CN104062430A - Kit box used for detecting influenza virus in sample and detection method and application thereof - Google Patents
Kit box used for detecting influenza virus in sample and detection method and application thereof Download PDFInfo
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- CN104062430A CN104062430A CN201410308507.3A CN201410308507A CN104062430A CN 104062430 A CN104062430 A CN 104062430A CN 201410308507 A CN201410308507 A CN 201410308507A CN 104062430 A CN104062430 A CN 104062430A
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- monoclonal antibody
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- influenza virus
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
Abstract
The invention provides a kit box used for detecting an influenza virus in a sample. The kit box comprises a buffer solution supply unit and a detection test strip. The detection test strip sequentially comprises a substrate supply area, a sample supply area and a detection area in the longitudinal direction. The substrate supply area comprises a substrate pad (3), a dry enzyme substrate is adsorbed to the substrate pad, and the substrate pad (3) makes contact with a nitrocellulose membrane (1). The sample supply area comprises an enzyme mark pad (2), a rat resistance avian influenza virus nucleoprotein antibody marking monoclonal antibody 1 is adsorbed to the enzyme mark pad (2), and the enzyme substrate can product a chromogenic reaction with enzymes marked on the marking monoclonal antibody 1. The detection area is provided with a rat resistance avian influenza virus nucleoprotein antibody fixed monoclonal antibody 2 in an immobilization mode. The invention further provides a method for detecting the influenza virus in the sample through the kit box. The kit box and the detection method of the kit box have the advantages of being rapid and accurate in detection, high in specificity, sensitive and good in stability.
Description
Technical field
The invention belongs to biological technical field.
Background technology
A type influenza virus comprises avian influenza virus (Avian Influenza Vitus, AIV), swine influenza virus (Swine Influenza Vitus, SIV), equine influenza virus (Equine InfluenzaVitus, EIV) and canine influenza virus (Canine Influenza Vitus, CIV) etc., (the Office of International Education of OIE, OIE) classify the caused disease of A type influenza virus as category-A animal epidemic, China is classified as a class animal epidemic.
Wherein, avian influenza virus can cause bird flu, and bird flu is a kind of hyperinfection disease, according to it, is pathogenicly divided into highly pathogenic bird flu and low pathogenicity bird flu, and highly pathogenic bird flu has been brought crushing blow to aviculture especially; Swine influenza virus can cause swine flu, even causes pig dead, and it is healthy that swine flu not only endangers pig body, affects the Time To Market of growing and fattening pigs, also increased feeding cost, caused the loss can not be ignored to pig industry; Equine influenza virus can cause equine influenza, and horse breathing, pulse frequency are increased, and appetite reduces, and spirit is tired, and conjunctival congestion oedema, sheds tears in a large number, at fb dur, often occurs muscular tremor, has also increased and has raised and train cost; Canine influenza virus causes dog influenza, shows as persistent cough, with yellow nasal discharge, there will be the symptom of the similar pneumonia such as high fever, respiratory rate quickening when serious, also public health has been caused to tremendous influence.
In the preventing and controlling of A type influenza virus, diagnosis, monitoring are vital links.At present, A type influenza virus detection method mainly comprises nucleic acid detection method and collaurum method for quick.Though nucleic acid detection method specificity is good, highly sensitive, but detecting, it needs expensive PCR instrument and technical professional, and can only carry out in the laboratory that possesses detection of nucleic acids ability, and most basic unit clinical condition does not possess the ability of detection of nucleic acids, the sample gathering need to be delivered to the laboratory that possesses detectability and detect, lack ageingly, be unfavorable for the control of epidemic situation, also limited its application aspect extensive examination.The advantages such as collaurum quick detection reagent is quick, accurate, easy with it, naked eyes interpretation, the easy preservation of experimental result are widely used in the fast detecting and field diagnostic of A type influenza virus, but its sensitivity is lower.Therefore, quick, accurate, highly sensitive, easy A type influenza test method is the emphasis of current research.
Summary of the invention
For realize A type influenza virus quick, easy, detect and detect the problem that medium sensitivity is lower exactly, the invention provides the enzyme chromatography detection kit of two kinds of anti-influenza type A virus nucleoprotein monoclonal antibodies and preparation thereof, this kit not only has higher sensitivity than conventional colloidal gold fast detecting test paper strip, and detect quick, easy and simple to handlely, be applicable to the fast detecting of A type influenza virus.
Fundamental purpose of the present invention is to provide a kind of kit for detection of A type influenza virus in sample, and wherein, described kit comprises damping fluid feeding unit and test strip; Described damping fluid feeding unit is for supplying with described test strip by damping fluid; Described test strip comprises nitrocellulose filter (1), and described test strip comprises substrate supply area, sample supply area, detection zone in the vertical successively; Described substrate supply area comprises substrate pad (3), on it, absorption has dry zymolyte, described substrate pad (3) contacts with nitrocellulose filter (1), and described zymolyte is dissolved in damping fluid and in the upper distal migration to the described damping fluid feeding unit of distance of nitrocellulose filter (1); Described sample supply area comprises enzyme mark pad (2), on it, absorption has mouse anti-influenza type A virus nucleoprotein antibody labeling monoclonal antibody 1, described zymolyte can produce chromogenic reaction with the enzyme of mark on described labeled monoclonal antibody 1, described enzyme mark pad (2) contacts with nitrocellulose filter (1), and described labeled monoclonal antibody 1 is dissolved in damping fluid and in the upper distal migration to the described damping fluid feeding unit of distance of nitrocellulose filter (1); And the immobilization of described detection zone has mouse anti-influenza type A virus nucleoprotein antibody immobilized monoclonal antibody 2.
Preferably, described damping fluid feeding unit comprises expansion fluid cushion (5), substrate buffer solution groove (8), substrate buffer solution (9) and damping fluid button, described substrate buffer solution (9) is placed in substrate buffer solution groove (8), described damping fluid button is positioned at the top of substrate buffer solution groove (8), presses and described expansion fluid cushion (5) can be immersed in damping fluid (9); Described detection zone comprises detection line (6), nature controlling line (7), wherein, described nature controlling line (7) compared with detection line (6) further from described sample supply area, in described detection line (6) immobilization, there is mouse anti-influenza type A virus nucleoprotein antibody immobilized monoclonal antibody 2, in described nature controlling line (7) immobilization, have sheep anti mouse how anti-, and to be adsorbed on labeled monoclonal antibody 1 on enzyme mark pad (2) be excessive for being fixed on the immobilized monoclonal antibody 2 of detection line (6); The full section of described nitrocellulose filter (1) sticks on above support (10), stilt (10) connects described damping fluid feeding unit, substrate supply area, sample supply area, detection zone and adsorptive pads (4), described adsorptive pads (4) is in the distal-most end apart from described damping fluid feeding unit; And the position that detection sample (11) adds is the position of described enzyme mark pad (2).
As one embodiment of the present invention, described enzyme chromatography test strip comprises the nitrocellulose filter 1 of solid phase, the enzyme mark pad 2 that contains labelled reagent, substrate pad 3, adsorptive pads 4, launches fluid cushion 5, detection line 6, nature controlling line 7, substrate buffer solution groove 8, substrate buffer solution 9, stilt 10, wherein 2 belong to sample supply area, 3 belong to substrate supply area, 5,8 belong to damping fluid feeding unit, and 6,7 belong to detection zone.The position that detection sample 11 adds is the position of enzyme mark pad 2.Enzyme chromatography test strip is fixed in plastic casing, on it, from left to right fixes successively and launches fluid cushion 5, substrate pad 3, enzyme mark pad 2, adsorptive pads 4.Nitrocellulose filter 1 sticks to complete section of stilt 10; Adsorptive pads 4 is stuck in the top of nitrocellulose filter 1, and has overlapping with nitrocellulose filter 1; Enzyme mark pad 2 is positioned at the stage casing of nitrocellulose filter 1, above the dry mouse anti-influenza type A virus nucleoprotein antibody 1 that has enzyme labeling; Substrate pad 3 is stuck in the bottom of nitrocellulose filter 1, has dry zymolyte on it.The upper end of launching fluid cushion 5 has covered substrate pad 3, and its lower end is positioned at the bottom of substrate buffer solution groove 8.Above substrate buffer solution groove 8, covered one deck aluminium-foil paper, to prevent substrate buffer solution 9 seepages, on it, had damping fluid button, pressed damping fluid button and can puncture aluminium-foil paper, and the lower end of launching fluid cushion 5 has been immersed in substrate buffer solution 9.Detection line 6 is arranged in the upper end of nitrocellulose filter 1, and on it, immobilization has mouse anti-influenza type A virus nucleoprotein antibody 2.Nature controlling line 7 is arranged in the upper end of nitrocellulose filter 1, the upstream of detection line 6, and on it, immobilization has sheep anti mouse how anti-.
Preferably, the amino acid sequence of described labeled monoclonal antibody 1 its variable region of heavy chain is that the amino acid sequence of SEQID No.2, variable region of light chain is SEQ ID No.3; And the amino acid sequence of described immobilized monoclonal antibody 2 its variable region of heavy chain is that the amino acid sequence of SEQ ID No.4, variable region of light chain is SEQ ID No.5.
Term used herein " monoclonal antibody " refers to that the antibody individuality that forms this colony is all identical available from the antibody of the antibody population of homology substantially, except there is a small amount of possible spontaneous mutation.Therefore, modifier " monoclonal " refers to that the character of this antibody is not the potpourri of discrete antibody.Preferably, described monoclonal antibody comprises derivant, function equivalent and homologue unit price or single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody and above-mentioned antibody, also comprises antibody fragment and any polypeptide that contains antigen binding structural domain.
" antibody " of the present invention should be interpreted as containing any specific binding factor with required specific binding structural domain.Thereby function equivalent and the homologue of the antibody fragment of homology, derivant, humanized antibody and antibody with it contained in this term, also comprises any polypeptide that contains antigen binding structural domain, no matter be natural or synthetic generation.The example of antibody is immunoglobulin (Ig) hypotype (as IgG, IgE, IgM, IgD and IgA) and hypotype subclass thereof; Also can be comprise antigen binding structural domain fragment as Fab, scFv, Fv, dAb, Fd; With two strands anti-(diabodies).Fusion to chimera molecule or equivalent another polypeptide, that comprise antigen binding structural domain is also included within wherein.The cloning and expression of chimeric antibody is described in EP.A.0120694 and EP.A.0125023.
" antibody " of the present invention can be modified by many modes, can produce and retain specific other antibody of original antibody or chimeric molecule with DNA recombinant technique.This technology can comprise that constant region or constant region that the DNA of the immune globulin variable region of encoding antibody or complementarity-determining region (CDRs) is introduced to different immunoglobulin (Ig)s add framework region.Referring to, EP.A.184187, GB2188638A or EP.A.239400.Can also carry out genetic mutation or other change to other cell of hybridoma or generation antibody, this can change or not change the binding specificity of produced antibody.
For " monoclonal antibody " of the present invention also available hybridoma method make, because the DNA sequence dna of code book invention humanized antibody can be used conventional means well known to those skilled in the art, as manually synthesized according to amino acid sequence disclosed by the invention or obtaining with the amplification of PCR method, thereby also available recombinant DNA method, available the whole bag of tricks well known in the art is connected into this sequence in suitable expression vector.Finally, be applicable to, under the condition of antibody expression of the present invention, cultivating the host cell that transforms gained, the conventional separation and purification means purifying that then those skilled in the art's application is known obtains monoclonal antibody of the present invention.
" variable region " of term used herein heavy chain of antibody or light chain is that the N of this chain holds ripe region.All Ranges, CDRs and residue numbering all take sequence alignment, by existing structure knowledge, as basis, define.The evaluation of framework region and CDRs residue and numbering are pressed Chothia and (Chothia described in other people, Structural determinants in the sequences of immunoglob μ linvariable domain.J.Mol.Biol., 1998:278,457).
Those of ordinary skills obviously know, the present invention on the heavy chain and light chain variable region amino acid sequence basis of concrete disclosed monoclonal antibody, can carry out by conventional genetic engineering and protein engineering method the modifications such as one or more amino acid whose interpolations, deletion, replacement, obtain conservative property variant or its fragment, and still can keep and A type influenza virus specific binding.
Term used herein " nucleoprotein " (Nuclear Protein, NP) is a kind of polypeptide of monomer phosphorylation, and the about 60kD of molecular weight is the major protein composition that forms virus nucleocapsid; This albumen has specificity, according to its antigenic difference, influenza virus is divided into A, B, C three types (Liu Hongqi etc., A type influenza virus protein progress, animal medicine progress, 2002,23 (2): 6-9).Described nucleoprotein is also its conservative property variant or active fragment, described " its conservative property variant or active fragment " compared with A type influenza virus ORF5 coding nucleoprotein amino acid sequence SEQ ID NO.1 of the present invention, can exist one or more conservative amino acid to replace, add or delete but still can keep and A type influenza virus specific binding.
Preferably, the enzyme of described mark comprises any of horseradish peroxidase, alkaline phosphatase and beta-D-galactosidase.
Preferably, the amino acid sequence that in described solid phase, anti-influenza type A virus nucleoprotein monoclonal antibody is variable region of heavy chain is that the amino acid sequence of SEQ ID No.4, variable region of light chain is the monoclonal antibody of SEQ IDNo.5, and the amino acid sequence that in described labelled reagent, anti-influenza type A virus nucleoprotein monoclonal antibody is variable region of heavy chain is that the amino acid sequence of SEQ ID No.2, variable region of light chain is the monoclonal antibody of SEQ ID No.3.Described labelled reagent can obtain by enzyme labeling; Preferably, described enzyme comprises any in horseradish peroxidase (HRP), alkaline phosphatase (ALP) and beta-D-galactosidase.
Preferably, described substrate buffer solution is aqueous solvent, comprises one or more of phosphate buffer, surfactant, antiseptic and carbohydrate.
Preferably, described kit also comprises sample processing tube, described sample processing tube contains sample preparation liquid, and described sample preparation liquid is surfactant solution, and described surfactant comprises any of stearic acid, neopelex, lecithin, fatty glyceride and CHAPS.
The material that term used herein " surfactant " can make target solution surface tension significantly decline, can reduce the surface tension between two kinds of liquid or liquid-solid.
Preferably, described kit also comprises Sample storage bottle, and described Sample storage bottle contains Sample storage liquid, and described Sample storage liquid is phosphate buffer, is preferably the phosphate buffer of pH7.4.
Term used herein " phosphate buffer " refers to the solution that contains phosphoric acid or its salt and be adjusted to ideal pH value, is the most widely used a kind of damping fluid in biochemical research.Usually, phosphate buffer is prepared from phosphoric acid or phosphate (including but not limited to sodium and sylvite).Some phosphate have been known in this area, for example sodium dihydrogen phosphate and potassium dihydrogen phosphate, sodium hydrogen phosphate and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate.Known that phosphate is that hydrate forms with salt exists.Due to the secondary dissociation of damping fluid, the pH value scope of buffering is very wide, and for example about pH4 is to the scope of about pH10, and the scope of preferred about pH5 to pH9, more has and select about pH6 to the scope of about pH8, most preferably from about pH7.4.Further preferably, the phosphate buffer that described phosphate buffer is sodium chloride-containing and potassium chloride.
Another object of the present invention is to provide a kind of method of using described kit to detect influenza virus in sample, described method comprises: (1) is used described sample processing tube pretreatment sample; (2) pretreatment sample in described step (1) is added to the position of described enzyme mark pad 2; (3) press damping fluid button, observations; And (4) if nature controlling line place there is no band, no matter whether detection line place has band, and testing process is all invalid, the in the situation that of having band at nature controlling line place, if there is band at detection line place, positive, otherwise negative.
As one embodiment of the present invention, the detection method of A type influenza virus enzyme chromatography of the present invention, for: (1) is inserted into the microorganism swab having gathered in sample processing tube, and sample is dissolved in solution as far as possible, finally make microorganism swab head fracture to stay with bottle in; (2) the sample processing tube lid head that contains sample is fractureed, extrude a sample and add well, press rapidly damping fluid button simultaneously; After (3) 30 minutes in the detection zone of described test strip observed and recorded result.
A further object of the present invention is to provide the application in influenza virus in detecting sample of described kit.
The present invention also provides a kind of anti-influenza type A virus nucleoprotein monoclonal antibody 1, and the amino acid sequence of described monoclonal antibody 1 its variable region of heavy chain is that the amino acid sequence of SEQ ID No.2, variable region of light chain is SEQ ID No.3.
The present invention also provides a kind of anti-influenza type A virus nucleoprotein monoclonal antibody 2, and the amino acid sequence of described monoclonal antibody 2 its variable region of heavy chain is that the amino acid sequence of SEQ ID No.4, variable region of light chain is SEQ ID No.5.
Based on this, outstanding advantages of the present invention is:
(1) enzyme chromatography test strip has completed ELISA testing process on nitrocellulose filter, realized enzyme linked immunological cascade enlarge-effect, therefore its sensitivity can reach ELISA level, higher more than 10 times than colloidal gold strip, suitable with PCR detection sensitivity in the detection of some cause of disease;
(2) have the advantages that result is simple, easy to use, detection efficiency is high.
Accompanying drawing explanation
Fig. 1 is the side schematic view of enzyme chromatography test strip, in figure: 1-nitrocellulose filter, 2-enzyme mark pad, 3-substrate pad, 4-adsorptive pads, 5-launches fluid cushion, 6-detection line, 7-nature controlling line, 8-substrate buffer solution groove, 9-substrate buffer solution, 10-stilt, 11-detects sample.
Fig. 2 is the purity that 2 strain anti-influenza type A virus nucleoprotein monoclonal antibodies are identified in SDS-PAGE gel electrophoresis, wherein swimming lane 1 is monoclonal antibody 2, comprises the heavy chain of upper end and the light chain of lower end, and swimming lane 2 is monoclonal antibody 1, comprise the heavy chain of upper end and the light chain of lower end, swimming lane 3 is albumen Maker.
in sequence table:
Sequence 1 is the amino acid sequence of A type influenza virus nucleoprotein;
The weight chain variable region amino acid sequence of the anti-influenza type A virus nucleoprotein monoclonal antibody 1 that sequence 2 is mark;
The light chain variable region amino acid sequence of the anti-influenza type A virus nucleoprotein monoclonal antibody 1 that sequence 3 is mark;
Sequence 4 is the weight chain variable region amino acid sequence of fixing anti-influenza type A virus nucleoprotein monoclonal antibody 2;
Sequence 5 is the light chain variable region amino acid sequence of fixing anti-influenza type A virus nucleoprotein monoclonal antibody 2.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with describing.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
Carbonate buffer solution used in the embodiment of the present invention, pH value is 9.6, its 1L volume formula is: Na
2cO
31.59g, NaHCO
32.93g, but no matter this embodiment does not under any circumstance all form limitation of the invention.
In example of the present invention, Sample storage liquid used is phosphate buffer, and pH value is 7.4, and its 1L volume formula is: NaC18.0g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.24g, the kanamycins of the penicillin that final concentration is 10000IU/mL, the streptomysin of 8000IU/mL and 5000IU/mL, but no matter this embodiment does not under any circumstance all form limitation of the invention.
Enzyme labelled antibody in the embodiment of the present invention be take horseradish peroxidase (HRP) and is carried out mark as example, but no matter this embodiment does not under any circumstance all form limitation of the invention.
In the embodiment of the present invention, A type avian flu strain A/Ck/HK/Yu22/02 strain is open in patented claim CN1814623A.
In the embodiment of the present invention, A type avian flu strain A/California/04/2009 strain is open in patented claim CN101974489A.
In the embodiment of the present invention, Type B strains of influenza viruses VR788 strain is open in patented claim CN102337351A.
It is pure that in the present invention, chemical reagent used is analysis, purchased from traditional Chinese medicines group.
In this example, the criterion of A type influenza virus enzyme chromatography used is: having or not of nature controlling line determined that whether testing process is effective, if nature controlling line place there is no band, no matter whether detection line place has band, and testing process is all invalid; In the situation that there is band at nature controlling line place, if there is band at detection line place, positive, otherwise negative.
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention.Experimental technique of the present invention, if without specified otherwise, is conventional method; Described biomaterial, if without specified otherwise, all can obtain from commercial channels.
Preparation, purifying, evaluation and the check of embodiment 1 anti-influenza type A virus nucleoprotein monoclonal antibody
Preparation and the purifying of 1.1 anti-influenza type A virus nucleoprotein monoclonal antibodies
Preparation and the purifying of anti-influenza type A virus nucleoprotein monoclonal antibody, comprise the steps:
1.1.1 animal immune: the inactivation of viruses 1mL of A type influenza virus A/California/04/2009 (H1N1) strain is added to the complete Freund's adjuvant of equivalent and through fully emulsified, the 6-8 BALB/c healthy mice in age in week of myeloma cell's homology immune and used, A type influenza virus A/California/04/2009 (H1N1) strain 500 μ L after every multi-point injection emulsification, interval is strengthened once for 2 weeks; With indirect ELISA, measure its antiserum:
After carrying out, adopt indirect ELISA to measure antiserum, the method is comprised of following operation steps:
A, coated:
Carbonate buffer solution with 20mmol/L, pH9.6 dilutes A type influenza virus nucleoprotein 1:2000, coated 96 hole polyethylene boards, and vacuum is drained, and seals 4 ℃ and saves backup.
B, sealing:
Every hole adds the phosphate buffer 200 μ L of pH7.4 to wash, include 10% (V/V) calf serum;
C, application of sample:
Every hole adds the clear 50 μ L of mouse peripheral blood of the 1:5000 of immune latter 3 days (V/V) dilution for the third time, and every plate is established a normal control, positive control and phosphate buffer blank, washing;
D, add the anti-every hole 100 μ L of enzyme mark sheep anti mouse two, washing;
E, colour developing:
Every hole adds substrate 100 μ L;
F, colorimetric:
With blank zeroing, 450nm wavelength is measured optical density (OD);
G, result judgement: P/N=measure sample OD average/negative serum OD average, and P/N >=2.1 are positive.
Polyethylene board specification in step a be 200 μ L/ holes, vacuum to drain temperature be 4 ℃, washing adopts 0.05% Tween-20 phosphate buffer washing 3 times.
Technological parameter in step b is that every hole adds pH7.4, contains 10% calf serum phosphate buffer 200 μ L, and temperature is 37 ℃, time 2 h, washs 1 time.
Process conditions in step c are that every hole adds the clear 50 μ L of the mouse peripheral blood of immune latter 3 days for the third time after 1:5000 (V/V) dilution, every plate is established a normal control, positive control and phosphate buffer blank, temperature is that 37 ℃, time are 1 hour, washs 3 times.
Process conditions in steps d add the anti-every hole 100 μ L of enzyme mark sheep anti mouse two, and temperature is that 37 ℃, time are 1 hour, washs 3 times.
Process conditions in step e are that room temperature, time are 15 minutes, then use stop buffer cessation reaction, through microplate reader, at wavelength 450nm place, read optical density value.
1.1.2 separating Morr. cell: get non-immune BALB/c healthy mice peritoneal macrophage, paving 96 well culture plates, are adjusted into 9 * 10 by the Sp2/0 cell changing after liquid 15 hours
5/ mL cell suspension, the mouse boosting cell of separating immune is also made cell suspension;
1.1.3 Fusion of Cells: the Sp2/0 myeloma cell in logarithmic phase is mixed in 1: 10 (V/V) ratio with splenocyte suspension, add PEG-1500 that cell is merged each other, in the mixed cell suspension of two kinds of cells, within the 1st minute, drip 4.5mL nutrient solution; Interval 2 minutes drips 5mL nutrient solution, then adds nutrient solution 50mL, and the HAT of take selects nutrient culture media to carry out cell cultivation by 36% hole as 1 cells/well;
1.1.4 screen hybridoma: when the cell merging is cultured to the 7th day, be that cell is cultured to while covering at the bottom of 10% hole, draw and in the hole of 96 well culture plates, occur that the culture supernatant of clone cell bunch detects antibody content with indirect ELISA, through limiting dilution, carry out three subclone screenings, according to the secretion situation of antibody, filter out the cell line of high-titer, high specific, expand cultivation frozen.
1.1.5 monoclonal antibody purification storage: select BALB/c mouse or its parental generation mouse, first with the capable mouse peritoneal injection of whiteruss, after one week by 5 * 10
5hybridoma is inoculated in mouse peritoneal, collects the ascites of mouse in inoculation after one week, and every mouse can be collected the ascites of 10mL, use AKTA protein purification instrument and DE-52 ion exchange column by mouse IgG monoclonal antibody ascites solution at A
280during nm, collect.
Finally obtain two strain of hybridoma system, respectively secrete monoclonal antibody 1 and monoclonal antibodies 2.
According to (Li Huijin etc. such as Li Huijin, nest-type PRC amplification and the sequential analysis of H1N1virus hemagglutinin monoclonal antibody light chain and heavy chain variable region gene, biotechnology communication, 2013,24 (1): 61-64) method of operating of document is determined variable region of heavy chain and the variable region of light chain of monoclonal antibody 1 and monoclonal antibody 2.
Result: the amino acid sequence of the variable region of heavy chain of monoclonal antibody 1 is that the amino acid sequence of SEQ ID No.2, variable region of light chain is SEQ ID No.3; The amino acid sequence of the variable region of heavy chain of monoclonal antibody 2 is that the amino acid sequence of SEQ ID No.4, variable region of light chain is SEQ IDNo.5.
The evaluation of 1.2 anti-influenza type A virus nucleoprotein monoclonal antibodies
1.2.1 the evaluation of monoclonal antibody type and subclass
1.2.1.1 according to (Fang Shisong etc. such as Fang Shisong, the clonal expression of influenza A virus nucleoprotein gene and purifying, China's experiment and clinical virology magazine, 2005,19 (2): 165-168) method of operating of document is prepared A type influenza virus nucleoprotein.
1.2.1.2 with the A type influenza virus nucleoprotein after the restructuring of carbonic acid buffer dilution gene engineering, coated by 100 μ g/ holes, cells and supernatant to monoclonal antibody detects, according to KoenigR. (Koenig R.Indirect ELISA methods for the broad specificity detection ofplant viruses.Journal of General Virology, 1981, 55 (1): 53-62) indirect elisa method in document carries out, the enzyme adding is respectively the HRP-mountain sheep anti mouse IgM (HRP-IgM) of dilution, HRP-mountain sheep anti mouse IgG1 (HRP-IgG1), HRP-mountain sheep anti mouse IgG2a (HRP-IgG2a), HRP-mountain sheep anti mouse IgG2b (HRP-IgG2b) and HRP-mountain sheep anti mouse IgG3 (HRP-Ig G3) enzyme-labeled antibody two are anti-, the results are shown in Table 1.
The evaluation of each monoclonal antibody type of table 1
Note :+representing the positive ,-expression is negative.
As shown in Table 1: the subclass of monoclonal antibody 1 is IgG1, the subclass of monoclonal antibody 2 is IgG2b.
1.2.2 the evaluation of monoclonal antibody specificity
1.2.2.1 with above-mentioned indirect ELISA method, measure respectively the reactivity of monoclonal antibody 1, monoclonal antibody 2 and A type influenza virus nucleoprotein.
1.2.2.2 with above-mentioned indirect ELISA method, measure respectively monoclonal antibody 1, monoclonal antibody 2 and Type B influenza virus nucleoprotein and whether there is cross reactivity.
Measurement result shows: 2 strain monoclonal antibodies and A type influenza virus nucleoprotein have good reactivity, and with the equal no cross reaction of Type B influenza virus nucleoprotein, show that this 2 strain monoclonal antibody is all monoclonal antibody specifics of anti-influenza type A virus nucleoprotein.
The check of 1.3 monoclonal antibody purifications
1.3.1 the outward appearance of monoclonal antibody purification
Under light-illuminating, the visible antibody purification of visual inspection is achromaticity and clarification state, has no floccus precipitation.
1.3.2 sterility test
Adopt Sterility Test, get each 2 pipe of nutrient agar slant medium, improvement Martin's nutrient culture media and sulphur glycollate culture medium (inoculum concentration is 0.5mL/ pipe) that monoclonal antibody raw material after purifying is seeded to 10mL/ pipe.Each pipe of postvaccinal sulphur glycollate culture medium and nutrient agar slant medium is placed in 30-35 ℃ of cultivation, and another pipe is in 20-25 ℃ of cultivation.Improvement Martin nutrient culture media is put 20-25 ℃ of cultivation.With 0.9% aseptic NaCl solution, with method operation, do negative control simultaneously.Cultivate and all do not observe in nutrient culture media and have growth of microorganism after 14 days, show that monoclonal antibody raw material meets aseptic requirement.
1.3.3 monoclonal antibody purity
Use 12%SDS-PAGE protein electrophoresis, the applied sample amount of every swimming lane is 10 μ g, after electrophoresis, with coomassie brilliant blue staining, through the scanning of gel imaging instrument, and records concentration by software.As shown in Figure 2, wherein swimming lane 1 is monoclonal antibody 2 to testing result, comprises the heavy chain that is greater than 44.3kD and the light chain that is greater than 20.1kD; Swimming lane 2 is monoclonal antibody 1, comprises the heavy chain that is greater than 44.3kD and the light chain that is greater than 20.1kD; Swimming lane 3 is albumen Maker.
Result shows: the purity of 2 strain monoclonal antibodies is all not less than 90%.
The pairing of embodiment 2 monoclonal antibodies
The selection of 2.1 monoclonal antibody pairing modes
During screening monoclonal antibody combinations of pairs, mainly consider following factor: the one, the activity of monoclonal antibody; The 2nd, whether monoclonal antibody and non-A type influenza virus there is nonspecific reaction; The 3rd, colour developing background.
2.2 monoclonal antibodies are active to be detected
Select A type avian flu strain A/Ck/HK/Yu22/02, be diluted to 0.01HA, for different collocation modes, detect, the results are shown in Table 2:
The collocation of table 2 monoclonal antibody and active detection
Note :+representing the positive ,-expression is negative.
Result shows: Yu22 viral dilution liquid fixing except monoclonal antibody 1 and monoclonal antibody 2 enzyme marks detection 0.01HA is negative, other collocation is positive findings, but monoclonal antibody 1, monoclonal antibody 2 itself fixing be marked in application certainly infeasible.
The non-specific detection of 2.3 monoclonal antibody
Select Type B strains of influenza viruses VR788 to cultivate, virus titer is 256HA, for different collocation modes, detects, and the results are shown in Table 3:
The collocation of table 3 monoclonal antibody and non-specific detection
Note :+representing the positive ,-expression is negative.
Result shows: monoclonal antibody 1 is fixing fixes and monoclonal antibody 2 marks with monoclonal antibody 1 mark, monoclonal antibody 2, and nonspecific reaction all appears in these two kinds of collocation.
According to above experimental result: monoclonal antibody 1 antibody that serves as a mark, select monoclonal antibody 2 as sessile antibody.
Structure and the use of embodiment 3 A type influenza virus enzyme chromatography detection kit
Fixing of 3.1 monoclonal antibodies
With the three-dimensional specking platform of BioDot XYZ3050, the monoclonal antibody 2 of embodiment 1 preparation is fixed on nitrocellulose filter.
The horseradish peroxidase of 3.2 monoclonal antibodies (HRP) mark
According to the monoclonal antibody 1 of embodiment 1 preparation, according to (Tijssen P such as Tijssen P, Kurstak E.Highly efficient and simple methods for the preparation ofperoxidase and active peroxidase antibody conjugates for enzymeimmunoassays (J) .Anal Biochem, 1984, document 136:451-457) carries out horseradish peroxidase (HRP) mark to anti-influenza type A virus monoclonal antibody.
The composition of 3.3 detection kit
Detection kit includes enzyme chromatography test strip, sample preparation liquid, sample processing tube, Sample storage liquid.Wherein,
(1) enzyme chromatography test strip, as shown in Figure 1.
(2) sample preparation liquid, in sample preparation bottle, is the phosphate buffer of the pH7.4 containing 0.5%-3.5%CHAPS.
(3) sample processing tube.
(4) Sample storage liquid, in Sample storage bottle, is the phosphate buffer of pH7.4.
Described enzyme chromatography test strip comprises sample supply area, and sample supply area comprises enzyme mark pad 2, and on it, absorption has the labeled monoclonal antibody 1 of embodiment 3.2 preparations; Substrate supply area, substrate supply area comprises substrate pad 3; Damping fluid feeding unit, damping fluid feeding unit comprises expansion fluid cushion 5, substrate buffer solution groove 8, substrate buffer solution 9; Detection zone, detection zone comprises detection line 6, nature controlling line 7, has the immobilized monoclonal antibody 2 of embodiment 1 preparation in detection line 6 immobilizations, in nature controlling line 7 immobilizations, has sheep anti mouse how anti-.Nitrocellulose filter 1 sticks to complete section of above support 10; And the position that detection sample 11 adds is the position of described enzyme mark pad 2.
Enzyme chromatography test strip is fixed in plastic casing, on it, from left to right fixes successively and launches fluid cushion 5, substrate pad 3, enzyme mark pad 2, adsorptive pads 4.Adsorptive pads 4 is stuck in the top of nitrocellulose filter 1; Enzyme mark pad 2 is positioned at the stage casing of nitrocellulose filter 1; Substrate pad 3 is stuck in the bottom of nitrocellulose filter 1, has dry zymolyte on it.The upper end of launching fluid cushion 5 has covered substrate pad 3, and its lower end is positioned at the bottom of substrate buffer solution groove 8.Above substrate buffer solution groove 8, covered one deck aluminium-foil paper, to prevent substrate buffer solution 9 seepages, top is damping fluid button (not marking in Fig. 1).Detection line 6 is arranged in the upper end of nitrocellulose filter 1, and nature controlling line 7 is arranged in the upper end of nitrocellulose filter 1, and is positioned at the upstream of detection line 6.
When using detection kit, first use sample processing tube pretreatment sample; Then pretreatment sample is added to the position of enzyme mark pad 2, press damping fluid button simultaneously, damping fluid button control damping fluid 9 is by launching fluid cushion 5 to the chromatography process of adsorptive pads 4.If in pretreatment sample, contain A type influenza virus its will with 1 combination of enzyme labeling monoclonal antibody, A type influenza virus and enzyme labeling monoclonal antibody 1 combination and excessive enzyme labeling monoclonal antibody 1 under capillarity to adsorptive pads 4 directions migrations.When the A type influenza virus moving and enzyme labeling monoclonal antibody 1 combination arrival detection line 6 position, the immobilized monoclonal antibody 2 that is fixed in detection line 6 positions is caught, when the substrate coming from the migration of substrate supply area arrives detection line 6, produce chromogenic reaction with enzyme labeling monoclonal antibody 1.Meanwhile, excessive enzyme labeling monoclonal antibody 1 continues to move to nature controlling line 7, is fixed on how anti-the catching of sheep anti mouse of nature controlling line 7 positions, and produces chromogenic reaction with the substrate coming from the migration of substrate supply area.
Examination criteria: whole reaction is carried out approximately 30 minutes, after reaction finishes, if nature controlling line place there is no band, no matter whether detection line place has band, and testing process is all invalid, in the situation that there is band at nature controlling line place, if there is band at detection line place, positive, in interpret sample, there is A type influenza virus, otherwise negative, in interpret sample, there is not A type influenza virus.
Embodiment 4 sample pre-treatments
The collection of 4.1 samples
The main sample gathering comes from pharynx nasalis, pars oralis pharyngis, cloaca portion, and Details as Follows:
While using microorganism swab to gather pharynx nasalis sample, rotate gently and promote microorganism swab, making microorganism swab head deeply be positioned at the pharynx nasalis of nasal cavity root, gently turning several circles to obtain the Nasopharyngeal swabs sample that viral abundance is higher;
While using the pharyngeal sample of microorganism swab acquisition port, appropriateness is firmly wiped away pharynx rear wall and both sides, should avoid touching tongue;
While using microorganism swab to gather cloaca portion sample, microorganism swab is partly stretched into cloaca 1.5-2cm place, adherent rotation was taken out after 3 weeks.
The processing of 4.2 samples
The microorganism swab having gathered is inserted in sample processing tube, near tube wall rotation, repeatedly makes sample be dissolved in as far as possible in solution, finally make microorganism swab head fracture to stay with bottle in.
The application of embodiment 5 A type influenza virus enzyme chromatography detection kit
The detection method of A type influenza endonuclease chromatography is:
(1) the sample processing tube lid head that contains sample is fractureed, extrude a sample and add well, press rapidly damping fluid button simultaneously;
After (2) 30 minutes in view window observed and recorded result.
5.1 sensitivity and susceptibility
With the kit of three continuous lot numbers, the different dilutabilitys of 70 parts of test samples are detected, statistics detects lower limit, in order to evaluate sensitivity and the susceptibility of the method.A type avian influenza virus (H5 hypotype: 10 to known viruse content simultaneously
8.5eID
50/ 100 μ L, H9 hypotype: 10
9.5eID
50/ 100 μ L) carry out a series of dilutions, dilutability is followed successively by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, then detect, determine its lowest detection lower limit.What the kit of three continuous lot numbers detected 70 parts of different dilutability samples the results are shown in Table 4.
The testing result of the A type influenza virus of table 4 pair different subtype
Note :-representing feminine gender, ND represents not detect.
Result shows: kit detects down and is limited to 0.01-0.1HA, confirms that this kit has higher sensitivity; To 70 duplicate samples, all can detect, confirm that this kit susceptibility is good.
A type avian influenza virus (H5 hypotype: 10 to known viruse content
8.5eID
50/ 100 μ L, H9 hypotype: 10
9.5eID
50/ 100 μ L) carry out a series of dilutions, dilutability is followed successively by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, then detecting, assay is in Table 5.
The detection lower limit of table 5 A type influenza virus enzyme chromatography detection kit
Note :+representing the positive ,-expression is negative, the-/weak positive of+expression.
As shown in Table 5: the kit of three continuous lot numbers is virus stock solution used dilution 10 to the lowest detection lower limit of H5, H9 hypotype A type avian influenza virus
4times, the lowest detection lower limit that obtains this kit through converting is respectively 10
4.5eID
50/ 100 μ L (H5 hypotype) and 10
5.5eID
50/ 100 μ L (H9 hypotype).
The pharynx of SPF chicken, cloaca swab sample to H5 subtype influenza virus-positive; Pharynx, the cloaca swab sample of the pharynx of SPF chicken, cloaca swab sample and the clinical onset chicken of inoculation H9 subtype influenza virus; The nasal cavity swab sample of the pig of clinical infection H1, H3 hypotype swine influenza virus; The Nasopharyngeal swabs sample of the dog of clinical infection H3 hypotype canine influenza virus detects, the susceptibility of kits for evaluation.Testing result is in Table 6.
Table 6 A type influenza virus enzyme chromatography detection kit swab sample sensitivity Detection result
Note :+representing the positive ,-expression is negative ,-/+representing the weak positive, ND represents not detect.
As shown in Table 6: the kit of three continuous lot numbers can both effectively detect above-mentioned swab sample, illustrate that kit has good susceptibility.
5.2 specificity
With three continuous lot number kits to the pharynx of negative SPF chicken, cloaca swab sample, the pharynx of healthy chicken, cloaca swab sample, the Nasopharyngeal swabs sample of health pig, the Nasopharyngeal swabs sample of Healthy Dogs detects, and evaluates its specificity.Three continuous lot number kits are to the pharynx of negative SPF chicken, cloaca swab sample, the pharynx of healthy chicken, cloaca swab sample, and the Nasopharyngeal swabs sample of health pig, the assay of the Nasopharyngeal swabs sample of Healthy Dogs is in Table 7.
The negative swab specific detection of table 7 A type influenza virus enzyme chromatography detection kit result
Note :+representing the positive ,-expression is negative.
As shown in Table 7: specificity can reach 100%, proved kit has higher specificity.
The kit of three continuous lot numbers represents that to the avian influenza virus of the popular or separated Main Subtype of China (the H5 subtype avian influenza virus that comprises various years and geographical separation represents strain), H1 and H3 hypotype swine influenza virus strain, H3 hypotype canine influenza virus represent that strain, H6, H7 and H9 subtype avian influenza virus represent 10 of other bird viruses such as strain and Avian pneumo-encephalitis virus, IBV, infectious bursa of Fabricius virus, infectious laryngotracheitis
-1, 10
-2, 10
-3deng serial dilution degree, detect, evaluate the specificity of its avian influenza virus for Main Subtype and other bird viruses.Assay is in Table 8.
Table 8 A type influenza virus enzyme chromatography detection kit specific detection result
Note :+representing the positive ,-expression is negative ,-/+representing the weak positive, ND represents not detect.
As shown in Table 8: three continuous lot number kit Jun Keyu China's Major Epidemics or separated antigen group (clade0, clade1, clade2.2, clade2.3.2, clade2.3.4 class RE-5 vaccine strain, clade2.5, clade3, clade4, clade5, clade7 class RE-4 vaccine strain, H5N1 subtype avian influenza virus generation specific reaction clade9); Can represent that strain, H3 hypotype canine influenza virus represent that strain, H6, H7 and H9 subtype avian influenza virus represent strain generation specific reaction with H1 and H3 hypotype swine influenza virus; , with Avian pneumo-encephalitis virus, IBV, infectious bursa of Fabricius virus, infectious laryngotracheitis generation nonspecific reaction, specificity is not good.
5.3 repeatable
With three continuous lot number kits by 3 people to comprising 30 parts of test sample duplicate detection three times of strong positive reference material 10 parts (P1-P10), weak positive reference material 10 parts (P11-P20) and specificity reference material 10 parts (N1-N10), coincidence rate between statistics testing result, with 5 single lot number kits, by 1 people, 30 parts of test samples are detected, in order to evaluate three batches of kits batch between, batch in repeatable, assay is in Table 9-11.
30 parts of test samples of table 9 batch between testing result
Note :+representing the positive ,-expression is negative.
Batch interior testing result of 30 parts of test samples of table 10
Note :+representing the positive ,-expression is negative.
That three batches of kits of table 11 are criticized is interior, batch between coincidence rate
From table 9-11: three continuous lot number kits are to 30 parts of test sample duplicate detection three times, positive coincidence rate between three lot number kits are criticized by statistics, between batch interior each testing result is respectively 100%, 100%, negative match-rate is respectively 96.8%, 96.2%, illustrate three batches of goods criticize between, batch in all there is good repeatability.
5.4 with the comparison of other diagnostic method
Select respectively 3 parts of avian influenza virus (H5, H9 hypotype) strong positive, the weak positive, negative samples, the avian influenza virus of known viruse content (H5, H9 hypotype) serial dilution is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7wait dilutability sample, by inoculation SPF chicken embryo virus separation method and RT-PCR method and influenza A virus enzyme chromatography test strip, detect the situation that meets of three kinds of detection methods of comparison.
This kit the results are shown in Table 12-13 with the parallel comparison test of egg inoculation isolated viral method and RT-PCR.
The comparison test 1 of table 12 influenza A virus enzyme chromatography test strip and egg inoculation isolated viral method and RT-PCR
Note :+representing the positive ,-expression is negative, the-/weak positive of+expression.
The comparison test 2 of table 13 influenza A virus enzyme chromatography test strip and egg inoculation isolated viral method and RT-PCR
Note :+representing the positive ,-expression is negative, the-/weak positive of+expression.
From showing 12-13: detect corresponding strong positive, the weak positive and ' negative ' specimens and avian influenza virus, although egg inoculation virus separation method is more responsive than this kit, but the method takes time and effort, can not realize, fast detection easy to A type influenza virus; Although the sensitivity of RT-PCR is slightly higher than this kit, the method needs expensive instrument and the technical professionals such as PCR instrument, can not meet easy, diagnostic requirements fast.
This kit is compared with the colloidal gold strip of the detection avian influenza virus of domestic certain company, and specificity is consistent, but remolding sensitivity it is high 10 times.
5.5 storage life
Three continuous lot number kits are placed 6 days and 7 days laggard line stabilization tests, i.e. kit heat stabilization tests respectively at 37+1 ℃; In order to prevent there are differences between 37 ± 1 ℃ of heat stabilization tests and 2-8 ℃ of storage condition, we deposit kit respectively 6 months, 9 months, 12 months and 15 months at 2-8 ℃ again, i.e. the real-time stabilization of kit test.The kit of different preservation conditions is tested with 35 parts of test samples, in order to evaluate the storage life of these goods.
Table 14 kit heat stabilization test result
Table 15 kit real-time stabilization test findings
This kit is preserved under 37 ± 1 ℃ of conditions, stores 7 days; Under 2-8 ℃ of rated condition, preserve, store 15 months, still can pass through finished product kit quality control standard, show kit term of validity duration of test steady quality, storage life at least can reach 12 months.
From above result, kit of the present invention and detection method tool thereof have the following advantages:
1, quick, testing process only needs 30 minutes;
2, accurate, high specificity, with Type B influenza virus generation non-specific responding, not with Avian pneumo-encephalitis virus, IBV, infectious bursa of Fabricius virus, ILTV generation non-specific responding;
3, susceptibility is high, can detect A type influenza virus and represent strain, comprises China's Major Epidemic or separated H5N1 hypotype antigen group (clade0, clade1, clade2.2, clade2.3.2 class RE-6 vaccine strain, clade2.3.4 class RE-5 vaccine strain, clade2.5, clade3, clade4, clade5, clade7.2 class RE-4 vaccine strain, clade8, clade9) avian influenza virus; Other subtype avian influenza virus such as H6, H7 and H9 hypotype represent strain; H1 and H3 swine influenza virus and equine influenza virus represent strain; H3 hypotype canine influenza virus represents strain;
4, sensitive, its sensitivity can reach ELISA level, higher more than 10 times than colloidal gold strip, suitable with PCR detection sensitivity in the detection of some cause of disease, under detection, is limited to 0.01HA-0.1HA;
5, easy, do not need to be equipped with expensive device and the technical professionals such as PCR instrument, can under clinical condition, detect, simple to operate, easy to carry;
6, good stability, can preserve at 2-8 ℃ long-term, and storage life is 12 months.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (11)
1. for detection of a kit for influenza virus in sample, wherein, described kit comprises damping fluid feeding unit and test strip;
Described damping fluid feeding unit is for supplying with described test strip by damping fluid;
Described test strip comprises nitrocellulose filter (1), and described test strip comprises substrate supply area, sample supply area, detection zone in the vertical successively;
Described substrate supply area comprises substrate pad (3), on it, absorption has dry zymolyte, described substrate pad (3) contacts with nitrocellulose filter (1), and described zymolyte is dissolved in damping fluid and in the upper distal migration to the described damping fluid feeding unit of distance of nitrocellulose filter (1);
Described sample supply area comprises enzyme mark pad (2), on it, absorption has mouse anti-influenza type A virus nucleoprotein antibody labeling monoclonal antibody 1, described zymolyte can produce chromogenic reaction with the enzyme of mark on described labeled monoclonal antibody 1, described enzyme mark pad (2) contacts with nitrocellulose filter (1), described labeled monoclonal antibody 1 is dissolved in damping fluid, and upper to the distal migration apart from described damping fluid feeding unit at nitrocellulose filter (1); And
The immobilization of described detection zone has mouse anti-influenza type A virus nucleoprotein antibody immobilized monoclonal antibody 2.
2. kit according to claim 1, wherein, described damping fluid feeding unit comprises expansion fluid cushion (5), substrate buffer solution groove (8), substrate buffer solution (9) and damping fluid button, described substrate buffer solution (9) is placed in substrate buffer solution groove (8), described damping fluid button is positioned at the top of substrate buffer solution groove (8), presses and described expansion fluid cushion (5) can be immersed in damping fluid (9);
Described detection zone comprises detection line (6), nature controlling line (7), wherein, described nature controlling line (7) compared with detection line (6) further from described sample supply area, in described detection line (6) immobilization, there is mouse anti-influenza type A virus nucleoprotein antibody immobilized monoclonal antibody 2, in described nature controlling line (7) immobilization, have sheep anti mouse how anti-, and to be adsorbed on labeled monoclonal antibody 1 on enzyme mark pad (2) be excessive for being fixed on the immobilized monoclonal antibody 2 of detection line (6);
The full section of described nitrocellulose filter (1) sticks on above support (10), stilt (10) connects described damping fluid feeding unit, substrate supply area, sample supply area, detection zone and adsorptive pads (4), described adsorptive pads (4) is in the distal-most end apart from described damping fluid feeding unit; And
The position that detection sample (11) adds is the position of described enzyme mark pad (2).
3. kit according to claim 1, wherein, the amino acid sequence of described labeled monoclonal antibody 1 its variable region of heavy chain is that the amino acid sequence of SEQ ID No.2, variable region of light chain is SEQ ID No.3; And
The amino acid sequence of described immobilized monoclonal antibody 2 its variable region of heavy chain is that the amino acid sequence of SEQ IDNo.4, variable region of light chain is SEQ ID No.5.
4. kit according to claim 1, wherein, the enzyme of described mark comprises any in horseradish peroxidase, alkaline phosphatase and beta-D-galactosidase.
5. kit according to claim 2, wherein, described substrate buffer solution is aqueous solvent, comprises one or more in phosphate buffer, surfactant, antiseptic and carbohydrate.
6. kit according to claim 1, wherein, described kit also comprises sample processing tube, described sample processing tube contains sample preparation liquid, described sample preparation liquid is surfactant solution, and described surfactant comprises any in stearic acid, neopelex, lecithin, fatty glyceride and CHAPS.
7. kit according to claim 1, wherein, described kit also comprises Sample storage bottle, and described Sample storage bottle contains Sample storage liquid, and Sample storage liquid is phosphate buffer, is preferably the phosphate buffer of pH7.4.
8. right to use requires kit described in 1～7 any one to detect a method for influenza virus in sample, and described method comprises:
(1) use described sample processing tube pretreatment sample;
(2) pretreatment sample in described step (1) is added to the position of described enzyme mark pad (2);
(3) press damping fluid button; And
(4) if nature controlling line place there is no band, no matter whether detection line place has band, and testing process is all invalid, the in the situation that of having band at nature controlling line place, if there is band at detection line place, positive, otherwise negative.
9. according to the application in influenza virus in detecting sample of kit described in claim 1～7 any one.
10. an anti-influenza type A virus nucleoprotein monoclonal antibody 1, the amino acid sequence of described monoclonal antibody 1 its variable region of heavy chain is that the amino acid sequence of SEQ ID No.2, variable region of light chain is SEQ ID No.3.
11. 1 kinds of anti-influenza type A virus nucleoprotein monoclonal antibodies 2, the amino acid sequence of described monoclonal antibody 2 its variable region of heavy chain is that the amino acid sequence of SEQ ID No.4, variable region of light chain is SEQ ID No.5.
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