CN114324896A

CN114324896A - Application of reagent specifically binding to MUCORICIN protein and trichoderma detection kit

Info

Publication number: CN114324896A
Application number: CN202111680018.7A
Authority: CN
Inventors: 谢洋洋; 代双; 夏良雨; 于秀玲; 罗沛
Original assignee: Fapon Biotech Inc
Current assignee: Fapon Biotech Inc
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-12

Abstract

The invention provides an application of a reagent specifically binding to MUCORICIN protein and a trichoderma detection kit. Wherein the use comprises: the reagent for detecting mucorales infection is prepared by using the reagent which is specifically combined with the MUCORICIN protein. Solves the problems of specificity and sensitivity of mucor detection in the prior art, and is suitable for the field of mucor detection.

Description

Application of reagent specifically binding to MUCORICIN protein and trichoderma detection kit

Technical Field

The invention relates to the field of mucor detection, and in particular relates to an application of a reagent specifically binding to MUCORICIN protein and a mucor detection kit.

Background

Mucormycosis is a fungal pathogen that causes mucormycosis, a fatal vascular invasive disease. Mucormycosis has become the third most common invasive fungal infection in hematological malignancies and organ transplant patients over the last 20 years. This fatal disease is mainly caused by fungal infection of the order mucorales in the genus rhizopus.

Several studies have shown that the incidence of mucormycosis is increasing. Early diagnosis of mucormycosis is therefore of paramount importance, and early diagnosis can affect the therapeutic outcome, improve survival rates, and reduce surgical excision, disfigurement and pain. This is of great importance when high suspicion is due to the rare occurrence of mucormycosis. Current diagnostics include identification of risk factors, assessment of clinical manifestations, early use of imaging methods, and rapid initiation of histopathology, tissue culture, and molecular technology-based diagnostic methods.

At present, the clinical diagnosis methods of mucormycosis, such as histopathology, specimen fiber observation, sample culture and other technologies, need invasive surgery to obtain samples, are long in time consumption and poor in sensitivity and specificity, and cannot be used for diagnosis in early disease. Molecular detection and development are important tools to identify infections and identify the strains involved. Serum mucorales PCR has proven to be a highly reliable tool for diagnosing invasive mucormycosis in immunocompromised patients. Although the serum qPCR detection method has high specificity, the sensitivity is poor, the equipment is expensive, and the time consumption is long compared with the immunological method. The diagnosis products developed based on immunology in the market generally have the problems of low specificity and low sensitivity, and can not effectively and accurately identify whether an individual is infected by mucor or not at an early stage, so that the diagnosis and treatment time is missed.

Based on the above problems, the novel protein marker of the present invention is proposed to solve the problems of specificity and sensitivity in the immunoassay of mucor.

Disclosure of Invention

The invention mainly aims to provide an application of a reagent specifically binding to MUCORICIN protein and a mucor detection kit, so as to solve the problems of specificity and sensitivity to mucor in the prior art.

In order to achieve the above object, according to a first aspect of the present invention, there is provided the use of an agent that specifically binds to a mucrocicin protein for the preparation of an agent for detecting mucorales infection.

Further, the agent that specifically binds to the mucrocicin protein may be an antibody or antigen-binding fragment thereof that binds to mucrocicin.

Further, provided is the use of a reagent which specifically binds to the mucrocicin protein and a reagent which specifically binds to COTH3 protein in combination for the preparation of a reagent for the detection of mucorales infection.

Further, provided is a use of an antibody or an antigen-binding fragment thereof which specifically binds to mucrocicin and an antibody or an antigen-binding fragment thereof which specifically binds to COTH3 for preparing a reagent for detecting mucorales infection.

Further, the samples were tested for the presence of mucrocicin using the mucrocicin antibody and for the presence of COTH3 using the COTH3 antibody.

Further, the detection method is selected from any one or more of the following: radioimmunoassay, enzyme-linked immunosorbent assay, fluoroimmunoassay, immunodiafiltration, immunochromatography, chemiluminescence or time-resolved analysis.

Further, the detection method adopts double antibody sandwich detection; preferably, the mucrocinin protein is detected using an antibody against mucrocicin, further preferably using a monoclonal antibody against mucrocicin; preferably, the COTH3 protein is detected using an antibody against COTH3, more preferably a monoclonal antibody against COTH 3.

Further, the above-mentioned detection reagent is used for measuring whether a sample taken from an individual is mucrocinin protein and/or COTH3 protein in blood, plasma or serum.

Further, when the COI of the MUCORICIN protein is more than 1, the COI of the COTH3 protein is more than 1, or the COI of the combined MUCORICIN protein and the COTH3 protein is more than 1, the individual is determined to be infected with mucor.

In order to achieve the above object, according to a second aspect of the present invention, there is provided a mucor detection kit comprising a reagent for detecting a mucrocidin protein.

Further, the kit further comprises a reagent for detecting the COTH3 protein in combination with a reagent for detecting the MUCORICIN protein; preferably, the reagent for detecting the mucrocicin protein comprises an antibody against mucrocicin, and further comprises a monoclonal antibody against mucrocicin; preferably, the reagent for detecting the COTH3 protein comprises an antibody against COTH3, and further comprises a monoclonal antibody against COTH 3.

Further, the kit is selected from an enzyme-linked immunoassay kit, a fluorescence immunoassay kit or a chemiluminescence immunoassay kit.

By applying the technical scheme of the invention, whether the sample to be detected contains the mucorales specific product MUCORICIN protein or not is detected, and if the mucorales specific product MUCORICIN protein is detected, the source of the sample is infected with mucorales. By using the immunological method, whether the sample to be detected has the trichoderma specific product or not and whether the source of the sample is infected with the trichoderma or not can be quickly known.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

As mentioned in the background art, the current clinical diagnosis methods of mucormycosis, such as histopathology, observation of specimen fibers, and culture of specimens, require invasive surgery to obtain specimens, are time-consuming, have poor sensitivity and specificity, and cannot be used for early diagnosis of mucormycosis. Molecular detection and development are important tools to identify infections and identify the strains involved. Serum mucorales PCR has proven to be a highly reliable tool for diagnosing invasive mucormycosis in immunocompromised patients. Although the serum qPCR detection method has high specificity, the sensitivity is poor, the equipment is expensive, and the time consumption is long compared with the immunological method.

Thus, in the present application, the inventors tried to detect mucor using an immunological method. The application discovers that the MUCORICIN protein is a specific protein of mucor, so that the MUCORICIN protein can be used as a biomarker of mucor and is used for detecting whether a sample to be detected contains mucor. In addition, further experiments show that the detection sensitivity is greatly improved on the premise that the detection specificity is kept unchanged by using the protein COTH3 as a marker for detection. On the basis of the research results, the applicant proposes a series of protection schemes of the application.

In a first exemplary embodiment of the present application, there is provided a method for detecting mucor, the method including: the mucrocicin protein is detected in the sample.

The sample to be detected is derived from serum, plasma or blood of an organism, if the organism is infected by mucor, mucor or a specific product thereof exists in the sample to be detected, an immunological method is utilized to detect whether the sample to be detected contains mucor, and if the detection result is positive, the source organism of the sample is infected by mucor. Mucor-specific products include mucrocicin protein. Whether the mucor exists in the sample and organisms from which the sample is derived can be judged by detecting whether the mucrocicin protein exists in the sample.

In a preferred embodiment, the mucrocicin protein and COTH3 protein are detected in a sample.

Mucorales-specific products also include COTH3 protein. The 2 mucor specificity products are used for carrying out combined detection on mucor in the sample, so that the accuracy of the detection method can be further improved.

In a preferred embodiment, the detection method is selected from any one or more of the following: radioimmunoassay, enzyme-linked immunosorbent assay, fluoroimmunoassay, immunodiafiltration, immunochromatography, chemiluminescence or time-resolved analysis.

In the method, the mucorales can be detected by using methods such as radioisotope measurement, enzyme catalysis, fluorescent labeling and the like, and can be flexibly selected according to detection conditions, detection requirements and the like.

In a preferred embodiment, the detection method employs a double antibody sandwich assay; preferably, the reagent for detecting the mucrocicin protein comprises an antibody against mucrocicin, and further comprises a monoclonal antibody against mucrocicin; preferably, the reagent for detecting the COTH3 protein comprises an antibody against COTH3, and further comprises a monoclonal antibody against COTH 3.

The double antibody sandwich assay refers to the simultaneous binding of one unit of antigen to two units of antibody to form a "sandwich". Catalyzing a substrate of the enzyme by using an enzyme-linked antibody in the antibody, and judging the content of the antigen according to the color depth of a product. In the detection method, the double-antibody sandwich detection method is utilized, so that the content of the antigen related to mucor in the sample to be detected can be accurately measured, and whether the mucor exists in the sample or the sample source is judged. If mucor exists in the sample to be detected, the antibody against MUCORICIN can be combined with MUCORICIN protein, and the antibody against COTH3 can be combined with COTH3 protein. The detection method has high judgment accuracy, mature and simple operation method, and can quickly process a large number of samples and draw conclusions.

In a second exemplary embodiment of the present application, the use of an agent that specifically binds to a mucrocicin protein for the preparation of an agent for detecting mucor infection.

In a preferred embodiment, the MUCORICIN protein and the detection COTH3 protein are combined to be used as a marker for preparing a detection product for detecting mucor; preferably, the reagent for detecting the mucrocicin protein comprises an antibody against mucrocicin, and further comprises a monoclonal antibody against mucrocicin; preferably, the reagent for detecting the COTH3 protein comprises an antibody against COTH3, and further comprises a monoclonal antibody against COTH 3.

Mucor-specific products include mucrocicin protein. The antibody against the MUCORICIN is utilized to be capable of being combined with the MUCORICIN protein to generate immunological reaction, and the antibody against the MUCORICIN is applied to prepare a detection product for detecting mucor. Antibodies against mucrocicin include monoclonal antibodies against mucrocicin. If the sample to be detected contains mucor, the detection product containing the antibody against the MUCORICIN can detect that the sample contains mucor by detecting specific MUCORICIN protein.

Mucor-specific products include not only the mucrocicin protein but also the COTH3 protein. The antibody against COTH3 can be specifically combined with COTH3 protein. The antibody in the step 2 is used in a detection product for detecting mucor, so that the detection accuracy and sensitivity can be improved, and the occurrence of missed detection and false positive can be reduced.

In a preferred embodiment, the reagent is used for measuring the mucrocicin protein and/or the COTH3 protein in a blood, plasma or serum sample taken from an individual.

In a preferred embodiment, an individual is identified as infected with mucor when the MUCORICIN protein has a COI > 1 measurement, or the COTH3 protein has a COI > 1 measurement, or the MUCORICIN protein and the COTH3 protein are combined to have a COI > 1 measurement.

In a preferred embodiment, the detection product is a detection kit, and preferably, the kit is selected from an enzyme-linked immunoassay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

The detection product can be produced, sold and used in the form of a detection kit, and other materials or reagents required in detection can be included in the detection kit, including but not limited to empty ELISA plates, antibody-coated ELISA plates, substrates, substrate solutions, distilled water, washing solutions and stop solutions. Providing materials or reagents required for the assay in the form of a kit can reduce the preparation time required for the assay, improve the assay efficiency, and also reduce errors or errors caused by the user preparing the materials or reagents by himself/herself.

In a third exemplary embodiment of the present application, a mucor detection kit is provided, which comprises a reagent for detecting a mucrocinin protein.

The mucor detection kit comprises a reagent for detecting the MUCORICIN protein, and can detect mucor in a sample by utilizing the characteristic that the reagent can be combined with the specific product MUCORICIN protein of mucor.

In a preferred embodiment, the kit further comprises a reagent for detecting the COTH3 protein in combination with a reagent for detecting the mucrocinin protein; preferably, the reagent for detecting the mucrocicin protein comprises an antibody against mucrocicin, and further comprises a monoclonal antibody against mucrocicin; preferably, the reagent for detecting the COTH3 protein comprises an antibody against COTH3, and further comprises a monoclonal antibody against COTH 3.

An anti-COTH 3 reagent which can be specifically combined with another specific product COTH3 protein of the mucor is added into the kit, and the 2 reagents are used for simultaneously detecting the mucor, so that the detection reliability of the mucor detection kit can be improved. Reagents for detecting specific products include antibodies. If mucor exists in the sample to be detected, the antibody against MUCORICIN can be combined with MUCORICIN protein, and the antibody against COTH3 can be combined with COTH3 protein.

In a preferred embodiment, the kit is selected from an enzyme-linked immunoassay kit, a fluorescent immunoassay kit or a radioimmunoassay kit.

According to different detection conditions, detection requirements and the like, the kit can be flexibly adjusted into different kits including but not limited to an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a radioimmunoassay kit, and the mucor is detected by using methods such as radioisotope determination, enzyme catalysis and fluorescent labeling.

The advantageous effects of the present application will be explained in further detail below with reference to specific examples.

Example 1: preparation of monoclonal antibody against COTH3

1. Immunizing animals

A BALB/c mouse which is 8-12 weeks old and has the same line with myeloma cells is taken, 100 mu g of COTH3 protein and an equivalent amount of Freund's complete adjuvant are fully and uniformly mixed, and the mixture is injected into the abdominal cavity of the mouse. Thereafter, 100. mu.g of COTH3 protein was mixed well with an equal amount of Freund's incomplete adjuvant every 2 weeks, and injected into the abdominal cavity of the mouse several times to boost the immunity. By detecting mouse serum (indirect ELISA method), the antibody with titer above 1: 2000 can be used for fusion, 3 days before fusion, the antibody can be boosted again in abdominal cavity of mouse, and the dosage is 50 μ g/mouse.

2. Preparation of feeder cells

BALB/c mouse peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mouse neck-pulled to be killed, 75% alcohol soaked in whole body, in super clean bench, using scissors to cut off abdominal skin under aseptic operation, exposing peritoneum, using syringe to inject 5mL of RPMI1640 basic culture solution into abdominal cavity, repeatedly washing, recovering washing solution, 1000rpm, centrifuging for 5min, leaving precipitate, using RPMI1640 to screen culture solution (in complete culture solution of RPMI1640 containing HAT)) Resuspending and adjusting the cell concentration to 1X 10⁵Add to 96-well plate at 150. mu.L/well, 37 ℃ with 5% CO₂The culture was carried out overnight.

3. Preparation of immune spleen cells

Three days after the last immunization of the mice, the spleen is taken out under the aseptic condition, placed in a plate, washed once by RPMI1640 basic culture solution, placed on a nylon net of a small beaker, ground and filtered to prepare cell suspension. Centrifuging, discarding supernatant, resuspending RPMI1640 basic culture solution, repeating the steps three times, and counting.

4. Cell fusion

(1) Respectively putting 40mL HAT culture solution, 15mL DMEM serum-free culture solution and 1mL 50% PEG (M12000) in a water bath at 37 ℃ for pre-warming;

(2) respectively taking mouse myeloma cells Sp2/0 (stored by Roc biology Ltd.) (2-5 multiplied by 10)⁷) The above immune spleen cell (10)⁸) The suspension is added into a 50mL centrifuge tube for even mixing, and DMEM serum-free culture solution is added to the centrifuge tube until the volume is 40 mL. Centrifuging for 10 min, pouring out the supernatant, and mixing;

(3) the centrifuge tube was placed in pre-warmed water at 37 ℃ and 0.7mL of pre-warmed 50% PEG solution was allowed to stand for 90 seconds. 15mL of prewarmed serum-free medium at 37 ℃ was immediately added dropwise.

(4) DMEM serum-free culture solution is supplemented to 40mL, centrifugation is carried out for 10 minutes, and the supernatant is poured out. Adding 40mL HAT culture solution containing 15% -20% fetal calf serum. Mixing with a pipette, dropping 2 drops into small holes of 4 pieces of 96-well cell culture plate containing feeder cells, placing at 37 deg.C and 7% CO₂Cultured in an incubator.

5. Selective culture of hybridoma cells

After being treated by PEG, the immune mouse spleen cell and the mouse myeloma cell form a mixture of a plurality of cell components, wherein the cell components comprise unfused myeloma cells and immune spleen cells; a corekaryon of myeloma cells and a corekaryon of immune spleen cells, and a corekaryon of myeloma cells and immune spleen cells. Only the latter can form hybridoma cells. For this purpose, it is necessary to remove unfused cells and homologously fused coenckaryons from the various cell mixtures and to select for true hybrid cells. Therefore, the cells were cultured with the HAT medium on days 1, 3, 5, and 7 after cell fusion.

6. Detection of specific antibodies and hybridoma cell cloning

The supernatant of each culture well was aspirated, and culture wells containing an antibody specific to COTH3 in the culture were detected by indirect ELISA. Hybridoma cells were cloned by limiting dilution. After being cultured, the single cell can be proliferated into homologous cell clone; 4 cell strains which stably secrete the monoclonal antibody against COTH3 are obtained through reactivity screening, and the titer of the cell strains is 10⁵～10⁷In the meantime.

Numbering	Potency of the drug
		4A7	2.6×10⁶
5B3	6.8×10⁵
		9B6	3.5×10⁷
3C9	4.1×10⁶

Example 2: preparation of monoclonal antibody against MUCORICIN

In the same way as in the example, the immunogen was replaced by MUCORICIN protein to obtain 5 cell lines stably secreting monoclonal antibodies against MUCORICIN, all at a titer of 10⁵～10⁷In the meantime.

Numbering	Potency of the drug
		3A2	6.9×10⁵
5A7	7.6×10⁵
		7B3	5.3×10⁶
2C6	4.4×10⁷
		6C4	3.1×10⁶

Example 3:

the ELISA plate was coated with mucor specific protein COTH3 and MUCORICIN monoclonal antibody, and horseradish peroxidase HRP was labeled with another monoclonal antibody of COTH3 and MUCORICIN.

Firstly, human serum is added into an ELISA plate, mucor specific protein COTH3 and protein MUCORICIN possibly contained in the human serum are firstly combined with a monoclonal antibody of anti-COTH 3 and MUCORICIN coated on the plate to form a compound, and then unbound protein is washed away; in the second step, monoclonal antibodies against COTH3 and mucrocicin labeled with HRP were combined with the complex of the first step to form a double antibody sandwich.

1. Monoclonal antibody (5B3) against COTH3 coated ELISA plate

Monoclonal antibody 1 against COTH3 was prepared by the method of example 1.

Monoclonal antibody 1 against COTH3 was diluted with phosphate buffer ph9.6, and monoclonal antibody 1 against COTH3 was coated at a concentration of 2 μ g/ml.

The diluted monoclonal antibody 1 against COTH3 was applied to ELISA plates in an amount of 100. mu.L/well and coated overnight at 4 ℃. Washed 4 times with PBST solution and blocked with 1% BSA solution at 37 ℃ for 2 h. And (3) washing the substrate for 4 times by using a PBST solution, and drying and storing the substrate to obtain the ELISA plate coated by the monoclonal antibody resisting COTH 3.

2. anti-MUCORICIN monoclonal antibody (5A7) coated ELISA plate

Monoclonal antibody 1 against mucrocicin was prepared by the method of example 2.

The anti-MUCORICIN monoclonal antibody 1 was diluted with phosphate buffer pH9.6, and the coating concentration of the anti-MUCORICIN monoclonal antibody 1 was 3. mu.g/mL.

The diluted anti-MUCORICIN monoclonal antibody 1 was applied to ELISA plates at 100. mu.L/well and coated overnight at 4 ℃. Washed 4 times with PBST solution and blocked with 1% BSA solution at 37 ℃ for 2 h. And (3) washing the substrate for 4 times by using a PBST solution, drying and storing to obtain the ELISA plate coated by the MUCORICIN-resistant monoclonal antibody.

3. Combined coating ELISA plate of monoclonal antibody against COTH3 and MUCORICIN

The coating concentration of monoclonal antibody 1 against COTH3 was 2. mu.g/mL, and the coating concentration of monoclonal antibody 1 against MUCORICIN was 3. mu.g/mL, each diluted to the coating concentration with phosphate buffer solution of pH9.6, and added to an ELISA plate in an amount of 100. mu.L/well. Coating was carried out overnight at 4 ℃. Washed 4 times with PBST solution and blocked with 1% BSA solution at 37 ℃ for 2 h. And (3) washing the substrate for 4 times by using a PBST solution, drying and storing the substrate to obtain the ELISA plate jointly coated by the monoclonal antibodies resisting COTH3 and MUCORICIN.

4. Monoclonal antibody (4A7)2 against COTH3 labeled HRP

In the experiment, an HRP (horse radish peroxidase) labeled antibody is carried out by adopting a sodium periodate method, and the HRP is purchased from Sigma-Aldrich. (5 mg HRP was weighed out and dissolved in 1mL distilled water, 0.2mL of fresh 0.1M NaIO was added₄Solutions ofStirred at room temperature for 20 minutes in the dark.

The above solution was filled into a dialysis bag and dialyzed against 1mM sodium acetate buffer pH4.4 at 4 ℃ overnight.

The liquid in the dialysis bag was transferred to a beaker, and 20. mu.l of 0.2M carbonate buffer solution at pH9.5 was added to raise the pH of the above reaction solution to 9.0 to 9.5, followed immediately by addition of 10mg of the antibody to be labeled, i.e., monoclonal antibody 2 against COTH 3. The reaction mixture was added to 1mL of 0.01M carbonate buffer, and the mixture was gently stirred at room temperature for 2 hours in the dark.

Adding 0.1mL of newly formulated 4mg/mL NaBH₄Mixing the solutions, and standing at 4 deg.C for 2 hr.

The above solution was put into a dialysis bag and dialyzed against 0.15M PBS pH7.4 at 4 ℃ overnight.

An equal volume of saturated ammonium sulfate was added dropwise with stirring and left at 4 ℃ for 1 hour.

Centrifuge at 3000rpm for half an hour, and discard the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate and finally dissolved in a small amount of 0.15M PBS pH 7.4.

Putting the solution into a dialysis bag, dialyzing 0.15M PBS (phosphate buffer saline) with pH7.4, removing ammonium ions (detected by a naphthalene reagent), centrifuging at 10000rpm for 30 minutes to remove precipitates, and subpackaging and freezing the supernatant, wherein the supernatant is HRP (horse radish peroxidase) marked by the antibody against COTH3 monoclonal.

5. anti-MUCORICIN monoclonal antibody (2C6)2 labeled HRP

The labeling method is the same as above, and the HRP labeled by the anti-MUCORICIN monoclonal antibody is obtained, subpackaged and frozen for storage. 6. ELISA detection steps of detecting COTH3 protein and MUCORICIN protein by using constructed ELISA plates coated by the monoclonal antibody resisting COTH3, the monoclonal antibody resisting MUCORICIN and the monoclonal antibody resisting COTH3 and MUCORICIN:

50 μ L of the test sample and the control sample were added to the respective wells of the above-mentioned ELISA plate coated with the monoclonal antibody, and the test sample was diluted with PBS buffer by a double dilution method. The ELISA plates after the addition were incubated at 37 ℃ for 30min, the solution was spun off after incubation, washed 5 times with PBS buffer and spun dry.

And adding 100 mu L of enzyme-labeled working solution containing HRP labeled by the monoclonal antibody resisting COTH3 and HRP labeled by the monoclonal antibody resisting MUCORICIN into each well, placing the ELISA plate at 37 ℃ for incubation for 30min after adding, throwing off the solution after incubation, washing for 5 times by PBS buffer solution, and then drying by spinning.

Add 50. mu.L of substrate A solution (10% H) to each well₂O₂) And 50. mu.L of substrate B solution (0.05 m/v% TMB, 3,3 ', 5, 5' -tetramethylbenzidine), shaking, mixing, and incubating at room temperature in the dark for 15 min.

Add 50. mu.L of stop solution (2mol/L sulfuric acid) to each well and determine OD₄₅₀The absorbance value of (c). The results are given in table 1 below.

Table 1:

7. clinical specificity verification by using constructed monoclonal antibody combined coated ELISA plate resisting COTH3 and MUCORICIN

Sample preparation: 10 samples of fresh normal serum and 10 samples of serum infected with fungi other than mucor.

50 μ L of the sample to be tested and the control sample were added to the respective wells, and the sample to be tested was diluted with PBS buffer by a double dilution method. After the addition, the ELISA plate was incubated at 37 ℃ for 30min, after which the solution was spun off, washed 5 times with PBS buffer and spun dry.

And adding 50 mu L of substrate A liquid and 50 mu L of substrate B liquid into each hole, shaking, and incubating for 15min at room temperature in a dark place.

Add 50. mu.L of stop buffer to each well and determine OD₄₅₀The absorbance value of (c). The results are given in table 2 below.

And (4) judging a result: cut Off (OD) as negative control₄₅₀The value x 3+0.1,

COI-OD 450/cut off COI < 1 of the sample is negative.

Table 2:

note: COI: critical index (cut off index).

8. Clinical sensitivity verification is carried out on the constructed monoclonal antibody combination coated ELISA plate resisting COTH3 and MUCORICIN

Sample preparation: 20 positive serum samples

50 mu L of the sample to be detected and the control sample are respectively added into corresponding holes, the ELISA plate is placed at 37 ℃ for incubation for 30min after the addition, the solution is thrown off after the incubation, and the solution is spun-dry after 5 times of washing.

And adding 100 mu L of enzyme-labeled working solution containing HRP labeled by the monoclonal antibody resisting COTH3 and HRP labeled by the monoclonal antibody resisting MUCORICIN into each well, placing the ELISA plate at 37 ℃ for incubation for 30min after adding, throwing off the solution after incubation, washing for 5 times, and then drying by spinning.

Add 50. mu.L of stop buffer to each well and determine OD₄₅₀The absorbance value of (c). The results are given in Table 3 below.

And (4) judging a result: cut off as negative control OD₄₅₀The value x 3+0.1,

COI-OD 450/cut off COI < 1 of the sample is negative.

Table 3:

note: COI: critical index (cut off index).

In addition, the application also uses commercial antibodies or other antibodies to carry out combined detection on the COTH3 protein and the MUCORICIN protein, and the detection effect is consistent with the effect obtained in the tables 2 and 3.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: and (3) judging whether the source of the sample is infected with mucor by detecting whether the sample to be detected contains mucor specific products MUCORICIN protein and/or COTH3 protein.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a reagent that specifically binds to a mucrocicin protein in the preparation of a reagent for detecting mucorales infection.

2. The use according to claim 1, wherein the agent that specifically binds to the MUCORICIN protein is an antibody or antigen-binding fragment thereof that binds to the MUCORICIN protein.

3. Use according to claim 1, comprising the use of an agent that specifically binds to the COTH3 protein for the preparation of an agent for the detection of mucorales infection;

preferably, the agent that specifically binds to a COTH3 protein is an antibody or antigen-binding fragment thereof that binds to the COTH3 protein.

4. Use according to any one of claims 1 to 3, wherein the agent for detecting mucorales infection is selected from any one of the following methodologies: radioimmunoassay, enzyme-linked immunosorbent assay, fluoroimmunoassay, immunodiafiltration, immunochromatography, chemiluminescence or time-resolved analysis.

5. The use according to any one of claims 1 to 3, wherein the agent for detecting mucorales infection is a double antibody sandwich assay;

preferably, the mucrocinin protein is detected using an antibody against mucrocicin, further preferably using a monoclonal antibody against mucrocicin;

preferably, the COTH3 protein is detected using an antibody against COTH3, further preferably a monoclonal antibody against COTH 3.

6. Use according to any one of claims 1 to 5, characterized in that said reagent is used for measuring said MUCORICIN protein and/or said COTH3 protein in a blood, plasma or serum sample taken from an individual.

7. Use according to any one of claims 1 to 5, wherein an individual is deemed to be infected with mucor when the MUCORICIN protein has a COI > 1 measurement or the COTH3 protein has a COI > 1 measurement or a combination of the MUCORICIN protein and the COTH3 protein has a COI > 1 measurement.

8. A mucor detection kit is characterized by comprising a reagent for detecting MUCORICIN protein.

9. The kit of claim 8, wherein the kit further comprises reagents for detecting the COTH3 protein;

preferably, the reagent for detecting the MUCORICIN protein comprises an antibody against MUCORICIN, and further comprises a monoclonal antibody against MUCORICIN;

preferably, the reagent for detecting the COTH3 protein comprises an antibody against COTH3, and further comprises a monoclonal antibody against COTH 3.

10. The kit of claim 8, wherein the kit is selected from an enzyme-linked immunoassay kit, a fluorescent immunoassay kit, or a chemiluminescent immunoassay kit.