CN1826355A - Monoclonal antibody against influenza B virus and immunoassay instrument using the antibody - Google Patents
Monoclonal antibody against influenza B virus and immunoassay instrument using the antibody Download PDFInfo
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- CN1826355A CN1826355A CNA2004800211308A CN200480021130A CN1826355A CN 1826355 A CN1826355 A CN 1826355A CN A2004800211308 A CNA2004800211308 A CN A2004800211308A CN 200480021130 A CN200480021130 A CN 200480021130A CN 1826355 A CN1826355 A CN 1826355A
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Abstract
A highly specific monoclonal antibody against influenza B virus and an immunoassay instrument using the same whereby influenza B virus can be specifically detected. This monoclonal antibody against influenza B virus undergoes an antigen-antibody reaction with a nucleoprotein of influenza virus B having a molecular weight of from 60 to 75 kD but substantially shows no antigen-antibody reaction with influenza A virus.
Description
Technical field
The present invention relates to anti-Type B influenza 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.
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, it is low to measure sensitivity, operates miscellaneously, 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-Type B influenza virus monoclonal antibody in the past, its majority are viral surface antigen HA of identification and NA's, and specificity and reactive low can not satisfy highly sensitive immunoassay.In addition, the circulation type device, though the antibody in the reagent uses the antibody at influenza virus nucleoprotein, measurement operation is miscellaneous, is not easy determinator, the ratio height that false positive occurs when detecting the Type B influenza virus is (with reference to J.Clin.Microbiol; 40:1675-1680,2002).
On the other hand, in order to select curative, a kind of device of demand, this device uses and does not react with A type influenza virus, but antibody with the reaction of the Type B influenza virus of the variant that comprises influenza virus, in the differentiation of influenza A C-type virus C or Type B virus, do not need special diagnostic instrumentation and determinator, and obtain measurement result at short notice, just can differentiate A type or Type B with 1 operation.
Therefore, the purpose of this invention is to provide the high anti-Type B influenza virus monoclonal antibody of a specific specificity.In addition, the present invention also provides a kind of immunoassay instrument that can detect the Type B influenza virus specifically.
The result of inventor's lucubrate, the nucleoprotein of successfully making with the Type B influenza virus is antigen, with the Type B influenza virus the anti-Type B influenza virus monoclonal antibody of specific reaction takes place, thereby has finished the present invention.In addition, by utilizing this Type B influenza virus monoclonal antibody, expected providing can distinguish with A type influenza virus turn up the soil, detect the immunoassay instrument of Type B influenza virus.
That is, the invention provides the nucleoprotein generation antigen antibody reaction with the molecular weight 60-75kD of Type B influenza virus, anti-Type B influenza virus monoclonal antibody or its antigen-binding fragment of antigen antibody reaction do not take place basically with A type 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-Type B Antibody of Influenza of mark movably to be set on the matrix and to make anti-Type B Antibody of Influenza be fixed on the utensil of the Type B influenza virus detection zone on the matrix, and the anti-Type B Antibody of Influenza of above-mentioned mark and at least a antibody that is fixed on the anti-Type B Antibody of Influenza 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 the Type B influenza virus, but the anti-Type B influenza virus monoclonal antibody that does not react with A type influenza virus.The anti-Type B influenza virus monoclonal antibody of the application of the invention is carried out immunoassay, can distinguish with A type influenza virus turn up the soil, detect Type B influenza virus or it is quantitative.According to the present invention, also provide the immunoassay instrument of the anti-Type B influenza virus monoclonal antibody of having used the invention described above.The immunoassay instrument of the application of the invention, can be easy and distinguish with A type influenza virus turn up the soil, detect the Type B 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 the schematic cross-section of an embodiment of expression mensuration utensil of the present invention.
Fig. 3 is the vertical view that expression is placed in mensuration utensil of the present invention an example in the box.
Fig. 4 is the sectional view of the A-A ' of Fig. 3.
The preferred plan that carries out an invention
As mentioned above, the nucleoprotein generation antigen antibody reaction of the molecular weight 60-75kD of monoclonal antibody of the present invention and Type B influenza virus.With the nucleoprotein generation antigen antibody reaction of the molecular weight 60-75kD of Type B influenza virus can by with the Type B influenza virus as sample, the Western blotting that carries out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirms.When carrying out the Western trace when (with reference to following embodiment), the nucleoprotein of monoclonal antibody identification molecular weight 60-75kD of the present invention.
According to above-mentioned, antigen antibody reaction does not take place in monoclonal antibody of the present invention and A type 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 the Type B influenza virus, with the Type B influenza virus antigen antibody reaction does not take place and refers to the degree reaction only understood this area practitioner.In addition, at this, so-called " antigen antibody reaction not taking place basically with A type 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 A type influenza virus.In preferred scheme, monoclonal antibody of the present invention and C type influenza virus do not react basically yet.
In preferred scheme, monoclonal antibody of the present invention is with the nucleoprotein generation antigen antibody reaction of each hypotype of Type B influenza virus.That is,, be classified as all hypotypes according to the structure of these H and N is different though the Type B 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 the hypotype (with reference to table 3) of following embodiment record, and then the nucleoprotein generation antigen antibody reaction of the hypotype of preferred and known all Type B influenza viruses.
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 Type B influenza virus.The nucleoprotein of the Type B 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 Type B influenza virus is as the immunogen immune animal, it is produced cell of Type B 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 Type B 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 selecting to produce the hybridoma of the antibody of discerning Type B 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 Type B 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 Type B 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 FrB1-03, hybridoma FrB1-07 and hybridoma FVB2-16 respectively.These hybridomas are speciallyyed 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) based on budapest treaty to Independent Administrative Leged Industrial Technology Complex Inst respectively and have been submitted international preservation to, wherein, FrB1-03 is represented in the evaluation of hybridoma FrB1-03, preserving number is FERM BP-10070 (preservation day: on July 18th, 2003, transfer international preservation on July 14th, 2004 to by domestic preservation FERM P-19441); Hybridoma FrB1-07 identifies expression FrB1-07, and preserving number is FERM BP-10071 (preservation day: on July 18th, 2003, transfer international preservation on July 14th, 2004 to by domestic preservation FERM P-19442); FVB2-16 is represented in the evaluation of hybridoma FVB2-16, and preserving number is FERMBP-10069 (preservation day: on July 18th, 2003, transfer international preservation on July 14th, 2004 to by domestic preservation FERM P-19440).
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 Type B influenza virus, and various virus strain reactions of Type B influenza virus.Preservation and getable Type B influenza virus preservation strain now (with reference to following table 3) 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 that is used for the Type B influenza virus or quantitative immunoassay.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 A type influenza virus and to turn up the soil, to detect the Type B 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-Type B Antibody of Influenza be fixed on the utensil of the Type B influenza virus detection zone on the matrix with anti-Type B Antibody of Influenza of mark movably, the anti-Type B Antibody of Influenza of above-mentioned mark and to be fixed at least a in the anti-Type B Antibody of Influenza 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 Type B influenza virus detection zone., be fixed on anti-Type B Antibody of Influenza 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 Type B influenza virus detection zone by having is unmarkedly judged.The developping solution that passes through in Type B 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 Type B 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 Type B 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 Type B influenza virus detection zone, also can detect the Type B 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 Type B influenza virus test section of having fixed anti-Type B Antibody of Influenza on the above-mentioned matrix at detection zone.Be fixed on the anti-Type B Antibody of Influenza of this test section and the anti-Type B Antibody of Influenza of mark described later, at least a is anti-Type B influenza virus monoclonal antibody of the present invention, and preferably these two kinds of antibody are anti-Type B influenza virus monoclonal antibodies.The above-mentioned anti-Type B Antibody of Influenza 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 anti-Type B influenza virus polyclonal antibody, can from market sale, and the polyclonal antibody that can obtain easily suitable the selection use.
The anti-Type B Antibody of Influenza of this detection zone is above-mentioned antibody, monoclonal antibody can also be used separately or mixed and use.Anti-Type B Antibody of Influenza can be IgG antibody, IgM antibody, can also be antigen-binding fragment F (ab), the F (ab ') of these antibody
2Deng.
The anti-Type B Antibody of Influenza 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-Type B Antibody of Influenza 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-Type B influenza virus monoclonal antibody in conjunction with the time, can be undertaken by above-mentioned chemical bond or physical adsorption.
In detection zone, except the Type B influenza virus test section that adopts anti-Type B Antibody of Influenza, the A type influenza virus test section that adopts anti-influenza type A virus antibody can be set also.This A type influenza virus test section, if above-mentioned Type B influenza virus test section near, then the transfusion direction at developping solution can be the either side in above-mentioned Type B influenza virus test section upstream side or downstream side.At A type influenza virus detection zone fixed anti-influenza type A virus antibody, also can be polyclonal antibody or monoclonal antibody, same with above-mentioned anti-Type B Antibody of Influenza, can be fixed on the matrix by chemical bond or physical adsorption.
As above-mentioned, the detection zone on matrix is provided with the Type B influenza virus test section that adopts anti-Type B Antibody of Influenza at least, and then A type influenza virus test section is set, and measures at the same time on Type B influenza virus and the A type 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-Type B Antibody of Influenza 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-Type B Antibody of Influenza that serves as a mark and the above-mentioned antibody that is arranged on detection zone, at least a is anti-Type B influenza virus monoclonal antibody, preferably these two kinds of antibody are anti-Type B influenza virus monoclonal antibodies.The antibody of the anti-Type B Antibody of Influenza that serves as a mark similarly can also use its fragment with the antibody of above-mentioned detection zone.
The anti-Type B Antibody of Influenza 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-Type B Antibody of Influenza 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-Type B Antibody of Influenza of mark.In addition, as non-covalent bonded method, can enumerate physisorphtion etc.
According to above-mentioned, except detecting the Type B influenza virus, when also detecting A type influenza virus simultaneously, add the anti-influenza type A virus antibody of mark in the labelled reagent district and prepare utensil.The anti-Type B Antibody of Influenza of the mark that contains in the labelled reagent district and the anti-influenza type A virus antibody of mark, in matrix or water-absorbent pad wherein a kind of, contain, perhaps can make matrix and water-absorbent pad contain the underlined anti-Type B Antibody of Influenza and the anti-influenza type A virus antibody of mark in the two.When measuring when carrying out Type B influenza virus and A type 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 of mark and the anti-influenza type A virus antibody amount of 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 mark and the anti-influenza type A virus antibody of mark contain 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 4).
Measure the using method of utensil
Can utilize mensuration utensil of the present invention to measure Type B 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 Type B influenza virus in the sample liquid to measure the marker that is fixed on test section, can carry out the mensuration of Type B 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 Type B influenza virus.And, can also utilize colorimeter etc. that the colour developing of detection zone is quantized, thereby carry out quantitatively.
With Fig. 2-preferred immunoassay instrument of an example of the present invention shown in Figure 4 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 Type B influenza virus detection zone 6b, react with the anti-Type B Antibody of Influenza that is fixed among the Type B influenza virus detection zone 6b, be fixed among the Type B influenza virus detection zone 6b.When containing the Type B influenza virus in sample, the reaction by marker enzyme and matrix causes colour developing.On the other hand, when not containing the Type B influenza virus in sample, antigen antibody reaction can not take place in traget antibody, because mark is not fixed among the Type B influenza virus detection zone 6b, so do not cause colour developing.Therefore, whether develop the color just to know whether contain the Type B influenza virus in the sample according to A type influenza virus detection zone 6b.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-influenza type A virus antibody, on the other hand, if the A type influenza virus detection zone 6a that has fixed anti-influenza type A virus antibody is set, then also can carry out the detection of A type influenza virus simultaneously, can be Type B or A type 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 immunogen use contains the antigenic influenza HA vaccine of nucleoprotein influenza (B/ sorb/166/98 strain) or Type B influenza reorganization nucleoprotein (derives from (r-NP/B) of B/ sorb/166/98; The DDBJ/GeneBank database).
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 screening the time used with influenza HA vaccine, influenza Type B reorganization nucleoprotein (r-NP/B) immobilization enzyme-linked immunosorbent assay (ELISA method).Promptly, the influenza HA vaccine with 0.1M carbonic acid buffer pH9.6 be diluted to * 300 times, 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), 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.
About being judged to be the male culture supernatant, adopt with influenza A type 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) carries out 2 screenings for antigenic ELISA, finally obtained generation and Type B influenza virus nucleoprotein antigen reacts, with hybridoma 3 strains (hybridoma FrB1-03, hybridoma FrB1-07 and hybridoma FVB2-16) of the nonreactive antibody of influenza A type nucleoprotein antigen.The subclass of the monoclonal antibody that is obtained by these hybridoma 3 strains all is IgGl.Table 1 is expressed the result.
The reactivity of table 1 monoclonal antibody
| Monoclonal antibody | Antigen | |||
| r-NP/B | Vaccine | r-NP/H1N1 | r-NP/H3N2 | |
| FrB1-03 FrB1-07 FVB2-16 | 1.682 1.721 1.404 | 0.578 0.596 0.431 | 0.020 0.024 0.019 | 0.022 0.023 0.016 |
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.
For monoclonal antibody FrB1-03, FrB1-07, bands of a spectrum near 60-75kD, have been seen.Fig. 1 expresses this result.
The antigen measuring of embodiment 3 in the ELISA that has used monoclonal antibody
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 more equal and even exceed 4 times reactivity 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 | ||
| Enzyme labelling | Solid phase | B/ sorb/166/98 | B/ is triple/and 1/93 |
| FrB1-03 | FrB1-03 FrB1-07 FVB2-16 | ×40 ×20 ×40 | ×80 ×80 ×80 |
| FrB1-07 | FrB1-03 FrB1-07 FVB2-16 | ×40 ×40 ×40 | ×80 ×80 ×160 |
| FVB2-16* | FrB1-03 FrB1-07 FVB2-16 | ×80 ×80 ×80 | ×320 ×320 ×160 |
| Commercially available antibody * | Commercially available antibody * | ×20 | ×80 |
*: Fab ' mark
*: commercially available monoclonal antibody
The anti-Type B influenza virus of reference example 1 alkali phosphatase enzyme mark MONOCLONAL ANTIBODIES SPECIFIC FOR
Above-mentioned anti-Type B influenza virus monoclonal antibody (FrB1-03) is carried out pepsin, cut off the Fc district and obtain 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-Type B Antibody of Influenza of alkali phosphatase enzyme mark.
The preparation of reference example 2 anti-influenza type A virus monoclonal antibodies
Immunogen used contain the antigenic influenza HA vaccine of nucleoprotein influenza (change the blood development: (IVR-116) strain of A/New Caledonia/20/99 (H1N1), A/ Panama/2007/99 (H3N2) (NIB-41), B/ sorb/166/98 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 with influenza HA vaccine, influenza A type reorganization nucleoprotein antigen (derive from A/New Caledonia/20/99 (r-NP/H1N1), derive from (r-NP/H3N2) of A/ Kitakyushu/159/93) immobilization enzyme-linked immunosorbent assay (ELISA method).Promptly, the influenza HA vaccine with 0.1M carbonic acid buffer pH9.6 be diluted to * 300 times, 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 is carried out 2 screenings with influenza Type B reorganization nucleoprotein antigen (B/ sorb/166/98 (r-NP/B)) 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.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 FVA2-3 is FERM BP-10066, the preserving number of hybridoma FVA2-6 is FERM BP-10067, and the preserving number of hybridoma FVA2-11 is FERM BP-10068.
The preparation of reference example 3 alkali phosphatase enzyme mark anti-influenza type A virus monoclonal antibodies
The anti-influenza type A virus monoclonal antibody (FVA2-11) that use prepares in above-mentioned reference example 2 repeats the processing identical with above-mentioned reference example 1, has obtained the alkali phosphatase enzyme mark anti-influenza type A virus monoclonal antibody.
The utensil of embodiment 3 while immunoassay A type influenza virus and Type B influenza virus
As shown in Figure 2, 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 3, 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 1, 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. 3 and the utensil 1 of immunoassay A type influenza virus and Type B influenza virus the time shown in Figure 4.
Reference example 4 is the utensil (in the past measuring utensil) 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, thus 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 4 A type influenza viruses and Type B influenza virus
The hypotype sample of the Type B 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 3 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.
The measurement result of table 3 Type B strains of influenza viruses
| Sample | Dilution ratio | Mensuration utensil in the past | The present invention measures utensil | ||
| Colour developing 6a | Colour developing 6b | Colour developing 6a | Colour developing 6b | ||
| Sorb/166/98 | ×100 | - | + | - | + |
| ×200 | - | +/- | - | + | |
| ×400 | - | - | - | + | |
| ×800 | - | - | - | - | |
| Shandong/7/97 | ×400 | - | +w | - | + |
| ×800 | - | - | - | +/- | |
| ×1,600 | - | - | - | - | |
| Triple/1/93 | ×400 | - | + | - | + |
| ×800 | - | - | - | + | |
| ×1,600 | - | - | - | +/- | |
| B/Lee/40 | ×100 | - | + | - | + |
| ×1,000 | - | + | - | + | |
| ×10,000 | - | - | - | +w | |
| ×100,000 | - | - | - | - | |
| B/ Hokkaido/26/99 * | ×100 | - | + | - | + |
| ×1,000 | - | + | - | + | |
| ×10,000 | - | + | - | + | |
| ×100,000 | - | - | - | - | |
| Chorioallantois liquid | - | - | - | - | |
| MEM medium | - | - | - | - | |
*: owing to egg is not tamed, so measurement result for adopting the MDCK culture supernatant to obtain
In addition, in contrast, utilize the said sample 30 μ l that in the past measure 3 records of utensil his-and-hers watches that in reference example 4, make similarly to carry out viral detection.Table 3 is expressed its measurement result.
From the result, about the various samples of in the past the non-detectable Type B influenza virus of mensuration utensil, 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 3, 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.
Claims (5)
1. an anti-Type B influenza virus monoclonal antibody or its antigen-binding fragment, antigen antibody reaction does not take place with A type influenza virus in the nucleoprotein generation antigen antibody reaction of the molecular weight 60-75kD of itself and Type B influenza virus basically.
2. monoclonal antibody according to claim 1 or its antigen-binding fragment, its by according to budapest treaty with preserving number FERM BP-10070 carried out international preservation hybridoma FrB1-03, carried out the hybridoma FrB1-07 of international preservation or produced with preserving number FERM BP-10071 with the hybridoma FVB2-16 that preserving number FERM BP-10069 has carried out international preservation.
3. immunoassay instrument, this utensil is to be provided with labelled reagent district, sample drop district, developping solution drainage area, the developping solution uptake zone with anti-Type B Antibody of Influenza of mark movably on the matrix and to make anti-Type B Antibody of Influenza be fixed on the utensil of the Type B influenza virus detection zone on the matrix, and the anti-Type B Antibody of Influenza of above-mentioned mark and at least a antibody that is fixed on the anti-Type B Antibody of Influenza in the detection zone are claim 1 or 2 described monoclonal antibodies.
4. immunoassay instrument according to claim 3, wherein, in the labelled reagent district, also further contain underlined anti-influenza type A virus antibody, and near the Type B influenza virus detection zone of having fixed Type B influenza virus monoclonal antibody, also further have the anti-influenza type A virus detection zone of having fixed A type influenza virus monoclonal antibody.
5. according to claim 3 or 4 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 (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003278528 | 2003-07-23 | ||
| JP278528/2003 | 2003-07-23 |
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| Publication Number | Publication Date |
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| CN1826355A true CN1826355A (en) | 2006-08-30 |
| CN100441597C CN100441597C (en) | 2008-12-10 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004800211308A Expired - Fee Related CN100441597C (en) | 2003-07-23 | 2004-07-23 | Monoclonal antibody against influenza B virus and immunoassay instrument using the antibody |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPWO2005007698A1 (en) |
| KR (1) | KR20060054333A (en) |
| CN (1) | CN100441597C (en) |
| HK (1) | HK1096975A1 (en) |
| RU (1) | RU2366663C2 (en) |
| WO (1) | WO2005007698A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102112878A (en) * | 2008-06-06 | 2011-06-29 | 国立大学法人富山大学 | Device for detection of influenza virus |
| CN103052884A (en) * | 2010-08-03 | 2013-04-17 | 田中贵金属工业株式会社 | Immunochromatography reagent composition, and measurement method using same |
| CN105683756A (en) * | 2013-09-10 | 2016-06-15 | 电化生研株式会社 | Method for measuring influenza b virus |
| CN105793707A (en) * | 2013-11-29 | 2016-07-20 | 积水医疗株式会社 | Immunochromatography-assisted detection method |
| CN109485723A (en) * | 2017-09-12 | 2019-03-19 | 松下知识产权经营株式会社 | Can in conjunction with the nuclear protein matter of influenza virus antibody, use its composite material, detection device and method |
| CN114276441A (en) * | 2020-09-27 | 2022-04-05 | 东莞市朋志生物科技有限公司 | Anti-influenza B virus antibody, preparation method thereof and detection kit |
| CN114276440A (en) * | 2020-09-27 | 2022-04-05 | 东莞市朋志生物科技有限公司 | Antibody and detection kit for influenza B virus |
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| CN102388310B (en) | 2009-04-09 | 2014-07-16 | 爱科来株式会社 | Sample analyzing tool, method of manufacturing sample analyzing tool, and method of minimizing drop in solution permeability of deployment member |
| JP2010261912A (en) * | 2009-05-11 | 2010-11-18 | Bl:Kk | Immunology-detection method of human influenza virus h3 subtype |
| RU2491338C2 (en) * | 2011-06-30 | 2013-08-27 | Федеральное государственное бюджетное учреждение "Научно-исследовательский Институт гриппа" Министерства здравоохранения и социального развития Российской Федерации (ФГБУ "НИИ гриппа" Минздравсоцразвития России) | Using monoclonal antibodies for identifying yamagata or victorian influenza b viral evolution line, hybridoma 4h7 strain for preparing monoclonal antibodies used to identify influenza b viruses in yamagata branch, hybridoma b/4h1 strain for preparing monoclonal antibodies used to identify influenza b viruses in victorian branch |
| TW201346040A (en) | 2012-03-30 | 2013-11-16 | Tanaka Precious Metal Ind | Kit for identifying influenza a virus |
| JP6262969B2 (en) | 2013-09-10 | 2018-01-17 | デンカ生研株式会社 | Method for measuring influenza A virus |
| WO2017082213A1 (en) * | 2015-11-09 | 2017-05-18 | 国立大学法人京都工芸繊維大学 | SEPARATING AGENT FOR HUMAN SERUM-DERIVED IgG POLYCLONAL ANTIBODIES, AND METHOD FOR SEPARATING HUMAN SERUM-DERIVED IgG POLYCLONAL ANTIBODIES USING SAME |
| CN117384279A (en) * | 2022-07-05 | 2024-01-12 | 东莞市朋志生物科技有限公司 | Anti-influenza B virus antibody, and reagent and kit for detecting influenza B virus |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2518607B2 (en) * | 1985-08-29 | 1996-07-24 | 雪印乳業株式会社 | Monoclonal antibody against influenza A virus HA antigen and method for producing the same |
| JP3334558B2 (en) * | 1997-04-23 | 2002-10-15 | 富士レビオ株式会社 | Enzyme immunoassay and test strips |
-
2004
- 2004-07-23 JP JP2005511914A patent/JPWO2005007698A1/en active Pending
- 2004-07-23 KR KR1020067001431A patent/KR20060054333A/en not_active Application Discontinuation
- 2004-07-23 WO PCT/JP2004/010476 patent/WO2005007698A1/en active Application Filing
- 2004-07-23 CN CNB2004800211308A patent/CN100441597C/en not_active Expired - Fee Related
- 2004-07-23 RU RU2006103302/13A patent/RU2366663C2/en not_active IP Right Cessation
-
2007
- 2007-02-12 HK HK07101652.0A patent/HK1096975A1/en unknown
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102112878A (en) * | 2008-06-06 | 2011-06-29 | 国立大学法人富山大学 | Device for detection of influenza virus |
| CN103052884A (en) * | 2010-08-03 | 2013-04-17 | 田中贵金属工业株式会社 | Immunochromatography reagent composition, and measurement method using same |
| CN103052884B (en) * | 2010-08-03 | 2015-07-15 | 田中贵金属工业株式会社 | Immunochromatography reagent composition, and measurement method using same |
| CN105683756A (en) * | 2013-09-10 | 2016-06-15 | 电化生研株式会社 | Method for measuring influenza b virus |
| CN105683756B (en) * | 2013-09-10 | 2017-10-24 | 电化生研株式会社 | The assay method of influenza B virus |
| CN105793707A (en) * | 2013-11-29 | 2016-07-20 | 积水医疗株式会社 | Immunochromatography-assisted detection method |
| CN105793707B (en) * | 2013-11-29 | 2018-05-08 | 积水医疗株式会社 | The detection method of immunochromatography auxiliary |
| CN109485723A (en) * | 2017-09-12 | 2019-03-19 | 松下知识产权经营株式会社 | Can in conjunction with the nuclear protein matter of influenza virus antibody, use its composite material, detection device and method |
| CN114276441A (en) * | 2020-09-27 | 2022-04-05 | 东莞市朋志生物科技有限公司 | Anti-influenza B virus antibody, preparation method thereof and detection kit |
| CN114276440A (en) * | 2020-09-27 | 2022-04-05 | 东莞市朋志生物科技有限公司 | Antibody and detection kit for influenza B virus |
| CN114276440B (en) * | 2020-09-27 | 2022-12-27 | 东莞市朋志生物科技有限公司 | Antibody and detection kit for influenza B virus |
| CN114276441B (en) * | 2020-09-27 | 2022-12-27 | 东莞市朋志生物科技有限公司 | Anti-influenza B virus antibody, preparation method thereof and detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2366663C2 (en) | 2009-09-10 |
| RU2006103302A (en) | 2007-09-10 |
| JPWO2005007698A1 (en) | 2008-01-17 |
| KR20060054333A (en) | 2006-05-22 |
| WO2005007698A1 (en) | 2005-01-27 |
| CN100441597C (en) | 2008-12-10 |
| HK1096975A1 (en) | 2007-06-15 |
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