information technology) to obtain an activated antibody; the alkaline phosphatase was activated with SMCC to obtain activated alkaline phosphatase.

(3) The preparation process of the enzyme-labeled 1, 3-beta-D glucan antigen derivative comprises the following steps:

separately activating the 1,3- β -D glucan antigen derivative of claim 2 and alkaline phosphatase, and mixing the activated glucan antigen derivative and alkaline phosphatase at 2-8 ℃ for 18 hours; purifying the reactant by a chromatographic column, collecting a peak I and a peak II, mixing, and preparing the mixture into working solution by using a buffer solution; further 1, 3-beta-D dextran antigen derivatives were dialyzed against 0.1 × PBS and then activated with 2IT to obtain activated antigen. The alkaline phosphatase was activated with SMCC to obtain activated alkaline phosphatase.

(4) The preparation process of the biotinylation 1, 3-beta-D glucan mouse monoclonal antibody comprises the following steps:

calculated from the molecular weight of 1,3- β -D glucan murine mab (150000) and the molecular weight of biotin-NHS (587) in a molar ratio of 1: weighing biotin-NHS at the feeding amount of 20, dissolving the biotin-NHS with DMSO, adding the dissolved biotin-NHS into 1, 3-beta-D dextran mouse monoclonal antibody, fully and uniformly mixing, reacting at room temperature for 2 hours, dialyzing a reaction product with a dialysis bag, and diluting the dialyzed product with a buffer solution to the concentration of a working solution.

(5) Subpackaging the biotin 1, 3-beta-D glucan antigen derivative, an antibody enzyme labeling reagent, an enzyme labeled 1, 3-beta-D glucan antigen derivative, a biotinylation 1, 3-beta-D glucan murine monoclonal antibody, a calibration solution and a quality control product.

A method for measuring the content of 1, 3-beta-D glucan is characterized by comprising the following steps:

p1: taking 15 mul of sample, calibration solution and quality control product, adding 30ul of biotinylated 1, 3-beta-D glucan antigen derivative, adding 30 mul of antibody enzyme labeling reagent, and incubating for 15 min;

p2: adding 30 μ l of streptomycin affinity magnetic beads into the P1 step, and incubating for 5 min;

p3 magnetic separation for 2min, removing supernatant;

p4 washing 3 times, 300 mul washing liquid each time;

p5-add 200. mu.l of substrate, found.

Further, the magnetic beads are streptavidin magnetic beads.

A method for measuring the content of 1, 3-beta-D glucan is characterized by comprising the following steps:

p1, taking 15 mul of sample, calibration solution and quality control product, adding 30 mul of enzyme-labeled 1, 3-beta-D glucan antigen derivative, adding 30 mul of biotinylated 1, 3-beta-D glucan mouse monoclonal antibody, and incubating for 15 min;

p2: adding 30 μ l of streptomycin affinity magnetic beads into the P1 step, and incubating for 5 min;

p3 magnetic separation for 2min, removing supernatant;

p4 washing 3 times, 300 mul washing liquid each time;

p5-add 200. mu.l of substrate, found.

Further, the magnetic beads are streptavidin magnetic beads.

The invention aims to provide a simple, stable and high-specificity 1, 3-beta-D glucan magnetic particle chemiluminescence immunoassay kit which is developed by combining a magnetic particle separation technology, a biotin-avidin amplification technology and a chemiluminescence analysis technology.

The invention simultaneously uses the 1, 3-beta-D glucan antigen derivative to prepare the biotin 1, 3-beta-D glucan antigen derivative and the enzyme-labeled 1, 3-beta-D glucan antigen derivative, so that 2 systems (1, biotinylation 1, 3-beta-D glucan antigen derivative, enzyme-labeled 1, 3-beta-D glucan mouse monoclonal antibody and a calibration solution, and 2, enzyme-labeled 1, 3-beta-D glucan antigen derivative, biotinylation 1, 3-beta-D glucan mouse monoclonal antibody and a calibration solution) are constructed, and the test of the 1, 3-beta-D glucan in a sample can be carried out. For enterprises, enzymatic luminescence systems need to optimize each anti-reagent component, and coupling of biotin, enzyme labeling, even protein and other different conditions may need to be performed on 1, 3-beta-D glucan antigens under different conditions, so that different enterprises can select more flexibly during development of kits, and the preparation can be realized only by preparing an intermediate of the 1, 3-beta-D glucan antigen derivative.

The invention has the advantages that:

compared with the limulus reagent method and the enzyme immunoassay method, the invention has the following advantages: the whole reaction system overcomes the defects that the specificity of the limulus reagent is not high and the reaction conditions are harsh, inherits the advantage of high specificity of the enzyme immunoassay, improves the defects of low sensitivity and narrow linear range of the enzyme immunoassay, and simplifies the experimental operation process.

Detailed Description

The invention will be further illustrated by the following detailed description of specific embodiments, which are not to be construed as limiting the invention but are intended to be exemplary only. The reagents such as kit, buffer, etc. used herein are only reagents specifically selected in this specific example, and it is understood that those skilled in the art can select corresponding reagents of other companies as needed to achieve the purpose of the present invention.

Examples

A1, 3-beta-D glucan antigen derivative is prepared by the following steps:

1) preparing 0.1M HCl solution;

2) preparing 0.01M NaHCO3 buffer solution with pH being 9.0 and 0.15M buffer solution with pH being 9.5 respectively;

3) weighing about 10mg of 1,3- β -D-glucan and dissolving to 10mg/mL with N, N-Dimethylformamide (DMF) buffer;

4) weighing 50mg of succinic anhydride and dissolving the succinic anhydride in 10mL of DMF to 5 mg/mL;

5) weighing 20mg of lysine and dissolving the lysine in 1mL of 0.01M NaHCO3 buffer solution with pH 9.0 to 20 mg/mL;

6) adding 0.5mL of 1, 3-beta-D glucan-DMF solution into 10mL of succinic anhydride-DMF solution, and reacting for 6h at 65 ℃;

7) after the reaction is finished, adding 1.2mL of tributylamine and 0.65mL of isobutyl chloroformate, and reacting at room temperature for 30 min;

8) adding 1mL of lysine solution with the concentration of 20mg/mL into the DMF solution of the 1, 3-beta-D glucan-succinic anhydride which is reacted before, and reacting for 3 hours at room temperature;

9) the reaction was dialyzed overnight against water (dialysis molecular weight 500);

10) adjusting pH of the dialysate to 4.8-5.1 with 0.1MHCl to generate maximum precipitate, and standing the precipitate at room temperature for 1 hr and 4 deg.C for 3 hr;

11) the precipitate was centrifuged at 4 ℃ for 10-15min and dissolved to 2mg/mL in 0.15M Na2CO3 buffer at pH 9.5.

The kit comprises a biotinylation 1, 3-beta-D glucan antigen derivative, an antibody enzyme labeling reagent, an enzyme labeling 1, 3-beta-D glucan antigen derivative, a biotinylation 1, 3-beta-D glucan mouse monoclonal antibody, a calibration solution and a quality control product. The preparation method of the kit comprises the following steps:

the main components in the kit are prepared according to the following preparation method:

(1) the biotinylation 1, 3-beta-D glucan antigen derivative is prepared by the following steps:

1) measuring 0.5ml1, 3-beta-D dextran antigen derivative (1 mg);

2) calculated from the antigen molecular weight (605) and the biotin-NHS molecular weight (587), in a molar ratio of 1: weighing biotin (19.4mg) at a dosage of 20;

3) weighing about 19.4mg of biotin-NHS, and dissolving with DMSO to a final concentration of 30 mg/mL;

4) adding biotin dissolved in DMSO into the antigen, fully and uniformly mixing, and reacting at room temperature for 2 h;

5) dialyzing the reaction product of step 4) against a dialysis bag with a molecular weight cut-off of 1000, the dialysis buffer being 0.1M NaHCO3 buffer at PH 9.0;

6) the mixture was diluted to 0.5. mu.g/ml with an anti-reagent buffer and used as a working solution.

(2) The enzyme labeling reagent of the antibody comprises the following preparation steps:

1) dialyzing 0.5mg1,3- β -D-glucan murine mab against 0.1 × PBS followed by activation with 2 IT;

2) activating 0.5mg of alkali phosphatase by using SMCC;

3) mixing the activated 1, 3-beta-D glucan mouse monoclonal antibody and the activated alkali phosphatase, and reacting for 18 hours at 2-8 ℃;

4) purifying the reactant by a chromatographic column, collecting a peak I and a peak II, and mixing;

5) diluting the antibody-alkali phosphatase to 0.1 mu g/ml by using an anti-reagent buffer solution to serve as a working solution;

(3) the enzyme-labeled 1, 3-beta-D glucan antigen derivative is prepared by the following steps:

1) dialyzing 0.5mg1,3- β -D glucan antigen derivative against 0.1 × PBS followed by activation with 2 IT;

2) activating 0.5mg of alkali phosphatase by using SMCC;

3) mixing the activated 1, 3-beta-D glucan antigen derivative with the activated alkali phosphatase, and reacting for 18 hours at 2-8 ℃;

4) purifying the reactant by a chromatographic column, collecting a peak I and a peak II, and mixing;

5) diluting the 1, 3-beta-D glucan antigen derivative-alkali phosphatase conjugate to 0.3 mu g/ml by using an anti-reagent buffer solution to serve as a working solution;

(4) biotinylated 1, 3-beta-D glucan antibody was prepared as follows:

1) measuring 0.5mg of 1, 3-beta-D dextran mouse monoclonal antibody;

2) calculated from the antibody molecular weight (150000) and biotin-NHS molecular weight (587) in a molar ratio of 1: weighing biotin with the feeding amount of 20;

3) weighing about 1mg of biotin-NHS, and dissolving with DMSO to a final concentration of 20 mg/ml;

4) adding 0.04mg of biotin dissolved in DMSO into the antibody, fully and uniformly mixing, and reacting at room temperature for 2 h;

5) dialyzing the reaction product of step 4) against a dialysis bag with a molecular weight cut-off of 10000, the dialysis buffer being 0.1M NaHCO3 buffer at PH 9.0;

6) the 1, 3-. beta. -D-dextran murine monoclonal antibody-biotin conjugate was diluted to 0.5. mu.g/ml with an anti-reagent buffer and used as a working solution.

(5) The preparation of the calibration solution and the quality control product comprises the following steps:

1) preparing a 1, 3-beta-D glucan calibration solution buffer solution: 5% BSA, 0.1% preservative were added to 0.05M PBS buffer (pH 7.4).

2) The 1, 3-beta-D dextran antigen was diluted with the buffer of the calibration solution to prepare a calibration spot of 0.5 ng/mL.

3) Bovine serum was used as a buffer to prepare concentration points with concentrations of 0ng/mL and 3ng/mL as quality control substances.

(6) Subpackaging the biotin 1, 3-beta-D glucan antigen derivative, an antibody enzyme labeling reagent, an enzyme labeled 1, 3-beta-D glucan antigen derivative, a biotinylation 1, 3-beta-D glucan murine monoclonal antibody, a calibration solution and a quality control product.

A method for measuring the content of 1, 3-beta-D glucan comprises the following steps:

p1: taking 15 ul of a sample, a calibration solution or a quality control product, adding 30ul of biotinylated 1, 3-beta-D glucan antigen derivative, adding 30ul of the antibody enzyme-labeled reagent according to claims 3-6, and incubating for 15 min;

p2: adding 30 mu l of streptavidin magnetic beads into the step P1, and incubating for 5 min;

p3 magnetic separation for 2min, removing supernatant;

p4 washing 3 times, 300 mul washing liquid each time;

p5-add 200. mu.l of substrate, found. Or the following method is adopted for detection:

p1, taking a sample, a calibration solution and 15 mul of a quality control product, adding 30 mul of enzyme-labeled 1, 3-beta-D glucan antigen derivative, adding 30 mul of biotinylation 1, 3-beta-D glucan mouse monoclonal antibody as claimed in claims 3-6, and incubating for 15 min;

p2: adding 30 mu l of streptavidin magnetic beads into the step P1, and incubating for 5 min;

p3 magnetic separation for 2min, removing supernatant;

p4 washing 3 times, 300 mul washing liquid each time;

p5-add 200. mu.l of substrate, found.

To examine the differences between the measured values of the samples in these 2 systems, we tested the same group of samples in these 2 systems and compared them with the given values, and the data are shown in the following table:

TABLE 1 comparison of measured values with given values and comparison between measured values for different systems

As can be seen from the results, if the given value <1 is negative and >1 is positive, the negative and positive result coincidence rate is 100% in comparison with the given values and measured values between the system 1, the system 2, and the system 1 and the system 2. The coincidence rate of the measured values of different systems and the given value is 100%, which indicates that the measured values of 2 systems are consistent. Therefore, the technical scheme of the invention is completely feasible and has higher flexibility.