CN1934133A - Anti-influenza type A virus monoclonal antibody and immunoassay instrument using the antibody - Google Patents
Anti-influenza type A virus monoclonal antibody and immunoassay instrument using the antibody Download PDFInfo
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- CN1934133A CN1934133A CNA2004800211740A CN200480021174A CN1934133A CN 1934133 A CN1934133 A CN 1934133A CN A2004800211740 A CNA2004800211740 A CN A2004800211740A CN 200480021174 A CN200480021174 A CN 200480021174A CN 1934133 A CN1934133 A CN 1934133A
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- 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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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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Abstract
An anti-influenza type A virus monoclonal antibody of high specificity; and an immunoassay instrument comprising the same that is capable of specifically detecting influenza type A viruses. This anti-influenza type A virus monoclonal antibody performs an antigen antibody reaction with the nucleoprotein of 55 to 70 kD molecular weight of influenza type A virus but substantially does not perform any antigen antibody reaction with influenza type B viruses.
Description
Technical field
The present invention relates to anti-influenza type A virus monoclonal antibody and use the immunoassay instrument of this antibody.
Background technology
Before the influenza with regard to the cycle repeatedly at world pop, whenever influenza pandemic, a lot of dead persons are just arranged, be detected in the virus as the influenza reason in 1931, opened and led to the road of illustrating the influenza reason.Influenza pathogens virus is according to being positioned at the inner SNP (nucleoprotein of virus; NP) antigenicity is classified as 3 types on A type, Type B or C type.A type influenza is further according to the hemagglutinin (hemagglutinin that is present in virus surface; HA) and neuraminidase (neuraminidase; NA) antigenicity of these 2 kinds of envelope glycoproteins is told hypotype.
Since 1972 for flu-prevention, use the vaccine of handling back usefulness formalin passivation through ether.In recent years, beyond the vaccine that is used to prevent, also found antiviral drug as curative, and be widely used.As these curatives, be virofral of adaptation A type influenza virus and the zanamivir that adapts to A type influenza virus and Type B influenza virus, phosphoric acid Olympic Competition Ta Miwei etc.
When selecting these curatives, importantly detect the influenza virus in the sample, and determine that infection is that caused, the also definite Virus Type of influenza virus is A type or Type B.In addition, A type influenza virus and Type B influenza virus ratio, therefore infectivity is strong, causes serious symptoms, infective virus definite even more important in the treatment in early days.
In the past, utilize the resisiting influenza virus antibody test to go out influenza virus.Up to now at the antibody of A type influenza virus, in the hypotype of differentiating anticipation popular virus and preparation vaccine, work, understood identification virus specifically HA or NA, be used to discern the antibody (for example with reference to patent documentation 1, patent documentation 2) of the hypotype of virus.In addition, determined the nucleoprotein aminoacid sequence of each hypotype of A type influenza virus, at National Library of Medicine; Among the Entrez Nucleotides etc. report is arranged.
And, as the device that detects influenza virus, for example know: preparation can be in conjunction with the solid phase of influenza antigen, after influenza virus in the sample is reacted with it, and then make in conjunction with the A type Antibody of Influenza of first enzyme with in conjunction with Type B Antibody of Influenza and the above-mentioned solid phase of second enzyme and react, add matrix and react, the circulation type of the colour developing on the visual observations solid phase (flow-through) device (with reference to patent documentation 3).This device, though the antibody in the reagent uses the antibody at influenza virus nucleoprotein, measurement operation is miscellaneous, is not easy determinator.Also know the immunoassay instrument that used at the monoclonal antibody of influenza virus nucleoprotein (below be called " immune chromatograph utensil ") (with reference to non-patent literature 1) in addition.
On the other hand, developed the immune chromatograph utensil that uses transfusable zonal matrix, drop sample on the position of defined only, do not need special proofing unit, skilled determination techniques and can be at short notice antigen or the antibody in the working sample easily.For the immune chromatograph utensil, developed according to its determination object material: for example can be at detection zone in conjunction with a plurality of treponemal antigen (TP antigen), and detect the immune chromatograph utensil (with reference to patent documentation 4) of a plurality of anti-TP antibody in the sample at each detection zone.In the detection zone of this utensil, have and combine the antigenic a plurality of districts of different TP, in 1 time mensuration, just detect different anti-TP antibody respectively, determine infection period, be used for the treatment of the selection of medicine etc.
Patent documentation 1: the spy opens flat 6-100594 number
Patent documentation 2: the spy opens flat 7-304799 number
Patent documentation 3: the spy opens 2001-124775 number
Patent documentation 4: the spy opens flat 9-229938 number
Non-patent literature 1: infect disease will 2001; 75; 792-799
Summary of the invention
Anti-influenza type A virus monoclonal antibody in the past, though can as in the immunochromatography etc. at the antibody of nucleoprotein because low, can not satisfy highly sensitive immunoassay to specificity and the reactivity of A type influenza virus.In addition, in order to select to control the influenza medicine, demand with the reaction of Type B influenza virus, but the antibody of all A type influenza viruses reactions of the hypotype that has made a variation with HA that comprises influenza virus and NA.
In addition, demand is not used special diagnostic instrumentation and determinator in the differentiation of A type influenza virus or Type B influenza virus, and obtains measurement result at short notice, just can differentiate the device of A type or Type B through 1 operation.
Therefore, the purpose of this invention is to provide the high anti-influenza type A virus monoclonal antibody of a specific specificity.In addition, the present invention also provides a kind of immunoassay instrument that can detect A type influenza virus specifically.
The result of inventor's lucubrate, the nucleoprotein of successfully making with A type influenza virus is antigen, with A type influenza virus the anti-influenza type A virus monoclonal antibody of specific reaction takes place, thereby has finished the present invention.In addition, by utilizing this A type influenza virus monoclonal antibody, expected to provide to distinguish to turn up the soil with the Type B influenza virus detecting the immunoassay instrument of A type influenza virus.
That is, the invention provides the nucleoprotein generation antigen antibody reaction with the molecular weight 55-70kD of A type influenza virus, anti-influenza type A virus monoclonal antibody or its antigen-binding fragment of antigen antibody reaction do not take place basically with the Type B influenza virus.In addition, the invention provides a kind of immunoassay instrument, this utensil is labelled reagent district, sample drop district, developping solution drainage area, the developping solution uptake zone with anti-influenza type A virus antibody of mark movably to be set on the matrix and to make anti-influenza type A virus antibody be fixed on the utensil of the A type influenza virus detection zone on the matrix, and the anti-influenza type A virus antibody of above-mentioned mark and at least a antibody that is fixed on the anti-influenza type A virus antibody in the detection zone are the monoclonal antibodies of the invention described above.
The invention effect
According to the present invention, provide and immune response takes place A type influenza virus, but the anti-influenza type A virus monoclonal antibody that does not react with the Type B influenza virus.The anti-influenza type A virus monoclonal antibody of the application of the invention carries out immunoassay, can turn up the soil, detect A type influenza virus or it is quantitative to distinguish with the Type B influenza virus.According to the present invention, also provide the immunoassay instrument of the anti-influenza type A virus monoclonal antibody that has used the invention described above.The immunoassay instrument of the application of the invention can be easy and distinguish with the Type B influenza virus and to turn up the soil, to detect A type influenza virus.Therefore, the present invention that waits in expectation has big contribution in the diagnosis of influenza and treatment.
The simple declaration of accompanying drawing
Fig. 1 is the result of expression when making monoclonal antibody of the present invention and adopting the isolating antigen-reactive of Western blotting figure.
Fig. 2 is illustrated in the figure that the restriction map of plasmid (pW6A) is used in the expression of using among the embodiment 4.
Fig. 3 is expression with the A fragment of influenza H1N1 further segmentation and the amino acid whose figure of Segment A 1～A7 of obtaining.
Fig. 4 is that expression is confirmed and the reactive CBB dyeing pattern of FVA2-11 and the figure of Western trace.
Fig. 5 is the figure of the nucleoprotein aminoacid sequence (80～140) of expression A type influenza virus sub-strain.
Fig. 6 is the schematic cross-section of an embodiment of expression mensuration utensil of the present invention.
Fig. 7 is the vertical view that expression is placed in mensuration utensil of the present invention an example in the box.
Fig. 8 is the sectional view of the A-A ' of Fig. 7.
The preferred plan that carries out an invention
As mentioned above, the nucleoprotein generation antigen antibody reaction of the molecular weight 55-70kD of monoclonal antibody of the present invention and A type influenza virus.With the nucleoprotein generation antigen antibody reaction of the molecular weight 55-70kD of A type influenza virus can by with A type influenza virus as sample, carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirm by the Western blotting.When carrying out the Western trace when (with reference to following embodiment), the nucleoprotein of monoclonal antibody identification molecular weight 55-70kD of the present invention.
According to above-mentioned, antigen antibody reaction does not take place in monoclonal antibody of the present invention and Type B influenza virus basically.At this, so-called " antigen antibody reaction does not take place basically " is meant: even can antigen antibody reaction not take place or causing antigen antibody reaction under the detected level, the degree of its reaction also obviously is weaker than the antigen antibody reaction with A type influenza virus, with A type influenza virus antigen antibody reaction does not take place and is meant the degree reaction of only understanding this area practitioner.In addition, at this, so-called " antigen antibody reaction not taking place basically with the Type B influenza virus " is to go up the meaning that antigen antibody reaction does not take place substantially with the protein of each integrant of this virus of the nucleoprotein that comprises the Type B influenza virus.In preferred scheme, monoclonal antibody of the present invention is not reacted with C type influenza virus basically yet.
In preferred scheme, monoclonal antibody of the present invention is reacted with the epi-position in the 59th (following put down in writing " 59aa ")～130 amino acids residue zones of the aminoacid sequence of A type influenza virus nucleoprotein.The aminoacid sequence of A type influenza virus nucleoprotein is known, for example is documented among GenBank Accession No.ITY238021 etc.Fig. 5 expresses the 59aa～140aa aminoacid sequence of nucleoprotein of each hypotype of A type influenza virus.In addition, in preferred scheme, monoclonal antibody of the present invention, because nucleoprotein generation antigen antibody reaction with the A type influenza virus of each hypotype, therefore, to consensus sequence be arranged in the aminoacid sequence shown in Figure 5, zones such as for example 90aa～95aa, the 111aa～115aa of hydrophilic structure or 120aa～123aa be arranged, the zone that perhaps comprises these zones and have a consensus sequence be as epi-position.
In preferred scheme, monoclonal antibody of the present invention is with the nucleoprotein generation antigen antibody reaction of each hypotype of A type influenza virus.That is,, be classified as all hypotypes according to the structure of these H and N is different though A type influenza virus has hemagglutinin (H) and neuraminidase (N) on its virus particle surface.Monoclonal antibody of the present invention, preferred at least with the nucleoprotein generation antigen antibody reaction of following hypotype, and then the nucleoprotein generation antigen antibody reaction of the hypotype of preferred and known all A type influenza viruses.H1N1、H2N2、H3N2、H3N8、H4N6、H5N2、H6N2、H7N7、H8N4、H9N2、H10N7、H11N6、H12N5、H13N6、H14N5、H15N8、H5N1。
In addition, in preferred scheme, antigen antibody reaction does not take place with the similar pathogenic agent that other infect diseases of influenza with symptom in monoclonal antibody of the present invention at least in part basically yet.For example with adenovirus (1～7 type), Coxsackie virus (A16, B1～B6 type), herpes simplex virus type 1, Echo virus (3 types, 4 types, 7 types, 22 types, 30 types), enterovirus (71 type), mumps virus, poliovirus (1～3 type), RS virus (subgroup A, subgroup B), parainfluenza virus (1～3 type), intestinal bacteria, pneumobacillus, the green pus bacterium, serratia marcescens, staphylococcus epidermidis, Bacillus proteus, staphylococcus aureus, the rod bacillus, corynebacterium diphtheriae, Candida albicans (Candida albicans), streptococcus pyogenes (Streptococcus pyogenes), the bacterium of streptococcus (group B, C, G, F), streptococcus pneumoniae (Streptococcus pneumoniae), hemophilus influenzae (Haemophilus influenzae), Listeria monocytogenes (Listeriamonocytogenes), mycoplasma pneumoniae (Mycoplasmal pneumonia), chlamydia trachomatis (Chlamydia trachomatis), antigen antibody reaction does not take place in Chlamydia pneumoniae (Chlamydia pneumonia) basically.
In addition, well-known, by decomposing antibody, can obtain Fab fragment and F (ab ') with papoid or stomach en-
2Fragment and so on have a antibody fragment (being called " antigen-binding fragment " in this manual) with corresponding antigenic associativity; but the antigen-binding fragment of monoclonal antibody of the present invention also can similarly be used with monoclonal antibody of the present invention, is included in protection scope of the present invention.
Monoclonal antibody of the present invention as immunogen, adopts the preparation of conventional hybridization knurl method with the nucleoprotein of A type influenza virus.The nucleoprotein of the A type influenza virus of using as immunogen if nucleoprotein exists in a large number, can be brought into play its immunogen effect, then cultivates viral liquid, influenza HA vaccine, HI and all can use with HA antigen, recombinant antigen.In order to suppress high HA of the contained immunogenicity of antigen and the effect of NA, also can be by super centrifugal nucleoprotein purifying (for example with reference to J.Biochem.; 102:1241-1249,1987) or protease treatment (for example with reference to J.Immunol.Methods; 180:107-116,1995) antigen.
Said monoclonal antibody can produce by following manner, promptly, the antigen of nucleoprotein that will comprise above-mentioned A type influenza virus is as the immunogen immune animal, it is produced cell of A type influenza virus monoclonal antibody and swollenly merges with oncocyte, utilizes the hybridoma of gained to produce said monoclonal antibody.
Above-mentioned hybridoma can adopt following method to obtain.That is, the above-mentioned antigenic A type influenza virus nucleoprotein that obtains as described above is divided into bestowing by intraperitoneal or vein every 2-3 week for several times with Freund's complete adjuvant animals such as mouse are carried out immunity.Then, make the generation antibody cell that derives from spleen etc. and merge from the reproducible tumour cell in developmental tube of the cell (myeloma cell) of myeloma cell line etc.
As above-mentioned fusion method, can utilize polyoxyethylene glycol to carry out according to the ordinary method (Nature, 256 volumes, 495 pages, 1975) of Kohler and Milstein, perhaps can adopt Sendai virus to wait and carry out.
As the method for the hybridoma of selecting to produce the antibody of discerning A type influenza virus nucleoprotein from above-mentioned fused cell, for example can carry out as follows.That is, be chosen in the cell of surviving in HAT substratum and/or the HT substratum as hybridoma by limiting dilution assay by above-mentioned fused cell.Then, after the nutrient solution of the above-mentioned hybridoma of utilization cultivation reacts on the check-out console of the A type influenza virus nucleoprotein of having fixed the high purity purifying, by with the further EIA method etc. of reaction such as anti-mouse immuning ball protein (Ig), can select to produce the hybridoma of the monoclonal antibody of discerning A type influenza virus nucleoprotein specifically.
In the following embodiments, obtained the preferred monoclonal antibody of multiple the present invention, with wherein 3 kinds called after hybridoma FVA2-3, hybridoma FVA2-6 and hybridoma FVA2-11 respectively.These hybridomas are preserved in Independent Administrative Leged Industrial Technology Complex Inst according to budapest treaty respectively and specially permit biological preservation center (a kind of ground 1 of 1 fourth order, ripple east, central authorities the 6th are built in the Ibaraki, Japan), wherein, hybridoma FVA2-3 identifies expression FVA2-3, its preserving number is FERMBP-10066 (preservation day: on July 18th, 2003, transfer international preservation on July 14th, 2004 to from domestic preservation FERM P-19437); Hybridoma FVA2-6 identifies expression FVA2-6, and its preserving number is FERM BP-10067 (preservation day: on July 18th, 2003, transfer international preservation on July 14th, 2004 to from domestic preservation FERM P-19438); Hybridoma FVA2-11 differentiates expression FVA2-11, and preserving number is FERM BP-10068 (preservation day: on July 18th, 2003, transfer international preservation on July 14th, 2004 to from domestic preservation FERM P-19439).
Above-mentioned each hybridoma by cultivating at the substratum that is used for cell cultures, can reclaim monoclonal antibody from culture supernatant.In addition, by bestowing the animal that produces hybridoma, can also store ascites, and from this ascites, reclaim.
As the recovery method of said monoclonal antibody, can use the purification process that carries out usually, for example enumerate the affinity chromatography of gel filtration chromatography, ion exchange chromatography, a-protein etc.
Zhi Bei monoclonal antibody according to the method described above, through confirm can with the nucleoprotein of A type influenza virus, and various hypotypes reactions of A type influenza virus.Now the A type influenza virus preservation strain 38 kinds (with reference to following tables 3) of preservation and getable people, horse, wild duck, firebird, sea dog, chicken, sea-gull, mallard etc. also reacts with it, and they can detect with monoclonal antibody of the present invention.On the other hand, as the microorganism of considering cross reactivity, for example confirmed and adenovirus (1～7 type), Coxsackie virus (A16, B1～B6 type), herpes simplex virus type 1, Echo virus (3 types, 4 types, 7 types, 22 types, 30 types), enterovirus (71 type), mumps virus, poliovirus (1～3 type), RS virus (subgroup A, subgroup B), parainfluenza virus (1～3 type), intestinal bacteria, pneumobacillus, the green pus bacterium, serratia marcescens, staphylococcus epidermidis, Bacillus proteus, staphylococcus aureus, the rod bacillus, corynebacterium diphtheriae, Candida albicans, streptococcus pyogenes, the bacterium of streptococcus (group B, C, G, F), streptococcus pneumoniae, hemophilus influenzae, Listeria monocytogenes, mycoplasma pneumoniae, chlamydia trachomatis, the reactivity of Chlamydia pneumoniae etc., but reaction is not found.
Monoclonal antibody of the present invention can be used in detection or the quantitative immunoassay of A type influenza virus.Method of immunity is well-known, can adopt known any method of immunity.That is, by measuring formal classification sandwiching, competition law, coacervation, Western blotting etc. are arranged, the labeled bracketing by using has fluorescent method, enzyme process, radioactive method, vitamin H method etc., can use the wantonly a kind of method in these methods.And, can also dye by immuning tissue and diagnose.During applying marking antibody, the marking method of antibody is well-known, can adopt known any method in method of immunity.Well-known in addition, antibody is decomposed with papoid or decompose with stomach en-, can obtain as Fab fragment and F (ab ')
2Fragment such, have the antibody fragment (being called " antigen-binding fragment " in this manual) that associativity is arranged with corresponding antigen, the antigen-binding fragment of monoclonal antibody of the present invention also can similarly be used with monoclonal antibody of the present invention.
These method of immunity self are well-known, do not need to illustrate in this manual, be briefly described as follows, for example in sandwiching, antibody of the present invention or its antigen-binding fragment are fixed in solid phase as the 1st antibody, make it and example reaction, after cleaning, make it and with of the present invention and the 2nd antibody response enzyme generation antigen antibody reaction, after cleaning, measure the 2nd antibody that is incorporated into solid phase.By with mark the 2nd antibody such as enzyme, fluorescent substance, radioactive substance, vitamin Hs, can measure the 2nd antibody that is incorporated into solid phase.Utilize aforesaid method to measure to the known a plurality of standard test specimens of concentration, make typical curve based on the relation of the enzyme of the present invention in labelled amount of being measured and the standard test specimen, be used for this typical curve by measurement result cover with the tested sample of unknown concentration, can the enzyme of the present invention in the tested sample is quantitative.Also can be with the 1st antibody and the 2nd antibody mutual alternative in the above description.In addition, in coacervation, on the particle of latex etc., fix antibody of the present invention or its antigen-binding fragment, make it and example reaction, measure absorbancy.Utilize aforesaid method to measure to the known a plurality of standard test specimens of concentration, make typical curve based on the relation of the enzyme of the present invention in labelled amount of being measured and the standard test specimen, be used for this typical curve by measurement result cover with the tested sample of unknown concentration, can the enzyme of the present invention in the tested sample is quantitative.
The present invention also provides a kind of immunoassay instrument, and this utensil utilizes the monoclonal antibody of the invention described above, can be easy and distinguish with the Type B influenza virus and to turn up the soil, to detect A type influenza virus.
Immunoassay instrument of the present invention, be to be provided with labelled reagent district, sample drop district, developping solution drainage area, developping solution uptake zone on the matrix and to make anti-influenza type A virus antibody be fixed on the utensil of the A type influenza virus detection zone on the matrix with anti-influenza type A virus antibody of mark movably, the anti-influenza type A virus antibody of above-mentioned mark and to be fixed at least a in the anti-influenza type A virus antibody in the detection zone be monoclonal antibody of the present invention wherein.Point drops in sample spot and drips sample in the district, with the traget antibody generation antigen antibody reaction that the labelled reagent district is contained, forms the immune complex that has been labeled.This immune complex flows with the developping solution of being supplied with by the developping solution drainage area, arrives A type influenza virus detection zone., be fixed on anti-influenza type A virus antibody and above-mentioned labelled antigen-antibody camplex generation antigen antibody reaction on the matrix here, above-mentioned labelled antigen-antibody camplex is fixed on the matrix.Whether labelled antigen-antibody camplex is fixed in the A type influenza virus detection zone by having is unmarkedly judged.The developping solution that passes through in A type influenza virus detection zone is unfolded the liquid uptake zone and absorbs.Sample drop district and labelled reagent district can be same (sample are dropped in the labelled reagent district by point in such cases).In immunoassay instrument of the present invention, traget antibody or be fixed on antibody in the A type influenza virus detection zone any is a monoclonal antibody of the present invention at least.In addition, wherein a kind of antibody also can be polyclonal antibody.The nucleoprotein of A type influenza virus usually in conjunction with or adhering to a plurality of molecules, even so traget antibody or be fixed on the two identical monoclonal antibody of antibody in the A type influenza virus detection zone, also can detect A type influenza virus.
Each constitutive requirements of immunoassay instrument of the present invention below are described respectively.
Matrix
Ribbon base in the immunoassay instrument of the present invention adopts the absorbent material formation of utilizing capillary action and carrying liquid.As this absorbent material, for example be preparations such as the Mierocrystalline cellulose or derivatives thereof that uses Mierocrystalline cellulose, Nitrocellulose etc. separately, glass fibre or their are mixed use and paper, film, the porous material of preparation.The size of this matrix without limits, but the ribbon base about wide 3mm-10mm, about long 30mm-100mm because of processing ease so preferred.But the matrix used thickness is 100 μ m-1mm's.In addition, matrix is for the absorption due to the non-specific responding of protein on matrix that prevents to derive from sample when measuring, and the animal serum of for example available bovine serum albumin (BSA) etc.s, casein, sucrose etc. are with its part or all seal (blocking) and use.
Detection zone
Can be arranged on the A type influenza virus test section of having fixed anti-influenza type A virus antibody on the above-mentioned matrix at detection zone.Be fixed on the anti-influenza type A virus antibody of this test section and the anti-influenza type A virus antibody of mark described later, at least a is anti-influenza type A virus monoclonal antibody of the present invention, and preferably these two kinds of antibody are anti-influenza type A virus monoclonal antibodies.The above-mentioned anti-influenza type A virus antibody of test section on the matrix with the orthogonal direction of transfusion direction (matrix vertically) of unfolded liquid in matrix on, can provide sensitivity to measure well when being provided with, so preferably linear.As the anti-influenza type A virus polyclonal antibody, can from market sale, and the polyclonal antibody that can obtain easily suitable the selection use.
The anti-influenza type A virus antibody of this detection zone is above-mentioned antibody, monoclonal antibody can also be used separately or mixed and use.Anti-influenza type A virus antibody can be IgG antibody, IgM antibody, can also be antigen-binding fragment F (ab), the F (ab ') of these antibody
2Deng.
The anti-influenza type A virus antibody that is fixed on test section can make it on the detection zone that direct physical is adsorbed on matrix, thereby also can utilize chemical bonds such as covalent linkage to fix setting.In addition, anti-influenza type A virus antibody is combined with water-insoluble carrier, thereby make matrix contain this antibody.As this insoluble carrier, can enumerate the mixture of being made up of gelatin, gum arabic and Sodium hexametaphosphate 99 is insoluble and the particle that obtains (special public clear 63-29223), polystyrene latex particle, glass fibre etc.Make insoluble carrier and anti-influenza type A virus monoclonal antibody in conjunction with the time, can be undertaken by above-mentioned chemical bond or physical adsorption.
In detection zone, except the A type influenza virus test section that adopts anti-influenza type A virus antibody, the Type B influenza virus test section that adopts anti-Type B Antibody of Influenza can be set also.This Type B influenza virus test section, if above-mentioned A type influenza virus test section near, then the transfusion direction at developping solution can be the either side in above-mentioned A type influenza virus test section upstream side or downstream side.In the anti-Type B Antibody of Influenza of Type B influenza virus detection zone fixed, also can be polyclonal antibody or monoclonal antibody, same with above-mentioned anti-influenza type A virus antibody, can be fixed on the matrix by chemical bond or physical adsorption.
As above-mentioned, the detection zone on matrix is provided with the A type influenza virus test section that adopts anti-influenza type A virus antibody at least, and then Type B influenza virus test section is set, and measures at the same time on A type influenza virus and the Type B influenza virus to preferred.These test sections are the downstream sides of enzyme labelling reagent area, sample drop district and developping solution drainage area on the transfusion direction of matrix, are the upstream sides of annex solution uptake zone.Test section can be provided with many line at the linear that approaches on the matrix about wide 0.5mm～5mm.If the matrix about wide 5mm then usually with about above-mentioned antibody and antigen difference drop 0.1 μ g-10 μ g, makes it dry, thereby can make test section.
The labelled reagent district
The labelled reagent district, thus to be arranged on the matrix the district movably the anti-influenza type A virus antibody of drop mark be provided with.This district can be arranged on the upstream side of above-mentioned detection zone in the transfusion direction from the developping solution of developping solution drainage area.This district utilizes following method to constitute: to matrix drop enzyme labelling compositions and methods; The water-absorbent bed course that will contain enzyme labelling reagent is stacked in the method on the matrix; Perhaps make part or all contain the enzyme labelling compositions and methods with pad with filling up the adherent body portion.As the water-absorbent pad, can use the pad in sample drop described later district.
The antibody of the anti-influenza type A virus antibody that serves as a mark and the above-mentioned antibody that is arranged on detection zone, at least a is anti-influenza type A virus monoclonal antibody, preferably these two kinds of antibody are anti-influenza type A virus monoclonal antibodies.The antibody of the anti-influenza type A virus antibody that serves as a mark similarly can also use its fragment with the antibody of above-mentioned detection zone.
The anti-influenza type A virus antibody of above-mentioned mark, make above-mentioned antibody and marker in conjunction with and prepare.The thing that serves as a mark can be enumerated enzyme, metallic colloid particle, painted latex particle, luminophore, fluorescent substance etc.As enzyme, be the employed various enzymes of enzyme immunoassay (EIA), for example can enumerate alkaline phosphatase, peroxidase, beta-D-galactosidase etc. as this kind of enzyme.In addition, for example can use colloidal gold particle, electroselenium particle etc. as the metallic colloid particle.
In addition, the combining method of marker and anti-influenza type A virus antibody can utilize the method preparation of known formation covalent linkage or non covalent bond.The bonded method for example can be enumerated glutaraldehyde (グ Le ア Le デ ヒ De) method, periodic acid method, maleimide method, pyridyl disulfide method, use (for example No. 31, reference " protein nucleic acid enzyme " supplementary issue, 37-45 page or leaf (1985)) such as methods of various linking agents.In the combining method that uses linking agent, for example can use N-succinimido (ス Network シ Application イ ミ ジ Le)-4-dimaleoyl imino butyric acid (GMBS), N-succinimido-6-dimaleoyl imino caproic acid, N-succinimido-4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylic acid etc. as linking agent.In covalently bound method, except using the functional group that in antibody, exists, for example adopt ordinary method to import after the functional groups such as thiol group, amino, carboxyl, hydroxyl, utilize above-mentioned combined techniques can prepare the anti-influenza type A virus antibody of mark.In addition, as non-covalent bonded method, can enumerate physisorphtion etc.
According to above-mentioned, except detecting A type influenza virus, when also detecting the Type B influenza virus simultaneously, add the anti-Type B Antibody of Influenza of mark in the labelled reagent district and prepare utensil.The anti-Type B Antibody of Influenza of the anti-influenza type A virus antibody of the mark that contains in the labelled reagent district and mark, in matrix or water-absorbent pad wherein a kind of, contain, perhaps can make matrix and water-absorbent pad contain the anti-Type B Antibody of Influenza of underlined anti-influenza type A virus antibody and mark in the two.When measuring when carrying out A type influenza virus and Type B influenza virus, because more ground applying marking reagent, therefore contain enzyme labelling reagent in the two separately or at matrix and pad is favourable on carrying out high sensitivity measuring with mixing.The anti-Type B Antibody of Influenza amount of the anti-influenza type A virus antibody of mark and mark can change corresponding to the premeasuring of checking object is suitable usually, but dry weight is about 0.01 μ g-5 μ g usually.The anti-Type B Antibody of Influenza of the anti-influenza type A virus antibody of mark and mark contains sometimes in the enzyme labelling district, can be with coatings such as the stablizer of reagent, dissolution regulator.
Sample drop district
Sample drop district especially can not contain reagent etc. and is arranged on the matrix of upstream side of downstream side, detection zone of transfusion direction of developping solution of developping solution drainage area.And sample drop district can be arranged on: the 1) position of the defined of the upstream side of the downstream side of the developping solution transfusion direction of expansion liquid zone, enzyme labelling reagent area; 2) position of the defined of the upstream side of the downstream side in labelled reagent district, detection zone; 3) position of the defined in the labelled reagent district etc.In addition, in above-mentioned enzyme labelling reagent area, be provided with in the device in sample drop district,, set up the water-absorbent pad that contains enzyme labelling and carrying out expeditiously analytically for preferred as above-mentioned.In the device that has added this pad, can a large amount of sample liquid of drop, therefore can carry out the mensuration of trace ingredients in the sample with good detection sensitivity.As this water-absorbent pad, from to selecting in the few material of the absorption of the influenza virus labelled reagent and the sample, for example can use individually by polyvinyl alcohol (PVA) thereby, the materials that porous is synthetic or natural macromolecular compound is formed such as non-woven fabrics, Mierocrystalline cellulose constitute or with these combinations of materials formations.The size of this pad, thickness, density etc. do not limit, usually uses long and wide as about 3mm-10mm, the pad of thickness about as 0.5mm-4mm can measure expeditiously, so preferably.
The developping solution drainage area
The developping solution drainage area is arranged on an end longitudinally of matrix, supplies with the district of developping solution.When beginning to measure, this district can be immersed in the container of the developping solution that the amount that arrives the developping solution uptake zone at least is housed.And, in order to supply with developping solution,, developping solution is contacted with matrix by the cover of abolishing this liquid bath launching the additional liquid bath that developping solution is housed in liquid zone, begin to measure.Can in developping solution, suit to contain tensio-active agent, buffer reagent, stablizer, antiseptic-germicide etc.In addition, when the thing that serves as a mark also uses enzyme, can in developping solution, add matrix with matrix agent described later district.As the damping fluid that contains buffer reagent, for example can enumerate acetate buffer, borate buffer, Tris-hydrochloride buffer, diethanolamine buffer etc.In addition,, supply with developping solution to matrix, can set up the expansion fluid cushion for stable also enforcement continuously in the developping solution drainage area.For example can use the filter paper of Mierocrystalline cellulose or derivatived cellulose etc. as launching fluid cushion.
The developping solution uptake zone
The developping solution uptake zone is arranged on the matrix the other end with respect to the above-mentioned expansion liquid zone that is arranged on matrix one end.This district also analyzes smoothly and is provided with in order to absorb the developping solution of supplying with to matrix.The developping solution uptake zone can form matrix longways to guarantee this district.In addition, also can on matrix, set up water-absorbing material to promote expansion.This water-absorbing material can use the high filter paper of the water-retentivity that is made of natural high moleculer eompound, synthetic macromolecular compound etc., sponge etc.The developping solution uptake zone has and has the volumetrical cushion absorbent material that absorbs developping solution fully, by absorbent material is layered on the matrix or under just can prepare the immunoassay instrument of miniaturization.
The matrix agent district
And, when the marker of the reagent area that serves as a mark uses enzyme,, can make and contain matrix in the developping solution or the matrix agent district is arranged near the above-mentioned developping solution drainage area of matrix according to above-mentioned.The matrix agent district, when comprising, being arranged on the above-mentioned expansion fluid cushion that is attached to the developping solution drainage area, when increasing the matrix amount and carry out high sensitivity measuring for preferred.
As matrix, can use various colour developing matrix shown below, fluorescence matrix, luminous host etc. accordingly with the enzyme of labelled reagent.
(a) colour developing matrix peroxidase is used: with 2 of hydrogen peroxide combination, 2 '-azino-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) (ABTS), 3,3 ', 5,5 '-tetramethyl benzidine (TMB), diaminobenzidine (DAB) alkaline phosphatase are used: 5-bromo-4-chloro-3-indyl phosphoric acid (BCIP)
(b) fluorescence matrix alkaline phosphatase is used: 4-methyl umbrella shape phenyl-phosphoric acid ester (4MUP) beta-D-galactosidase is used: 4-methyl umbrella shape phenyl-β-D-galactoside (4MUG)
(c) the luminous host alkaline phosphatase is used: 3-(2 '-spiral shell diamantane)-4-methoxyl group-4-(3 "-the phosphorus acyloxy) phenyl-1; 2-dioxetane disodium salt (AMPPD) beta-D-galactosidase is used: 3-(2 '-spiral shell diamantane)-4-methoxyl group-4-(3 "-β-D-semi-lactosi pyrans glycosyl (ガ ラ Network ト ピ ラ ノ シ Le)) phenyl-1,2-dioxetane (AMGPD) peroxidase is used: with luminol,3-aminophthalic acid cyclic hydrazide, the different luminol,3-aminophthalic acid cyclic hydrazide of hydrogen peroxide combination.
When above-mentioned matrix is set to the matrix district, usually with above-mentioned stromatolysis in the aqueous solution, reach the standard grade after strip ground is coated with launching fluid cushion, make it drying and can form thus, also can add the signal toughener, stablizer, dissolution regulator of matrix etc. as required.The matrix district does not then limit especially if be attached in the expansion fluid cushion of matrix end.Developping solution and the matrix amount of adding on the expansion fluid cushion can be determined according to condition determination, but per 1 utensil can use about 5～500 μ g usually.
The configuration in each district
Fig. 6-Fig. 8 schematically expresses the preferred immunoassay instrument of 1 example of the present invention.Among Fig. 6, numbering 2 is matrixes, the 4th, and the labelled reagent district, the 8th, sample drop district, the 3rd, the developping solution drainage area, the 5th, the developping solution uptake zone, 6a is an A type influenza virus detection zone, the 7th, matrix agent district, the 9th, sample.In this example, sample drop district 8 and labelled reagent district 4 are same.Numbering 6b is a Type B influenza virus detection zone, the 10th, and developping solution is confirmed the district, the 11st, launch liquid bath (with reference to following embodiment 6).
Measure the using method of utensil
Can utilize mensuration utensil of the present invention to measure A type influenza virus in the various sample specimens.Measure following carrying out, at first after the sample drop district with sample supply mensuration utensil of the present invention, supply with developping solution, on matrix, launch to launching fluid cushion.As dilution of sample liquid, can use the damping fluid that contains tensio-active agent as the sample diluent.Developping solution moves in matrix by capillary action, arrives the developping solution uptake zone, not with detection zone bonded sample in composition, enzyme labelling reagent etc. be absorbed, finished expansion.Behind specific time (being generally 10 fens～20 minutes), observe detection zone, utilize the A type influenza virus in the sample liquid to measure the marker that is fixed on test section, can carry out the mensuration of A type influenza virus thus.This detection can according to marker or and the corresponding respectively determinator of range estimation or colorimeter, fluorophotometer, photon counter, photosensory membrane etc. that uses of the enzyme that uses of marker implement.In mensuration, for example the method for the colour developing by measuring detection zone is easy.In addition, in the method,, can carry out semiquantitative analysis by using the colour code (colour chart) corresponding with the concentration of A type influenza virus.And, can also utilize colorimeter etc. that the colour developing of detection zone is quantized, thereby carry out quantitatively.
With Fig. 6-preferred immunoassay instrument of an example of the present invention shown in Figure 8 is example, further specifies measuring principle.9 in sample being dropped in when sample spot drips in the district 8, the developping solution that launches in the liquid bath 11 is supplied with to developping solution drainage area 3.Developping solution utilizes capillary phenomenon to move towards the horizontal white direction of arrow in matrix 2.The matrix that contains in the matrix agent district 7 is dissolved in the developping solution, moves with developping solution.On the other hand, the traget antibody reaction in sample 9 and the labelled reagent district 4, the immune complex of formation mark.When developping solution reaches here, immune complex one side of mark reacts with the matrix in the developping solution, in matrix 2, move on one side, when arriving A type influenza virus detection zone 6a, react with the anti-influenza type A virus antibody that is fixed among the A type influenza virus detection zone 6a, be fixed among the A type influenza virus detection zone 6a.When containing A type influenza virus in sample, the reaction by marker enzyme and matrix causes colour developing.On the other hand, when not containing A type influenza virus in sample, antigen antibody reaction can not take place in traget antibody, because mark is not fixed among the A type influenza virus detection zone 6a, so do not cause colour developing.Therefore, whether develop the color just to know whether contain A type influenza virus in the sample according to A type influenza virus detection zone 6a.Confirm in the district 10 at developping solution, react with reagent in the developping solution and the enzyme that develops the color is fixed,, know that then developping solution reaches the there if developping solution is confirmed district's 10 colour developings.Perhaps also can confirm that locking pin is to the antibody of marker enzyme in the district 10 at developping solution, arrive developping solution and confirm that the labelled reagent in district 10 is by this antibody capture, and then when developping solution arrives, the colour developing thereby the matrix in captive mark and the developping solution reacts (in the following embodiments, the alkali-resistivity phosphatase antibody being fixed).In addition, in labelled reagent district 4, contain underlined anti-Type B Antibody of Influenza, on the other hand, if the Type B influenza virus detection zone 6b that has fixed anti-Type B Antibody of Influenza is set, then also can carry out the detection of Type B influenza virus simultaneously, can be A type or Type B with single measurement operation investigation influenza virus.
In addition, above-mentioned matrix can also be stacked, on the bracing member that is fixed on plastics, metal, paper etc. and use.Above-mentioned matrix is set up the liquid bath that contains developping solution by in the shell that is fixed on plastics etc. in the developping solution drainage area, above-mentioned each district's part is protected with the shell of having opened the hole, thereby can be constituted the utensil of easy handling.As the sample that in immunoassay instrument of the present invention, uses, from collections such as people, animals, be considered to contain the sample of influenza virus, can enumerate various body fluid, for example nasal cavity and wipe away liquid, nasal cavity imbibition, pharynx and wipe away body fluid extracting solutions such as liquid.
Below further specify the present invention by reference example, embodiment, but the present invention is not by these
Embodiment limits.
Embodiment
The preparation of embodiment 1 anti-influenza type A virus monoclonal antibody
Immunogen has been used and has been contained the antigenic influenza HA vaccine of nucleoprotein influenza (change blood development: (IVR-116) strain of A/New Caledonia/20/99 (H1N1), A/ Panama/2007/99 (H3N2) NIB-41) strain).The Freund's complete adjuvant (ヤ ト ロ Application corporate system) that adds equivalent in immunogen after mixing fully, carries out immunity 4 times to the Balb/c mouse in 2 weeks at interval.Injecting immune is former in 3 days forward direction mouse peritoneals of cytogamy, uses PEG to merge mouse boosting cell and myeloma cell (P3U1).Nutrient solution uses HAT to select substratum, gathers culture supernatant and use for screening after about 2 weeks.1 when screening use will contain the antigenic influenza HA vaccine of nucleoprotein influenza, A type influenza reorganization nucleoprotein antigen (derive from A/New Caledonia/20/99 (r-NP/H1N1), derive from (r-NP/H3N2) of A/ Kitakyushu/159/93; The DDBJ/GeneBank database) immobilization enzyme-linked immunosorbent assay (ELISA method).Promptly, the influenza HA vaccine is diluted to x300 doubly with 0.1M carbonic acid buffer pH9.6, and same A type reorganization nucleoprotein antigen is diluted to the concentration of 1 μ g/ml, respectively adds 100 μ l in each hole of miniature culture plate module (Nunc corporate system), hatch an evening, immobilization at 4 ℃.Secondly, wash each hole with the PBS (PBS-Tween) that contains 0.1% Tween20 (trade(brand)name) after, add 1% bovine serum albumin (BSA) 300 μ l with the PBS dilution, spend the night 4 ℃ of sealings.Remove after the confining liquid, add 100 μ l culture supernatant, 37 ℃ of reactions 1 hour.After fully cleaning with PBS-Tween, to add 100 μ l with the every hole of the anti-mouse Igs of enzyme labelling antibody (DAKO corporate system) of 2000 times of the 0.05M phosphoric acid buffer pH7.5 that contains 0.2%BSA, 0.2% エ マ Le ゲ Application 985,1% sucrose, 1%KCl (reaction liquid) dilutions, 37 ℃ of reactions 1 hour.After the reaction, with fully clean with PBS-Tween, every hole adds 2 of 100 μ l, 2 '-azino-two-3-ethyl benzo thiazole phenanthroline-6-sulfonic acid (ABTS), at room temperature reaction after 30 minutes, every hole adds the reaction terminating liquid of 100 μ l, has measured the colour developing level under predominant wavelength 415nm commplementary wave length 490nm.
For being judged to be the male culture supernatant, employing is carried out 2 screenings with influenza Type B reorganization nucleoprotein (B/ sorb/166/98 (r-NP/B) DDBJ/GeneBank database) for antigenic ELISA, finally obtained producing and the reaction of A type influenza virus nucleoprotein antigen, with hybridoma 3 strains (hybridoma FVA2-3, hybridoma FVA2-6 and hybridoma FVA2-11) of the nonreactive antibody of influenza Type B nucleoprotein antigen.These hybridomas are in above-mentioned patent microbial preservation center preservation.The subclass of the monoclonal antibody that is obtained by these hybridoma 3 strains all is IgGI.Table 1 is expressed reactive result of hybridoma.
The reactivity of table 1 monoclonal antibody
| Monoclonal antibody | Antigen | |||
| r-NP/H1N1 | r-NP/H3N2 | Vaccine | r-NP/B | |
| FVA2-3 FVA2-6 FVA2-11 | +++ +++ +++ | +++ +++ +++ | 2.206 2.183 2.223 | 0.018 0.017 0.018 |
+++, relates to
The reactivity of embodiment 2 monoclonal antibodies in the Western blotting
Use recombinant antigen, influenza HA vaccine (containing nucleoprotein antigen), viral liquid to utilize the Western blotting to confirm reactivity.In the 0.01M Tris-HCl (pH8.0) that contains 0.001M EDTA, 1%SDS, 5%2ME (2 mercapto ethanol), 10% glycerol, 0.005%BPB (tetrabromophenol sulfonphthalein), after 100 ℃ of heat treated, the power supply swimming is used with each sample dissolution.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) uses e-PAGEL10% gel strength (ATTO corporate system) and follows ordinary method.Transfer printing is on pvdf membrane (ATTO corporate system) behind the electrophoresis, seals at 4 ℃ with Block Ace (big Japanese drugmaker system) and spends the night.After removing confining liquid and cleaning with PBS-Tween, add the monoclonal antibody of the concentration that is adjusted to 10 μ g/ml, reaction is 45 minutes under the room temperature.After fully cleaning with PBS-Tween, at room temperature reacted 45 fens with reacting the anti-mouse IgG antibody (Cappel corporate system) that is diluted to 4000 times enzyme labelling with liquid.After fully cleaning with PBS-Tween, use ECLWestern trace detection system (ア マ シ ヤ system life science corporate system) with X ray film (kodak corporate system) exposure 1～15 minute, detection signal.
Monoclonal antibody FVA2-3, FVA2-6, FVA2-11 are for all antigen, being that bands of a spectrum have been seen at the center near the 55-70kD.Fig. 1 expresses this result.
Adopt 0.1M phosphoric acid buffer pH7.5 in each hole of miniature culture plate module (Nunc corporate system), respectively to add 100 μ l 3 kinds of monoclonal antibody purifications, at 4 ℃ of overnight incubation, immobilization with the concentration of 2 μ g/ml.Clean each hole with the PBS (PBS-Tween) that contains 0.1%Tween20 (trade(brand)name) then, add 1% the bovine serum albumin (BSA) of 300 μ l, spend the night 4 ℃ of sealings with the PBS dilution.To in each hole, respectively add 100 μ l with the A type influenza virus liquid that liquid dilutes with reaction, 37 ℃ of reactions 1 hour.After fully cleaning with PBS-Tween, will in each hole, respectively add 100 μ l with 3 kinds of alkali phosphatase enzyme mark monoclonal antibodies of liquid adjustment with reaction, 37 ℃ of reactions 1 hour.After the reaction, fully clean with PBS-Tween, every hole adds the 4-nitrophenyl phosphoric acid ester (4-NPP) of 100 μ l, and after 30 minutes, every hole adds the reaction terminating liquid of 100 μ l at room temperature reaction, has measured the colour developing level under predominant wavelength 415nm commplementary wave length 490nm.The positive is decided to be more than 2.1 times of colour developing level of damping fluid blank (buffer blank), represents reactive power with becoming the final dilution ratio of male.
All demonstrate the reactivity higher 2～8 times in be combined to form with 3 kinds of monoclonal antibodies of gained whole 9 groups than the anti-influenza type A virus monoclonal antibody of market sale.Table 2 is expressed this result.
Table 2
Table 2 antigen measuring ELISA
| Monoclonal antibody | Antigen | ||||
| The enzymic-labelled antibody insolubilized antibody | A/シドニ— /5/97 | A/ Beijing/352/89 | A/New caledonia /20/99 | A/ Panama/2007/99 | |
| The antibody * that FVA2-3 FVA2-6 FVA2-11 is commercially available | The commercially available antibody * of FVA2-3 FVA2-6 FVA2-11 FVA2-3 FVA2-6 FVA2-11 FVA2-3 FVA2-6 FVA2-11 | x640 x640 x640 x640 x640 x640 x640 x640 x640 x320 | x80 x160 x160 x80 x160 x80 x80 x160 x160 x40 | x320 x320 x320 x320 x320 x320 x320 x320 x320 x80 | x640 x640 x640 x640 x640 x640 x640 x640 x640 x80 |
* commercially available monoclonal antibody
The mensuration of the reactive site of embodiment 4 anti-influenza type A virus monoclonal antibodies
The preparation of 4-1 plasmid
In order to determine the recognition site of monoclonal antibody FVA2-11, will amplify 3 fragments of 1-159 amino acid (A fragment), 162-327 amino acid (B fragment) and 327-498 amino acid (C fragment) with the PCR method by the influenza H1N1 strain (New Caledonia/20/99) that 498 amino acid constitute.EcoRI, BamHI site on primer, have been added in advance.PCR purification kit purifying amplified fragments with QIAGEN company, and be inserted into expression shown in Figure 2 and integrated on the EcoRI-BamHI site of expression that GST forms preparation plasmid pWGInf.H1N1-A, pWGInf.H1N1-B and pWGInf.H1N1-C with the NdeI-EcoRI site of plasmid pW6A with plasmid pWG6A.With they transformed into escherichia coli BL21 (DE3) (available from Brookhaven National Laboratory), obtain transformant e. coli bl21 (DE3) pWGInf.H1N1-A, BL21 (DE3) pWGInf.H1N1-B and BL21 (DE3) pWGInf.H1N1-C of amicillin resistance.
The expression of 4-2 recombinant protein (GST+H1N1-A, GST+H1N1-B and GST+H1N1-C)
In containing the LB substratum of penbritin of 50 μ g/ml, 2ml cultivates the transformant for preparing among the 4-1 in 37 ℃.The pre-cultivation reaches after the 0.6-0.8 OD under the 600nm, adds 0.4mM IPTG, carries out induced expression, cultivates 3 hours again.With the centrifugation 2 minutes under 5000rpm of the bacterial culture fluid of 1.5ml, collect thalline, be suspended in the damping fluid (10mM Tris-hydrochloric acid, pH8.0,0.1M sodium-chlor, 1mM EDTA) of 100 μ l the ultrasonic disruption through 15 minutes, fully broken thalline.With it as the thalline sample.
Sds polyacrylamide damping fluid (0.15MTris-hydrochloric acid, pH6.8,6%SDS, 24% glycerol, 6mM EDTA, 2%2-mercaptoethanol, 0.03% tetrabromophenol sulfonphthalein) the 4 μ l that in 8 μ l thalline samples, add 3 times of concentration, after fully stirring, carried out the SDS-polyacrylamide gel electrophoresis.The Western trace is transferred on the Nitrocellulose filter membrane, utilizes the 1%BSA sealing, makes to adopt phosphoric acid buffer (10mM phosphoric acid, pH7.4,0.15M sodium-chlor) to be diluted to 1000 times monoclonal antibody FVA2-11 reaction.Make anti-rat immune globulin rabbit polyclonal antibody (the ダ コ corporate system) reaction of peroxidase labelling again, the matrix colour developing liquid (0.01% aquae hydrogenii dioxidi, 0.6mg/ml 4-chloro-1-naphthols) of cleaning back interpolation 10ml makes it colour developing.Its result determines that monoclonal antibody FVA2-11 only reacts with GST+H1N1-A.
The preparation of 4-3 plasmid
Secondly, the A fragment of influenza H1N1 is further segmented, resulting Segment A 1 (1～90 amino acid), A2 (85～159 amino acid), A3 (44～130 amino acid), A4 (59～103 amino acid), A5 (44～103 amino acid), A6 (59～130 amino acid) and A7 (1～130 amino acid) (Fig. 3) are increased with the PCR method.PCR purification kit purifying amplified fragments with QIAGEN company, and be inserted on the EcoRI-BamHI site of above-mentioned expression with plasmid pWG6A preparation plasmid pWGInf.H1N1-A1, pWGInf.H1N1-A2, pWGInf.H1N1-A3, pWGInf.H1N1-A4, pWGInf.H1N1-A5, pWGInf.H1N1-A6.The A7 fragment is inserted the EcoRI-BamHI site of pW6A shown in Figure 2, preparation plasmid pWInf.H1N1-A7.With they transformed into escherichia coli BL21 (DE3) (obtaining), obtain transformant e. coli bl21 (DE3) pWGInf.H1N1-A1, BL21 (DE3) pWGInf.H1N1-A2, BL21 (DE3) pWGInf.H1N1-A3, BL21 (DE3) pWGInf.H1N1-A4, BL21 (DE3) pWGInf.H1N1-A5, BL21 (DE3) pWGInf.H1N1-A6 and BL21 (DE3) pWInf.H1N1-A7 of amicillin resistance from Brookhaven National Laboratory.
The expression of 4-4 recombinant protein (GST+H1N1-A1, GST+H1N1-A2, GST+H1N1-A3, GST+H1N1-A4, GST+H1N1-A5, GST+H1N1-A6 and H1N1-A7)
In containing the LB substratum of penbritin of 50 μ g/ml, 2ml cultivates the transformant for preparing among the 4-3 in 37 ℃.After OD is 0.6-0.8 under being cultured to 600nm in advance, add 0.4mMIPTG, carry out induced expression, cultivated again 3 hours.With the centrifugation 2 minutes under 5000rpm of the bacterial culture fluid of 1.5ml, collect thalline, be suspended in the damping fluid (10mMTris-hydrochloric acid, pH8.0,0.1M sodium-chlor, 1mM EDTA) of 100 μ l the ultrasonic disruption through 15 minutes, fully broken thalline.With it as the thalline sample.
The sds polyacrylamide damping fluid (0.15MTris-hydrochloric acid, pH6.8,6%SDS, 24% glycerol, 6mM EDTA, 2%2-mercaptoethanol, 0.03% tetrabromophenol sulfonphthalein, molecular weight marker (117.6,113.9,81.2,60.7,47.4,36.1,25.3,19.0,14.7,6.1KD) that in 8 μ l thalline samples, adds 3 times of concentration) 4 μ l, after fully stirring, carried out the SDS-polyacrylamide gel electrophoresis.The Western trace is transferred on the Nitrocellulose filter membrane, utilize 1%BSA sealing after, make and adopt phosphoric acid buffer (10mM phosphoric acid, pH7.4,0.15M sodium-chlor) to be diluted to 1000 times monoclonal antibody FVA2-11 reaction.Make anti-rat immune globulin rabbit polyclonal antibody (the ダ コ corporate system) reaction of peroxidase labelling again, the matrix colour developing liquid (0.01% aquae hydrogenii dioxidi, 0.6mg/ml 4-chloro-1-naphthols) of cleaning back interpolation 10ml makes it colour developing.Fig. 3 expresses each fragment.Fig. 4 expresses the CBB dyeing pattern behind the electrophoresis and the result of Western trace.Know that by above result the recognition site of anti-influenza type A virus monoclonal antibody (FVA2-11) is amino acid 59～130 districts of influenza H1N1.In addition, further the nucleoprotein aminoacid sequence from A type influenza subtype shown in Figure 5 is contemplated to: the reactive site at amino acid numbering 59-130 has consensus sequence, and the aminoacid sequence of for example 90-95, the 111-115 of possess hydrophilic property structure or 120-123 etc. is an epitope.
The preparation of reference example 1 alkali phosphatase enzyme mark anti-influenza type A virus monoclonal antibody
(FVA2-11) carries out pepsin with anti-influenza type A virus monoclonal antibody, cuts off the Fc district and obtains F (ab ')
2, secondly, use 2ME (2 mercapto ethanol, Na カ ラ イ テ ス Network corporate system), cut off disulfide linkage, obtain the F that free exposed thiol group (ab ').The alkaline phosphatase of dimaleoyl imino and F (ab ') are introduced in coupling then, by the gel-filtration purifying, obtain the anti-influenza type A virus antibody of alkali phosphatase enzyme mark.
Reference example 2 anti-Type B influenza virus MONOCLONAL ANTIBODIES SPECIFIC FOR
Immunogen use contain the antigenic influenza HA vaccine of nucleoprotein influenza (B/ sorb/166/98 strain) or A type influenza reorganization nucleoprotein antigen (derive from A/New Caledonia/20/99 (r-NP/H1N1), derive from (r-NP/H3N2) of A/ Kitakyushu/159/93.
The Freund's complete adjuvant (ヤ ト ロ Application corporate system) that adds equivalent in immunogen after mixing fully, carries out immunity 4 times to the Balb/c mouse in 2 weeks at interval.Injecting immune is former in 3 days forward direction mouse peritoneals of cytogamy, uses PEG to merge mouse boosting cell and myeloma cell (P3U1).Nutrient solution uses HAT to select substratum, gathers culture supernatant and use for screening after about 2 weeks.1 when screening use with influenza HA vaccine, influenza Type B reorganization nucleoprotein (r-NP/B) immobilization enzyme-linked immunosorbent assay (ELISA method).Promptly, the influenza HA vaccine is diluted to x300 doubly with 0.1M carbonic acid buffer pH9.6, and same Type B reorganization nucleoprotein antigen is diluted to the concentration of 1 μ g/ml, respectively adds 100 μ l in each hole of miniature culture plate module (Nunc corporate system), at 4 ℃ of overnight incubation, immobilization.Clean each hole with the PBS (PBS-Tween) that contains 0.1% Tween20 (trade(brand)name) then, add 1% the BSA of 300 μ l, seal at 4 ℃ and spend the night with the PBS dilution.Remove after the confining liquid, add 100 μ l culture supernatant, 37 ℃ of reactions 1 hour.After fully cleaning with PBS-Tween, to add 100 μ l with the every hole of the anti-mouse Igs of enzyme labelling antibody (DAKO corporate system) of 2000 times of the 0.05M phosphoric acid buffer pH7.5 that contains 0.2%BSA, 0.2% エ マ Le ゲ Application 985,1% sucrose, 1%KCl (reaction liquid) dilutions, 37 ℃ of reactions 1 hour.After the reaction, fully clean with PBS-Tween, every hole adds 2 of 100 μ l, 2 '-azino-two-3-ethyl benzo thiazole phenanthroline-6-sulfonic acid (ABTS), after 30 minutes, every hole adds the reaction terminating liquid of 100 μ l at room temperature reaction, has measured the colour developing level under predominant wavelength 415nm commplementary wave length 490nm.
About being judged to be the male culture supernatant, employing with A type influenza reorganization nucleoprotein antigen (derive from A/New Caledonia/20/99 (r-NP/H1N1), derive from (r-NP/H3N2) of A/ Kitakyushu/159/93) carry out 2 screenings for antigenic ELISA, finally obtain producing and the reaction of Type B influenza virus nucleoprotein antigen, with hybridoma 3 strains (FrB1-03, FrB1-07, FVB2-16) of the nonreactive antibody of A type nucleoprotein influenza antigen.Above-mentioned each hybridoma is speciallyyed permit biological preservation center according to budapest treaty in the industrial technology synthetic study and has been carried out preservation, the preserving number of hybridoma FrB1-03 is FERM BP-10070, the preserving number of hybridoma FrB1-07 is FERM BP-10071, and the preserving number of hybridoma FVB2-16 is FERM BP-10069.The subclass of the monoclonal antibody that is obtained by these hybridoma 3 strains all is IgGI.
(reactivity of monoclonal antibody in the Western blotting)
Use recombinant antigen, influenza HA vaccine (containing nucleoprotein antigen), utilize the Western blotting to confirm reactivity.SDS-PAGE uses e-PAGE10% gel strength (ATTO-corporate system) and follows ordinary method.Transfer printing is spent the night 4 ℃ of sealings with Block Ace (big Japanese drugmaker system) on pvdf membrane (ATTO-corporate system) behind the electrophoresis.After removing confining liquid and cleaning with PBS-Tween, add the monoclonal antibody of the concentration that is adjusted to 10 μ g/ml, reaction is 45 minutes under the room temperature.After fully cleaning with PBS-Tween, be diluted to 4000 times the anti-mouse IgG of enzyme labelling antibody (Cappel corporate system) with liquid and at room temperature reacted 45 fens with reacting.After fully cleaning with PBS-Tween, use ECLWestern trace detection system (ア マ シ ヤ system life science corporate system) to X ray film (kodak corporate system) exposure 1 minute, detection signal.
Monoclonal antibody FrB1-03, FrB1-07 have seen strong bands of a spectrum near 60-75kD.
The preparation of the anti-Type B Antibody of Influenza of reference example 3 alkali phosphatase enzyme marks
The anti-Type B influenza virus monoclonal antibody (FrB1-03) that use prepares in above-mentioned reference example 3 repeats the processing identical with the foregoing description reference example 1, obtains the anti-Type B Antibody of Influenza of alkali phosphatase enzyme mark thus.
The utensil of embodiment 5 while immunoassay A type influenza virus and Type B influenza virus
As shown in Figure 6, on the position of the terminal 16mm of developping solution uptake zone 5 sides of Nitrocellulose film (the ミ リ Port ア corporate system) matrix 2 of distance wide 5mm, long 50mm and 13.5mm, respectively drop 0.7 μ l contain in embodiment 2 the anti-influenza type A virus antibody (FVA2-11) of preparation and in reference example 2 aqueous solution of the anti-Type B Antibody of Influenza (FrB1-3) of preparation made detection zone 6a and 6b on the Nitrocellulose film and make it dry.And then drop alkali-resistivity phosphatase antibody (ダ コ corporate system) and make it dry on the position of the terminal 11mm of the developping solution uptake zone of distance matrix 25 sides has made developping solution confirmation unit 10.Then, the mixed aqueous solution of the anti-influenza type A virus antibody (5 μ g/ml) of the alkali phosphatase enzyme mark that in reference example 1, prepares to the 5 μ l of drop on the matrix and the anti-Type B Antibody of Influenza (5 μ g/ml) of the alkali phosphatase enzyme mark of preparation in reference example 3, make it dry, made the labelled reagent district that constitutes by enzyme labelling reagent pad 4.
Go up at the filter paper (ミ リ Port ア corporate system) of wide 5mm, long 20mm 100 μ g are become the linear of wide 6.1mm as 5-bromo-4-chloro-3-indyl phosphoric acid (BCIP) drop of matrix, and make it dry system and launch fluid cushion 3.With above-mentioned matrix 2, launch fluid cushion 3, enzyme labelling reagent pad 4 and developping solution absorption pad 5 (filter paper of wide 10mm, long 20mm, thick 1mm (ミ リ Port ア corporate system)) and be fixed on and have in the plastic housing that launches liquid bath 11, prepare Fig. 7 and the utensil 1 of immunoassay A type influenza virus and Type B influenza virus the time shown in Figure 8.
Reference example 4 is the utensil (utensil of previous methods) of immunoassay A type influenza virus and Type B influenza virus simultaneously
The detection zone 6a and the 6b of the utensil 1 of immunoassay A type influenza virus and Type B influenza virus when use is made in embodiment 3, about alkali phosphatase enzyme mark resisiting influenza virus antibody, anti-influenza type A virus monoclonal antibody and anti-Type B influenza virus monoclonal antibody that use to buy respectively make, thereby have made utensil (the エ ス プ ラ イ Application Bing DuA ﹠amp of immunoassay A type influenza virus and Type B influenza virus simultaneously; B (レ of Fuji PVC オ corporate system); Mensuration utensil in the past).
The mensuration of embodiment 6A type influenza virus and Type B influenza virus
The hypotype sample of the A type influenza virus that drop table 3 is put down in writing in the sample drop district 8 of the utensil 1 (mensuration utensil of the present invention) of immunoassay A type influenza virus and Type B influenza virus in preparation in the foregoing description 5 is (as the sample diluent, use contains the damping fluid (pH8.0) of tensio-active agent) after each 30 μ l, pressurize downwards and make it distortion being arranged on press section 12 on the deformation element, to launch in the fluid cushion 3 insertion expansion liquid baths 11 by the jut 13 that is attached on the deformation element, supply with developping solution to launching fluid cushion 3, begin to measure.Measure beginning after 15 minutes, confirmed that according to the colour developing in subject assay district 10 developping solution launches after, measure the colour developing of detection zone 6a and 6b by range estimation.Table 3 is expressed its result.
In addition, in contrast, utilize the said sample 30 μ l of 3 records of mensuration utensil his-and-hers watches in the past that in reference example 4, make similarly to carry out viral detection.Table 3 is expressed its measurement result.
The measurement result of table 3A type influenza virus sub-strain strain
| The strain name | Hypotype | Mensuration utensil in the past | The present invention measures utensil | ||
| Colour developing 6a | Colour developing 6b | Colour developing 6a | Colour developing 6b | ||
| A/Puerto Rico/8/34 | H1 N1 | + | - | + | - |
| A/Singapore/1/57 | H2 N2 | + | - | + | - |
| A/Aichi/2/68 | H3 N2 | + | - | + | - |
| A/equine/Miami/1/63 | H3 N8 | + | - | + | - |
| A/equine/Tokyo/2/71 | H3 N8 | + | - | + | - |
| A/equine/Kentucky/1/81 | H3 N8 | - | - | + | - |
| A/equine/Suffork/89 | H3 N8 | + | - | + | - |
| A/equine/Alaska/1/91 | H3 N8 | + | - | + | - |
| A/equine/Kentucky/1/91 | H3 N8 | - | - | + | - |
| A/equine/Rome/5/91 | H3 N8 | - | - | + | - |
| A/equine/Taby/91 | H3 N8 | - | - | + | - |
| A/equine/Lambourn/22778/92 | H3 N8 | - | - | + | - |
| A/equine/Hong Kong/92 | H3 N8 | - | - | + | - |
| A/equine/Avesta/1/93 | H3 N8 | - | - | + | - |
| A/equine/La Plata/1/93 | H3 N8 | + | - | + | - |
| A/equine/Kentucky/1/94 | H3 N8 | - | - | + | - |
| A/equine/La Plata/1/95 | H3 N8 | + | - | + | - |
| A/equine/La Plata/1/96 | H3 N8 | + | - | + | - |
| A/duck/Czech/56 | H4 N6 | - | - | + | - |
| A/duck/Pennsylvania/10128/83 | H5 N2 | + | - | + | - |
| A/turkey/Massachusetts/3740/65 | H6 N2 | + | - | + | - |
| A/seal/Massachusetts/1/80 | H7 N7 | + | - | + | - |
| A/equine/Prague/1/56 | H7 N7 | - | - | + | - |
| A/equine/Newmarket/1/77 | H7 N7 | - | - | + | - |
| A/turkey/Ontario/67 | H8 N4 | - | - | + | - |
| A/turkey/Wisconsin/66 | H9 N2 | - | - | + | - |
| A/chicken/Germany/N/49 | H10 N7 | - | - | + | - |
| A/duck/England/56 | H11 N6 | - | - | + | - |
| A/duck/Alberta/60/76 | H12 N5 | - | - | + | - |
| A/gull/Maryland/704/77 | H13 N6 | - | - | + | - |
| A/mallard/Astrakhan/263/82 | H14 N5 | + | - | + | - |
| A/duck/Australia/341/83 | H15 N8 | + | - | + | - |
| A/Hokkaido/11/02 | H1 N1 | + | - | + | - |
| A/Hokkaido/1/03 * | H3 N2 | + | - | + | - |
| A/swine/Miyagi/5/03 | H1 N2 | + | - | + | - |
| A/HongKong/483/97 | H5 N1 | - | - | + | - |
| A/HongKong/156/97 | H5 N1 | - | - | + | - |
| A/HongKong/reverse genetics-911 | H5 N1 | + | - | + | - |
| Measure contrast | - | - | - | - | |
*: because egg do not tame, so the measurement result that obtains for employing MDCK culture supernatant from the result, about the hypotype sample of in the past the non-detectable A type of mensuration utensil influenza virus, tester prodigiosin of the present invention enough all detects.
Measure the utensil 1 (mensuration utensil of the present invention) of A type influenza virus and Type B influenza virus when use prepares in the foregoing description 5, investigated similarly to Example 6 and adenovirus (1～7 type), Coxsackie virus (A16, B1～B6 type), herpes simplex virus type 1, Echo virus (3 types, 4 types, 7 types, 22 types, 30 types), enterovirus (71 type), mumps virus, poliovirus (1～3 type), RS virus (subgroup A, subgroup B), parainfluenza virus (1～3 type), intestinal bacteria, pneumobacillus, the green pus bacterium, serratia marcescens, staphylococcus epidermidis, Bacillus proteus, staphylococcus aureus, the rod bacillus, corynebacterium diphtheriae, Candida albicans, streptococcus pyogenes, the bacterium of streptococcus (group B, C, G, F), streptococcus pneumoniae, hemophilus influenzae, Listeria monocytogenes, mycoplasma pneumoniae, chlamydia trachomatis, the cross reactivity of Chlamydia pneumoniae.Its result does not react with wherein any virus or bacterium.
Sequence table
<110>FUJIREBIO INC.
＜120〉anti-influenza type A virus monoclonal antibody and use the immunoassay instrument of this antibody
<130>04PF0292-PCT
<150>JP2003-278527
<151>2003-07-23
<150>JP2003-364316
<151>2003-10-24
<150>JP2004-109763
<151>2004-04-02
<160>8
<170>PatentIn version 3.1
<210>1
<211>82
<212>PRT
＜213〉A type influenza virus H10N7
<400>1
Asn Ser Ile Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Met Arg
35 40 45
Glu Leu Thr Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Glu
50 55 60
Ala Asn Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met Ile
65 70 75 80
Trp His
<210>2
<211>82
<212>PRT
＜213〉A type influenza virus H11N6
<400>2
Asn Ser Ile Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg
35 40 45
Glu Leu Ile Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln
50 55 60
Ala Asn Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met Ile
65 70 75 80
Trp His
<210>3
<211>82
<212>PRT
＜213〉A type influenza virus H13N6
<400>3
Asn Ser Ile Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Thr Gly Arg Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg
35 40 45
Glu Leu Val Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Glu
50 55 60
Ala Asn Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met Ile
65 70 75 80
Trp His
<210>4
<211>82
<212>PRT
＜213〉A type influenza virus H14N5
<400>4
Asn Ser Ile Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Met Arg
35 40 45
Glu Leu Ile Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln
50 55 60
Ala Asn Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met Ile
65 70 75 80
Trp His
<210>5
<211>82
<212>PRT
＜213〉A type influenza virus H1N1
<400>5
Asn Ser Leu Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Arg Arg Val Asn Gly Lys Trp Met Arg
35 40 45
Glu Leu Ile Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Glu
50 55 60
Ala Asn Asn Gly Asp Asp Ala Thr Ala Gly Leu Thr His Met Met Ile
65 70 75 80
Trp His
<210>6
<211>82
<212>PRT
＜213〉A type influenza virus H2N2
<400>6
Asn Ser Leu Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Lys Arg Val Asn Gly Lys Trp Met Arg
35 40 45
Glu Leu Val Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln
50 55 60
Ala Asn Asn Gly Asp Asp Ala Thr Ala Gly Leu Thr His Met Met Ile
65 70 75 80
Trp His
<210>7
<211>82
<212>PRT
＜213〉A type influenza virus H4N6
<400>7
Asn Ser Ile Thr Ile Glu Arg Met Leu Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg
35 40 45
Glu Leu Ile Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln
50 55 60
Ala Asn Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met Ile
65 70 75 80
Trp His
<210>8
<211>82
<212>PRT
＜213〉A type influenza virus H7N7
<400>8
Asn Ser Ile Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg
1 5 10 15
Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys
20 25 30
Lys Thr Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Met Arg
35 40 45
Glu Leu Ile Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln
50 55 60
Ala Asn Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met Ile
65 70 75 80
Trp His
Claims (7)
1. an anti-influenza type A virus monoclonal antibody or its antigen-binding fragment, antigen antibody reaction does not take place with the Type B influenza virus in the nucleoprotein generation antigen antibody reaction of the molecular weight 55-70kD of itself and A type influenza virus basically.
2. monoclonal antibody according to claim 1 or its antigen-binding fragment, the epitope of the amino acid region that the aminoacid sequence of itself and A type influenza virus nucleoprotein is 59～130 reacts.
3. monoclonal antibody according to claim 1 and 2 or its antigen-binding fragment, each subtype virus generation antigen antibody reaction of itself and A type influenza virus.
4. monoclonal antibody according to claim 1 or its antigen-binding fragment, its by according to budapest treaty be with the preserving number FERM BP-10066 carrying out the hybridoma FVA2-3 of international preservation, with the preserving number be FERM BP-10067 carrying out international preservation hybridoma FVA2-6 or with the preserving number be FERM BP-10068 carrying out the hybridoma FVA2-11 of international preservation produce.
5. immunoassay instrument, this utensil is to be provided with labelled reagent district with anti-influenza type A virus antibody of mark movably, sample drop district, developping solution drainage area, developping solution uptake zone on the matrix and anti-influenza type A virus antibody is fixed on the utensil of the A type influenza virus detection zone on the matrix, and the anti-influenza type A virus antibody of above-mentioned mark and at least a antibody that is fixed on the anti-influenza type A virus antibody in the detection zone are each described monoclonal antibodies among the claim 1-4.
6. immunoassay instrument according to claim 5, wherein, in the labelled reagent district, also further contain underlined anti-Type B Antibody of Influenza, and near the A type influenza virus detection zone of having fixed A type influenza virus monoclonal antibody, also further have the Type B influenza virus detection zone of having fixed Type B influenza virus monoclonal antibody.
7. according to claim 5 or 6 described immunoassay instruments, wherein, marker is an enzyme, in developping solution and/or the developping solution drainage area nearby contain matrix at enzyme.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP278527/2003 | 2003-07-23 | ||
| JP2003278527 | 2003-07-23 | ||
| JP2003364316 | 2003-10-24 | ||
| JP364316/2003 | 2003-10-24 | ||
| JP109763/2004 | 2004-04-02 | ||
| JP2004109763 | 2004-04-02 | ||
| PCT/JP2004/010475 WO2005007697A1 (en) | 2003-07-23 | 2004-07-23 | Anti-influenza type a virus monoclonal antibody and immunoassay instrument using the antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1934133A true CN1934133A (en) | 2007-03-21 |
| CN1934133B CN1934133B (en) | 2011-12-14 |
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ID=34084275
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2004800211740A Expired - Fee Related CN1934133B (en) | 2003-07-23 | 2004-07-23 | Anti-influenza type A virus monoclonal antibody and immunoassay instrument using the antibody |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JP4595810B2 (en) |
| KR (1) | KR101133910B1 (en) |
| CN (1) | CN1934133B (en) |
| HK (1) | HK1105005A1 (en) |
| RU (1) | RU2366662C2 (en) |
| WO (1) | WO2005007697A1 (en) |
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| JPH05149951A (en) * | 1991-05-02 | 1993-06-15 | Fujirebio Inc | Test piece for simply measuring immune and measuring method using it |
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| EP1081496A1 (en) * | 1999-08-31 | 2001-03-07 | Becton, Dickinson and Company | Flow-through assay for visually detecting the presence of influenza A and B |
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2004
- 2004-07-23 JP JP2005511913A patent/JP4595810B2/en not_active Expired - Fee Related
- 2004-07-23 WO PCT/JP2004/010475 patent/WO2005007697A1/en active Application Filing
- 2004-07-23 RU RU2006103301/13A patent/RU2366662C2/en not_active IP Right Cessation
- 2004-07-23 CN CN2004800211740A patent/CN1934133B/en not_active Expired - Fee Related
- 2004-07-23 KR KR1020067001430A patent/KR101133910B1/en not_active IP Right Cessation
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2007
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Also Published As
| Publication number | Publication date |
|---|---|
| RU2366662C2 (en) | 2009-09-10 |
| HK1105005A1 (en) | 2008-02-01 |
| WO2005007697A1 (en) | 2005-01-27 |
| RU2006103301A (en) | 2007-09-10 |
| JPWO2005007697A1 (en) | 2008-01-10 |
| JP4595810B2 (en) | 2010-12-08 |
| KR20060054332A (en) | 2006-05-22 |
| KR101133910B1 (en) | 2012-04-13 |
| CN1934133B (en) | 2011-12-14 |
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