CN113655220A - Time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection and preparation method thereof - Google Patents
Time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection and preparation method thereof Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
Abstract
The invention provides time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection and a preparation method thereof, belonging to the technical field of in-vitro diagnostic reagents. The time-resolved fluorescence immunochromatographic test paper provided by the invention is used for detection based on a capture method detection principle. The test paper provided by the invention is used for detecting trace aspergillus antibodies in blood, so that 10 mul of serum, plasma or whole blood samples can be detected, the detection time is short, the detection result is obtained within 10-15 minutes, and the test paper can timely judge whether the aspergillus infection is invasive infection or not and timely guide the medication. Meanwhile, the test paper can realize field detection and timely discovery, and can be operated by medical personnel and non-medical personnel, so that the universality is strong.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection and a preparation method thereof.
Background
Aspergillus (Aspergillus) belongs to the filamentous fungus, is a common conditionally pathogenic fungus, and the common Aspergillus causing human diseases include Aspergillus fumigatus and Aspergillus flavus. Aspergillus widely spreads in nature, and can parasitize normal skin and upper respiratory tract, and is a conditioned pathogen. The normal people have certain resistance to aspergillus and do not cause diseases. Aspergillosis is mostly secondary, when the resistance of an organism is reduced, pathogenic bacteria can pass through a skin mucous membrane injury part or be inhaled into a respiratory tract, and then enter blood circulation to other tissues or organs to cause diseases. Therefore, whether the aspergillus develops into invasive infection can be detected in time, and timely symptomatic medication plays a vital role in recovering the health of patients.
The existing detection means is an enzyme-linked immunosorbent assay, but the detection period of the method is long, usually 1-2 hours, the operation is complex, professional technicians are required to operate the method, and the reagent is usually stored under refrigeration and is not easy to store and transport. At present, no product or literature adopting the time-resolved immunochromatography technology is published.
Disclosure of Invention
In view of this, the invention aims to provide a time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection and a preparation method thereof, and the test paper has the characteristics of short detection time and simple and convenient operation.
The invention provides time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection, which comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered on the bottom plate from bottom to top, wherein the nitrocellulose membrane comprises a quality control line C and a detection line T, and the combination pad is sprayed with a time-resolved fluorescence microsphere solution marked by an aspergillus antigen; the quality control line C is coated with a mouse anti-aspergillus antibody; the detection line T is coated with an anti-human IgG antibody.
Preferably, the preparation method of the aspergillus antigen labeled time-resolved fluorescent microsphere solution comprises the following steps:
1) sequentially adding N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) into the washed time-resolved fluorescent microsphere solution for activation reaction, separating the precipitate, washing and dispersing to obtain an activated time-resolved fluorescent microsphere solution;
2) diluting the activation time-resolved fluorescent microsphere solution, dropwise adding an aspergillus antigen solution for coupling reaction, and obtaining the aspergillus antigen marked time-resolved fluorescent microsphere solution through sealing, separation, precipitation and redissolution.
Preferably, the mass concentration of the time-resolved fluorescent microsphere solution is 0.8-1.2%;
the final adding concentration of the N-hydroxysuccinimide or the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is independently 1-5 mg/ml;
the time of the activation reaction is 25-35 min.
Preferably, the dilution multiple of the activation time-resolved fluorescent microsphere solution in the step 2) is 10-100 times.
Preferably, the mass ratio of the volume of the activation time-resolved fluorescent microsphere solution to the aspergillus antigen is 1 ml: 2-5 μ g;
the concentration of the aspergillus antigen solution is 0.01-0.1 mg/ml;
the dropping speed is 1-5 s/drop; the volume of each dropping liquid is 30-50 mu l.
Preferably, the spraying amount of the aspergillus antigen marked time-resolved fluorescent microsphere solution on the bonding pad is 0.1-3 mul/cm.
Preferably, the coating concentration of the mouse anti-aspergillus antibody or the mouse anti-human IgG antibody is 0.1-3 mg/ml independently.
Preferably, the murine anti-Aspergillus antibody is prepared by immunizing a mouse with an Aspergillus antigen.
Preferably, the sample pad is obtained by soaking the sample pad in a treatment solution and then drying the sample pad;
the treatment solution is 9-11 mM PBS buffer solution with pH of 7.3-7.5, which contains BSA with the mass percentage of 0.09-0.11%, sucrose with the mass percentage of 9-11% and Tween-20 with the volume percentage of 0.09-0.11%.
The invention provides a preparation method of the time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection, which comprises the following steps;
and sticking the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on a bottom plate, and cutting into strips.
The invention provides time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection, which comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered on the bottom plate from bottom to top, wherein the nitrocellulose membrane comprises a quality control line C and a detection line T, and the combination pad is sprayed with a time-resolved fluorescence microsphere solution marked by an aspergillus antigen; the quality control line C is coated with a mouse anti-aspergillus antibody; the detection line T is coated with an anti-human IgG antibody. The time-resolved fluorescence immunochromatographic test paper is used for detection based on a capture method detection principle. The test paper provided by the invention is used for detecting trace aspergillus antibodies in blood, so that 10 mul of serum, plasma or whole blood samples can be detected, the detection time is short, the detection result is obtained within 10-15 minutes, and the test paper can timely judge whether the aspergillus infection is invasive infection or not and timely guide the medication. Meanwhile, the test paper can realize field detection and timely discover that medical personnel and non-medical personnel can operate, has strong universality and can realize the detection of aspergillus fumigatus and aspergillus flavus at the same time.
Detailed Description
The invention provides time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection, which comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered on the bottom plate from bottom to top, wherein the nitrocellulose membrane comprises a quality control line C and a detection line T, and the combination pad is sprayed with a time-resolved fluorescence microsphere solution marked by an aspergillus antigen; the quality control line C is coated with a mouse anti-aspergillus antibody; the detection line T is coated with an anti-human IgG antibody.
In the present invention, the preparation method of the aspergillus antigen labeled time-resolved fluorescent microsphere solution preferably comprises the following steps:
1) sequentially adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the washed time-resolved fluorescent microsphere solution for activation reaction, separating the precipitate, washing and dispersing to obtain an activation time-resolved fluorescent microsphere solution;
2) diluting the activation time-resolved fluorescent microsphere solution, dropwise adding an aspergillus antigen solution for coupling reaction, and obtaining the aspergillus antigen marked time-resolved fluorescent microsphere solution through sealing, separation, precipitation and redissolution.
The method comprises the steps of sequentially adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to a washed time-resolved fluorescent microsphere solution for activation reaction, separating precipitates, washing and dispersing to obtain the activated time-resolved fluorescent microsphere solution.
In the present invention, the mass concentration of the time-resolved fluorescent microsphere solution is preferably 0.8% to 1.2%, and more preferably 1%. The particle size of the time-resolved fluorescent microsphere is preferably 100-500 nm, more preferably 150-450 nm, further preferably 200-400 nm, and further preferably 300 nm. The source of the time-resolved fluorescent microspheres is not particularly limited in the present invention, and the time-resolved fluorescent microspheres known in the art can be used. In the examples of the present invention, the time-resolved fluorescent microspheres were purchased from siemer feishel technologies (china) ltd. Preferably, the washing method comprises the steps of centrifuging the time-resolved fluorescent microsphere solution, discarding the supernatant, re-suspending the supernatant to the original volume by using a morpholine ethanesulfonic acid (MES) buffer solution, and performing ultrasonic treatment to obtain the washed time-resolved fluorescent microsphere solution. The centrifugal force of the centrifugal machine is preferably 7500-8500 g, and more preferably 8000 g. The time for centrifugation is preferably 8-12 min, and most preferably 10 min. The concentration of the morpholine ethanesulfonic acid buffer solution is preferably 10 mM. The power of the ultrasonic wave is preferably 150-200W, and more preferably 180W. The time of the ultrasonic treatment is preferably 0.1-1 min, and more preferably 0.5 min.
In the present invention, the final concentration of the N-hydroxysuccinimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide added is independently preferably 1 to 5mg/ml, more preferably 1.5 to 4.5mg/ml, even more preferably 2 to 4mg/ml, and most preferably 3 mg/ml. The N-hydroxysuccinimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is preferably formulated with 10mM pH 6MES buffer, initially at a concentration greater than 20 mg/ml. The time of the activation reaction is preferably 25-35 min, and more preferably 30 min. The temperature of the activation reaction is preferably carried out under room temperature conditions. The method for separating the precipitate is preferably performed by centrifugation. The centrifugal force of the centrifugal machine is preferably 7500-8500 g, and more preferably 8000 g. The time for centrifugation is preferably 8-12 min, and most preferably 10 min. And preferably, after washing, dispersing, namely, resuspending and redissolving the precipitate obtained by centrifugation to the original volume by using 10mM pH 6MES buffer solution, and forming the activation time-resolved fluorescent microspheres after uniform ultrasonic treatment.
After the activation time-resolved fluorescent microsphere solution is obtained, the activation time-resolved fluorescent microsphere solution is diluted, an aspergillus antigen solution is dripped to carry out coupling reaction, and the aspergillus antigen-labeled time-resolved fluorescent microsphere solution is obtained after sealing, separation, precipitation and redissolution.
In the invention, the dilution multiple of the activation time-resolved fluorescent microsphere solution is preferably 10-100 times, more preferably 20-80 times, even more preferably 30-60 times, and most preferably 40 times. The dilution solution is preferably 10mM PBS buffer pH 7.4. The Aspergillus antigen is preferably diluted to 0.01-0.1 mg/ml, more preferably 0.02-0.08 mg/ml, even more preferably 0.04-0.07 mg/ml, and most preferably 0.05mg/ml with 10mM pH7.4 PBS buffer. The Aspergillus antigens preferably include Aspergillus flavus antigens and Aspergillus fumigatus antigens. The source of the Aspergillus antigen is not particularly limited in the present invention, and any source of Aspergillus antigen known in the art may be used. In the embodiment of the invention, the aspergillus antigens are purchased from Beijing Daviny Biotechnology limited and belong to common epitope of aspergillus fumigatus and aspergillus flavus, so that the test paper prepared by the invention can be used for simultaneously detecting aspergillus fumigatus and aspergillus flavus. The volume of the activation time-resolved fluorescent microsphere solution and the mass ratio of the aspergillus antigen are preferably 1 ml: 2-5 μ g, more preferably 1 ml: 3-4 μ g, most preferably 1 ml: 3.5. mu.g. The dripping speed is preferably 1-5 s/drop, more preferably 2-4 s/drop and most preferably 3 s/drop. The volume of each drop is preferably 30 to 50. mu.l, and more preferably 40. mu.l. The coupling reaction lasts for preferably 1-4 h, more preferably 2-3 h, and most preferably 2.5 h. The method of blocking is not particularly limited in the present invention, and a blocking method known in the art may be used. In the embodiment of the invention, the blocking is preferably performed by adding 20% BSA solution into the coupling reaction solution to a final concentration of 0.1-0.2%. The closing time is preferably 0.5-1 h, and more preferably 0.7-0.9 h. The separation and precipitation are preferably carried out by centrifugation. The reconstitution is preferably performed by using 10mM pH7.4 PBS buffer containing 0.1% -1% BSA, 5-10% sucrose, and 0.1-1% Tween-20, more preferably 0.5% BSA, 8% sucrose, and 0.5% Tween-20, 10mM pH7.4 PBS buffer. The volume of reconstitution is preferably up to the volume of the time resolved fluorescent microsphere solution prior to reaction. After the aspergillus antigen marked time-resolved fluorescent microsphere solution is obtained, a film spraying instrument is preferably adopted to spray the paper bonding pad. The spraying amount of the aspergillus antigen marked time-resolved fluorescent microsphere solution on the bonding pad is preferably 0.1-3 mu l/cm, more preferably 0.5-2.5 mu l/cm, further preferably 1-2 mu l/cm, and most preferably 1.5 mu l/cm. The material of the conjugate pad preferably comprises a fiberglass film or a polyester film.
In the invention, a quality control line C and a detection line T are arranged on the nitrocellulose membrane. The quality control line C is coated with a mouse anti-aspergillus antibody. The detection line T is coated with a mouse anti-human IgG antibody. The coating concentration of the mouse anti-aspergillus antibody is 0.1-2 mg/ml, more preferably 0.5-1.5 mg/ml, and most preferably 1.2 mg/ml. The coating concentration of the mouse anti-human IgG antibody is preferably 0.1-3 mg/ml, more preferably 0.5-2.5 mg/ml, even more preferably 0.6-2 mg/ml, and most preferably 0.8 mg/ml. The source of the mouse anti-Aspergillus antibody or mouse anti-human IgG antibody is not particularly limited in the present invention, and a mouse anti-Aspergillus antibody or a mouse anti-human IgG antibody known in the art may be used. In the present examples, the murine anti-Aspergillus antibody was prepared from mice immunized with an Aspergillus antigen. The murine anti-human IgG antibody was purchased from sigma, Inc. under product designation 12136. The solvent for the murine anti-Aspergillus antibody or murine anti-human IgG antibody is preferably 10mM PBS buffer pH 7.4. The murine anti-human IgG antibody or murine anti-Aspergillus antibody is preferably coated on a nitrocellulose membrane at a rate of 1. mu.l/cm. The climbing speed of the nitrocellulose membrane is preferably 120-150 sec/4cm, the coating is finished, and the nitrocellulose membrane is dried for more than 4 hours for later use.
In the invention, the sample pad is obtained by drying after being soaked in the treatment solution. The treatment solution is 10mM PBS buffer solution with pH7.4, which contains BSA with the mass percentage of 0.1%, sucrose with the mass percentage of 10% and Tween-20 with the volume percentage of 0.1%.
In the present invention, the test paper preferably further includes a card case.
The invention provides a preparation method of the time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection, which comprises the following steps; and sticking the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on a bottom plate, and cutting into strips.
In the invention, the cutting width of the test paper is preferably 3-5 mm. And when the test paper card comprises a card shell, the cut test paper is put into the card shell to form the test paper card.
The detection method of the time-resolved fluorescence immunochromatographic test paper is completed based on a capture method, when an aspergillus antibody exists in a sample, the aspergillus antibody moves upwards along with water flow under a chromatography acting force to reach a binding pad to be combined with aspergillus flavus antigen-labeled time-resolved fluorescence microspheres to form a binary complex, when the binary complex moves upwards along with the chromatography acting force to reach a nitrocellulose membrane, the antibody in the binary complex is combined with a mouse anti-human IgG antibody to form a ternary complex, the mouse anti-human IgG antibody is fixedly coated on the nitrocellulose membrane, the ternary complex is fixed on a detection line T, meanwhile, redundant aspergillus flavus antigen-labeled time-resolved fluorescence microspheres on the binding pad reach a cellulose membrane nitric acid quality control line C along with the chromatography acting force, an antigen-antibody affinity reaction is carried out to form the binary complex, and a fluorescence signal is generated under the detection of a detector, the specific operation method comprises the following steps:
1) dripping a sample to be detected on a sample pad of the test paper, standing for 5-10 min, reading fluorescence signals of a quality control line C and a detection line T by using a time-resolved fluorescence immunochromatography test paper detector, and judging that the sample to be detected is positive and contains an aspergillus antibody when the quality control line C and the detection line T simultaneously display the fluorescence signals; when only the quality control line C displays a fluorescent signal and the detection line T does not display the fluorescent signal, judging that the sample to be detected is negative and does not contain an aspergillus antibody or the quantity of the aspergillus antibody contained in the sample is lower than the detection limit of the test paper; when only the detection line T displays a fluorescence signal and the quality control line C does not display the fluorescence signal, the detection is wrong, and the sample needs to be detected again. The experimental result shows that the detection result of the detection test paper prepared by the invention is consistent with that of an enzyme-linked immunosorbent assay, but the detection result is obtained within 10-15 minutes, and the operation is very simple.
The time-resolved fluoroimmunoassay test strip for detecting invasive aspergillus infection and the preparation method thereof provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A preparation method of time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection specifically comprises the following steps:
taking 100 mu l of time-resolved fluorescent microspheres with the concentration of 1% and the average particle size of 200nm, centrifuging for 10 minutes at 8000g, carefully sucking and discarding supernatant, re-suspending the obtained precipitate to 100 mu l by using 10mM pH 6MES (morpholine ethanesulfonic acid) buffer solution, and obtaining washed fluorescent microspheres for later use after uniform ultrasonic treatment;
NHS and EDC solutions were prepared at 50mg/ml concentrations using 10mM pH 6MES buffer, respectively. Adding 5 mul of NHS solution into the fluorescent microspheres, uniformly mixing, then adding 5 mul of EDC solution, uniformly mixing, reacting for 30 minutes, centrifuging for 10 minutes at 8000g, carefully sucking and discarding supernatant, re-suspending the precipitate with 10mM pH 6MES buffer solution to 100 mul, and uniformly performing ultrasonic treatment to form activation time-resolved fluorescent microspheres for later use;
diluting 0.5mg of aspergillus antigen to 0.01mg/ml by using 10mM PBS buffer solution with pH7.4, diluting 100 mu l of activation time-resolved fluorescent microspheres to 2ml by using 10mM PBS buffer solution with pH7.4, transferring to a clean beaker, putting into a clean rotor, dropwise adding all 0.01mg/ml aspergillus antigen while stirring, reacting for 2 hours after dropwise adding, then adding 0.5ml of 20% BSA solution for sealing, reacting for 1 hour, transferring the solution to a 30ml centrifuge tube, centrifuging for 45 minutes at 8000g, and discarding the supernatant; re-dissolving the precipitate to 1ml by using 10mM PBS buffer solution with pH7.4 and containing 0.1% BSA, 10% sucrose and 0.1% Tween-20 to form an aspergillus antigen labeled antigen time-resolved fluorescent microsphere solution;
and (3) spraying the antigen time-resolved fluorescent microsphere solution marked by the aspergillus antigen onto a polyester fiber membrane by using a membrane spraying instrument according to the membrane spraying amount of 2 mu l/cm, and drying to form a binding pad coated by the time-resolved microspheres.
Diluting a mouse anti-aspergillus antibody to 2mg/ml and a mouse anti-human IgG antibody to 0.8mg/ml by using 10mM PBS buffer solution with pH7.4, preparing a quality control line C coating solution and a detection line T coating solution, coating the solution on a nitrocellulose membrane by using a membrane scratching instrument according to the membrane scratching amount of 1 mu l/cm, wherein the climbing speed of the nitrocellulose membrane is 120-150 sec/4cm, and drying the coated solution at 37 ℃ for more than 4 hours for later use;
immersing a blank glass fiber membrane into 10mM PBS (phosphate buffer solution) with pH7.4 containing 0.1% BSA, 10% sucrose and 0.1% Tween-20, taking out and drying after the glass fiber membrane is completely immersed, and preparing a sample pad;
and sequentially sticking the nitrocellulose membrane, the time-resolved microsphere conjugate pad, the sample pad and the absorbent paper on a bottom plate, cutting to 3.8mm, and putting into a card shell to obtain the test strip.
Example 2
The test paper prepared in example 1 was used to detect whether the negative and positive samples (including positive samples of aspergillus fumigatus and positive samples of aspergillus flavus) and the test samples with the sensitivities S1-S6 (see table 1) contained aspergillus antibodies, and simultaneously, the same samples were detected by an enzyme linked immunosorbent assay (elisa) kit (enzyme linked immunosorbent assay, national institute of technology 20183400509) and the results of the two detection methods were compared.
TABLE 1 sensitivity S1-S6 detection of Aspergillus antibody concentration in samples
| Test sample | Concentration of Aspergillus antibody | Test sample | Concentration of Aspergillus antibody |
| S1 | 1:1 | S4 | 1:1000 |
| S2 | 1:10 | S5 | 1:10000 |
| S3 | 1:100 | S6 | 1:100000 |
The results are shown in Table 2.
TABLE 2 statistical table of results obtained by two detection methods
| Quality control plate | Enzyme immunoassay results | Test results of the invention |
| 20 negative samples | 20/20 | 20/20 |
| 20 positive samples | 20/20 | 20/20 |
| Sensitivity S1 ~ S6 (concentration decreases in order) | Positive S1-S4, negative S5 and S6 | Positive S1-S4, negative S5 and S6 |
The test result of the test strip prepared by the invention is consistent with the test result of the enzyme-linked immunosorbent assay, but the test result can be obtained within 10-15 minutes, and the operation is very simple.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered on the bottom plate from bottom to top, wherein the nitrocellulose membrane comprises a quality control line C and a detection line T; the quality control line C is coated with a mouse anti-aspergillus antibody; the detection line T is coated with an anti-human IgG antibody.
2. The time-resolved fluorescence immunochromatographic test strip for detecting invasive aspergillus infection according to claim 1, wherein the preparation method of the aspergillus antigen-labeled time-resolved fluorescence microsphere solution comprises the following steps:
1) sequentially adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the washed time-resolved fluorescent microsphere solution for activation reaction, separating the precipitate, washing and dispersing to obtain an activation time-resolved fluorescent microsphere solution;
2) diluting the activation time-resolved fluorescent microsphere solution, dropwise adding an aspergillus antigen solution for coupling reaction, and obtaining the aspergillus antigen marked time-resolved fluorescent microsphere solution through sealing, separation, precipitation and redissolution.
3. The time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection according to claim 2, wherein the mass concentration of the time-resolved fluorescence microsphere solution is 0.8% -1.2%;
the final adding concentration of the N-hydroxysuccinimide or the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is independently 1-5 mg/ml;
the time of the activation reaction is 25-35 min.
4. The time-resolved fluorescence immunochromatographic test strip for detecting invasive aspergillus infection according to claim 2, wherein the dilution factor of the activated time-resolved fluorescence microsphere solution in step 2) is 10 to 100 times.
5. The time-resolved fluorescence immunochromatographic test strip for detecting invasive aspergillus infection according to claim 4, wherein the volume of the activated time-resolved fluorescence microsphere solution and the mass ratio of aspergillus antigen are 1 ml: 2-5 μ g;
the concentration of the aspergillus antigen solution is 0.01-0.1 mg/ml;
the dropping speed is 1-5 s/drop; the volume of each dropping liquid is 30-50 mu l.
6. The time-resolved fluoroimmunoassay test paper for detecting invasive aspergillus infection according to any one of claims 2 to 4, wherein the spraying amount of the aspergillus antigen-labeled time-resolved fluorescent microsphere solution on the bonding pad is 0.1 to 3 μ l/cm.
7. The time-resolved fluorescence immunochromatographic test strip for detecting an invasive aspergillus infection according to claim 1, wherein the coating concentration of the murine anti-aspergillus antibody or the murine anti-human IgG antibody is 0.1 to 3mg/ml independently.
8. The time-resolved fluoroimmunoassay test strip for detecting invasive aspergillus infection according to claim 1 or 7, wherein said murine anti-aspergillus antibody is prepared from an aspergillus antigen immunized mouse.
9. The time-resolved fluorescence immunochromatographic test strip for detecting invasive aspergillus infection according to any one of claims 1 to 5 and 7, characterized in that the sample pad is obtained by drying after being soaked in a treatment solution;
the treatment solution is 9-11 mM PBS buffer solution with pH of 7.3-7.5, which contains 0.09-0.11% of BSA (bovine serum albumin), 9-11% of sucrose and 0.09-0.11% of Tween-20 by volume.
10. The method for preparing the time-resolved fluorescence immunochromatographic test strip for detecting invasive aspergillus infection according to any one of claims 1 to 9, characterized by comprising the following steps;
and sticking the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on a bottom plate, and cutting into strips.
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