CN108107207A - The detection kit of A type H9 subtype influenza virus and its application - Google Patents
The detection kit of A type H9 subtype influenza virus and its application Download PDFInfo
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- CN108107207A CN108107207A CN201711219303.2A CN201711219303A CN108107207A CN 108107207 A CN108107207 A CN 108107207A CN 201711219303 A CN201711219303 A CN 201711219303A CN 108107207 A CN108107207 A CN 108107207A
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Abstract
Detection kit and its application the invention discloses A type H9 subtype influenza virus.Wherein, the detection kit of the influenza virus includes：Basal layer；First coated film, first coated film is formed on the basal layer, and is coated with the first antibody of fluorescent marker, and the first antibody specifically binds the A types H9 subtype influenza virus；Second coated film, second coated film is formed on the basal layer, and it is connected with first coated film, separated first area and second area are provided on second coated film, and first area is located at the nearly first coated film end, wherein, the first area is coated with secondary antibody, the secondary antibody and the first antibody can specifically bind the A types H9 subtype influenza virus, the second area is coated with antiantibody, and the antiantibody can specifically bind the first antibody.The detection sensitivity height of the kit, high specificity, speed are fast.
Description
Technical field
The present invention relates to field of virus detection, and in particular, to the detection kit of influenza virus and its application, more specifically
Ground is related to the detection kit of A type H9 subtype influenza virus and the method for detection A type H9 subtype influenza virus
Background technology
Influenza A, that is, influenza A virus is common influenza virus, and influenza A virus easily morphs, sub-
Type is often known as " bird flu ".Bird flu is to seriously endanger the crushing epidemic disease of world's aviculture, and cause of disease is easily mutated,
With multiple serotypes, difficulty is brought to the sick prevention and control, can infect the mankind, metainfective symptom master after viral gene variation
High fever, cough, runny nose, myalgia etc. are shown as, majority is led with a variety of organ failures such as serious pneumonia, severe patient's heart, kidney
It is lethal to die.Flu-A H9 hypotypes are high to human pathogenic, counted according to WHO, and the death rate that people infects H9 hypotypes is up to 60%.
The detection method of A type H9 subtype influenza virus common at present mainly has：Detection of nucleic acids and Virus Isolation.Core
Acid detection have RT-PCR detection and real-time RT-PCR detection methods, detection of nucleic acids dependent on sample form RNA matter with
Amount, operation requirement is stringent, it is necessary to which a large amount of instrument and equipments and professional's operation, detection time is longer, and detection sensitivity is high.Disease
Poison separation identification is usually used in identifying, it is necessary to which specialized laboratories could carry out the hypotype of positive virus.
The detection method of A types H9 subtype influenza virus has much room for improvement as a result,.
The content of the invention
It is contemplated that at least solve one of technical problem in the prior art.For this purpose, one object of the present invention
It is to propose a kind of detection kit of influenza virus, detection sensitivity height, high specificity, the energy of the kit are quick, easy
Detect A type H9 subtype influenza virus in ground.
Thus, according to an aspect of the present invention, the present invention provides a kind of detection kits of influenza virus.According to this
The embodiment of invention, the kit include：Basal layer；First coated film, first coated film are formed on the basal layer,
And the first antibody of fluorescent marker is coated with, the first antibody specifically binds the A types H9 subtype influenza virus；Second bag
Envelope, second coated film are formed on the basal layer, and are connected with first coated film, on second coated film
Separated first area and second area are provided with, and first area is located at the nearly first coated film end, wherein, described first
Region is coated with secondary antibody, and the secondary antibody and the first antibody can specifically bind the A types H9 subtype influenzas
Virus, the second area are coated with antiantibody, and the antiantibody can specifically bind the first antibody.
Kit according to embodiments of the present invention, inventor are carried out by the antibody to a large amount of A types H9 subtype influenza virus
Screening obtains the two kinds of antibody that can specifically bind to A type H9 subtype influenza virus simultaneously, i.e. two kinds of antibody of the invention (i.e.
First antibody and secondary antibody) it can be specifically bound with the different surfaces determinant of antigen, it is compound so as to form double-antibody sandwich
Object, the fluorescence intensity based on the fluorescent marker of band on the first antibody in detection compound are described whether there is in judgement sample
Antigen.The detection sensitivity of the kit is high, high specificity, and detection time is short, and testing cost is low, easily operated and popularization.
In addition, the detection kit of influenza virus according to the above embodiment of the present invention, can also have following additional
Technical characteristic：
According to an embodiment of the invention, this method further comprises：Water absorption pad, the water absorption pad and second coated film
The remote first coated film end be connected.
According to an embodiment of the invention, the first antibody and the secondary antibody are monoclonal antibody.
According to an embodiment of the invention, the first antibody is F9-17's for the number purchased from the big monoclonal antibody center of Kunming cloud
Monoclonal antibody, the monoclonal antibody that the secondary antibody is F9-27 for the number purchased from the big monoclonal antibody center of Kunming cloud.
According to an embodiment of the invention, the first antibody is F9-17's for the number purchased from the big monoclonal antibody center of Kunming cloud
Monoclonal antibody, the monoclonal antibody that the secondary antibody is F9-47 for the number purchased from the big monoclonal antibody center of Kunming cloud.
According to an embodiment of the invention, the first antibody is F9-19's for the number purchased from the big monoclonal antibody center of Kunming cloud
Monoclonal antibody, the monoclonal antibody that the secondary antibody is F9-27 for the number purchased from the big monoclonal antibody center of Kunming cloud.
According to an embodiment of the invention, the fluorescent marker marks for fluorescent microsphere.
According to an embodiment of the invention, the average diameter of the fluorescent microsphere is 10-500nm.It is according to the present invention preferred
Embodiment, the average diameter of the fluorescent microsphere is 20-300nm.More preferred embodiment according to the present invention, the fluorescent microsphere
Average diameter be 100-120nm.
According to an embodiment of the invention, the first antibody is marked with the fluorescent microsphere and combined by covalent peptide bonds.
According to an embodiment of the invention, the antiantibody is sheep anti-mouse igg antibody.
According to an embodiment of the invention, first coated film is glass fibre element film, and second coated film is nitric acid
Cellulose membrane.
According to another aspect of the present invention, the present invention provides a kind of methods for detecting influenza virus.It is according to the present invention
Embodiment, this method include, and sample to be tested is contacted with the first coated film in the detection kit of foregoing influenza virus；Detection
The fluorescence intensity of first area and second area in the kit, obtains first area fluorescent measurement and second area is glimmering
Light detected value；Based on the first area fluorescent measurement and second area fluorescent measurement, judge be in the sample to be tested
It is no that there are A type H9 subtype influenza virus.As a result, using this method detect A type H9 subtype influenza virus, the high sensitivity of detection,
High specificity, detection time is short, and testing cost is low, easily operated and popularization.
According to an embodiment of the invention, by fluorescence intensity ratio compared with threshold value, judge be in the sample to be tested
It is no there are A type H9 subtype influenza virus, wherein, the fluorescence intensity ratio=first area fluorescent measurement/second area is glimmering
Light detected value.
According to an embodiment of the invention, the threshold value is 0.001-0.02.According to a preferred embodiment of the invention, the threshold
It is worth for 0.008.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
It obtains substantially or is recognized by the practice of the present invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Substantially and it is readily appreciated that, wherein：
Fig. 1 shows the main structure diagram of the detection kit of influenza virus according to an embodiment of the invention；
Fig. 2 shows the side structure schematic view of the detection kit of influenza virus according to an embodiment of the invention；
Fig. 3 shows the main structure diagram of the detection kit of influenza virus according to an embodiment of the invention；
Fig. 4 shows that the flow of the method for the detection kit of preparation influenza virus according to an embodiment of the invention is shown
It is intended to；
Fig. 5 shows the detected value of the detection kit of influenza virus according to an embodiment of the invention and the song of concentration
Line schematic diagram.
Specific embodiment
The embodiment of the present invention is described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or has the function of same or like element.Below with reference to attached
The embodiment of figure description is exemplary, and is only used for explaining the present invention, and is not considered as limiting the invention.
In the description of the present invention, term " longitudinal direction ", " transverse direction ", " on ", " under ", "front", "rear", "left", "right", " perpendicular
Directly ", the orientation of the instructions such as " level ", " top ", " bottom " or position relationship are based on orientation shown in the drawings or position relationship, are only
The present invention rather than require the present invention therefore it is not intended that right with specific azimuth configuration and operation for ease of description
The limitation of the present invention.
It should be noted that term " first ", " second " are only used for description purpose, and it is not intended that instruction or hint phase
To importance or the implicit quantity for indicating indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the present invention, unless otherwise saying
Bright, " multiple " are meant that two or more.
Kit
According to an aspect of the present invention, the present invention provides a kind of detection kits of influenza virus.Inventor passes through
The antibody of a large amount of A types H9 subtype influenza virus is screened, acquisition can specifically bind to A type H9 subtype influenza virus simultaneously
Two kinds of antibody, i.e. the two of the embodiment of the present invention kind antibody (i.e. first antibody and secondary antibody) can determine with the different surfaces of antigen
Determine group-specific combination, so as to form double-antibody sandwich compound, the fluorescence based on band on the first antibody in detection compound
The fluorescence intensity of mark to whether there is described antigen in judgement sample.The detection sensitivity of the kit is high, high specificity,
Detection time is short, and testing cost is low, easily operated and popularization.
For the ease of understanding the kit of the embodiment of the present invention, with reference to Fig. 1 and 2, according to an embodiment of the invention, to the examination
Agent box is explained, specific as follows：
Basal layer 100：According to an embodiment of the invention, the type of basal layer 100 is not particularly limited, as long as not with treating
Sample reacts or does not influence antigen-antibody combination, and those skilled in the art can voluntarily be selected as needed
It selects.Some specific embodiments according to the present invention can choose inert material as basal layer, such as cardboard and plastic plate etc..
Wherein, it is necessary to illustrate, the term " coating " in the present invention is immune technics, includes fixed and/or adsorbs
Meaning.
First coated film 200：First coated film 200 is formed on basal layer 100, and is coated with the first of fluorescent marker
Antibody, first antibody specific binding A type H9 subtype influenza virus.Wherein, it is necessary to which explanation, the first coated film 200 can
To be that subregion can also be the first antibody that whole region is coated with fluorescent marker.According to a particular embodiment of the invention,
The region for being coated with fluorescent marker first antibody in first coated film is to be about 2cm, the rectangle of width about 3mm.It is to be measured as a result,
Sample and first antibody combination effect are good.
According to an embodiment of the invention, first antibody is monoclonal antibody.As a result, first antibody specificity with A types H9
Subtype influenza virus combines, and the sensitivity and accuracy of kit are high.
According to an embodiment of the invention, fluorescent marker marks for fluorescent microsphere.Fluorescent microsphere is to be stimulated energy by outside energy
Inspire the solia particle of fluorescence.According to some embodiments of the present invention, the fluorescent material of fluorescent microsphere load can be doping
Fluorescent dye, rare-earth complex and quantum dot etc. it is fluorescent nano material converted.It is easy to inspire by outside energy stimulation as a result,
Fluorescence, the sensitivity higher of detection.
According to an embodiment of the invention, in order to improve the stability of fluorescent marker, the fluorescent material of fluorescent microsphere load
Active functional group group can be modified with by high molecular polymer, for example, the active functional group group can be carboxyl, amino, hydroxyl
Base, sulfydryl.According to some embodiments of the present invention, first antibody is marked with fluorescent microsphere and combined by covalent peptide bonds.It is glimmering as a result,
The stability of signal object is high, avoids the influence of antibody steric hindrance, is conducive to improve sensitivity and specificity.
According to an embodiment of the invention, the average diameter of fluorescent microsphere is 10-500nm.It is according to the present invention to be preferably implemented
Example, the average diameter of the fluorescent microsphere is 20-300nm.More preferred embodiment according to the present invention, the fluorescent microsphere are put down
A diameter of 100-120nm.The diameter of fluorescent microsphere is small as a result, effectively prevents the influence of steric hindrance between antibody, makes to resist
Body is easily incorporated on A type H9 subtype influenza virus, is conducive to improve sensitivity and specificity.
A specific embodiment according to the present invention, it is public that fluorescent microsphere can be purchased from Bangs Laboratories, Inc.
Department, catalog number is that FC02F/10930 solid contents are 10mg/ml, is the fluorescent microsphere of carboxyl modified, which is averaged
A diameter of 110nm.
Second coated film 300：Second coated film 300 is formed on basal layer 100, and is connected with the first coated film 200,
Separated first area 310 and second area 320 are provided on second coated film 200, and first area 310 is located at nearly first
200 end of coated film, wherein, first area 310 is coated with secondary antibody, and secondary antibody and first antibody can be specifically bound
The A types H9 subtype influenza virus, second area 320 is coated with antiantibody, and the antiantibody can specifically bind first and resist
Body.
According to some embodiments of the present invention, the first area in the second coated film and second area are all wire, wherein,
First area can be referred to as detection line (T lines), and second area can be referred to as nature controlling line (C lines), the line where two regions
Shape plane is parallel to the bonding side of two coated films.Some specific embodiments according to the present invention, wire first or second region
Width be about 0.5-1mm, the width of a length of film.The width suitable in the first and second regions as a result, convenient for first antibody
One region combines to form double antibodies sandwich compound with the compound that influenza virus combines to form in first area and secondary antibody, together
When, the first antibody convenient for not forming double antibodies sandwich compound is combined in second area with antiantibody, and specific binding, i.e., with
Extra band fluorescent marker first antibody combines to form immune complex, it is ensured that the validity of first antibody, by exciting fluorescence,
So as to it make the sensitivity higher of kit.
According to an embodiment of the invention, first antibody and secondary antibody are monoclonal antibody.First antibody and as a result,
Two antibody are specifically combined with influenza virus, and the accuracy of detection is high.Due to the antigen table between influenza A different subtype
Position variation is complicated, and the binding site of monoclonal antibody and the steric hindrance of itself etc., which directly affect first antibody and secondary antibody, is
It is no can be in combination with to the more difficult acquisition of specific antibody on influenza virus, made for H9 subtype influenza virus different epitopes, invention
People screens the monoclonal antibody of substantial amounts of A types H9 subtype influenza virus, having obtained can be same by substantial amounts of experimental study
When be attached to first antibody and secondary antibody on influenza virus, and first antibody and secondary antibody are to A type H9 subtype influenzas
The specificity of virus is high.According to an embodiment of the invention, first antibody is F9-25 for the number purchased from the big monoclonal antibody center of Kunming cloud
Monoclonal antibody, the monoclonal antibody that secondary antibody is F9-26 for number purchased from the big monoclonal antibody center of Kunming cloud.According to this hair
Bright embodiment, the monoclonal antibody that first antibody is F9-14 for the number purchased from the big monoclonal antibody center of Kunming cloud, secondary antibody are
The monoclonal antibody that number purchased from the big monoclonal antibody center of Kunming cloud is F9-12.According to an embodiment of the invention, first antibody is purchase
The monoclonal antibody that number from the big monoclonal antibody center of Kunming cloud is F9-25, secondary antibody are purchased from the big monoclonal antibody center of Kunming cloud
The monoclonal antibody that number is F9-12.Wherein, the list that first antibody is F9-17 for the number purchased from the big monoclonal antibody center of Kunming cloud
Clonal antibody, the kit for the monoclonal antibody that secondary antibody is F9-47 for the number purchased from the big monoclonal antibody center of Kunming cloud, detection
Specificity and sensitivity more preferably.
According to an embodiment of the invention, antiantibody is sheep anti-mouse igg antibody.The antiantibody can be with first antibody as a result,
Specific binding, the extra band fluorescent marker first antibody with not forming double antibodies sandwich compound combines to form immune compound
Object excites fluorescence, so as to qualitative and/or quantitatively detect the immune complex.
According to an embodiment of the invention, the first coated film 200 is glass fibre element film.Since glass fibre membrane is lazy in chemistry
Property, it without adhesive, is fabricated using 100% pyrex fiber, is coated with the first antibody of fluorescent marker thereon, profit
It is specifically bound in first antibody and the target antigen in sample to be tested.A specific embodiment according to the present invention, glass
Glass tunica fibrosa can be purchased from Millipore companies, cat. no GF-CP20300.
According to an embodiment of the invention, the second coated film 300 is nitrocellulose filter.Since nitrocellulose filter has added
Surfactant is added, hydrophilic ability is strong, and there are certain buffer system, has capillary fiber structure, can adsorb than same
The more moisture of cellulosic filter papers are waited, flow velocity is fast, high temperature resistant, anti-beneficial to coated secondary antibody thereon and fluorescent marker first
Specific binding reaction occurs for body-influenza antigen, excites fluorescence.A specific embodiment according to the present invention, nitric acid are fine
The plain film of dimension can be purchased from Millipore companies, cat. no is Hi-Flow Plus HF135.
With reference to figure 3, according to an embodiment of the invention, which further comprises：Water absorption pad 400, the water absorption pad 400 with
Remote first coated film, 200 end of second coated film 300 is connected, for example, the first coated film 200, the second coated film 300 and water absorption pad
400 are sequentially connected.According to an embodiment of the invention, water absorption pad can be strong absorbent material, when detecting liquid sample, water absorption pad
400, which can give directed forces, makes liquid sample from the first coated film orientation chromatography to the second coated film.It is according to the present invention
One specific embodiment, water absorption pad can be purchased from Millipore companies, cat. no CF-SP22300.
The method for detecting influenza virus
According to another aspect of the present invention, the present invention provides a kind of methods for detecting influenza virus.Inventor has found, sharp
A type H9 subtype influenza virus is detected with this method, the high sensitivity of detection, high specificity, detection time is short, and testing cost is low,
Easily operated and popularization.
With reference to figure 4, according to an embodiment of the invention, the method for detecting influenza virus is explained, this method bag
It includes：
S100 is loaded
According to an embodiment of the invention, by the first coated film in the detection kit of sample to be tested and foregoing influenza virus
Contact.And then acted on by chromatography, sample to be tested is moved to the second coated film, so as to be combined with corresponding antibodies.
S200 fluoroscopic examinations
According to an embodiment of the invention, the fluorescence intensity of the first area in detection kit and second area obtains the
One region fluorescent measurement and second area fluorescent measurement.For example, hand-held instrument fluorescence intensity can be passed through.
S300 judges
According to an embodiment of the invention, based on first area fluorescent measurement and second area fluorescent measurement, judge to treat
It whether there is A type H9 subtype influenza virus in test sample sheet.
According to an embodiment of the invention, by fluorescence intensity ratio compared with threshold value, judge whether deposited in sample to be tested
In A type H9 subtype influenza virus, wherein, fluorescence intensity ratio=first area fluorescent measurement/second area fluorescent measurement.
Inventor is by reference to utilizing the condition of known double-antibody sandwich immune response and commercial reagent box product, largely
It measures normal sample and adds the normal sample threshold value of positive sample, with first area and second area fluorescent measurement
For the average of ratio as threshold value, it is positive or negative that can detect sample with accurate judgement.According to an embodiment of the invention, the threshold
It is worth for 0.001-0.02.According to a preferred embodiment of the invention, the threshold value is 0.008.Fluorescence intensity ratio is more than threshold value
The positive, that is, detect A type H9 subtype influenza virus, and fluorescence intensity ratio is less than threshold value for feminine gender, that is, A type H9 hypotypes are not detected
Influenza virus.The kit of the embodiment of the present invention is utilized as a result, can be realized to influenza A H9 hypotypes based on the threshold value
Quick and highly sensitive measure.
For the ease of understanding foregoing influenza virus kit, the conventional method for preparing the kit is provided herein, including
Following steps：
(1) preparation of the fluorescent microsphere label probe of carboxyl modified
Using the fluorescent microsphere of suitable carboxyl modified, after the carboxyl for activating its surface, divided by the way of covalent coupling
A type H9 subtype influenza virus mark secondary antibody orientation is not connected to the fluorescent microsphere surface of the carboxyl modified.
The coating of (2) first and second domain antibodies
Using spray film instrument, A types H9 subtype influenza virus coated second is sprayed at the first area of the second coated film
Antibody, the first area are detection line (T lines), sheep anti-mouse igg antibody are sprayed at second area, which is control line
(C lines).
The coating of label probe at (3) first coated films
Using spraying apparatus, in the first coated film (being referred to as sample pad) specific location spraying fluorescent microsphere mark
A type H9 subtype influenza virus mixtures of antibodies, which is one piece of region on the first coated film, the region, that is, conduct
Subsequent " sample-adding end ".
(4) assembled formation of kit
With reference to figure 3, the second coated film as test section is pasted among basal layer, is glued in the T line ends of the second coated film
Sample pad is pasted, C line ends paste water absorption pad.Using test paper cutting machine, the paper slip for certain broadband, such as 4mm wide are cut, and
Intermediate plate is packed into, is packed with the aluminium foil bag equipped with drier.
(5) formation of antigen-antibody fluorescent immune complex
Sample to be tested is added at the sample-adding end of above-mentioned assembled formation, A type H9 subtype influenza virus and fluorescence in sample
The A type H9 subtype influenza virus bags that chromatography is sprayed at T lines after the A type H9 subtype influenza virus labelled antibody of microballoon mark combines
By antibody, coated antibody-antigen-fluorescent microsphere labelled antibody immune complex, extra fluorescent microsphere mark are formed at T lines
The fluorescent mark immunity compound that A type H9 subtype influenza virus antibody is then formed at C lines with sheep anti-mouse igg.
(6) fluorescent mark immunity complex fluorescence intensity detection
It is measured with fluorescence detector at T lines and fluorescence intensity at C lines, compares to determine that its is positive by the threshold value with setting
Or negative findings, the C lines measurement result then Quality Control internal standard as the assay method.Wherein, fluorescent mark immunity compound is glimmering
Luminous intensity refers to that the quantity of the combination fluorescent microsphere under being detained respectively at T lines, C lines is acquired after being measured with fluorescence detector
Numerical value.By the condition of double-antibody sandwich immune response, can determine that through largely measuring normal sample and addition positive sample
The measure average of variant normal sample determines the positive or negative knot of detection sample in this, as critical value (cutoff)
Fruit.The C lines measurement result then Quality Control internal standard as the assay method.
Below with reference to specific embodiment, the present invention will be described, it is necessary to which explanation, these embodiments are only explanation
Property, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it (such as is translated according to the described technology of document in the art or condition with reference to the works such as J. Pehanorm Brookers, Huang Peitang etc.
's《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument
Production firm person is not specified, being can be with conventional products that are commercially available, such as can purchase from Millipore companies.
Embodiment 1
Using the method for the embodiment of the present invention, it is as follows to prepare the step of detecting A type H9 subtype influenza virus kits：
1st, prepared by buffer solution
(1) pH is 7.4 0.02M PBS buffer solution：Weigh 2.3g Na2HPO4、0.524gNaH2PO4.H2O、8.77g
NaCl is dissolved in pure water, is settled to 1L with pure water, adjusts pH to 7.4, obtains the 0.02M PBS buffer solution that pH is 7.4.
(2) film process buffer solution：Tween-20, BSA, sucrose are dissolved in the 0.02M PBS buffer solution that above-mentioned pH is 7.4 makes
The mass percentage of Tween-20 is 0.2%, the mass percentage of BSA is 1%, the mass percentage of sucrose is 2%,
PH to 7.4 is adjusted, obtains film process buffer solution.
(3) 50mM pH are 8.5 borate buffer solution：Weigh 1.9g Na2B4O7.10H2O is dissolved in 100ml pure water, adjusts pH extremely
8.5 obtain the borate buffer solution that 50mM pH are 8.5.
(4) sample treatment liquid：Tween-20 is dissolved in the 0.02M PBS buffer solution that above-mentioned pH is 7.4 makes Tween-20's
Mass percentage is 0.2%, adjusts pH to 7.4, obtains sample treatment liquid.
2nd, the preparation of fluorescent microsphere mark A type H9 subtype influenza virus labelled antibodies
Using average diameter as 110nm, carboxyl modified fluorescent microsphere (purchased from Bangs Laboratories, Inc. companies,
Catalog number is FC02F/10930), the first monoclonal antibody of anti-A type H9 subtype influenza virus (is purchased from the big monoclonal antibody of Kunming cloud
Center, number F9-17), fluorescent microsphere mark A type H9 subtype influenza virus mark first antibodies are prepared by the following method：
(1) after the fluorescent microsphere of the above-mentioned carboxyl modified of 5mg is taken to be washed and centrifuged with MES buffer solutions (0.1M, pH4.7),
It is resuspended with 1ml MES buffer solutions (0.1M, pH4.7), adds in 1- ethyls-(3- dimethylaminopropyls) carbodiimide (EDC) extremely
Final concentration of 5mM, NHS (N- hydroxysuccinimides) to final concentration of 10mM is added in, room temperature is protected from light, and reaction half an hour is lived
The fluorescent microsphere of carboxyl modified after change.
(2) fluorescent microsphere of carboxyl modified, takes respectively after washing the activation with the borate buffer solution of 50mM pH8.5
Carboxyl modified after the above-mentioned A types H9 subtype influenza virus first antibodies (number F9-17) to be marked of 0.37mg and the above-mentioned activation of 5mg
Fluorescent microsphere be mixed into abundant mixing in the borate buffer solution of 50mM pH8.5.Room temperature be protected from light lower reaction 2 it is small when, allow the antibody
Stable covalent peptide bonds are formed with fluorescent microsphere to combine, and obtain the conjugate of fluorescent microsphere and A type H9 subtype influenza virus antibody.
After reaction, the BSA solution of final concentration of 1% (mass percentage) is added in fluorescent microsphere and A type H9 subtype influenzas disease
Residual activity carboxyl site is closed on the conjugate of malicious antibody, room temperature be protected from light 0.5 it is small when.After finishing, with pH9.4's
The washing of 0.02M PBS buffer solution, resuspension obtain 5mg/ml fluorescent microspheres mark A type H9 subtype influenza virus antibody liquid, 4 DEG C of guarantors
It deposits for use.
3rd, the preparation of A type H9 subtype influenza virus kits is detected
(1) secondary antibody is coated with A type H9 subtype influenza virus, the second coated film is prepared with sheep anti-mouse igg antibody, specifically
Method is as follows：
(a) the 0.02M PBS buffer solution of pH7.4 is used, sheep anti-mouse igg antibody (is won into the limited public affairs of excellent biotechnology in Changsha
Department, ABGAM-0500) concentration 1mg/ml solution is formulated as, A type H9 subtype influenza virus is coated with secondary antibody (is purchased from Kunming cloud
Big monoclonal antibody center, number F9-47) compound concentration be 2mg/ml solution.
(b) select the XYZ3050 spray membranous systems of BioDot that step (1) is obtained sheep anti-mouse igg antibody solution and be sprayed onto second
A type H9 subtype influenza virus coated antibody buffer solutions are sprayed onto inspection by nature controlling line (C lines) position of coated film (nitrocellulose filter)
Survey line (T lines) position, in relative humidity be less than 10% drying plant carry out dehumidifier 4 it is small when after dried for standby, had
The coated film of detection line and nature controlling line.
(2) the all-glass paper half an hour obtained with film process buffer solution soaking step 3- (1), the temperature of immersion is 37
DEG C, in same dehumidifier condition carry out dehumidifier 4 it is small when after, with 1 gained fluorescent microsphere of film process buffer solution dilution step mark A types
H9 subtype influenza virus antibody liquid to fluorescent microsphere mark A type H9 subtype influenza virus first antibodies content is 0.05mg/ml
Mixed liquor after, using BioDot XYZ3050 spray membranous system be sprayed on above-mentioned processed glass fibre element film, make
It is standby to form sample pad, it is dried in same dehumidifier condition.Above-mentioned dried in 100,000 grades of cleanings and dry workshop
The second coated film, sample pad, water absorption pad and the basal layer with detection line and nature controlling line carry out collocation assembling as shown in Figure 3
Afterwards, width of the CM4000 cutting systems of BioDot by the Paperboard cutting posted for 4mm/ items is used, detection is packed into and is treated with intermediate plate
With.
Embodiment 2
The detection A type H9 subtype influenza virus kits that embodiment 1 obtains are assessed, specific method is as follows：
1st, detection sensitivity
Antigen is tested as sample to be tested using A type H9 subtype influenza virus HI to measure the detection A type H9 hypotypes of embodiment 1
The sensitivity of influenza virus kit.
A type H9 subtype influenza virus HI experiment antigens respectively and are mixed with the 0.02M PBS buffer solution that pH is 7.4 simultaneously
Be configured to series concentration (64,32,16,8,4,2,1,0.5,0.25,0.125,0HA unit), be separately added by embodiment 1
To detection A type H9 subtype influenza virus kits sample-adding end in, and using fluorescence detector detect.Detecting step：Detection
It is preceding that detected sample is first recovered into room temperature (25 DEG C), with accurate pipettor 60 μ l of detected sample is taken vertically to be slowly dropped into above-mentioned obtain
To detection A type H9 subtype influenza virus kits sample-adding end on, tested after ten minutes with fluorescence detector.
Its testing result is as shown in table 1 below.It can show that mentioned reagent box detects A type H9 subtype influenzas from testing result
The sensitivity of virus be that 0.25HA unit, T/C Cut-Off values are the positive more than 0.008.
A type H9 subtype influenza virus kit detected values and concentration curve are as shown in Figure 5.
The kit detected value of 1 A type H9 subtype influenza virus difference sample concentrations of table
2nd, accuracy detects
Choose the sample of 3 parts of various concentrations, duplicate measurements 10 times according to the method described in the present invention respectively, according to 10 times
As a result batch interior average deviation CV% values are calculated.It is dense to choose 3 parts of differences for the 3 batches of kits prepared according to the method described in the present invention
The sample of degree, respectively duplicate measurements 10 times calculate average deviation CV% values between criticizing according to result.Its testing result such as the following table 2 institute
Show, it can be seen that high using kit detection precision.
The interior difference between batch of 2 batches, table measures
3rd, cross reaction detects
It is 7.4 with pH by influenza A hypotype HI experiment antigens and other common Respirovirus HI experiments antigens
0.02M PBS buffer solution is formulated as concentration 64HA unit, with accurate pipettor 60 μ l is taken vertically to be slowly dropped into embodiment 1 respectively
The sample-adding end of obtained detection A type H9 subtype influenza virus kits, is tested after ten minutes with fluorescence detector.It is detected
As a result it is as shown in table 3 below.The A types H9 subtype influenza virus kit and other hypotypes of influenza A and other common breathings
There is no cross reaction between road virus, there is very strong specificity to H9 hypotypes.
3 A type H9 subtype influenza virus kits cross reaction of table detects
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms is not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle of the present invention and objective a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (10)
1. a kind of detection kit of influenza virus, which is characterized in that including：
Basal layer；
First coated film, first coated film is formed on the basal layer, and is coated with the first antibody of fluorescent marker, institute
It states first antibody and specifically binds the A types H9 subtype influenza virus；
Second coated film, second coated film are formed on the basal layer, and are connected with first coated film, and described
Separated first area and second area are provided on two coated films, and first area is located at the nearly first coated film end,
In,
The first area is coated with secondary antibody, and the secondary antibody and the first antibody can specifically bind the A
Type H9 subtype influenza virus,
The second area is coated with antiantibody, and the antiantibody can specifically bind the first antibody.
2. kit according to claim 1, which is characterized in that further comprise：
Water absorption pad, the water absorption pad are connected with the remote first coated film end of second coated film.
3. kit according to claim 1, which is characterized in that the first antibody and the secondary antibody are Dan Ke
Grand antibody.
4. kit according to claim 1, which is characterized in that the first antibody is purchased from the big monoclonal antibody center of Kunming cloud
Number be F9-17 monoclonal antibody, the list that the secondary antibody is F9-27 for number purchased from the big monoclonal antibody center of Kunming cloud
Clonal antibody,
Optionally, the monoclonal antibody that the first antibody is F9-17 for number purchased from the big monoclonal antibody center of Kunming cloud, described the
The monoclonal antibody that two antibody are F9-47 for the number purchased from the big monoclonal antibody center of Kunming cloud,
Optionally, the monoclonal antibody that the first antibody is F9-19 for number purchased from the big monoclonal antibody center of Kunming cloud, described the
The monoclonal antibody that two antibody are F9-27 for the number purchased from the big monoclonal antibody center of Kunming cloud.
5. kit according to claim 1, which is characterized in that the fluorescent marker marks for fluorescent microsphere,
Optionally, the average diameter of the fluorescent microsphere is 10-500nm, it is preferable that is 20-300nm, it is highly preferred that being 100-
120nm。
6. the kit of claim 5, which is characterized in that the first antibody passes through covalent peptide bonds with fluorescent microsphere mark
With reference to.
7. kit according to claim 1, which is characterized in that the antiantibody is sheep anti-mouse igg antibody.
8. kit according to claim 1, which is characterized in that first coated film is glass fibre element film, described
Second coated film is nitrocellulose filter.
A kind of 9. method for detecting influenza virus, which is characterized in that including,
Sample to be tested is contacted with the first coated film in the detection kit of any one of the claim 1-8 influenza viruses；
It detects the first area in the kit and the fluorescence intensity of second area, obtains first area fluorescent measurement and the
Two region fluorescent measurements；And
Based on the first area fluorescent measurement and second area fluorescent measurement, judge to whether there is in the sample to be tested
A type H9 subtype influenza virus.
10. according to the method described in claim 9, it is characterized in that, by fluorescence intensity ratio compared with threshold value, institute is judged
It states with the presence or absence of A type H9 subtype influenza virus in sample to be tested, wherein, the fluorescence intensity ratio=first area fluoroscopic examination
Value/second area fluorescent measurement,
Optionally, the threshold value is 0.001-0.02, it is preferable that is 0.008.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120024313A (en) * | 2010-09-06 | 2012-03-14 | 대한민국(관리부서 : 농림수산식품부 농림수산검역검사본부) | Diagnostic kit for h9 type avian influenza virus using rapid immunochromatography and the method for diagnosing h9 type avian influenza by using the same |
| CN104407135A (en) * | 2014-11-03 | 2015-03-11 | 清华大学深圳研究生院 | Method and kit for detecting A type influenza virus H5 and H9 subtypes |
| CN107589253A (en) * | 2017-09-06 | 2018-01-16 | 詹爱军 | A kind of H9 subtype avian influenza virus fluorescent chromatographic test strips and its application |
| CN207472897U (en) * | 2017-11-09 | 2018-06-08 | 詹爱军 | Novel trivalent bird flu fluorescence immune chromatography test paper bar |
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2017
- 2017-11-28 CN CN201711219303.2A patent/CN108107207A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120024313A (en) * | 2010-09-06 | 2012-03-14 | 대한민국(관리부서 : 농림수산식품부 농림수산검역검사본부) | Diagnostic kit for h9 type avian influenza virus using rapid immunochromatography and the method for diagnosing h9 type avian influenza by using the same |
| CN104407135A (en) * | 2014-11-03 | 2015-03-11 | 清华大学深圳研究生院 | Method and kit for detecting A type influenza virus H5 and H9 subtypes |
| CN107589253A (en) * | 2017-09-06 | 2018-01-16 | 詹爱军 | A kind of H9 subtype avian influenza virus fluorescent chromatographic test strips and its application |
| CN207472897U (en) * | 2017-11-09 | 2018-06-08 | 詹爱军 | Novel trivalent bird flu fluorescence immune chromatography test paper bar |
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Application publication date: 20180601 |