CN109900913A - A kind of A type and influenza B virus antigen colloidal gold method combined detection kit and preparation method thereof - Google Patents
A kind of A type and influenza B virus antigen colloidal gold method combined detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of A types and influenza B virus antigen colloidal gold method combined detection kit and preparation method thereof, are related to influenza virus detection box field.The anti-influenza B virus monoclonal antibody of mouse of the mouse anti-influenza A virus monoclonal antibody and colloid gold label of colloid gold label is coated on the detection card that the combined detection kit includes；The nature controlling line location of C for detecting the NC film of card is coated with sheep anti mouse polyclonal antibody, and influenza B detection line T1 position is coated with the anti-influenza B virus monoclonal antibody of mouse, and the position Flu-A detection line T2 is coated with mouse anti-influenza A monoclonal antibody.The features such as kit can detect A type and B-mode two kinds of influenza viruses in sample simultaneously, which has detection speed fast, high sensitivity.
Description
Technical field
The present invention relates to influenza virus detection kit fields, in particular to a kind of A type and influenza B virus
Antigen colloidal gold method combined detection kit and preparation method thereof.
Background technique
Influenza virus, abbreviation influenza virus are that one kind causes people, fowl, pig, horse, dog etc. to suffer from influenza
A kind of RNA virus, Orthomyxoviridae family is belonged on taxology, clinically will lead to acute upper respiratory infection.With sky
Gas is that medium is propagated rapidly, is in usually eruption and prevalence, often has all over the world and be periodically very popular.Virus 1933 by
British Wilson's Smith (Wilson Smith) separates successfully for the first time, is named as H1N1.H represents hemagglutinin；N is represented
Neuraminidase.Arabic numerals after letter then represent the type of antigen.
According to the object of influenza infection, virus can be divided into human influenza virus, swine influenza virus, equine influenza disease
The monoids such as poison and avian influenza virus, wherein human influenza virus can be divided into three classes according to the antigenicity of its nucleoprotein: A type
Influenza virus (Influenza A virus), also known as influenza A；Influenza B virus (Influenza B virus),
Also known as Type B influenza virus；Influenza virus C (Influenza C virus), also known as c-type influenza virus.
Influenza virus is spherical in shape, and the strain newly separated is then mostly in filiform, and diameter is between 80 to 120 nanometers, filiform stream
The length of Influenza Virus is up to 4000 nanometers.Influenza structural can be divided into coating, stromatin and core three from outer to inner
Point.
Core: viral core contains the inhereditary material of storage Virus Info and replicates the necessary enzyme of these information.
The inhereditary material of influenza virus is sub-thread strand RNA, is abbreviated as ss-RNA, and ss-RNA is combined with nucleoprotein (NP), is wound in
Ribosome exists in the form of very high density.In addition to ribosome, there are also the RNA polies for being responsible for rna transcription
Enzyme.The RNA of A type and influenza B virus is made of 8 segments, influenza virus C then than their a few segment, the 1st, 2,
What 3 segments encoded is the more aggregation enzymes of RNA, and the 4th segment is responsible for encoding hemagglutinin；Encoding nuclear proteins are responsible in 5th segment, the
What 6 segments encoded is neuraminidase；7th segment encodes stromatin, and the 8th segment coding is a kind of to play spelling
The non-structural protein of RNA function is connect, the other function of this albumen is still unknown.What influenza virus C lacked is the 6th
The hemagglutinin of segment, fourth segment coding can exercise the function of neuraminidase simultaneously.
Stromatin: stromatin constitutes the outer casing framework of virus, in practical upper skeleton in addition to stromatin (M1) it
It is outer that there are also memebrane protein (M2).M2 albumen has ion (mainly Na+) effect of pH value in channel and adjusting film, but quantity is very
It is few.Stromatin and the outermost coating of virus, which are combined closely, to be played protection virus core and maintains the work of viral space structure
With.After influenza virus completes its breeding in host cell, stromatin is distributed across on host cell membrane inner wall
, molding virus core capsid can identify the position on host cell membrane containing stromatin, formation virus in combination
Structure, and the prominent mature virus of release in the form of budding.
Coating: coating is wrapped around one layer of phospholipid bilayer tunic except stromatin, this tunic is from host's
Cell membrane, mature influenza virus sprout from host cell, are detached from cell after the cell membrane of host is wrapped on one's body,
It goes to infect next target.In coating other than phospholipid molecule, also there are two types of very important glycoprotein: hemagglutinin and nerve
Propylhomoserin enzyme.The prominent virus of these two types of albumen is external, and length is about 10 to 40 nanometers, is referred to as furcella.A general influenza virus
500 hemagglutinin spikes and 100 neuraminidase furcellas can be distributed in surface.Hemagglutinin and nerve in influenza A virus
The antigenicity of propylhomoserin enzyme can change, this is to discriminate between the foundation of virus stain hypotype.
Hemagglutinin (HA): being in the form of a column, and can combine with the receptor on the animal erythrocytes surfaces such as people, bird, pig cavy and cause to coagulate
Blood, so it is referred to as hemagglutinin.It is divided into light chain and heavy chain two parts after hemagglutinin hydrolysis, the latter can be with host cell membrane
On sialic acid receptor combine, the former then can assist peplos mutually to merge with host cell membrane.Hemagglutinin is in virus
Key player is played during importing host cell.Hemagglutinin has immunogenicity, and antihemagglutinin antibody can be with neutralized stream
Influenza Virus.
Neuraminidase (NA): it is one in mushroom tetramer glycoprotein, there is the activity of hydrolysis sialic acid, treat as
After mode of the ripe influenza virus through sprouting is detached from host cell, the hemagglutinin of virus surface can be via sialic acid receptor and place
Mainly cell membrane is kept in touch, and needs to be hydrolyzed sialic acid by neuraminidase, and cutting virus is finally contacted with host cell, is made
Virus can smoothly be discharged from host cell, then infect next host cell.Therefore neuraminidase is also controlled as influenza
An action target spot for treating drug, the Oseltamivir for the design of this enzyme is one of foremost Tamiflu.
Naming method: the Influenza virus strain nomenclature amendment passed through for 1980 according to the World Health Organization, influenza poison
The name of strain includes 6 elements: type/host/separation area/strain serial number/separation time (HnNn), wherein flowing for the mankind
Influenza Virus omits hosted information, omits hypotype information for B-mode and influenza virus C.Such as A/swine/Lowa/15/30
(H1N1) what is indicated is the H1N1 subtype influenza virus poison with pig for host that nucleoprotein separates for A type, nineteen thirty in lowa
Strain, strain serial number 15, this is also first Influenza virus strain of mankind's separation.
Current diagnostic method: virus infection can inducing interferon expression and cellular immunity conditioning, cause it is some itself
Immune response, including high fever, headache, gastrocnemius and whole-body muscular pain etc., the toxin sample product and cell of viral metabolism are bad
Dead release product will also result in and aggravate above-mentioned reaction.It is removed since influenza infection can reduce respiratory mucosa epithelial cell
With stick the ability of foreign matter, so greatly reduce the ability that human body resists respiratory tract infection, thus influenza often will cause after
The sexy dye of hair, the secondary pneumonia as caused by influenza is lethal one of the underlying cause of death of influenza.In epizootic modeling combination clinical symptoms
It is not difficult to diagnose influenza, but to make a definite diagnosis or when Epidemiology monitor must carry out laboratory inspection, mainly include virus purification culture,
Serodiagnosis and fast diagnosis method.
The separation and identification of virus: the throat wash or throat swab of patient in morbidity 3 days is usually taken, after antibiotic treatment
It is inoculated in 9~11 age in days chick embryo amnoitic sacs and allantoic cavity, after 33 DEG C~35 DEG C are incubated for 3~4 days, collects amniotic fluid and allantoic fluid
Carry out hemagglutination test.Such as the hemagglutination test positive, then with known immune serum carry out blood clotting inhibition (Hemoagglutination
Inhibition, HI) test, identify type.If hemagglutination test is negative, with chicken embryo blind passage 3 times again, still there can be no
Blood clotting then judges virus purification for feminine gender.Tissue culture cells (such as human embryo kidney (HEK) or monkey kidney) isolated viral can also be used, is determined whether
Virus multiplication can use hemadsorption method or fluorescence antibody method.
Serodiagnosis: the Acute Stage (in morbidity 5 days) and convalescence (course of disease 2~4 weeks) paired sera are taken, often
With HI testing inspection antibody.If convalescence increases 4 times or more than acute phase serum antibody titer, diagnosis can be made.Normally
Often contain nonspecific inhibition in human serum, therefore can be clear with trypsase etc. regulating blood condition before carrying out HI test, Yi Mianying
Ring HI test result.HI test virus used should be the Strain closely related with current popular, and reaction result could really
It cuts.Complement fixation test (CFT) (compliment fixation, CF) can only detect the antibody of NP, MP.The appearance of these antibody is early, disappears
It loses fast.Therefore, CF test can only be as the index whether infected recently.
Quick diagnosis: carrying out quick diagnosis to patient, mainly using directly or indirectly immunofluorescence technique, ELISA method inspection
Survey viral antigen.Patient's turbinate mucosa printingout or respiratory tract Exfoliative cells smear often are taken, with fluorescein-labeled influenza disease
Malicious immune serum carries out immunofluorescence dyeing and checks antigen, or checks the antigen in patient's Pharyngeal aspirate with ELISA.It is anti-with monoclonal
Body only can quickly detect virion of the first, influenza B virus in infection cell with 24~72 hours through immunoenzyme labeling method
Or virus-associated antigen.The methods of PCR, nucleic acid hybridization or sequence analysis also be used to detect influenza nucleic acids or divided
Type.
Lack the kit that can quickly detect A type and influenza B virus simultaneously on the market at present.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of A type and influenza B virus antigen colloidal gold method combined detection kit,
The combined detection kit can detect A type and B-mode two kinds of influenza viruses in sample simultaneously, detect first using the kit
The features such as type and influenza B virus have detection speed fast, high sensitivity.
Another object of the present invention is to provide a kind of A types and influenza B virus antigen colloidal gold method joint-detection to try
The preparation method of agent box, combined detection kit made of the preparation method can detect A type in sample and two kinds B-mode simultaneously
The features such as influenza virus, the kit detect A type and influenza B virus, have detection speed fast, high sensitivity.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of A type and influenza B virus antigen colloidal gold method combined detection kit,
It includes detection card, and above-mentioned detection card includes shell and is fixed on above-mentioned enclosure interior for detecting A type and influenza B virus
Colloid gold chromatographic test paper strip；
Wherein, above-mentioned colloid gold chromatographic test paper strip include successively overlapped the sample pad being arranged on bottom plate, colloidal gold pad,
NC film and blotting paper；
The nature controlling line location of C of above-mentioned NC film is coated with sheep anti mouse polyclonal antibody, the influenza B detection line of above-mentioned NC film
The position T1 is coated with the anti-influenza B virus monoclonal antibody of mouse, and the position Flu-A detection line T2 of above-mentioned NC film is coated with mouse
Anti-influenza A monoclonal antibody；
Above-mentioned colloidal gold pad is coated with the mouse anti-influenza A virus monoclonal antibody and colloid gold label of colloid gold label
The anti-influenza B virus monoclonal antibody of mouse.
Further, in some embodiments of the present invention, above-mentioned combined detection kit further includes sample extraction liquid,
Above-mentioned sample extraction liquid is the phosphate buffer of 0.1M, pH7.3-7.7；
Above-mentioned sample extraction liquid contains the sodium chloride that concentration is 0.5%~1.0% and concentration is 0.1%~1.0%
Tween-20。
Further, in some embodiments of the present invention, above-mentioned combined detection kit further includes with lower component
It is one or two kinds of: sampling swab and sampling dropper；
Wherein, above-mentioned sampling swab length is 15~20cm, diameter is 1.0~2.0mm, material PP, PE or ABS, on
The end for stating sampling swab is stained with that the length for adsorbing pharyngeal sample is 1.0~2.0cm, diameter is the de- of 0.3~0.8cm
Rouge cotton or sponge.
Further, in some embodiments of the present invention, above-mentioned blotting paper with a thickness of 0.5mm~1.0mm, width
Degree is 3.84~3.86mm, length is 1.9~2.1cm；
Preferably, above-mentioned shell is plastic casing；
Preferably, the material of above-mentioned plastic casing is selected from one of HDPE, LDPE, PP, ABS or a variety of；
Preferably, the material of above-mentioned bottom plate is polyvinyl chloride；
Preferably, the material of above-mentioned colloidal gold pad be glass fibre, above-mentioned colloidal gold pad with a thickness of 0.2mm~1.0mm,
Width is 3.84~3.86mm, length is 0.5~0.8cm；
Preferably, the material of above-mentioned sample pad be glass fibre, above-mentioned sample pad with a thickness of 0.2mm~1.0mm, width
Degree is 3.84~3.86mm, length is 1.5~2.0cm；
Preferably, the material of above-mentioned NC film be nitrocellulose filter, above-mentioned NC film with a thickness of 0.1mm~0.5mm, width
It is 1.8~2.2cm for 3.84~3.86mm, length, the mean pore size on above-mentioned NC film is 5 μm~20 μm.
Further, in some embodiments of the present invention, above-mentioned plastic casing includes head components and base plate part,
There is the observation window for observing nature controlling line C, influenza B detection line T1 and Flu-A detection line T2 on above-mentioned head components；
Above-mentioned observation window includes the semicircle windows of a rectangular window and two, wherein rectangular window having a size of
(14.80~14.85) mm × (3.70~3.75) mm, it is (3.30~3.35) that the two sides of rectangular window, which are respectively connected a radius,
The semicircle window of mm, the semicircle windows of rectangular window and two collectively constitute having a size of (21.40~21.55) mm ×
The observation window of (3.70~3.75) mm；
Preferably, the outer profile size of above-mentioned plastic casing are as follows: (74.50~75.50) mm × (24.50~25.50) mm
× (3.84~3.86) mm, inside the notch that has for being adapted to width be 3.84~3.86mm, length be 5.8~
The above-mentioned colloid gold chromatographic test paper strip of 6.2cm.
Further, in some embodiments of the present invention, above-mentioned plastic shell is by being located at the top cover portion on top
Part and base plate part positioned at lower part fasten composition, the internal card slot formed for accommodating colloid gold chromatographic test paper strip.
Further, in some embodiments of the present invention, the color of upper header component is white.Surface can print coloured silk
Colored pattern and Text region information；The color of lower raft component is white.
On the other hand, the present invention provides a kind of A types and influenza B virus antigen colloidal gold method combined detection kit
Preparation method comprising following steps:
Assembling steps: the colloid gold chromatographic test paper strip for being used to detect A type and influenza B virus is fixed on enclosure interior
To constitute detection card,
Wherein, above-mentioned colloid gold chromatographic test paper strip include successively overlapped the sample pad being arranged on bottom plate, colloidal gold pad,
NC film and blotting paper；
The nature controlling line location of C of above-mentioned NC film is coated with sheep anti mouse polyclonal antibody, the influenza B detection line of above-mentioned NC film
The position T1 is coated with the anti-influenza B virus monoclonal antibody of mouse, and the position Flu-A detection line T2 of above-mentioned NC film is coated with mouse
Anti-influenza A monoclonal antibody；
Above-mentioned colloidal gold pad is coated with the mouse anti-influenza A virus monoclonal antibody and colloid gold label of colloid gold label
The anti-influenza B virus monoclonal antibody of mouse.
Further, in some embodiments of the present invention, before above-mentioned assembling steps, above-mentioned preparation method is also wrapped
Include following steps:
Colloidal gold pad preparation step:
By the Immuno gold solution A of the mouse anti-influenza A virus monoclonal antibody containing colloid gold label and contain colloidal gold
The mixed immunity gold solution that the Immuno gold solution B of the anti-influenza B virus monoclonal antibody of the mouse of label is mixed to get, will mix
Immune gold solution is adsorbed on glass fibre, be made be coated with colloid gold label mouse anti-influenza A virus monoclonal antibody and
The colloidal gold pad of the anti-influenza B virus monoclonal antibody of the mouse of colloid gold label；
NC film preparation step:
Dilute the anti-influenza B virus monoclonal antibody of sheep anti mouse polyclonal antibody, mouse and the anti-A type of mouse respectively with coating buffer
Monoclonal antibody against Influenza respectively obtains C line working solution, T1 line detection working solution and T2 line detection working solution；
By the different location of C line working solution, T1 line detection working solution and T2 line detection working solution on nitrocellulose filter
It carries out scribing line processing and forms the NC film with nature controlling line C, influenza B detection line T1 and Flu-A detection line T2, wherein matter
Line location of C is coated with sheep anti mouse polyclonal antibody, influenza B detection line T1 position is coated with the anti-influenza B virus list of mouse for control
Clonal antibody and the position Flu-A detection line T2 are coated with mouse anti-influenza A monoclonal antibody；
Colloid gold chromatographic test paper strip preparation step:
Sample pad, above-mentioned colloidal gold pad, above-mentioned NC film and blotting paper after being dried successively is overlapped on bottom plate,
Form colloid gold chromatographic test paper strip；
Preferably, above-mentioned shell is plastic casing；
Preferably, the material of above-mentioned plastic casing is selected from one of HDPE, LDPE, PP, ABS or a variety of；
Preferably, the material of above-mentioned bottom plate is polyvinyl chloride；
Preferably, the mixed immunity gold that adsorption volume is 25~35mL on the glass fibre of every 25cm × 30cm area；
Preferably, the OD of Immuno gold solution A540Greater than 4.0；The OD of Immuno gold solution B540Greater than 4.0；Mixed immunity gold is molten
The OD of liquid540Greater than 8.
Further, in some embodiments of the present invention, before colloidal gold pad preparation step, above-mentioned preparation method
Further include: Immuno gold solution preparation step；
Above-mentioned Immuno gold solution preparation step includes:
Mouse anti-influenza A virus monoclonal antibody or the anti-influenza B virus monoclonal of mouse are added into colloidal gold solution
Antibody and bovine serum albumin solution, centrifugation are precipitated, and above-mentioned precipitating is resuspended with gold mark dilution, obtain mark containing colloidal gold
The Immuno gold solution A of the mouse anti-influenza A virus monoclonal antibody of note or the anti-influenza B virus list of mouse containing colloid gold label
The Immuno gold solution B of clonal antibody；
Preferably, in above-mentioned Immuno gold solution preparation step, the 5 anti-A types of μ g mouse are added in every above-mentioned colloidal gold solution of 1mL
Influenza virus monoclonal antibody or the 5 anti-influenza B virus monoclonal antibodies of μ g mouse；
Preferably, in above-mentioned Immuno gold solution preparation step, the ultraviolet-visible absorption maximum of above-mentioned colloidal gold solution
Peak value is 523~540nm；
Preferably, in above-mentioned Immuno gold solution preparation step, above-mentioned gold marks the Tris-HCL that dilution is 10~30mM
Buffer, pH8.0-8.2 contain: 0.5%~1.5%BSA, 5%~10% sucrose, 1%~5% trehalose and
0.1%~1%TritonX-100.
Further, in some embodiments of the present invention, it in above-mentioned colloid gold chromatographic test paper strip preparation step, does
Dry treatment conditions are as follows: for relative humidity less than 37 DEG C~45 DEG C of 30%, environment temperature, drying time is 6~18 hours；
Preferably, the drying mode of above-mentioned drying process is forced air drying, one of dries, is dried in vacuo or a variety of.
Further, in some embodiments of the present invention, in above-mentioned NC film preparation step, coating buffer used is
The borate buffer solution of 12.5mM~50mM, pH9.5-9.7 contain 1%~5.0% trehalose；
Preferably, in above-mentioned NC film preparation step, sheep anti mouse Anti-TNF-α bulk concentration in C line working solution is 0.5~
It is 0.5~2.0mg/mL, T2 line that 2.0mg/mL, T1 line, which detect the anti-influenza B virus MAb concentration of mouse in working solution,
Detecting the mouse anti-influenza A MAb concentration in working solution is 0.5~2.0mg/mL.
It should be noted that unless otherwise indicated, percentage involved in the present invention is mass percent.
The invention has the following advantages:
When being detected using combined detection kit provided by the invention, when there is influenza A virus antigen in sample,
On NC film, Flu-A detection line T2 and nature controlling line location of C show a brownish red band respectively；When there is influenza B in sample
When viral antigen, a brownish red band is shown respectively in NC film influenza B detection line T1 and nature controlling line location of C；When in sample
When having two kinds of antigen of A type and influenza B virus, in NC film influenza B detection line T1, Flu-A detection line T2 and Quality Control
Line location of C shows a brownish red band respectively.When in sample without A type and when influenza B virus antigen, it is only aobvious on NC film
Show one brownish red band of nature controlling line location of C；Combined detection kit provided by the invention detection can be simultaneously to depositing in sample
Influenza A virus and influenza B virus detection, have the characteristics that detect speed fastly, high sensitivity.
The preparation method of combined detection kit detection provided by the invention, preparation method detection card are pressed from both sides using double antibody
The preparation of heart colloidal gold method, and detection card is packaged into using shell, detection card three judgement lines of setting are respectively nature controlling line C, B-mode
Influenza test line T1, Flu-A detection line T2, can be simultaneously to influenza A virus present in sample and influenza B disease
The joint-detection of poison detection, obtained combined detection kit have the spies such as detection speed is fast, high sensitivity, performance are stablized
Point.
In addition, the Optimization of preparation of combined detection kit provided by the invention detection antibody is coated with condition, Jin Biao
The mode of working solution condition, drying process condition etc., improves the storage stability of kit.It is protected from light condition at 4~30 DEG C
Lower preservation, validity period are 18 months；Effective life, is limited to 1 hour behind detection card Kaifeng；And kit is in -20 DEG C~45 DEG C rings
It can directly be transported at a temperature of border, the time was no more than 6 days.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the structural schematic diagram of the colloid gold chromatographic test paper strip in the embodiment of the present invention；
Fig. 2 is the bottom substance schematic diagram of the detection card in the embodiment of the present invention；
Fig. 3 is the top structure schematic diagram of the detection card in the embodiment of the present invention；
Fig. 4 is the judging result ideograph of the detection card provided in the embodiment of the present invention.
Icon: 100- detection card；101- colloid gold chromatographic test paper strip；102- polyvinyl chloride bottom plate；103- sample pad；104-
Colloidal gold pad；105-NC film；106- blotting paper；107- nature controlling line C；108- influenza B detection line T1；The inspection of 109- Flu-A
Survey line T2；120- plastic shell；121- head components；122- observation window；123- well；124- base plate part.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The preparation method of A type provided in this embodiment and influenza B virus antigen colloidal gold method combined detection kit
It is as follows.
1 prepares Immuno gold
(a) preparation of the mouse anti-influenza A virus monoclonal antibody of colloid gold label:
1000mL colloidal gold solution will be entered into 0.1M K under stiring2CO3It is adjusted to best pH value, it is anti-that mouse is added after five minutes
(be purchased from: Medix, article No.: 100082) 5mg is slowly stirred to react 60 minutes Flu-A monoclonal antibody；In above-mentioned reaction
20mL10% BSA is added in liquid, the reaction was continued 60 minutes and is slowly stirred；By the immune colloid gold prepared i.e. colloidal gold mark
The mouse anti-influenza A virus monoclonal antibody 9000rpm of note is centrifuged 30 minutes, collects precipitating.It is scanned, is surveyed with spectrophotometer
Peak value is received at its wavelength 540nm, calculates the OD540 value of measured object, the mouse anti-influenza A virus obtained containing colloid gold label
The Immuno gold solution A of monoclonal antibody is saved in 2~8 DEG C, spare.
(b) preparation of the anti-influenza B virus monoclonal antibody of the mouse of colloid gold label:
1000mL colloidal gold solution will be entered into 0.1M K under stiring2CO3It is adjusted to best pH value, it is anti-that mouse is added after five minutes
(be purchased from: Medix, article No.: 100472) 5mg is slowly stirred to react 60 minutes influenza B virus monoclonal antibody；Above-mentioned anti-
It answers and 20mL10%BSA is added in liquid, the reaction was continued 60 minutes and slowly stirs；By the immune colloid gold prepared i.e. colloidal gold
The anti-influenza B virus monoclonal antibody 9000rpm of the mouse of label is centrifuged 30 minutes, collects precipitating.It is scanned with spectrophotometer,
Receipts peak value at its wavelength 540nm is surveyed, the OD540 value of measured object, the anti-influenza B disease of the mouse obtained containing colloid gold label are calculated
The Immuno gold solution B of malicious monoclonal antibody is saved in 2~8 DEG C, spare.
The preparation of 2 colloidal gold pads
Prepare gold mark dilution: 30mM Tris-HCL buffer, pH8.1 ± 0.1 are contained: 1.5%BSA, 6% sugarcane
Sugar, 4% trehalose, 0.5%TritonX-100.
Above-mentioned Immuno gold solution A and above-mentioned Immuno gold solution B are marked into diluted with above-mentioned gold respectively, make its OD540
Value respectively reaches 4.0, and the two is mixed, and forms mixed immunity gold solution (OD540 value up to 8).Mixed immunity gold solution is poured on
On all-glass paper, fully absorb it.
The colloidal gold glass fibre for absorbing saturation is put into vacuum oven, relative humidity less than 30% under the conditions of dry 6
Hour or more, colloidal gold pad is made.It is cut into the size of needs as required, participates in subsequent assembling process.
The preparation of 3 sample pads:
Prepare solution: 50mM Tris-HCL buffer, pH8.1 ± 0.1 are contained: 0.5% casein-sodium, 0.5%
Tween-20,0.5%PVP.
Above-mentioned prepared solution is poured on all-glass paper, it is made to fully absorb buffer.The glass of saturation will be absorbed
Glass fiber sample pad is put into 45 DEG C of drying boxes, relative humidity less than 30% under the conditions of it is 12 hours dry or more, sample pad is made.It presses
It is required that being just cut into the size of needs, subsequent assembling process is participated in.
4NC film preparation:
Dilute sheep anti mouse polyclonal antibody (being purchased from: the east of Beijing foam, article No.: EM002), the anti-second of mouse respectively with coating buffer
Type influenza virus monoclonal antibody (be purchased from: Medix, article No.: 100117) and mouse anti-influenza A monoclonal antibody (be purchased from:
Article No.: 100081) Medix, respectively obtains C line working solution, T1 line detection working solution and T2 line detection working solution.
C line working solution, T1 line detection working solution and T2 line are detected into working solution on nitrocellulose filter using film instrument is drawn
Different location carry out scribing line processing, form the NC with nature controlling line C, influenza B detection line T1 and Flu-A detection line T2
Film.It is that 1 μ L/cm carries out NC film coated antibody that film instrument, which will be drawn, to be arranged to package amount；
Wherein, it is anti-that nature controlling line location of C is coated with sheep anti mouse polyclonal antibody, influenza B detection line T1 position is coated with mouse
Influenza B virus monoclonal antibody and the position Flu-A detection line T2 are coated with mouse anti-influenza A monoclonal antibody.
NC film after coated antibody is placed into 45 DEG C of drying boxes, and under the conditions of relative humidity is less than 30%, drying 12 is small
When more than.
In the present embodiment, the sheep anti mouse polyclonal antibody 1.5mg/mL of C line working solution；The anti-second of mouse of T1 line detection working solution
Type influenza virus monoclonal antibody 1.0mg/mL；The mouse anti-influenza A virus monoclonal antibody 1.5mg/ of T2 line detection working solution
mL。
In the present embodiment, coating buffer used is the borate buffer solution of 12.5mM, and pH9.6 ± 0.1 contains 2.0% sea
Algae sugar.
The assembling of 5 colloid gold chromatographic test papers:
Blotting paper is cut into the slice of 20.5mm × 30cm, colloidal gold all-glass paper cuts into the thin of 8mm × 30cm
Item, sample pad all-glass paper cut into the slice of 15mm × 30cm.It is successively glued from top to bottom in polyvinyl chloride bottom plate (PVC board)
Patch water suction paper slip, NC film item, colloid gold bar, sample filler strip, laminate mutually.Wherein the top edge at both ends is absorbed water respectively on NC film
Paper slip and colloidal gold pad are pushed down, and wherein colloid gold bar pressure NC film 1.0mm~2.0mm is advisable.
Structural reference Fig. 1 of assembled colloid gold chromatographic test paper strip 101, for Fig. 1, blotting paper 106, NC film
105, colloidal gold pad 104 and sample pad 103 are pasted on polyvinyl chloride bottom plate 102.It is successively blotting paper from left to right
106, NC film 105, colloidal gold pad 104 and sample pad 103；Wherein, be successively from left to right on NC film 105 nature controlling line C 107,
Influenza B detection line T1 108 and Flu-A detection line T2 109.Wherein the top edge at 105 both ends of NC film is absorbed water respectively
Paper 106 and colloidal gold pad 104 are pushed down, its stabilization is enhanced.
Assembled colloid gold chromatographic test paper strip is cut into the test paper of 3.85 ± 0.05mm width on slitting machine
Item is used for subsequent assembling finished product kit.
The preparation of 6 sample extraction liquid:
0.1M phosphate buffer, pH7.5 ± 0.2 are contained: 0.9% sodium chloride, 0.5% Tween-20.
It is dispensed into the drop bottle of 10mL according to 6mL/ bottles.
The assembling of 7 kits:
Colloid gold chromatographic test paper strip is placed into shell such as plastic shell and is assembled into colloidal-gold detecting-card, with desiccant one
It rises and is sealed in aluminium foil bag.By sample extraction liquid, colloidal-gold detecting-card, other matching component (such as sampling swab or sampling
Dropper etc.) finished product kit is formed by packing specification together.
The structure for detecting card is as shown in Figures 2 and 3.
Fig. 2 show detection card 100 bottom structure, plastic shell 120 by be located at lower part base plate part 124 and top
Head components 121 combine constitute.It internal is formed for by colloid layer gold after base plate part 124 and head components 121 fasten
Analysis test strips are fastened on internal card slot.Head components 121 have observation window 122, and observation window 122 includes a rectangular window
With two semicircle windows, wherein rectangular window size is about 14.82mm × 3.74mm, and the two sides of rectangular window are respectively connected
The semicircle window that one radius is 3.30mm, collectively constitutes the observation window 122 having a size of 21.42mm × 3.74mm.
Head components 121 also have well 123, and sample to be tested is added by well 123 to sample pad 103.
The technical principle of combined detection kit provided by the invention: the reagent belongs to quick diagnosis class in laboratory diagnosis
Reagent is designed using double antibody sandwich method principle.Sample to be examined is added drop-wise in the well of kit, sample first with immune glue
Immune colloid gold mixing on body gold paper, then again toward chromatographing on nitrocellulose filter.If there is influenza A virus in sample
In the presence of influenza A virus antigen is combined with the influenza A virus antibody of colloid gold label first, forms " influenza A virus
Antigen-influenza A virus antibody-colloidal gold compound " when compound is chromatographed to Flu-A detection line T2, can be wrapped in advance
By the influenza A virus antibody capture in Flu-A detection line T2, a red line is formed at Flu-A detection line T2
Item.
If with the presence of influenza B virus in sample, influenza B virus antigen first with the B-mode stream of colloid gold label
Influenza Virus antibody combines, and is formed " influenza B virus antigen-influenza B virus antibody-colloidal gold compound ", composite layer
When analysis to influenza B detection line T1 line, it can be coated on the influenza B antibody capture of influenza B detection line T1 in advance,
A red lines are formed at influenza B detection line T1.If there is no influenza A virus antigen or influenza B disease in sample
Malicious antigen then will not form lines at influenza B detection line T1 and Flu-A detection line T2.
Be coated with sheep anti mouse polyclonal antibody in the nature controlling line C line of kit, no matter in sample to be tested whether containing needing
Antigen is surveyed, the coated sheep anti mouse polyclonal antibody of nature controlling line C is all by the mouse anti-influenza A virus monoclonal with colloid gold label
Antibody and the anti-influenza B monoclonal antibody of mouse combine, and red lines occur in nature controlling line location of C.
The application method of the combined detection kit of the present embodiment is as follows:
(1) sample collection: avoiding tongue, pharyngeal after being wiped with swab and tonsillotome area, takes out swab；
(2) 400 μ L (about 7 drop) sample extraction liquid is vertically added in sample extraction pipe；
(3) in solution, it will be rotated about 10 times against inboard wall of test tube in the swab insertion sample extraction pipe after acquisition, make sample
It is eluted in solution as far as possible；
(4) it takes out and discards swab, cover sample extraction pipe water dropper；
(5) it tears aluminium foil bag and takes out detection card, lay flat.
(6) 2 drops are added dropwise into detection card well treated sample.
(7) result is observed in 15-20 minutes.
Testing result interpretation: (see Fig. 4)
If influenza B detection line T1, Flu-A detection line T2 and nature controlling line C develop the color, for A type (A), B-mode
(B) influenza antigen is positive；
It is positive for B-mode (B) influenza antigen if influenza B detection line T1 and nature controlling line C are displayed in red；
If Flu-A detection line T2 and nature controlling line C are displayed in red, for A type (A) influenza antigen positive；
It is negative for A type (A) and B-mode (B) influenza antigen if only nature controlling line C develops the color；
If nature controlling line C does not develop the color, then it represents that this testing result is invalid；
Note: "+" represents the positive, and "-" represents feminine gender.
The present embodiment is realized in such a way that three capture lines being arranged on test strips NC film are coated with different antibodies respectively
While detecting A type and influenza B virus respectively and the Quality Control function tested.In Fig. 4, matter in three capture lines
Line C is controlled, sheep anti mouse polyclonal antibody is coated with；Second streamline T1 is influenza B detection line T1, is coated with the anti-influenza B of mouse
Viral monoclonal antibodies；Swin flu line T2 line is Flu-A detection line T2, is coated with mouse anti-influenza A virus monoclonal antibody.
It, which puts in order, is followed successively by T2, T1, C from sample-adding nose end to blotting paper end.
In addition, the present embodiment is coated with the side of condition, gold mark working solution condition, drying process condition etc. by optimization antibody
Formula improves the storage stability of kit.It is saved under the conditions of being protected from light for 4~30 DEG C, and validity period is 18 months；Detection card is opened
Effective life of being honored as a queen, is limited to 1 hour；And kit can be transported directly under -20 DEG C~45 DEG C environment temperatures, the time should not surpass
Spend 6 days.
Embodiment 2
Present embodiments provide a kind of A type and influenza B virus antigen colloidal gold method combined detection kit, kit
Consist of the following parts: detection card (being provided by embodiment 1), sample extraction liquid (being provided by embodiment 1)), sampling swab, drop
Pipe/water dropper, specification, and can need to be assembled into different packing specifications, such as 20 person-portions and 50 person-portion packets according to different
Dress specification see the table below 1:
Table 1
Embodiment 3
Minimum detectability evaluation
It is real while realization using the kit that embodiment 1 provides to A type and influenza B virus while qualitative detection
The high sensitivity for having showed detection, to minimum detection limit index such as the following table 2 of A type and the different classes of strain of influenza B virus:
Table 2
Embodiment 4
Cross reactivity evaluation
The pathogen microorganism that following different type various concentration is detected using the kit that embodiment 1 provides, is not found
Cross reaction phenomenon, concrete outcome see the table below 3:
Table 3
Embodiment 5
With contrast agents Conformance Assessment
(lot number: producer: W05950402C inspires confidence in biology in Guangzhou ten thousand to the kit provided using embodiment 1 with contrast agents box
Technical concern Co., Ltd) 1012 throat swab samples are carried out and contrast agents testing result consistency in three medical institutions
It is studied, as a result such as the following table 4:
Table 4
As the result is shown: the positive concordance rate for the kit that embodiment 1 provides is 98.4%；Negative concordance rate is 98.6%；
Whole concordance rate is 98.5%.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of A type and influenza B virus antigen colloidal gold method combined detection kit, which is characterized in that it includes detection
Card, the detection card include shell and are fixed on the enclosure interior for detecting the colloid layer gold of A type and influenza B virus
Analyse test strips；
Wherein, the colloid gold chromatographic test paper strip includes successively overlapped sample pad, colloidal gold pad, the NC film being arranged on bottom plate
And blotting paper；
The nature controlling line location of C of the NC film is coated with sheep anti mouse polyclonal antibody, the influenza B detection line T1 position of the NC film
It sets and is coated with the anti-influenza B virus monoclonal antibody of mouse, the position Flu-A detection line T2 of the NC film is coated with the anti-first of mouse
Type monoclonal antibody against Influenza；
The colloidal gold pad is coated with mouse anti-influenza A virus monoclonal antibody and the mouse of colloid gold label of colloid gold label
Anti- influenza B virus monoclonal antibody.
2. A type according to claim 1 and influenza B virus antigen colloidal gold method combined detection kit, feature
It is, the combined detection kit further includes sample extraction liquid, and the sample extraction liquid is the phosphate buffer of 0.1M,
pH7.3-7.7；
The sample extraction liquid contains the sodium chloride that concentration is 0.5%~1.0% and concentration is 0.1%~1.0%
Tween-20。
3. A type according to claim 1 or 2 and influenza B virus antigen colloidal gold method combined detection kit, special
Sign is that the combined detection kit further includes the one or two with lower component: sampling swab and sampling dropper；
Wherein, the sampling swab length is 15~20cm, diameter is 1.0~2.0mm, material PP, PE or ABS, described to adopt
The end of sample swab is stained with the absorbent cotton that the length for adsorbing pharyngeal sample is 1.0~2.0cm, diameter is 0.3~0.8cm
Or sponge.
4. A type according to claim 1 or 2 and influenza B virus antigen colloidal gold method combined detection kit, special
Sign is, the blotting paper be 3.84~3.86mm with a thickness of 0.5mm~1.0mm, width, length is 1.9~2.1cm；
Preferably, the shell is plastic casing；
Preferably, the material of the plastic casing is selected from one of HDPE, LDPE, PP, ABS or a variety of；
Preferably, the material of the bottom plate is polyvinyl chloride；
Preferably, the material of the colloidal gold pad is glass fibre, and the colloidal gold pad is with a thickness of 0.2mm~1.0mm, width
3.84~3.86mm, length are 0.5~0.8cm；
Preferably, the material of the sample pad is glass fibre, and the sample pad is with a thickness of 0.2mm~1.0mm, width
3.84~3.86mm, length are 1.5~2.0cm；
Preferably, the material of the NC film is nitrocellulose filter, and the NC film is with a thickness of 0.1mm~0.5mm, width
3.84~3.86mm, length are 1.8~2.2cm, and the mean pore size on the NC film is 5 μm~20 μm.
5. A type according to claim 4 and influenza B virus antigen colloidal gold method combined detection kit, feature
It is, the plastic casing includes head components and base plate part, is had on the head components for observing nature controlling line C, second
The observation window of type influenza test line T1 and Flu-A detection line T2；
The observation window includes the semicircle windows of a rectangular window and two, wherein rectangular window having a size of (14.80~
14.85) mm × (3.70~3.75) mm, the two sides of rectangular window are respectively connected the semicircle that a radius is (3.30~3.35) mm
Shape window, rectangular window and two semicircle windows are collectively constituted having a size of (21.40~21.55) mm × (3.70~3.75)
The observation window of mm；
Preferably, the outer profile size of the plastic casing are as follows: (74.50~75.50) mm × (24.50~25.50) mm ×
(3.84~3.86) mm, inside the notch that has for being adapted to width be 3.84~3.86mm, length be 5.8~
The colloid gold chromatographic test paper strip of 6.2cm.
6. the preparation method of a kind of A type and influenza B virus antigen colloidal gold method combined detection kit, which is characterized in that
It includes the following steps:
Assembling steps: the colloid gold chromatographic test paper strip for being used to detect A type and influenza B virus is fixed on enclosure interior with structure
Block at detection,
Wherein, the colloid gold chromatographic test paper strip includes successively overlapped sample pad, colloidal gold pad, the NC film being arranged on bottom plate
And blotting paper；
The nature controlling line location of C of the NC film is coated with sheep anti mouse polyclonal antibody, the influenza B detection line T1 position of the NC film
It sets and is coated with the anti-influenza B virus monoclonal antibody of mouse, the position Flu-A detection line T2 of the NC film is coated with the anti-first of mouse
Type monoclonal antibody against Influenza；
The colloidal gold pad is coated with mouse anti-influenza A virus monoclonal antibody and the mouse of colloid gold label of colloid gold label
Anti- influenza B virus monoclonal antibody.
7. the preparation side of A type as claimed in claim 6 and influenza B virus antigen colloidal gold method combined detection kit
Method, which is characterized in that before the assembling steps, the preparation method further includes following steps:
Colloidal gold pad preparation step:
By the Immuno gold solution A of the mouse anti-influenza A virus monoclonal antibody containing colloid gold label and contain colloid gold label
The anti-influenza B virus monoclonal antibody of mouse the mixed immunity gold solution that is mixed to get of Immuno gold solution B, by mixed immunity
Gold solution is adsorbed on glass fibre, and the mouse anti-influenza A virus monoclonal antibody and colloid for being coated with colloid gold label is made
The colloidal gold pad of the anti-influenza B virus monoclonal antibody of mouse of gold label；
NC film preparation step:
Dilute the anti-influenza B virus monoclonal antibody of sheep anti mouse polyclonal antibody, mouse and mouse anti-influenza A respectively with coating buffer
Monoclonal antibody respectively obtains C line working solution, T1 line detection working solution and T2 line detection working solution；
The different location of C line working solution, T1 line detection working solution and T2 line detection working solution on nitrocellulose filter is carried out
Processing of crossing forms the NC film with nature controlling line C, influenza B detection line T1 and Flu-A detection line T2, wherein nature controlling line C
It is anti-that position is coated with sheep anti mouse polyclonal antibody, influenza B detection line T1 position is coated with the anti-influenza B virus monoclonal of mouse
Body and the position Flu-A detection line T2 are coated with mouse anti-influenza A monoclonal antibody；
Colloid gold chromatographic test paper strip preparation step:
Sample pad, the colloidal gold pad, the NC film and blotting paper after being dried successively is overlapped on bottom plate, is formed
Colloid gold chromatographic test paper strip；
Preferably, the shell is plastic casing；
Preferably, the material of the plastic casing is selected from one of HDPE, LDPE, PP, ABS or a variety of；
Preferably, the material of the bottom plate is polyvinyl chloride；
Preferably, the mixed immunity gold that adsorption volume is 25~35mL on the glass fibre of every 25cm × 30cm area；
Preferably, the OD of Immuno gold solution A540Greater than 4.0；The OD of Immuno gold solution B540Greater than 4.0；Mixed immunity gold solution
OD540Greater than 8.
8. the preparation side of A type as claimed in claim 6 and influenza B virus antigen colloidal gold method combined detection kit
Method, which is characterized in that before colloidal gold pad preparation step, the preparation method further include: Immuno gold solution preparation step；
The Immuno gold solution preparation step includes:
Mouse anti-influenza A virus monoclonal antibody or the anti-influenza B virus monoclonal antibody of mouse are added into colloidal gold solution
And bovine serum albumin solution, centrifugation are precipitated, the precipitating is resuspended with gold mark dilution, is obtained containing colloid gold label
The Immuno gold solution A of mouse anti-influenza A virus monoclonal antibody or the anti-influenza B virus monoclonal of mouse containing colloid gold label
The Immuno gold solution B of antibody；
Preferably, in the Immuno gold solution preparation step, 5 μ g mouse anti-influenza As are added in colloidal gold solution described in every 1mL
Viral monoclonal antibodies or the 5 anti-influenza B virus monoclonal antibodies of μ g mouse；
Preferably, in the Immuno gold solution preparation step, the ultraviolet-visible maximum absorption peak of the colloidal gold solution
For 523~540nm；
Preferably, in the Immuno gold solution preparation step, the Tris-HCL that the gold mark dilution is 10~30mM is buffered
Liquid, pH8.0-8.2 contain: 0.5%~1.5%BSA, 5%~10% sucrose, 1%~5% trehalose and 0.1%~
1%TritonX-100.
9. the preparation side of A type as claimed in claim 7 and influenza B virus antigen colloidal gold method combined detection kit
Method, which is characterized in that in the colloid gold chromatographic test paper strip preparation step, be dried condition are as follows: relative humidity is less than
30%, 37 DEG C~45 DEG C of environment temperature, drying time are 6~18 hours；
Preferably, the drying mode of the drying process is forced air drying, one of dries, is dried in vacuo or a variety of.
10. the preparation side of A type as claimed in claim 7 and influenza B virus antigen colloidal gold method combined detection kit
Method, which is characterized in that in the NC film preparation step, coating buffer used is the borate buffer solution of 12.5mM~50mM,
PH9.5-9.7 contains 1%~5.0% trehalose；
Preferably, in the NC film preparation step, sheep anti mouse Anti-TNF-α bulk concentration in C line working solution is 0.5~
It is 0.5~2.0mg/mL, T2 line that 2.0mg/mL, T1 line, which detect the anti-influenza B virus MAb concentration of mouse in working solution,
Detecting the mouse anti-influenza A MAb concentration in working solution is 0.5~2.0mg/mL.
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