WO2023155379A1 - Aspergillus galactomannan test strip and use thereof - Google Patents

Abstract

The present invention provides an aspergillus galactomannan test strip and a use thereof. The aspergillus galactomannan test strip uses a double-antibody sandwich fluorescence immunochromatography method to measure the content of aspergillus galactomannan in a sample. The aspergillus galactomannan test strip provided by the present invention overcomes the defects of the ELISA method, and has the advantages of point-of-care testing of products, low detection cost, point-of-care testing of samples, simple operation, low requirements for professional personnel and short detection time; the whole detection process can be completed only by three steps of simple sample processing, sample adding and reading; the test strip has the characteristics of rapid detection, high sensitivity and high accuracy.

Description

Aspergillus galactomannan detection test strip and application thereof technical field

The invention relates to the technical field of biological detection, in particular to an Aspergillus galactomannan detection test strip and an application thereof.

Background technique

Invasive fungal disease (IFD) refers to an infectious disease in which fungi invade the human body, grow and multiply in tissues, organs or blood, and cause inflammation and tissue damage. Epidemiological studies at home and abroad have shown that the incidence of invasive aspergillosis (IA) is increasing year by year. Patients with long-term neutropenia, hematopoietic stem cell transplantation, solid organ transplantation, patients receiving glucocorticoid therapy, patients receiving immunosuppressant therapy, and patients with advanced AIDS are all high-risk groups for IA.

Among patients with solid organ transplantation, the mortality rate caused by IA is as high as 67% to 82%. The main reason for the high mortality rate is the inability to diagnose and treat early, so early diagnosis is of great significance. The clinical manifestations of invasive aspergillosis are often non-specific, and the disease is easily covered by the primary disease, resulting in missed diagnosis, misdiagnosis and delayed treatment. Therefore, it is urgent to establish a rapid and specific IA diagnostic method in the early stage of disease development. The problem.

Aspergillus-specific antigen (Galactomannan, GM) is the main component of the cell wall of Aspergillus, and is the earliest marker antigen released into the blood after Aspergillus infection. The release amount is related to the degree of infection. GM detection test is the main method for early diagnosis of IA. The diagnostic methods of IA mainly include microscopic examination (histopathology or cytopathology), culture, imaging, serology (G test or GM test) and molecular biological detection.

At present, in the international market, the Aspergillus antigen detection kit (enzyme-linked immunoassay) of France Bio-Rad is mainly used for early diagnosis of invasive Aspergillus infection. Its sensitivity reaches 1ng/mL, which is currently an internationally recognized diagnostic method for invasive aspergillosis, and has been included in the latest IFI (invasive fungal disease) diagnostic criteria. At present, the domestic marketed kit products all use the ELISA detection method. The ELISA detection method has cumbersome operation steps, long detection time, high requirements on the operator, and cannot realize instant detection, which limits the user use scenarios.

Therefore, it is of great significance to develop an Aspergillus galactomannan detection method that can overcome the shortcomings of the ELISA method, can be detected instantly, is low-cost, simple to operate, and has a short detection time for the early diagnosis of IA.

Contents of the invention

In view of the deficiencies in the prior art, the object of the present invention is to provide a test strip for detection of Aspergillus galactomannan and its application. The test strip for detection of Aspergillus galactomannan provided by the invention overcomes the shortcomings of the ELISA method , has many advantages of real-time testing products, including low testing costs, sample inspection at any time, simple operation, low professional requirements for personnel, short testing time, and results can be obtained within 40 minutes, and only simple sample processing and sample addition are required. The whole detection process can be completed in three steps of reading and reading.

For reaching this purpose, the present invention adopts following technical scheme:

In the first aspect, the present invention provides a test strip for detection of Aspergillus galactomannan. The test strip for detection of Aspergillus galactomannan includes a bottom plate, and the bottom plate is sequentially provided with a sample pad, a fluorescent pads, nitrocellulose membranes and absorbent pads;

The fluorescent pad is embedded with fluorescent microsphere-labeled antibodies, including fluorescent microsphere-labeled Aspergillus galactomannan antibody and fluorescent microsphere-labeled chicken IgY antibody;

The Aspergillus galactomannan antibody labeled with fluorescent microspheres is obtained by linking avidin-labeled fluorescent microspheres and biotin-labeled antibodies;

The fluorescent microsphere-labeled chicken IgY antibody is obtained by connecting avidin-biotin-labeled fluorescent microspheres and biotin-labeled chicken IgY antibodies;

The nitrocellulose membrane is coated with detection lines and quality control lines;

The detection line is a detection line containing Aspergillus galactomannan antibody;

The quality control line is the quality control line containing goat anti-chicken IgY antibody.

In the present invention, the test strip uses double antibody sandwich fluorescent immunochromatography to detect Aspergillus galactomannan in the sample, and the test strip embeds the Aspergillus galactomannan antibody labeled with fluorescent microspheres on the fluorescent pad, Coated with Aspergillus galactomannan antibody in the test line. If the test sample is positive, the Aspergillus galactomannan in it combines with the fluorescent microsphere-labeled Aspergillus galactomannan antibody to form a complex. The complex moves forward along the paper strip under the action of chromatography, and when it passes the detection line Reaction with pre-coated Aspergillus galactomannan antibody to form immune complexes to present fluorescent bands. Aspergillus galactomannan is a multimeric antigen composed of repeating units released by Aspergillus, which contains multiple repeated antibody binding sites. When the complex formed by the combination of mannan antibody passes through the detection line, other sites of Aspergillus galactomannan react with the Aspergillus galactomannan antibody on the detection line to form a double-antibody sandwich complex. If the test sample is negative, no immune complexes will form and no bands will appear at the test line. Use a fluorescent immunoanalyzer to scan the detection area to obtain a fluorescent signal, and combine it with a standard curve to calculate the content of the corresponding Aspergillus galactomannan in the sample.

Preferably, the fluorescent microspheres include any one of time-resolved fluorescent microspheres, cy5 fluorescent microspheres, cy7 fluorescent microspheres, FITC fluorescent microspheres, or APC fluorescent microspheres.

Preferably, the surface of the fluorescent microspheres has functional groups including any one of carboxyl, amino, or sulfonic acid groups, preferably carboxyl time-resolved fluorescent microspheres.

Preferably, the particle size of the fluorescent microspheres is 50-200nm, for example, 50nm, 100nm, 120nm, 140nm, 160nm, 180nm or 200nm.

Preferably, the Aspergillus galactomannan antibody is a rabbit-derived antibody.

Preferably, the Aspergillus galactomannan detection test strip can detect samples derived from Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger.

In the present invention, the Aspergillus galactomannan antibody is a rabbit-derived monoclonal antibody, which has higher affinity and specificity than the mouse-derived anti-Aspergillus antibody, and the Aspergillus galactomannan antibody can recognize more novel Epitopes, easy recognition of small molecules or haptens. The Aspergillus galactomannan detection test strip can identify four kinds of Aspergillus, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger; Aspergillus galactomannan detection test strip detects four kinds of Aspergillus from different sources The sensitivity of Aspergillus galactomannan antigen reaction extracted from the strains was consistent.

In the present invention, the positive detection rate of the Aspergillus galactomannan detection test strip is above 95%, such as 95%, 96%, 97%, 98% or 99%; The negative detection rate of the mannan detection test strips reached 100%.

In a second aspect, the present invention provides a method for preparing the Aspergillus galactomannan detection test strip described in the first aspect, the preparation method of the Aspergillus galactomannan detection test strip comprises the following steps:

(1) processing the sample pad;

(2) Prepare avidin-labeled fluorescent microspheres and biotin-labeled antibodies;

(3) After mixing the avidin-labeled fluorescent microspheres and biotin-labeled antibodies, and embedding them on a fluorescent pad;

(4) Coating detection line and quality control line on the nitrocellulose membrane;

(5) Assemble the test strips.

Preferably, step (1) includes:

The sample pad is treated with the sample pad treatment liquid, and the treated sample pad is dried.

Preferably, the sample pad treatment solution includes buffer salt, sustained release agent, cosolvent and blocking agent.

Preferably, the buffer salt includes Tris, and the concentration of Tris in the sample pad treatment solution is 100-120mmol/L, such as 110mmol/L, 113mmol/L, 115mmol/L, 117mmol/L or 120mmol /L etc.

Preferably, the sustained-release agent includes PVP-K30, and the concentration of the PVP-K30 in the sample pad treatment solution is 0.3wt% to 0.8wt%, for example, it can be 0.3wt%, 0.4wt%, 0.5wt% %, 0.6wt%, 0.7wt% or 0.8wt%, etc.

Preferably, the co-solvent includes Tween-20, and the concentration of Tween-20 in the sample pad treatment solution is 0.01wt% to 1wt%, for example, it can be 0.01wt%, 0.05wt%, 0.1wt%, 0.5wt% or 1wt%, etc.

Preferably, the blocking agent includes BSA, and the concentration of the BSA in the sample pad treatment solution is 1wt% to 3wt%, such as 1wt%, 1.5wt%, 2wt%, 2.5wt% or 3wt%, etc. .

Preferably, the drying parameters are as follows: the humidity is lower than 30%, the temperature is 30-40° C., and the time is 1-24 hours.

In the present invention, use the sample pad treatment liquid to treat the sample pad evenly and put it into 30-40°C (such as 30°C, 33°C, 37°C or 40°C, etc.), low humidity (≤30%, such as 10%) , 15%, 20%, 25% or 30%, etc.) drying 1 ~ 24h (for example, can be 1h, 5h, 10h, 15h, 20h or 24h, etc.).

Preferably, step (2) comprises the following steps:

a. Mix the fluorescent microsphere solution and MES buffer solution to obtain the fluorescent microsphere mixed solution, add EDC solution and NHS solution to the fluorescent microsphere mixed solution to mix, shake the fluorescent microsphere to activate the fluorescent microsphere, collect the precipitate by centrifugation, and obtain the activated fluorescent microspheres;

b. diluting the activated fluorescent microspheres obtained in step a, mixing with avidin, and shaking and reacting to obtain avidin-labeled fluorescent microspheres;

c. Treat with blocking solution, collect the precipitate by centrifugation, and obtain the avidin-fluorescent microsphere complex;

d. The biotin activated by N-hydroxysuccinimide ester is mixed with the labeled antibody, shaken, and the unbound biotin is removed to obtain a biotin-antibody complex.

Preferably, in step a, the concentration of the MES buffer is 10-100mmol/L, such as 10mmol/L, 20mmol/L, 40mmol/L, 60mmol/L, 80mmol/L or 100mmol/L.

Preferably, in step a, the concentration of fluorescent microspheres in the fluorescent microsphere mixture is 0.1wt% to 1wt%, such as 0.1wt%, 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt% or 1wt%, etc.

Preferably, in step a, the concentration of the EDC solution is 5-50 mg/mL, such as 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL , 40mg/mL, 45mg/mL or 50mg/mL, etc.

Preferably, in step a, the concentration of the NHS solution is 5-50 mg/mL, such as 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL , 40mg/mL, 45mg/mL or 50mg/mL, etc.

Preferably, in step a, the volume ratio of fluorescent microsphere mixture, EDC solution and NHS solution is (20-100):1:1, for example, it can be 20:1:1, 30:1:1, 40:1 1:1, 50:1:1, 60:1:1, 70:1:1, 80:1:1, 90:1:1 or 100:1:1 etc.

In the present invention, EDC is used to activate the carboxyl groups on the fluorescent microspheres, and the combination with NHS can increase the coupling efficiency of the fluorescent microspheres. If the addition amount of EDC solution is too low, the activation time of fluorescent microspheres will be prolonged and the activation effect will be reduced; if the addition amount of EDC solution is too high, the residual EDC will cross-link with the antibody and affect the coupling reaction between fluorescent microspheres and antibodies.

Preferably, in step a, the temperature of the shaking reaction is 2-30°C, for example, 2°C, 5°C, 10°C, 15°C, 20°C, 25°C or 30°C, etc., and the time is 10-120 min, For example, it can be 10 min, 30 min, 60 min, 90 min or 120 min, etc.

Preferably, the centrifugation speed in step a is 10000-20000g, such as 10000g, 15000g, 18000g or 10000g, etc., and the centrifugation time is 10-30min, such as 10min, 15min, 20min, 25min or 30min.

Preferably, in step b, the diluent is a HEPES solution, and the concentration of the HEPES solution buffer is 10-100mmol/L, for example, it can be 10mmol/L, 20mmol/L, 40mmol/L, 60mmol/L, 80mmol /L or 100mmol/L, etc.

In the present invention, a large amount of EDC solution and NHS solution are added to fully activate the fluorescent microspheres. After activation, the obtained microspheres are washed with HEPES solution to remove excess EDC solution and NHS solution, which can prevent residual EDC Cross-linking with the Aspergillus galactomannan antibody facilitates the subsequent coupling of the antibody to the fluorescent microspheres.

Preferably, in step b, the avidin includes avidin or streptavidin.

Preferably, in step b, the mass ratio of the activated fluorescent microspheres mixed with avidin is (5-50):1, for example, it can be 5:1, 10:1, 15:1, 20:1 , 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1, etc.

Preferably, in step b, the temperature of the shaking reaction is 2-30°C, such as 2°C, 5°C, 10°C, 15°C, 20°C, 25°C or 30°C, etc., and the time is 0.5-18h, For example, it can be 0.5h, 1h, 2h, 5h, 10h, 15h or 18h, etc.

In the present invention, the blocking solution is a solution of amino acids, polypeptides or irrelevant proteins containing amino groups. Adding the blocking solution can prevent the non-specific adsorption of the Aspergillus galactomannan antibody and improve the detection of the Aspergillus galactomannan. Sensitivity of test strip detection.

Preferably, in step c, the blocking solution includes any one or a combination of at least two of glycine, polylysine, BSA, casein, skim milk powder, gelatin, protamine, and ovalbumin. The concentration of the blocking solution is 0.1wt%-3wt%, such as 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt% or 3wt%.

Preferably, in step d, the labeled antibody is an Aspergillus galactomannan antibody or a chicken IgY antibody, and the concentration of the labeled antibody is 1-10 mg/mL, for example, 1 mg/mL, 2 mg/mL, 4 mg/mL mL, 6mg/mL, 8mg/mL or 10mg/mL etc.

Preferably, in step d, the molar ratio of the labeled antibody to biotin activated by N-hydroxysuccinimide ester is (5-50):1, for example, it can be 5:1, 10:1, 15:1 1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1, etc., preferably (15-25):1.

Preferably, in step d, the shaking temperature is 2-30°C, for example, 2°C, 5°C, 10°C, 15°C, 20°C, 25°C or 30°C, etc., and the time is 0.5-18h, for example It can be 0.5h, 1h, 2h, 5h, 10h, 15h or 18h, etc.

Preferably, in step d, the method for removing unbound biotin includes desalting column desalting, dialysis or ultrafiltration.

Preferably, step (3) includes: mixing the avidin-fluorescent microsphere complex obtained in step (2) and the biotin-antibody complex, spraying it on the fluorescent pad, and drying the fluorescent pad.

The mass ratio of the avidin-fluorescent microsphere complex to the biotin-antibody complex in the mixture is (2-20):1, such as 2:1, 5:1, 10:1, 15:1 Or 20:1, etc., preferably (5-10):1.

Preferably, the amount of spraying is 2-20 μL/cm, such as 2 μL/cm, 4 μL/cm, 6 μL/cm, 8 μL/cm, 10 μL/cm, 12 μL/cm, 14 μL/cm, 16 μL/cm , 18 μL/cm or 20 μL/cm, etc., the pitch is 4 to 10 mm, such as 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 10 mm, etc.

Preferably, the drying parameters are humidity lower than 30%, temperature 30-40°C, time 1-4h.

In the present invention, the drying temperature is 30-40°C (such as 30°C, 33°C, 37°C or 40°C, etc.), low humidity (≤30%, such as 10%, 15%, 20% , 25% or 30%, etc.) drying 1 ~ 4h (for example, can be 1h, 2h, 3h or 4h, etc.).

Preferably, step (4) includes:

Use Aspergillus galactomannan antibody and goat anti-chicken IgY antibody to streak on nitrocellulose membrane and dry.

Preferably, the coating amounts of the Aspergillus galactomannan antibody and the goat anti-chicken IgY antibody are independently 0.5-2 μL/cm, for example, 0.5 μL/cm, 0.75 μL/cm, 1 μL/cm, 1.2 μL /cm, 1.4μL/cm, 1.6μL/cm, 1.8μL/cm or 2μL/cm, etc.

Preferably, the drying parameters are as follows: the humidity is lower than 30%, the temperature is 30-40° C., and the time is 1-24 hours.

In the present invention, the drying temperature in step (4) is 30-40°C (such as 30°C, 33°C, 37°C or 40°C, etc.), low humidity (≤30%, such as 10%, 15%, 20%, 25% or 30%, etc.) drying for 1-18h (for example, it can be 1h, 5h, 10h, 15h or 18h, etc.).

Preferably, step (5) includes:

Paste and assemble a nitrocellulose membrane, a water-absorbing pad, a fluorescent pad and a sample pad in sequence on the PVC bottom plate, and cut after assembling to obtain the Aspergillus galactomannan detection test strip.

In the present invention, the water-absorbing pad and the fluorescent pad cover the nitrocellulose membrane independently by 1-2mm, such as 1mm, 1.5mm or 2mm, etc., and the sample pad covers the fluorescent pad by 1-2mm, such as 1mm , 1.5mm or 2mm, etc.

As a preferred technical solution of the present invention, the preparation method of the Aspergillus galactomannan detection test strip comprises:

(1) Processing the sample pad

The sample pad is treated with the sample pad treatment liquid, the formula of the sample pad treatment liquid is 100～120mmol/L Tris, 0.3wt%～0.8wt% PVP-K30, 0.01wt%～1wt% Tween-20 and 1wt% ～3wt% BSA, dry the treated sample pad at 30～40°C and humidity not greater than 30% for 1～24h.

(2) Preparation of avidin-labeled fluorescent microspheres and biotin-labeled antibodies

a. Mix the fluorescent microsphere solution with 10-100mmol/L MES buffer solution to obtain a fluorescent microsphere mixed solution with a fluorescent microsphere concentration of 0.1wt%-1wt%, and add 5-50mg/L Mix mL of EDC solution with 5-50 mg/mL of NHS solution, wherein the volume ratio of fluorescent microsphere mixture, EDC solution and NHS solution is (20-100):1:1, shake and react at 2-30°C for 10- Activate the fluorescent microspheres for 120 minutes, centrifuge at 10000-20000 g for 10-30 minutes to collect the precipitate, and obtain the activated fluorescent microspheres;

b. Dilute the activated fluorescent microspheres obtained in step a with 10-100mM HEPES solution, and then add avidin to mix, wherein the mass ratio of the activated fluorescent microspheres to avidin is (5-50):1. Shaking reaction at 2-30°C for 0.5-18 hours to obtain avidin-labeled fluorescent microspheres;

c. Treat the avidin-labeled fluorescent microspheres with a blocking solution with a concentration of 0.1 wt% to 3 wt%, and collect the precipitate by centrifugation to obtain the avidin-fluorescent microsphere complex;

d. Mix the biotin activated by N-hydroxysuccinimide ester with the labeled antibody in a molar ratio of (5-50): 1, shake at 2-30°C for 0.5-18, remove unbound biotin, and obtain a biotin-antibody complex things.

(3) Mix the avidin-fluorescent microsphere complex obtained in step (2) and the biotin-antibody complex at a mass ratio of (2-20): 1, and use an amount of 2-20 μL/cm at a distance Spray it on the fluorescent pad with a thickness of 4-10mm, and dry it for 1-4 hours at 30-40°C and humidity not greater than 30%.

(4) Use the Aspergillus galactomannan antibody and the goat anti-chicken IgY antibody to streak on the nitrocellulose membrane respectively. The coating amount is 0.5-2μL/cm, and it is dried for 1-18 hours at 30-40°C and the humidity is not more than 30%.

(5) Paste nitrocellulose membrane, water-absorbing pad, fluorescent pad and sample pad successively on the PVC base plate, described water-absorbing pad and fluorescent pad cover nitrocellulose membrane 1～2mm independently respectively, and described sample pad covers fluorescent pad 1 ~2mm, assembled and cut to obtain the Aspergillus galactomannan detection test strip.

In a third aspect, the present invention provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip described in the first aspect.

In a fourth aspect, the present invention provides a method for using the Aspergillus galactomannan detection kit described in the third aspect, the method for using the Aspergillus galactomannan detection kit includes:

(1) prepare the sample to be tested;

(2) using the Aspergillus galactomannan detection test strip to detect the sample to be tested;

(3) Interpret the results after testing.

Preferably, in step (3), interpreting the result after the detection includes:

Import the standard curve of the test strip, and use the fluorescent immunoassay analyzer to scan the fluorescent signal in the detection area to determine whether the sample is negative or positive.

The numerical ranges described in the present invention not only include the above-mentioned point values, but also include any point values between the above-mentioned numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention will not exhaustively list the above-mentioned point values. Specific point values covered by the stated ranges.

Compared with prior art, the beneficial effect of the present invention is:

(1) The Aspergillus galactomannan detection test strip provided by the present invention overcomes the shortcomings of the ELISA method, and has many advantages of instant detection products, including low detection cost, sample inspection at any time, simple operation, and professionalism of personnel. The requirements are not high and the detection time is short, etc. The results can be obtained within 40 minutes, and the entire detection process can be completed with only three steps of simple sample processing, sample addition and reading.

(2) The microsphere labeling method provided by the present invention uses an avidin-biotin labeling system to directly label antibodies with the microspheres. The labeling method of the present invention can increase the effective antibody labeling amount of the microspheres, thereby improving the sensitivity of the antigen to be tested. Specifically, on the one hand, the amount of antibody labeling on the surface of the microspheres can be increased, and on the other hand, the combination of the labeled antibody and the small molecule biotin can reduce the damage to the activity of the antibody during the labeling process.

(3) When using the Aspergillus galactomannan detection kit provided by the present invention to detect the sample to be tested, use a fluorescent immunoanalyzer to scan the detection area to obtain a fluorescent signal, and then import the standard curve to calculate the corresponding Aspergillus galactomannan in the sample. The content of sugar can be detected quickly, with high sensitivity and high accuracy.

Detailed ways

The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the examples are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

The preparation method of the Aspergillus galactomannan antibody used in the present invention refers to the preparation method in the patent CN110894236A. The preparation method of the Aspergillus galactomannan antibody comprises the following steps:

(1) Construct the expression vector of Aspergillus galactomannan antibody

Use homologous recombination primers to add homologous recombination arms at both ends of the antibody heavy chain variable region gene and light chain variable region gene (see the patent for the sequence of the antibody heavy chain variable region gene and light chain variable region gene sequence) CN110894236A), using double enzymes to linearize the expression plasmid containing the gene sequence of the rabbit antibody heavy chain IgG1 constant region and the expression plasmid containing the gene sequence of the rabbit light chain IgG1 constant region to generate homologous recombination arms; The variable region gene fragment and the linearized plasmid are connected by homologous recombination to form a complete expression plasmid, and the expression plasmid of the heavy chain of the Aspergillus galactomannan antibody and the expression of the light chain of the Aspergillus galactomannan antibody are obtained. Plasmid, transform the expression plasmid into TOP10 Escherichia coli competent, and amplify the expression plasmid.

(2) Expression and purification of Aspergillus galactomannan antibody

Add the obtained expression plasmid of the heavy chain of the Aspergillus galactomannan antibody and the expression plasmid of the light chain of the Aspergillus galactomannan antibody to the Opti-Mem transfection medium in a ratio of 1:1, mix well and add 4 The transfection reagent PEI with twice the quality of DNA was mixed and kept at room temperature in the dark for 30 minutes, and then added to 293T cells. After incubation for 6 hours, the transfection system was removed, and FreeStyle ^TM 293 expression medium was added for cultivation. The supernatant of the medium was purified by using the AKTA protein purification system and the method of affinity purification (Protein A) to obtain the Aspergillus galactomannan antibody. Specifically The steps are:

(a) Centrifuge the medium supernatant at 2500g room temperature for 10min to remove the precipitate;

(b) Fully wash the affinity purification column with Protein A with 10 times the volume of binding buffer (Binding Buffer);

(c) passing the medium supernatant through the purification column at a flow rate of 5 mL/min;

(d) Use 20 times the volume of the purification column to fully wash the purification column with a binding buffer (Binding Buffer);

(e) elute the purification column with 0.1M pH3.2 citric acid buffer until the elution peak drops to an equilibrium state, and adjust the pH to 7.0 with 1M pH9.0 Tris-HCl buffer;

(f) Use a concentrated spin column to concentrate the purified Aspergillus galactomannan antibody, use PBS buffer as the buffer for Aspergillus galactomannan antibody preservation, and finally use the BSA protein concentration detection method to measure the concentrated Aspergillus galactoides Concentration of Mannan Antibody.

The sources of each component used in the following examples and comparative examples are as shown in table 1:

Table 1

components	factory	Item No.
PVP-K30	Sigma	81420
Tween-20	Sigma	P1379
BSA	Sigma	B2064
EDC	Sigma	E6383
NHS	Sigma	130672
fluorescent microspheres	bangs	FCEU002

Example 1

This embodiment provides a test strip for detection of Aspergillus galactomannan, and the preparation method of the test strip for detection of Aspergillus galactomannan comprises:

(1) Processing sample pad:

Use the sample pad treatment liquid to process the sample pad, the formula of the sample pad treatment liquid is 100mmol/L Tris, 0.5wt%PVP-K30, 0.5wt%Tween-20 and 1wt%BSA, the sample pad after processing Dry for 4 hours at 37°C and 25% humidity.

(2) Preparation of antibodies labeled with fluorescent microspheres:

a. The fluorescent microsphere solution is mixed with the MES buffer solution of 50mmol/L to obtain a fluorescent microsphere mixed solution with a fluorescent microsphere concentration of 0.1wt%. Add 10mg/mL EDC solution and 10mg/ Mix mL of NHS solution, wherein the volume ratio of the fluorescent microsphere mixture, EDC solution and NHS solution is 100:1:1, shake the reaction at 25°C for 120 minutes to activate the fluorescent microspheres, and centrifuge at 10000g for 30 minutes to collect the precipitate to obtain the activated fluorescent microspheres;

b. Dilute the activated fluorescent microspheres obtained in step a with 25mmol/L HEPES solution, then add avidin to mix, wherein the mass ratio of activated fluorescent microspheres to avidin is 20:1, and shake the reaction at 25°C 10h, get avidin-labeled fluorescent microspheres;

c. Treat the avidin-labeled fluorescent microspheres with a blocking solution with a concentration of 1 wt%, and collect the precipitate by centrifugation to obtain the avidin-fluorescent microsphere complex;

d. Mix biotin activated by N-hydroxysuccinimide ester with Aspergillus galactomannan antibody at a molar ratio of 20:1, shake at 25°C for 10 hours, remove unbound biotin, and obtain biotin-Aspergillus galactomannan Glycan-antibody complex: mix biotin activated by N-hydroxysuccinimide ester with chicken IgY antibody at a molar ratio of 20:1, shake at 25°C for 10 hours, remove unbound biotin, and obtain biotin-chicken IgY antibody complex;

(3) After mixing the avidin-labeled fluorescent microspheres and biotin-labeled antibodies, and embedding them on a fluorescent pad:

Mix the fluorescent microsphere-labeled avidin and antibody-labeled biotin obtained in step (2) at a mass ratio of 10:1, and the avidin-fluorescent microsphere complex and the biotin-Aspergillus galactomannan antibody complex The mass ratio of the avidin-fluorescent microsphere complex to the biotin-chicken IgY antibody complex is 10:1, sprayed on the fluorescent pad at a usage rate of 5 μL/cm and a distance of 6 mm, And dry at 37°C and 25% humidity for 2 hours.

(4) Coating detection line and quality control line on nitrocellulose membrane:

Use Aspergillus galactomannan antibody and goat anti-chicken IgY antibody to streak on the nitrocellulose membrane respectively, the coating volume of Aspergillus galactomannan antibody is 1 μL/cm, and the coating volume of goat anti-chicken IgY antibody is 1 μL/cm, and dried at 37°C and 25% humidity for 10h.

(5) Assemble test strips:

Paste nitrocellulose film, water-absorbing pad, fluorescent pad and sample pad successively on PVC base plate, described water-absorbing pad and fluorescent pad cover nitrocellulose membrane 2mm independently respectively, described sample pad covers fluorescent pad 2mm, cut after assembling, Obtain the Aspergillus galactomannan detection test strip.

Example 2

This embodiment provides a test strip for detection of Aspergillus galactomannan, the difference between the preparation method of the test strip for detection of Aspergillus galactomannan and that of Example 1 is that step (2) prepares the fluorescent microsphere label In the process of antibody, the volume ratio of fluorescent microsphere mixture solution, EDC solution and NHS solution in step a is 20:1:1.

Example 3

This embodiment provides a test strip for detection of Aspergillus galactomannan, the difference between the preparation method of the test strip for detection of Aspergillus galactomannan and that of Example 1 is that step (2) prepares the fluorescent microsphere label In the process of antibody, the volume ratio of fluorescent microsphere mixture, EDC solution and NHS solution in step a is 10:1:1.

Example 4

This embodiment provides a test strip for detection of Aspergillus galactomannan, the difference between the preparation method of the test strip for detection of Aspergillus galactomannan and that of Example 1 is that step (2) prepares the fluorescent microsphere label In the process of antibody, the volume ratio of fluorescent microsphere mixture solution, EDC solution and NHS solution in step a is 200:1:1.

Example 5

This embodiment provides a test strip for detection of Aspergillus galactomannan, the difference between the preparation method of the test strip for detection of Aspergillus galactomannan and that of Example 1 is that step (2) prepares the fluorescent microsphere label In the process of antibody, the mass ratio of biotin activated by N-hydroxysuccinimide ester in step d and the Aspergillus galactomannan antibody is 2:1.

Example 6

This embodiment provides a test strip for detection of Aspergillus galactomannan, the difference between the preparation method of the test strip for detection of Aspergillus galactomannan and that of Example 1 is that step (2) prepares the fluorescent microsphere label In the process of antibody, the mass ratio of biotin activated by N-hydroxysuccinimide ester in step d and the Aspergillus galactomannan antibody was 55:1.

Example 7

This embodiment provides a test strip for detection of Aspergillus galactomannan, the difference between the preparation method of the test strip for detection of Aspergillus galactomannan and that of Example 1 is that step (2) prepares the fluorescent microsphere label In the process of antibody, the mass ratio of biotin activated by N-hydroxysuccinimide ester and the Aspergillus galactomannan antibody in step d is 5:1.

Example 8

This embodiment provides a test strip for detection of Aspergillus galactomannan, the difference between the preparation method of the test strip for detection of Aspergillus galactomannan and that of Example 1 is that step (3) prepares the fluorescent microsphere label In the antibody process, the mass ratio of avidin-fluorescent microsphere complex to biotin-Aspergillus galactomannan antibody complex was 1:1, and the mass ratio of avidin-fluorescent microsphere complex to biotin-chicken The mass ratio of the IgY antibody complex is 1:1.

Comparative example 1

This embodiment provides a Aspergillus galactomannan detection test strip, and the preparation method of the Aspergillus galactomannan detection test strip comprises the following steps:

(1) Processing sample pad:

Use the sample pad treatment liquid to process the sample pad, the formula of the sample pad treatment liquid is 100mmol/L Tris, 0.5wt%PVP-K30, 0.5wt%Tween-20 and 1wt%BSA, the sample pad after processing Dry for 4 hours at 37°C and 25% humidity.

(2) Preparation of antibodies labeled with fluorescent microspheres:

a. The fluorescent microsphere solution is mixed with the MES buffer solution of 50mmol/L to obtain a fluorescent microsphere mixed solution with a fluorescent microsphere concentration of 0.1wt%. Add 10mg/mL EDC solution and 10mg/ Mix mL of NHS solution, wherein the volume ratio of the fluorescent microsphere mixture, EDC solution and NHS solution is 100:1:1, shake the reaction at 25°C for 120 minutes to activate the fluorescent microspheres, and centrifuge at 10000g for 30 minutes to collect the precipitate to obtain the activated fluorescent microspheres;

b. Dilute the activated fluorescent microspheres obtained in step a with 25mmol/L HEPES solution, then add Aspergillus galactomannan antibody to mix, shake and react at 25°C for 10h, and obtain fluorescent microsphere-labeled Aspergillus galactomannan antibody , the mixed mass ratio of the fluorescent microspheres and the Aspergillus galactomannan antibody is 20:1;

Dilute the activated fluorescent microspheres obtained in step a with 25mmol/L HEPES solution, then add chicken IgY to mix, shake and react at 25°C for 10h, to obtain chicken IgY labeled with fluorescent microspheres, the mass of the fluorescent microspheres mixed with chicken IgY The ratio is 20:1.

(3) Block the antibody labeled with the fluorescent microspheres and embed them on the fluorescent pad:

Use BSA solution to treat the Aspergillus galactomannan antibody labeled with fluorescent microspheres, and shake and react at 25°C for 10 hours to obtain an antibody blocking mixture. The concentration of BSA in the antibody blocking mixture is 1 wt%, and the antibody blocking mixture is The concentration of the Aspergillus galactomannan antibody labeled with fluorescent microspheres is 0.1 wt%, and the precipitated microspheres are collected by centrifugation at 10,000 g for 10 minutes to obtain the Aspergillus galactomannan antibody labeled with fluorescent microspheres after blocking;

Use BSA solution to treat chicken IgY labeled with fluorescent microspheres, and shake and react at 25°C for 10 hours to obtain an antibody blocking mixture. The concentration of BSA in the antibody blocking mixture is 1 wt%, and fluorescent microspheres are labeled in the antibody blocking mixture The concentration of the chicken IgY is 0.1wt%, and the precipitated microspheres are collected by centrifugation at 10,000 g for 10 minutes to obtain chicken IgY labeled with fluorescent microspheres after sealing;

The resulting blocked fluorescent microsphere-labeled Aspergillus galactomannan antibody and fluorescent microsphere-labeled chicken IgY were respectively prepared into 0.5 mg/mL dilutions, and the dilutions were used in an amount of 5 μL/cm with a spacing of 6 mm. Spray it on the fluorescent pad, and dry it for 2 hours at 37°C and 25% humidity.

(4) Coating detection line and quality control line on nitrocellulose membrane:

Use Aspergillus galactomannan antibody and goat anti-chicken IgY antibody to streak on the nitrocellulose membrane respectively, the coating volume of Aspergillus galactomannan antibody is 1 μL/cm, and the coating volume of goat anti-chicken IgY antibody is 1 μL/cm, and dried at 37°C and 25% humidity for 10h.

(5) Assemble the test strip:

Paste nitrocellulose film, water-absorbing pad, fluorescent pad and sample pad successively on PVC base plate, described water-absorbing pad and fluorescent pad cover nitrocellulose membrane 2mm independently respectively, described sample pad covers fluorescent pad 2mm, cut after assembling, Obtain the Aspergillus galactomannan detection test strip.

Application example 1

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 1.

Application example 2

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 2.

Application example 3

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 3.

Application example 4

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 4.

Application example 5

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 5.

Application example 6

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 6.

Application example 7

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 7.

Application example 8

This application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Example 7.

Comparative application example 1

This comparative application example provides an Aspergillus galactomannan detection kit, which includes the Aspergillus galactomannan detection test strip provided in Comparative Example 1.

Test example 1

The Aspergillus galactomannan detection kits obtained in Application Examples 1-8 and Comparative Application Example 1 were used to measure the accuracy of the standard solution, and the statistical test results are shown in Table 2.

Table 2

sample	Standard solution concentration	detection value	Recovery rate
Application example 1	5.0ng/mL	5.1ng/mL	102%
Application example 2	5.0ng/mL	4.9ng/mL	96%
Application example 3	5.0ng/mL	3.9ng/mL	78%
Application example 4	5.0ng/mL	4.2ng/mL	84%
Application example 5	5.0ng/mL	3.5ng/mL	70%
Application example 6	5.0ng/mL	2.3ng/mL	46%
Application example 7	5.0ng/mL	4.6ng/mL	92%
Application example 8	5.0ng/mL	3.7ng/mL	74%
Comparative application example 1	5.0ng/mL	4.6ng/mL	92%

Test example 2

Evaluation of diagnostic sensitivity and diagnostic specificity, using the Aspergillus galactomannan detection kit provided in Application Example 1 and the Aspergillus antigen detection kit (control) produced by Bio-Rad for two types of clinical samples (alveolar lavage fluid and serum) were tested, and the positive coincidence rate, negative coincidence rate and total coincidence rate were counted. The test results of the alveolar lavage fluid samples are shown in Table 3.

table 3

Positive coincidence rate=A/(A+C)×100%=92.4%

Negative coincidence rate = D/(B+D) × 100% = 95.5%

Total coincidence rate=(A+D)/(A+B+C+D)×100%=94.0%

The test results of serum samples are shown in Table 4.

Table 4

Positive coincidence rate = A/(A+C) × 100% = 90.4%

Negative coincidence rate = D/(B+D) × 100% = 97.7%

Total coincidence rate=(A+D)/(A+B+C+D)×100%=94.6%

Evaluation results of diagnostic sensitivity and diagnostic specificity: the positive coincidence rate of the two kits for alveolar lavage fluid samples was 92.4%, the negative coincidence rate was 95.5%, and the total coincidence rate was 94.0%; the positive coincidence rate of the two kits for serum samples The coincidence rate was 90.4%, the negative coincidence rate was 97.7%, and the total coincidence rate was 94.6%.

Comparing the Aspergillus galactomannan detection kit provided in Application Example 1 with the commercially available kit, the comparison between the kit of the present invention and the commercially available kit is shown in Table 5.

table 5

Result shows that described Aspergillus galactomannan detection kit bacterium can reach the detection standard of commercially available kit, simultaneously the Aspergillus galactomannan detection kit provided by the present invention has overcome the deficiency of ELISA method, has the advantage of immediate inspection product There are many advantages, such as low detection cost, short detection time, sample inspection at any time, simple operation, low professional requirements for personnel, and only three simple steps of sample processing, sample addition and reading can complete the entire detection process. The comprehensive performance of the kit of the invention is better than that of the existing aspergillus detection kits on the market.

Test example 3

Precision Evaluation:

Take 4 clinical serum samples (1 negative sample, 1 borderline positive sample, 1 moderately positive sample, 1 strong positive sample) and 4 clinical alveolar lavage fluid samples (1 negative sample, 1 borderline positive sample , 1 moderately positive sample, and 1 strongly positive sample), tested with 3 types of models, 2 operators for each type, 6 operators in total, and 3 batches of kits for each type , the kit was prepared with reference to Example 1, tested for 5 days, and each sample was repeated 5 times a day (3 models × 3 kit batches × 5 days × 5 repetitions/day = 225 results/day sample). The three models are model 1 (dry fluorescence immunoassay analyzer of Guangzhou Lanbo Biotechnology Co., Ltd., model AFS-1000), model 2 (dry fluorescence immunoassay analyzer of Guangzhou Lanbo Biotechnology Co., Ltd., Model AFS2000A), model 3 (dry fluorescent immunoassay analyzer of Suzhou Hemai Precision Instrument Co., Ltd., model FIC-Q100N).

Results of precision assessment of serum samples:

(Medium or strong) positive serum sample data analysis results are shown in Table 6.

Table 6

The data analysis results of critically positive serum samples are shown in Table 7.

Table 7

instrument	detection total mean	Number of results with I value ≥ 0.5	Number of results with I value < 0.5	positive detection rate
AFS-1000	0.60	73	2	97.3%
AFS2000A	0.60	74	1	98.7%
FIC-Q100N	0.61	74	1	98.7%

The results of data analysis of negative serum samples are shown in Table 8.

Table 8

instrument	detection total mean	Number of results with I value ≥ 0.5	Number of results with I value < 0.5	Negative detection rate
AFS-1000	0.18	0	75	100%
AFS2000A	0.18	0	75	100%
FIC-Q100N	0.18	0	75	100%

From the data analysis results in the above table, it can be seen that the CVs of the repeatability, indoor precision and batch-to-batch precision of the positive serum samples and strong positive serum samples in the detection of the three models are all less than 15%, which meets the requirements (repeatability CV≤15%) , CV ≤ 20% between batches); the positive detection rate of borderline positive serum samples was ≥95%, meeting the requirements; the negative detection rates of negative serum samples were all 100%, meeting the requirements.

Results of precision assessment of alveolar lavage fluid samples:

(Medium or strong) positive alveolar lavage fluid sample data analysis results are shown in Table 9.

Table 9

Table 10 shows the data analysis results of borderline positive alveolar lavage fluid samples.

Table 10

instrument	detection total mean	Number of results with I value ≥ 0.5	Number of results with I value < 0.5	positive detection rate
AFS-1000	0.60	74	1	98.67%
AFS2000A	0.59	73	2	97.33%
FIC-Q100N	0.59	73	2	97.33%

The data analysis results of negative alveolar lavage fluid samples are shown in Table 11.

Table 11

instrument	detection total mean	Number of results with I value ≥ 0.5	Number of results with I value < 0.5	Negative detection rate
AFS-1000	0.21	0	75	100%
AFS2000A	0.20	0	75	100%
FIC-Q100N	0.21	0	75	100%

From the data analysis results in the above table, it can be seen that the CV of the repeatability, indoor precision and batch-to-batch precision of positive alveolar lavage fluid samples and strong positive alveolar lavage fluid samples in the detection of the three models are all less than 15%, which meets the requirements ( Repeatability CV ≤ 15%, inter-assay CV ≤ 20%); the positive detection rate of borderline positive alveolar lavage fluid samples ≥ 95%, meeting the requirements; the negative detection rate of negative alveolar lavage fluid samples were all 100%, meet the requirements.

In this test example, the Aspergillus galactomannan detection kit described in Application Example 1 was used to detect Aspergillus galactomannan antigens extracted from four different sources of Aspergillus strains, and the antigens included Aspergillus fumigatus (ATCC : 16903), Aspergillus flavus (ATCC: 24133), Aspergillus terreus (ATCC: 1012) and Aspergillus niger (ATCC: 16888).

Table 12 shows the sensitivity results of the Aspergillus galactomannan detection kit reacting with Aspergillus galactomannan antigens extracted from four different sources of Aspergillus strains.

Table 12

Antigen concentration (ng/ml)	Aspergillus fumigatus antigen	Aspergillus flavus antigen	Aspergillus terreus antigen	Aspergillus niger antigen
1000	24.50	20.96	21.71	21.48
500	16.53	16.33	15.19	16.30
100	9.33	7.76	8.06	8.62
50	7.17	6.46	6.73	6.52

10	2.55	3.22	2.64	2.83
5	2.06	1.85	1.74	1.71
1	0.52	0.47	0.51	0.52
0.5	0.35	0.28	0.33	0.32
0	0.14	0.13	0.12	0.12

From the above results, it can be seen that the Aspergillus galactomannan detection kit has the same sensitivity as the Aspergillus galactomannan antigens extracted from four different sources of Aspergillus strains, and the Aspergillus galactomannan antibody can recognize Four different Aspergillus galactomannan antigens.

In summary, the Aspergillus galactomannan detection kit provided by the present invention overcomes the shortcomings of the ELISA method and has many advantages of instant detection products. The Aspergillus galactomannan detection kit provided by the invention can realize rapid, high-sensitivity, and high-accuracy detection.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A test strip for detection of Aspergillus galactomannan, characterized in that the test strip for detection of Aspergillus galactomannan comprises a bottom plate, and the bottom plate is sequentially provided with sample pads, fluorescent pads, nitric acid Cellulose membranes and absorbent pads;

   The fluorescent pad is embedded with fluorescent microsphere-labeled antibodies, including fluorescent microsphere-labeled Aspergillus galactomannan antibody and fluorescent microsphere-labeled chicken IgY antibody;

   The Aspergillus galactomannan antibody labeled with fluorescent microspheres is obtained by linking avidin-labeled fluorescent microspheres and biotin-labeled antibodies;

   The fluorescent microsphere-labeled chicken IgY antibody is obtained by connecting avidin-biotin-labeled fluorescent microspheres and biotin-labeled chicken IgY antibodies;

   The nitrocellulose membrane is coated with detection lines and quality control lines;

   The detection line is a detection line containing Aspergillus galactomannan antibody;

   The quality control line is the quality control line containing goat anti-chicken IgY antibody.
2. Aspergillus galactomannan detection test strip according to claim 1, wherein the fluorescent microspheres include time-resolved fluorescent microspheres, cy5 fluorescent microspheres, cy7 fluorescent microspheres, FITC fluorescent microspheres or APC Any one of fluorescent microspheres, etc.;

   Preferably, the surface of the fluorescent microspheres has functional groups including any one of carboxyl, amino or sulfonic acid groups;

   Preferably, the particle size of the fluorescent microspheres is 50-200 nm;

   Preferably, the Aspergillus galactomannan antibody is a rabbit-derived antibody;

   Preferably, the Aspergillus galactomannan detection test strip can detect samples derived from Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger.
3. A preparation method of the Aspergillus galactomannan detection test strip according to claim 1 or 2, characterized in that the preparation method of the Aspergillus galactomannan detection test strip comprises the steps:

   (1) processing the sample pad;

   (2) Prepare avidin-labeled fluorescent microspheres and biotin-labeled antibodies;

   (3) After mixing the avidin-labeled fluorescent microspheres and biotin-labeled antibodies, and embedding them on a fluorescent pad;

   (4) Coating detection line and quality control line on the nitrocellulose membrane;

   (5) Assemble the test strips.
4. The preparation method of Aspergillus galactomannan detection test strip according to claim 3, characterized in that, step (1) comprises: treating the sample pad with the sample pad treatment solution, drying the processed sample pad ;

   Preferably, the sample pad treatment solution includes buffer salts, sustained release agents, cosolvents and blocking agents;

   Preferably, the buffer salt includes Tris, and the concentration of Tris in the sample pad treatment solution is 100-120 mmol/L;

   Preferably, the slow-release agent includes PVP-K30, and the concentration of the PVP-K30 in the sample pad treatment solution is 0.3wt% to 0.8wt%;

   Preferably, the co-solvent includes Tween-20, and the concentration of Tween-20 in the sample pad treatment solution is 0.01wt%-1wt%;

   Preferably, the blocking agent includes BSA, and the concentration of the BSA in the sample pad treatment solution is 1wt%-3wt%;

   Preferably, the drying parameters are as follows: the humidity is lower than 30%, the temperature is 30-40° C., and the time is 1-24 hours.
5. The preparation method of Aspergillus galactomannan detection test strip according to claim 3 or 4, it is characterized in that step (2) comprises the following steps:

   a. Mix the fluorescent microsphere solution with the MES buffer to obtain a fluorescent microsphere mixture, add EDC solution and NHS solution to the fluorescent microsphere mixture, shake, and centrifuge to collect the precipitate to obtain activated fluorescent microspheres;

   b. diluting the activated fluorescent microspheres obtained in step a with a diluent, mixing with avidin, and shaking to obtain avidin-labeled fluorescent microspheres;

   c. Treat with blocking solution, collect the precipitate by centrifugation, and obtain the avidin-fluorescent microsphere complex;

   d. The biotin activated by N-hydroxysuccinimide ester is mixed with the labeled antibody, shaken, and unbound biotin is removed to obtain a biotin-antibody complex;

   Preferably, in step a, the concentration of the MES buffer is 10-100 mmol/L;

   Preferably, in step a, the concentration of fluorescent microspheres in the fluorescent microsphere mixture is 0.1wt%-1wt%;

   Preferably, in step a, the concentration of the EDC solution is 5-50 mg/mL;

   Preferably, in step a, the concentration of the NHS solution is 5-50 mg/mL;

   Preferably, in step a, the mixed volume ratio of fluorescent microsphere mixture, EDC solution and NHS solution is (20-100):1:1;

   Preferably, in step a, the shaking temperature is 2-30°C, and the time is 10-120 minutes;

   Preferably, in step b, the diluent is a HEPES solution, and the concentration of the HEPES solution is 10-100 mmol/L;

   Preferably, in step b, the avidin includes avidin or streptavidin;

   Preferably, in step b, the mass ratio of the activated fluorescent microspheres mixed with avidin is (5-50):1;

   Preferably, in step b, the shaking temperature is 2-30°C, and the time is 0.5-18h;

   Preferably, in step c, the blocking solution includes any one or a combination of at least two of glycine, polylysine, BSA, casein, skim milk powder, gelatin, protamine or ovalbumin, and The concentration of the blocking solution is 0.1wt% to 3wt%;

   Preferably, in step d, the labeled antibody is an Aspergillus galactomannan antibody or a chicken IgY antibody, and the concentration of the labeled antibody is 1-10 mg/mL;

   Preferably, in step d, the molar ratio of the labeled antibody and biotin activated by N-hydroxysuccinimide ester in the mixing is (5-50):1;

   Preferably, in step d, the shaking temperature is 2-30°C, and the time is 0.5-18h;

   Preferably, in step d, the method for removing unbound biotin includes desalting column desalting, dialysis or ultrafiltration.
6. According to the preparation method of the Aspergillus galactomannan detection test strip described in any one of claims 3 to 5, it is characterized in that step (3) comprises:

   Mixing the avidin-fluorescent microsphere complex and the biotin-antibody complex obtained in step (2), spraying it on the fluorescent pad, and drying the fluorescent pad;

   Preferably, the mass ratio of the avidin-fluorescent microsphere complex and the biotin-antibody complex in the mixture is (2-20):1;

   Preferably, the amount of spraying is 2-20 μL/cm, and the spacing is 4-10 mm;

   Preferably, the drying parameters are humidity lower than 30%, temperature 30-40°C, time 1-4h.
7. According to the preparation method of the Aspergillus galactomannan detection test strip described in any one of claims 3 to 6, it is characterized in that step (4) comprises:

   Use Aspergillus galactomannan antibody and goat anti-chicken IgY antibody to streak on nitrocellulose membrane;

   Preferably, the coating volumes of the Aspergillus galactomannan antibody and the goat anti-chicken IgY antibody are each independently 0.5-2 μL/cm;

   Preferably, the drying parameters are as follows: the humidity is lower than 30%, the temperature is 30-40° C., and the time is 1-24 hours.
8. According to the preparation method of the Aspergillus galactomannan detection test strip described in any one of claims 3 to 7, it is characterized in that step (5) comprises:

   Paste and assemble a nitrocellulose membrane, a water-absorbing pad, a fluorescent pad and a sample pad in sequence on the PVC bottom plate, and cut after assembling to obtain the Aspergillus galactomannan detection test strip.
9. An Aspergillus galactomannan detection kit, characterized in that the Aspergillus galactomannan detection kit comprises the Aspergillus galactomannan detection test strip according to claim 1 or 2.
10. A method for using the Aspergillus galactomannan detection kit according to claim 9, wherein the method for using the Aspergillus galactomannan detection kit comprises:

    (1) prepare the sample to be tested;

    (2) using the Aspergillus galactomannan detection test strip to detect the sample to be tested;

    (3) Interpret the results after testing;

    Preferably, in step (3), interpreting the result after the detection includes:

    Import the standard curve of the test strip, and use the fluorescent immunoassay analyzer to scan the fluorescent signal in the detection area to determine whether the sample is negative or positive.