CN106290882A - A kind of Candida albicans antigen near-infrared fluorescent detection kit and application thereof - Google Patents
A kind of Candida albicans antigen near-infrared fluorescent detection kit and application thereof Download PDFInfo
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- CN106290882A CN106290882A CN201510292089.8A CN201510292089A CN106290882A CN 106290882 A CN106290882 A CN 106290882A CN 201510292089 A CN201510292089 A CN 201510292089A CN 106290882 A CN106290882 A CN 106290882A
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- candida albicans
- monoclonal antibody
- antibody
- infrared fluorescent
- crown ether
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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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
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
- G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
Abstract
The invention discloses a kind of Candida albicans antigen near-infrared fluorescent detection kit and application thereof.The present invention provides a kind of Candida albicans antigen near-infrared fluorescent detection kit, including the reagent strip being positioned on backboard, reagent strip starts to be followed successively by sample pad, be marked with the glass fibre element film of immune fluorescent probe, the nitrocellulose filter being coated with anti-candida albicans monoclonal antibody and absorbent paper from sample-adding end.Detection kit provided by the present invention combines near-infrared fluorescent detection technique, can be quick, detects the Candida albicans antigen in sample accurately.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of Candida albicans antigen near-infrared fluorescent detection kit and
Purposes.
Background technology
Candida albicans (Canidia albicans), also known as candida albicans bacterium, is a kind of conditioned pathogen, is typically found in normal
Human mouth, upper respiratory tract, intestinal and vagina, typically in normal body, quantity is few, does not cause disease, works as body's immunity
Or typically phylactic power defensive power declines or the imbalance of normal flora mutual restrictive function, then easily breed at local raised growth, causes skin, sticks
Film even general candida albicans infection.Candida albicans can cause pudendum and the colpitis of women, mainly shows as vagina
Secretions thickness, yellow skin or cheese sample patch, the clinical symptoms such as pudendum, pruritus of vagina or burning sensation, bean dregs sample leucorrhea.Male
Patient is more rare, how through transmission through sex, the candidal balanitis of triggering male and Prepuce balanitis.
In recent years, monilial vaginitis prevalence remains high, it is considered to abuse, contraceptive device and the hygienic article of antibiotic
The factor such as unclean, improper is relevant.Estimate that the women of 75% at least occurs 1 vulvovaginal candidiasis, 5%～10% all one's life
Women occur every year 2 times, China's various places infection rate is 0.65～39%, and Candida albicans is vaginal candidiasis
Main pathogens.One of significant problem that the clinical drug-resistant problem of fungus always faces both at home and abroad, owing to antifungal agent is in clinic
On a large amount of use or irregularly apply, candidiasis Resistant strain also dramatically increases.
If pathogen is uncertain, therapeutic effect is the most undesirable, and relapse rate is high, and can induce drug resistance and dysbacteriosis, the soonest
Fast, sensitive, accurately Differential Diagnosis Candida albicans particularly significant to clinical treatment.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of Candida albicans antigen near-infrared fluorescent
Detection kit and application thereof, for setting up the most feasible Candida albicans antigen near-infrared fluorescent detection technique, solves existing
Problem in technology.
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of Candida albicans antigen near-infrared fluorescent
Detection kit and application thereof.Detection kit provided by the present invention combines near-infrared fluorescent detection technique, can be quick, accurately
The Candida albicans antigens detected in vagina secretions.
For achieving the above object and other relevant purposes, first aspect present invention provides a kind of Candida albicans antigen near-infrared fluorescent
Detection kit, including the reagent strip being positioned on backboard, reagent strip starts to be followed successively by sample pad from sample-adding end, to be marked with immunity glimmering
The glass fibre element film of light probe, the nitrocellulose filter being coated with anti-candida albicans monoclonal antibody and absorbent paper.
Preferably, described immune fluorescent probe is anti-candida albicans monoclonal antibody coated near-infrared fluorescent latex beads granule.
It is coated on coated anti-candida albicans monoclonal antibody and described near-infrared fluorescent latex beads granule on nitrocellulose filter
Anti-candida albicans monoclonal antibody can be identical antibody, it is also possible to for different antibody.
It is furthermore preferred that coated anti-candida albicans monoclonal antibody is on described nitrocellulose filterAnti-Candida
Albicans antibody [CDA] (ab82704), coated anti-candida albicans Dan Ke on described near-infrared fluorescent latex beads granule
Grand antibody isAnti-Candida albicans antibody [6401] (ab23368), or wrap on described nitrocellulose filter
The anti-candida albicans monoclonal antibody of quilt isAnti-Candida albicans antibody [6401] (ab23368), institute
Stating coated anti-candida albicans monoclonal antibody on near-infrared fluorescent latex beads granule isAnti-Candida
albicans antibody[CDA](ab82704).It is furthermore preferred that a diameter of 140-160nm of described fluorescent latex microsphere particle.
Near infrared light (NIR), is the electromagnetic wave between visible ray (VIS) and mid-infrared light (MIR), and ASTM is fixed
Justice NIR is wavelength electromagnetic wave in the range of 780nm-2526nm.In near-infrared region, organism self-absorption or fluorescence intensity
The least, ambient interferences can be avoided.Simultaneously because the biquadratic of scattered light intensity and wavelength is inverse ratio, the scattering interference of near infrared region
It is greatly reduced.
Second aspect present invention provides the preparation method of described Candida albicans antigen near-infrared fluorescent detection kit, including as follows
Step:
1) with buffer by fluorescent latex particles eccentric cleaning and by latex particle dispersion, in cleaned latex particle dispersion liquid
Add anti-candida albicans monoclonal antibody；Add EDCA and make antibody and granule coupling；Add confining liquid and close latex particle
Upper unnecessary group, prepares probe solution；
2) the buffer solution spray containing anti-candida albicans monoclonal antibody is added on nitrocellulose filter, dry for standby；
3) probe solution sized glass fibres thin film step 1 prepared, drying for standby；
4) by the nitrocellulose filter of step 2 gained, the glass fibre element film being marked with immune fluorescent probe of step 3 gained,
Sample pad, absorbent paper and backboard assemble and are prepared as Candida albicans antigen near-infrared fluorescent detection kit.
Preferably, in described step 1, add confining liquid and close group unnecessary on latex particle, and add crown ether, preparation
Obtain probe solution, the described crown ether group of one or more in two cyclohexyl-18-crown-s 6,15-crown ether-5, hexaoxacyclooctadecane-6-6
Close.
It is furthermore preferred that the concentration of crown ether is 0.1-20mg/ml, more preferably 4-8mg/ml in the probe solution for preparing of step 1.
Preferably, in described step 1, buffer is phosphate (PBS) buffer.
It is furthermore preferred that the PBS that described PBS is 0.009-0.011mol/L, PH7.25-7.35.
Preferably, in described step 1, a diameter of 140-160nm of fluorescent latex particles.
Preferably, in described step 1, latex particle, anti-candida albicans monoclonal antibody, the weight ratio of EDCA are 9-11:
0.9-1.1:0.9-1.1.
In an embodiment of the present invention, latex particle, anti-candida albicans monoclonal antibody, the inventory of EDCA be respectively 1mg,
100ug、100ug。
Preferably, described confining liquid is ethanolamine, and addition is appropriate, and those skilled in the art can adjust second according to practical situation
The consumption of hydramine.
Preferably, in described step 2, described buffer solution is PBS.
Those skilled in the art can choose the PBS of debita spissitudo and pH, more preferably 0.009-0.011mol/L,
The PBS of PH7.25-7.35.
Preferably, in described step 2, possibly together with crown ether in the buffer solution containing anti-candida albicans monoclonal antibody, described
The crown ether combination of one or more in two cyclohexyl-18-crown-s 6,15-crown ether-5, hexaoxacyclooctadecane-6-6.
It is furthermore preferred that the concentration of crown ether is 0.1-20mg/ml in the described buffer solution containing anti-candida albicans monoclonal antibody,
More preferably 4-8mg/ml.
Preferably, in described step 2, anti-candida albicans Dan Ke in the buffer solution containing anti-candida albicans monoclonal antibody
The concentration of grand antibody is 1.8-2.2mg/ml.
Preferably, in described step 2, the spray dosage of the buffer solution of anti-candida albicans monoclonal antibody is 0.9-1.1ul/cm.
Preferably, in described step 2, the actual conditions of drying is 36-38 DEG C of oven for drying.
Described spray adds process and utilizes trace albumin point membranous system.
Preferably, the anti-candida albicans monoclonal antibody in described step 1 isAnti-Candida albicans
Antibody [CDA] (ab82704), the anti-candida albicans monoclonal antibody in described step 2 isAnti-Candida
Albicans antibody [6401] (ab23368), or the anti-candida albicans monoclonal antibody in described step 1 isAnti-Candida albicans antibody [6401] (ab23368), the anti-candida albicans monoclonal in described step 2
Antibody isAnti-Candida albicans antibody[CDA](ab82704)。
Preferably, in described step 3, the actual conditions being dried is that vacuum drier is dried.
Third aspect present invention provides described Candida albicans antigen near-infrared fluorescent detection kit at Candida albicans detection field
Purposes.
Described purposes specially uses described Candida albicans antigen near-infrared fluorescent detection kit to the Candida albicans in sample
Detecting, described sample is vagina secretions.
Detection kit provided by the present invention can detect skill with vagina secretions for detection sample in conjunction with near-infrared fluorescent
Art, can be quick, detects the Candida albicans antigen in patient's specimen accurately.Described test kit uses immunofluorescence chromatography,
Its I is measured up to 0.05ng/ml, and easy to detect, low cost, highly sensitive, specificity is good, tries than similar quantitative Diagnosis
The low 20-30% of agent price, is greatly saved society's medical treatment cost, and during detection, sample collection and process are simple, can be quick and precisely
Acquisition result, be especially suitable for that accident is on-the-spot and basic unit uses, and may be used in Clinical detection, provide for clinical treatment
Qualitative foundation.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by disclosed by this specification
Content understand other advantages and effect of the present invention easily.The present invention can also be added by the most different detailed description of the invention
To implement or application, the every details in this specification can also be based on different viewpoints and application, in the essence without departing from the present invention
Various modification or change is carried out under god.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific
Specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments,
Rather than in order to limit the scope of the invention；In description of the invention and claims, unless literary composition additionally clearly refers to
Going out, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, two end points of each numerical range with
And any one numerical value all can be selected between two end points.Unless otherwise defined, all technology used in the present invention and section are academic
The same meaning that language and those skilled in the art of the present technique are generally understood that.In addition to the concrete grammar used in embodiment, equipment, material,
According to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use and the embodiment of the present invention
Described in method, any method, equipment and the material of the similar or equivalent prior art of equipment, material realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use the art normal
Rule molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell cultivate, recombinant DNA technology and
The routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the series
METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS
IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
In various embodiments of the present invention, test specimen is provided by Pudong New District the People's Hospital.Near-infrared fluorescent detector, by the triumphant wound in Shanghai
Bioisystech Co., Ltd's exploitation provides.
Embodiment 1
1. prepared by immunofluorescence anti-candida albicans antibody granule:
With 0.01mol/L, the phosphate buffer of PH7.3 ± 0.05 is by the fluorescent latex particles eccentric cleaning 2 of a diameter of 150nm
All over and by latex particle disperse, in cleaned latex particle addAnti-Candida albicans antibody
[CDA](ab82704)；Add EDCA and make antibody and granule coupling；Add confining liquid and close group unnecessary on latex particle,
Add two cyclohexyl-18-crown-s 6.By the antibody to be marked of every milligram of microsphere latex 100 microgram, the EDCA of 100 micrograms uses
Amount labelling.The microsphere latex of every milligram finally adds the confining liquid of the ethanolamine equivalent being equivalent to 20 micrograms and closes, the end of crown ether
Concentration is 6mg/ml.
2. the preparation of test kit:
Dilute with the PBS of 0.01M pH7.2Anti-Candida albicans antibody[6401](ab23368)
To concentration 2.0mg/ml, and adding crown ether, the final concentration of 6mg/ml of crown ether, solution is sprayed by recycling trace albumin point membranous system
Being added on the nitrocellulose filter in suitable aperture, carry out spraying film by the amount of 1ul/cm, 37 DEG C of oven for drying are standby.
With probe (prepared being marked with of step 1 preparedAnti-Candida albicans antibody[CDA]
(ab82704) near-infrared fluorescent latex beads) solution impregnation glass fiber membrane, drying for standby in vacuum drier.
The nitrocellulose filter of anti-candida albicans antibody will be coated, be marked with the glass fibre element film of immune fluorescent probe, sample
Pad, absorbent paper and backboard assemble and are prepared as Candida albicans antigen near-infrared fluorescent detection kit.
Embodiment 2
The sensitivity experiment of detection kit
The Candida albicans antigen detection kit that Example 1 prepares, is placed on test kit on level table, detects respectively
The Candida albicans antigen mark of 0.03ng/ml, 0.05ng/ml, 0.08ng/ml, 0.1ng/ml, 0.2ng/ml, 0.5ng/ml
Quasi-product (are respectively configured by Candida albicans antigen standard and PBS), and each concentration is repeated 5 times.Draw with suction pipe
Sample liquid 2-3 is added dropwise in sample cell, 8-15 minute sentence read result after dropping sample.
Table 1 test kit sensitivity results
Table 1, as a result, it was confirmed that Candida albicans antigen detection kit sensitivity≤0.05ng/ml of preparing of the present invention, has the highest
Sensitivity.
Embodiment 3
Candida albicans antigen near-infrared fluorescent detection method and antibacterial culturing contrast experiment:
Test kit detects:
Testing sample is vagina secretions, uses the PBS dilution of the 0.01M pH7.2 of 10 times of volumes.
Testing sample and test kit all balance and start detection to room temperature, drip 3 samples with dropper in the well of each test kit
This (about 120-150ul).When 15 minutes, detect fluorescence signal with near-infrared fluorescent detector, and judge with fluorescent instrument,
The detection range to fluorescence signal of analyser is AD value 0-10000, and according to the performance of instrument, CUTOFF value is 50, inspection
Surveying AD value more than or equal to 50 is positive findings.
Experimental result and bacteria cultivation results contrast verification.Candida albicans antigen near-infrared fluorescent detection method is thin with vaginal secretions
Bacterium cultivation results is as shown in table 2:
Table 2
Embodiment 4
Cross-over experiment:
Candida albicans antigen near infrared detection test kit drip respectively prepare enterococcus faecalis, enterococcus faecalis, large intestine angstrom wish
Bacterium, Acinetobacter bauamnnii, klebsiella, lactobacillus, Diplococcus gonorrhoeae, Pseudomonas aeruginosa, escherichia coli, human-like
Substance, ureaplasma urealyticum, staphylococcus aureus, streptococcus and helicobacter pylori bacterium solution (1 × 106CFU/ml), carry out intersecting in fact
Testing detection, detect whether above antibacterial produces impact to this test kit testing result, Candida albicans antigen near-infrared fluorescent detects
Reagent is as shown in table 3 with antibacterial cross-over experiment result, and in each antibacterial cross-over experiment, equal no cross reaction occurs:
Table 3
| Intersection thing | Result | Intersection thing | Result |
| Enterococcus faecalis | - | Diplococcus gonorrhoeae | - |
| Enterococcus faecalis | - | Pseudomonas aeruginosa | - |
| Escherichia coli | - | Escherichia coli | - |
| Acinetobacter bauamnnii | - | Mycoplasma hominis | - |
| Klebsiella | - | Ureaplasma urealyticum | - |
| Lactobacillus | - | Staphylococcus aureus | - |
| Helicobacter pylori | - | Streptococcus | - |
Embodiment 5
The stability experiment of Candida albicans antigen detection kit
Candida albicans antigen near-infrared fluorescent detection kit provided by the present invention is sealed and preserves, and deposit in 4 DEG C and room
Under temperature (about 25 DEG C), observe the different storage temperature impact on stabilization of kit.The test kit being stored in 4 DEG C takes weekly
Go out 4 boxes, detect the Candida albicans antigen standard of 0.03ng/ml, 0.05ng/ml, 0.08ng/ml, 0.1ng/ml respectively；Protect
The test kit being stored in room temperature (25 DEG C) takes out 4 boxes in every 3 days, detects 0.03ng/ml, 0.05ng/ml, 0.08ng/ml, 0.1ng/ml respectively
Candida albicans antigen standard.Empirical tests, paper box can preserve 24 months at 4 DEG C, and minimum detectability and repeatability
There is no significant change, at room temperature can preserve 16 months, and minimum detectability and repeatability do not have significant change；Preservable
In time limit, test kit all can reach the detection sensitivity of 0.08ng/ml.
In sum, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any it is familiar with this skill
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage of art.Therefore, such as
All that in art, tool usually intellectual is completed under without departing from disclosed spirit and technological thought etc.
Effect is modified or changes, and must be contained by the claim of the present invention.
Claims (8)
1. a Candida albicans antigen near-infrared fluorescent detection kit, including the reagent strip being positioned on backboard, reagent strip is from sample-adding end
Start to be followed successively by sample pad, be marked with the glass fibre element film of immune fluorescent probe, be coated with anti-candida albicans monoclonal anti
The nitrocellulose filter of body and absorbent paper, described immune fluorescent probe is the coated near-infrared of anti-candida albicans monoclonal antibody
Fluorescent latex microsphere particle.
2. test kit as claimed in claim 1, it is characterised in that coated anti-candida albicans Dan Ke on described nitrocellulose filter
Grand antibody can be identical with coated anti-candida albicans monoclonal antibody on described near-infrared fluorescent latex beads granule
Antibody, it is also possible to for different antibody.
3. test kit as claimed in claim 1, it is characterised in that a diameter of 140-160nm of described fluorescent latex microsphere particle.
4. the preparation method of the near-infrared fluorescent detection kit as described in claim 1-3 any claim, comprises the steps:
1) with buffer by fluorescent latex particles eccentric cleaning and by latex particle dispersion, in cleaned latex particle dispersion liquid
Add anti-candida albicans monoclonal antibody；Add EDCA and make antibody and granule coupling；Add confining liquid and close latex particle
Upper unnecessary group, prepares probe solution；
2) the buffer solution spray containing anti-candida albicans monoclonal antibody is added on nitrocellulose filter, dry for standby；
3) probe solution sized glass fibres thin film step 1 prepared, drying for standby；
4) by the nitrocellulose filter of step 2 gained, the glass fibre element film being marked with immune fluorescent probe of step 3 gained,
Sample pad, absorbent paper and backboard assemble and are prepared as Candida albicans antigen near-infrared fluorescent detection kit.
5. preparation method as claimed in claim 4, it is characterised in that in described step 1, adds confining liquid and closes latex particle
Upper unnecessary group, and add crown ether, preparing probe solution, described crown ether is selected from two cyclohexyl-18-crown-s 6,15-
The combination of one or more in crown ether-5, hexaoxacyclooctadecane-6-6；
And/or, in the probe solution that step 1 prepares, the concentration of crown ether is 0.1-20mg/ml；
And/or, in described step 1, buffer is PBS；
And/or, in described step 1, latex particle, anti-candida albicans monoclonal antibody, the weight ratio of EDCA are 9-11:
0.9-1.1:0.9-1.1；
And/or, described confining liquid is ethanolamine.
6. preparation method as claimed in claim 4, it is characterised in that in described step 2, containing Candida albicans monoclonal antibody
Buffer solution in possibly together with crown ether, described crown ether is in two cyclohexyl-18-crown-s 6,15-crown ether-5, the hexaoxacyclooctadecane-6-6
The combination of one or more；
And/or, in the described buffer solution containing anti-candida albicans monoclonal antibody, the concentration of crown ether is 0.1-20mg/ml；
And/or, in the buffer solution containing anti-candida albicans monoclonal antibody, the concentration of anti-candida albicans monoclonal antibody is
1.8-2.2mg/ml；
And/or, the spray dosage of the buffer solution of anti-candida albicans monoclonal antibody is 0.9-1.1ul/cm；
And/or, in described step 2, described buffer solution is PBS.
7. the near-infrared fluorescent detection kit as described in claim 1-3 any claim is in Candida albicans Detection of antigen field
Purposes.
8. purposes as claimed in claim 7, it is characterised in that described purposes specially uses described Candida albicans antigen near-infrared
Candida albicans antigen in sample is detected by fluorescence detection reagent kit.
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| CN111024947A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Candida albicans fluorescence immunochromatography assay kit and preparation method thereof |
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