WO2022170977A1 - Antifungal 1,3-beta-d-glucan monoclonal antibody and use thereof - Google Patents

Abstract

Provided in the present application are an antifungal 1,3-beta-D-glucan monoclonal antibody and the use thereof. The monoclonal antibody has the specific binding capacity to fungal 1,3-beta-D-glucan, and the constructed kit can be used to detect fungal 1,3-beta-D-glucan.

Description

A kind of antifungal 1,3-β-D-glucan monoclonal antibody and its application technical field

The application belongs to the technical field of fungal detection, and relates to an anti-fungal 1,3-β-D-glucan monoclonal antibody and applications thereof.

Background technique

Currently, the detection of fungal 1,3-β-D-glucan mainly adopts the Limulus reagent method. Form coagulase, which catalyzes the color reaction or turbidity reaction, generates free p-nitroaniline (pNA) and causes absorbance changes, and realizes 1,3-β-D-glucan by dynamically detecting the solution absorbance change rate Quantitative detection of concentration. The total detection time of this method was about 50 min, and the lower limit of linearity was 37.5 pg/mL.

However, the key raw materials for the Limulus Reagents come from the national second-class wild animal, Limulus, which has a long growth cycle and a low artificial breeding rate. Usually, it is necessary to catch wild Limulus and extract the raw materials of Limulus reagents from the blood of Limulus. Limulus is an endangered species. The detection of fungal 1,3-β-D-glucan by the Limulus reagent method can no longer meet the clinical needs, and the Limulus reagent prepared from natural Limulus blood varies greatly between batches, resulting in high production costs and poor reproducibility. Difference. In addition, under the action of bacterial endotoxin, the Limulus reagent will also activate coagulase to form coagulase, and bacterial endotoxin is widely present in nature, so the Limulus reagent method is very susceptible to the interference of bacterial endotoxin.

Chemiluminescence detection has been widely used in the field of biological macromolecule detection due to its good specificity, high sensitivity, short detection time, and easy automation. Take Roche's PCT detection kit as an example. The detection kit adopts the sandwich method. The sample, biotin-labeled antibody and ruthenium complex-labeled antibody form an antibody-antigen sandwich immune complex, and then streptavidin magnetic beads are added. The interaction between biotin and streptavidin binds to the immune complex; electromagnetic action is applied to the incubation mixture, the magnetic beads and the immune complex are adsorbed on the surface of the electrode, and the substances not bound to the magnetic beads are removed by ProCell; The electrode applies voltage to chemiluminesce the immune complex, and the concentration of the antigen in the sample is analyzed according to the intensity of the chemiluminescence. The detection time of the detection kit is short, only 18 minutes, but the biotin in the sample will interfere with the binding of the biotin-labeled antibody to the streptavidin magnetic beads, which will affect the accuracy of the results.

SUMMARY OF THE INVENTION

The present application provides an anti-fungal 1,3-β-D-glucan monoclonal antibody and application thereof, the anti-fungal 1,3-β-D-glucan monoclonal antibody is effective against fungal 1,3- β-D-glucan has specific binding ability, good stability and small batch-to-batch variation. The constructed chemiluminescence-based fungal 1,3-β-D-glucan detection kit achieves sensitivity, specificity and accuracy. The quantitative or semi-quantitative detection of fungal 1,3-β-D-glucan has important application prospects in judging the degree of fungal infection in patients.

In a first aspect, the present application provides an anti-fungal 1,3-β-D-glucan monoclonal antibody, the anti-fungal 1,3-β-D-glucan monoclonal antibody comprises a variable heavy chain regions and light chain variable regions;

The heavy chain variable region includes the heavy chain CDR1 shown in SEQ ID NO:1, the heavy chain CDR2 shown in SEQ ID NO:2 and the heavy chain CDR3 shown in SEQ ID NO:3;

The light chain variable region includes the light chain CDR1 shown in SEQ ID NO:4, the light chain CDR2 shown in SEQ ID NO:5 and the light chain CDR3 shown in SEQ ID NO:6;

SEQ ID NO: 1: SGGNTNQQK;

SEQ ID NO: 2: DSSDPPPMSLLTEV;

SEQ ID NO: 3: NNMDRAVKL;

SEQ ID NO: 4: GLCDETRFEC;

SEQ ID NO: 5: VPSPLAPISKQL;

SEQ ID NO: 6: QANIYSYCSE.

In the present application, the anti-fungal 1,3-β-D-glucan monoclonal antibodies comprising the CDRs of SEQ ID NOs: 1 to 6 have significant specific binding to fungal 1,3-β-D-glucan It is beneficial to construct a specific, sensitive, accurate and inexpensive fungal 1,3-β-D-glucan detection kit, which is of great significance in the field of fungal 1,3-β-D-glucan detection.

Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7;

SEQ ID NO: 7:

Preferably, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8;

SEQ ID NO: 8:

In this application, the antifungal 1,3-β-D-glucan monoclonal antibody containing the variable regions of SEQ ID NOs: 7-8 has good specificity and stability, and is compatible with fungal 1,3-β-D - Glucan has a strong affinity and can quickly combine with fungal 1,3-β-D-glucan, which is beneficial to shorten the detection time.

Preferably, the anti-fungal 1,3-β-D-glucan monoclonal antibody further comprises a constant region.

Preferably, the constant region comprises any one or a combination of at least two of the IgGl, IgG2, IgG3 or IgG4 constant regions.

Preferably, the antifungal 1,3-β-D-glucan monoclonal antibody is modified with a conjugate.

Preferably, the conjugate includes any one of a chemiluminescent group, horseradish peroxidase or a fluorescent group.

In a second aspect, the present application provides a preparation method of the antifungal 1,3-β-D-glucan monoclonal antibody described in the first aspect, the preparation method comprising the following steps:

(1) taking spleen cells from experimental animals immunized with fungal 1,3-β-D-glucan, and fusing them with myeloma cells to prepare hybridoma cells;

(2) Cloning the antifungal 1,3-β-D-glucan monoclonal antibody gene from the prepared hybridoma cells, and constructing a recombinant containing the antifungal 1,3-β-D-glucan monoclonal antibody gene expression vectors; and

(3) introducing the recombinant expression vector into competent cells, and separating and purifying the anti-fungal 1,3-β-D-glucan monoclonal antibody from the culture supernatant of the competent cells.

In the present application, a cell line stably expressing anti-fungal 1,3-β-D-glucan monoclonal antibody is constructed by genetic engineering technology, and the anti-fungal 1,3-β-D-glucan monoclonal antibody thus prepared The antibody has good stability, small batch-to-batch variation, and has strong specific binding to fungal 1,3-β-D-glucan.

In a third aspect, the application provides a fungal 1,3-β-D-glucan chemiluminescence detection kit, the fungal 1,3-β-D-glucan chemiluminescence detection kit includes a detection antibody and a signal antibody, the detection antibody and/or the signal antibody are the anti-fungal 1,3-β-D-glucan monoclonal antibody described in the first aspect.

In this application, an anti-fungal 1,3-β-D-glucan monoclonal antibody with strong specific binding force to fungal 1,3-β-D-glucan is used as a detection antibody and/or a signal antibody to construct The immunochemiluminescence detection kit has a wide range of raw materials, controllable, and good stability. The detection method has high sensitivity, good accuracy, short time, and simple sample processing method, which solves the problem that the traditional Limulus reagent method is susceptible to endotoxin interference.

Preferably, in the present application, both ethylenediaminetetraacetic acid (EDTA) solution can be used to pretreat serum samples, and acid-base sample release solution can be used to pretreat serum samples. For example, EDTA solution is added to serum samples, heated at 120°C for 6 minutes, 10000g Centrifuge for 10 minutes to obtain the supernatant as a test sample, or add an acidic sample release solution to the serum sample and let it stand for 5 minutes at room temperature, and then add an alkaline sample release solution for neutralization reaction to obtain a test sample. Compared with the sample processing method of the Limulus reagent method simpler.

Preferably, the detection antibody is coupled to a solid support, preferably a carboxylated solid support.

In the present application, the detection antibody is modified on the solid-phase carrier by the carboxylation coupling method, which avoids relying on the interaction of biotin-streptavidin, eliminates the interference of the sample biotin on the experimental results, and significantly improves the accuracy of the results.

Preferably, the solid phase carrier comprises any one or a combination of at least two of carboxylated magnetic particles, microtiter plates, microspheres, affinity membranes or liquid phase chips.

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

Preferably, the chemiluminescent group comprises any one of acridine sulfonamide, acridine ester, ruthenium complex, luminol, (diaramane)-1,2-dioxane or alkaline phosphatase one or a combination of at least two.

In this application, the chemiluminescence one-step sandwich method is used to detect the fungal 1,3-β-D-glucan in the sample, and the carboxylated magnetic bead-coupled detection antibody, the fungal 1,3-β-D-glucan and chemical The signal antibody is modified with a luminescent group to form an antibody-antigen sandwich immune complex. After magnetic separation and washing to remove substances not bound to the magnetic beads, an excitation solution is added to emit light, and the chemiluminescence intensity is detected to analyze the fungal 1,3-β-D- The content of glucan has achieved the effect of rapid and sensitive detection of fungal 1,3-β-D-glucan.

Preferably, the fungal 1,3-β-D-glucan chemiluminescence detection kit further includes any one or at least two of a positive control substance, a negative control substance, an excitation solution, a blocking solution or a washing solution. combination.

In a fourth aspect, the present application provides a fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit, the fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay reagent The cassette includes the antifungal 1,3-beta-D-glucan monoclonal antibody of the first aspect.

Preferably, the antifungal 1,3-β-D-glucan monoclonal antibody is modified with horseradish peroxidase.

Preferably, the fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit further comprises a solid phase carrier, preferably an enzyme label plate, magnetic particles, microspheres, affinity membranes or liquid phase chips. any one or a combination of at least two.

Preferably, the fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit further comprises any one or a combination of at least two of a blocking solution, a chromogenic solution or a stop solution.

In a fifth aspect, the present application provides a fungal 1,3-β-D-glucan immunofluorescence detection kit, the fungal 1,3-β-D-glucan immunofluorescence detection kit includes the first The antifungal 1,3-beta-D-glucan monoclonal antibody of the aspect.

Preferably, the antifungal 1,3-β-D-glucan monoclonal antibody is modified with a fluorescent group.

Preferably, the fungal 1,3-β-D-glucan immunofluorescence detection kit further includes a blocking solution and/or a washing solution.

In the sixth aspect, the present application provides the antifungal 1,3-β-D-glucan monoclonal antibody according to the first aspect, and the fungal 1,3-β-D-glucan chemistry according to the third aspect Luminescence detection kit, fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit according to the fourth aspect, or fungal 1,3-β-D-glucan immunization according to the fifth aspect The application of fluorescence detection kit in the preparation of fungal 1,3-β-D-glucan detection products.

Compared with the prior art, the present application has the following beneficial effects:

(1) The antifungal 1,3-β-D-glucan monoclonal antibody of the present application has a specific and strong affinity for fungal 1,3-β-D-glucan, and is expressed and prepared by genetic engineering methods. Good, small difference between batches, low price, suitable for mass production.

(2) The fungal 1,3-β-D-glucan detection kit based on the anti-fungal 1,3-β-D-glucan monoclonal antibody of this application has stable performance, high sensitivity, and the detection time is only 10 minutes. The results were in good correlation with the detection results of the Limulus reagent method.

(3) The fungal 1,3-β-D-glucan detection method of the present application is loose on the sample pretreatment conditions. It is treated with alkaline solution at 37 °C, EDTA at 100 °C or acid sample release solution at 2-40 °C After processing the samples, it does not affect the accuracy of the results, and the anti-biotin interference ability is strong, and the biotin concentration of 640ng/mL does not affect the negative and positive test results of the samples, which has important application prospects in the field of fungal detection technology.

Description of drawings

Figure 1 shows the correlation between the detection results of the chemiluminescence detection method and the Limulus reagent method.

Detailed ways

In order to further illustrate the technical means adopted in the present application and its effects, the present application will be further described below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through regular channels.

Example 1 Preparation of antifungal 1,3-β-D-glucan monoclonal antibody

Female BALB/c mice (50 μg/mice) were immunized subcutaneously in the abdomen with fungal 1,3-β-D-glucan and every other week with fungal 1,3-β-D- emulsified in incomplete Freund’s adjuvant Dextran was used for the second, third and fourth immunization; after the fourth immunization, the tail blood of the mice was collected, and the serum titer of serial dilution was determined by ELISA. Cell fusion with myeloma cells and culture with screening medium;

Screen the cell lines containing specific antibody activity in the culture medium, clone the specific antibody gene from these cell lines, and then construct a recombinant expression vector pCMVp-NEO-BAN-Ab containing the specific antibody gene. Transform into CHO cells for expression, purify the expressed antibody, and screen the antibody with good sensitivity and specificity as the final monoclonal antibody.

Example 2 Chemiluminescence detection based on antifungal 1,3-β-D-glucan monoclonal antibody

In this example, anti-fungal 1,3-β-D-glucan monoclonal antibodies whose variable regions are shown in SEQ ID NOs: 7 to 8 are used as detection antibodies and signal antibodies to construct a chemiluminescence system to detect fungi in samples. For the detection of 1,3-β-D-glucan, the steps are as follows:

(1) Add EDTA solution to the serum sample, heat at 120°C for 6 minutes, centrifuge at 10,000g for 10 minutes, and obtain the supernatant as the detection sample;

(2) Add EDTA pretreated sample, carboxyl magnetic bead-coupled detection antibody and acridine sulfonamide-labeled signal antibody to the reaction cup, and incubate for 6 minutes to form an antibody-antigen sandwich immune complex;

(3) placing the mixed solution on a magnetic separator, sucking off the supernatant, and adding a washing solution to wash to remove substances not bound to the magnetic beads;

(4) After placing the cuvette at the measurement point, add the excitation solution and mix it evenly to perform chemiluminescence, and use a photomultiplier to detect the luminescence intensity, and analyze according to the chemiluminescence value and the standard curve established in advance (the sample is a gradient-diluted positive standard) Concentration of fungal 1,3-beta-D-glucan in the sample.

In this example, the limulus reagent method was used to detect the fungal 1,3-β-D-glucan concentration in the same sample.

The results are shown in Table 1 and Figure 1. The samples with the detection concentration ≥ 90pg/mL are positive samples of fungal 1,3-β-D-glucan. It can be seen that based on the antifungal 1,3-β-D-glucan The chemiluminescence detection method of glycan monoclonal antibody and the detection results of the Limulus reagent method had a good correlation (R ² >0.98).

Table 1

sample number	Limulus reagent method (pg/mL)	Chemiluminescence (pg/mL)	Test results
N1	39.77	30.98	Negative / Negative
N2	37.83	31.55	Negative / Negative
N3	37.49	34.90	Negative / Negative

N4	39.27	34.74	Negative / Negative
N5	40.09	34.15	Negative / Negative
N6	48.16	50.17	Negative / Negative
N7	57.95	52.98	Negative / Negative
N8	65.16	58.89	Negative / Negative
N9	71.83	50.89	Negative / Negative
N10	73.32	57.54	Negative / Negative
P1	132.32	116.59	positive/positive
P2	154.86	137.62	positive/positive
P3	167.02	149.01	positive/positive
P4	184.26	206.32	positive/positive
P5	205.61	176.17	positive/positive
P6	253.35	258.19	positive/positive
P7	276.43	247.52	positive/positive
P8	302.95	274.85	positive/positive
P9	386.88	328.69	positive/positive
P10	517.49	452.45	positive/positive

Example 3 Influence of sample processing method on test results

In this example, different sample processing methods are used to pretreat the samples, and the limulus reagent method and the chemiluminescence method are respectively used to detect the concentration of fungal 1,3-β-D-glucan.

The results are shown in Table 2. The samples were treated with alkaline solution (1M KOH, 0.3M KCl) at 37°C, 0.12M EDTA-2Na at 100°C, or acid sample release solution (10%HCl, 2%Gly) at 2～ When treated at 40℃, the concentration of fungal 1,3-β-D-glucan (pg/mL) could be detected by chemiluminescence method, and the detection result had a good correlation with the Limulus reagent method (R ² >0.98).

Table 2

Example 4 Anti-interference effect of biotin

In this example, different concentrations of biotin were added to the sample, and the carboxylated magnetic bead-conjugated antibody was used as the detection antibody to detect the influence of biotin in the sample on the accuracy of the chemiluminescence system of Example 2.

The results are shown in Table 3. The detection time of the chemiluminescence method is significantly shorter than that of the Limulus reagent method (50min), and only 10min is required; adding biotin to the sample near the critical value to a concentration of 640ng/mL did not change the negative and positive test results of the sample. , it can be seen that the use of carboxylated magnetic beads coupled with antibodies eliminates the interference of biotin in the sample on the results.

table 3

Example 5 Enzyme-linked immunosorbent assay based on antifungal 1,3-β-D-glucan monoclonal antibody

In this example, anti-fungal 1,3-β-D-glucan monoclonal antibodies whose variable regions are shown in SEQ ID NOs: 7-8 are used as coating antibodies and enzyme-labeled antibodies to construct an ELISA detection system, and the samples For the detection of 1,3-β-D-glucan in fungi, the steps are as follows:

(1) The anti-fungal 1,3-β-D-glucan monoclonal antibody was prepared into a coating solution with a concentration of 1000ng/mL with 0.01M carbonate buffer (CBS), and added to the microtiter at 100μL/well. In the well plate, coat overnight at 4°C, remove the coating solution the next day, block for 1 h, and dry for 30 min to prepare an enzyme labeling plate;

(2) The HRP-labeled anti-fungal 1,3-β-D-glucan monoclonal antibody was prepared with a conjugate stabilizer into an enzyme-labeled antibody with a concentration of 1000 ng/mL;

(3) Heat the sample with EDTA-2Na, add 100 μL/well to the ELISA plate, incubate at 37°C for 20 min, after washing, add 100 μL/well of the enzyme-labeled antibody to the ELISA plate, incubate at 37°C for 20 min, wash ;

(4) Add 100 μL/well of chromogenic substrate to the ELISA plate, incubate at 37°C for 10 min, and stop reading.

Example 6 Immunofluorescence detection based on antifungal 1,3-β-D-glucan monoclonal antibody

In this example, anti-fungal 1,3-β-D-glucan monoclonal antibodies whose variable regions are shown in SEQ ID NOs: 7-8 are used as binding antibodies and detection antibodies to construct an immunofluorescence detection reagent card, and the samples For the detection of 1,3-β-D-glucan in fungi, the steps are as follows:

(1) Scratch 1 mg/mL fluorescent microsphere-labeled antifungal 1,3-β-D-glucan monoclonal antibody on the conjugate release pad, and 1 mg/mL antifungal 1,3-β-D-glucan Saccharide monoclonal antibody is drawn on the nitrocellulose membrane, and the reagent card is assembled after drying;

(2) After heat-treating the sample with EDTA-2Na, add 100 μL of the sample to the sample tank of the reagent card, let it stand for 10-15 minutes, and detect with a fluorescence analyzer.

To sum up, the antifungal 1,3-β-D-glucan monoclonal antibody of the present application has good specificity and stability, and can rapidly bind to fungal 1,3-β-D-glucan, The fungal 1,3-β-D-glucan detection method based on the anti-fungal 1,3-β-D-glucan monoclonal antibody has short detection time, high sensitivity and good accuracy, and has potential application value.

The applicant declares that the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation. Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

Claims (11)

1. An anti-fungal 1,3-β-D-glucan monoclonal antibody comprising a heavy chain variable region and a light chain variable region;

   The heavy chain variable region includes the heavy chain CDR1 shown in SEQ ID NO:1, the heavy chain CDR2 shown in SEQ ID NO:2 and the heavy chain CDR3 shown in SEQ ID NO:3;

   The light chain variable region includes the light chain CDR1 shown in SEQ ID NO:4, the light chain CDR2 shown in SEQ ID NO:5 and the light chain CDR3 shown in SEQ ID NO:6.
2. The antifungal 1,3-β-D-glucan monoclonal antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7.
3. The antifungal 1,3-β-D-glucan monoclonal antibody according to claim 1 or 2, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.
4. The antifungal 1,3-β-D-glucan monoclonal antibody according to any one of claims 1 to 3, wherein the antifungal 1,3-β-D-glucan monoclonal antibody further including the constant region;

   Preferably, the constant region comprises any one or a combination of at least two of the IgGl, IgG2, IgG3 or IgG4 constant regions.
5. The antifungal 1,3-β-D-glucan monoclonal antibody according to any one of claims 1 to 4, wherein the antifungal 1,3-β-D-glucan monoclonal antibody is modified has a conjugate;

   Preferably, the conjugate includes any one of a chemiluminescent group, horseradish peroxidase or a fluorescent group.
6. A method for preparing an antifungal 1,3-β-D-glucan monoclonal antibody according to any one of claims 1-5, comprising the following steps:

   (1) taking spleen cells from experimental animals immunized with fungal 1,3-β-D-glucan, and fusing them with myeloma cells to prepare hybridoma cells;

   (2) Cloning the antifungal 1,3-β-D-glucan monoclonal antibody gene from the prepared hybridoma cells, and constructing a recombinant containing the antifungal 1,3-β-D-glucan monoclonal antibody gene expression vectors; and

   (3) introducing the recombinant expression vector into competent cells, and separating and purifying the anti-fungal 1,3-β-D-glucan monoclonal antibody from the culture supernatant of the competent cells.
7. A fungal 1,3-β-D-glucan chemiluminescence detection kit, comprising a detection antibody and a signal antibody, wherein the detection antibody and/or the signal antibody are described in any one of claims 1-5 antifungal 1,3-β-D-glucan monoclonal antibody;

   Preferably, the detection antibody is coupled to a solid support, preferably a carboxylated solid support;

   Preferably, the solid phase carrier comprises any one or a combination of at least two of carboxylated magnetic particles, microtiter plates, microspheres, affinity membranes or liquid phase chips;

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

   Preferably, the chemiluminescent group comprises any one of acridine sulfonamide, acridine ester, ruthenium complex, luminol, (diaramane)-1,2-dioxane or alkaline phosphatase one or a combination of at least two.
8. The fungal 1,3-β-D-glucan chemiluminescence detection kit according to claim 7, wherein the fungal 1,3-β-D-glucan chemiluminescence detection kit further comprises a positive control Any one or a combination of at least two of the sample, negative control, challenge solution, blocking solution, or washing solution.
9. A fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit, comprising the antifungal 1,3-β-D-glucan monoclonal described in any one of claims 1-5 Antibody;

   Preferably, the antifungal 1,3-β-D-glucan monoclonal antibody is modified with horseradish peroxidase;

   Preferably, the fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit further comprises a solid phase carrier, preferably an enzyme label plate, magnetic particles, microspheres, affinity membranes or liquid phase chips. any one or a combination of at least two;

   Preferably, the fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit further comprises any one or a combination of at least two of a blocking solution, a chromogenic solution or a stop solution.
10. A fungal 1,3-β-D-glucan immunofluorescence detection kit, comprising the anti-fungal 1,3-β-D-glucan monoclonal antibody according to any one of claims 1-5;

    Preferably, the antifungal 1,3-β-D-glucan monoclonal antibody is modified with a fluorescent group;

    Preferably, the fungal 1,3-β-D-glucan immunofluorescence detection kit further includes a blocking solution and/or a washing solution.
11. The antifungal 1,3-β-D-glucan monoclonal antibody according to any one of claims 1-5, the fungal 1,3-β-D-glucan chemiluminescence according to claim 7 or 8 Detection kit, the fungal 1,3-β-D-glucan enzyme-linked immunosorbent assay kit according to claim 9 or the fungal 1,3-β-D-glucan immunofluorescence according to claim 10 The application of the detection kit in the preparation of fungal 1,3-β-D-glucan detection products.

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