CN101220097A - Monoclone antibody of H5 subtype avian influenza virus haemagglutinin protein, or its combination liveness fragment and uses thereof - Google Patents

Abstract

The invention provides a McAb which can be specifically binded with the hemagglutinin (HA) protein of H5N1 avain influenza virus and a McAb which can prevent the binding activity of at least 50 percent of McAb with the hemagglutinin (HA) protein of H5N1 avain influenza virus. The McAb can be used for detecting, diagnosing, preventing and treating the avain influenza virus, in particular to the H5N1 avain influenza virus. The McAb of the invention also provides the relating hybridoma cell lines, the separated nucleic acid molecule and short peptide, as well as the drug combination, the medicine diagnosis equipment and the kit with the McAb included.

Description

The monoclonal antibody of H5 subtype avian influenza virus hemagglutinin or it is in conjunction with active fragments and uses thereof

Related application

[0001] the present patent application requires to be derived from the right of priority of the Chinese patent application of submitting on January 26th, 2,006 200610002312.1, and the full content of this application is contained among the present patent application.

Invention field

[0002] but the present invention relates to the monoclonal antibody of specificity in conjunction with H5 subtype avian influenza virus hemagglutinin (HA) albumen, and conservative property varient or active fragments, or the correlative coding sequence of its polypeptide or polypeptide analog, or produce the cell strain of described monoclonal antibody, and use the method that this antibody or fragment are used for Clinics and Practices.

Background technology

[0003] from the bird flu of H5 type in 1996 (Xu X et al. since the gaggle on farm, China Guangdong Province breaks out at first, 1999, Virology), virus is drilled born another strain H5 virus and is broken out in the bird farm (in April, 1997) and the market (in November, 1997) in Hong Kong thus, caused that avian influenza virus is for the first time directly by the birds-to-human transmission incident in history, there are 6 people's death front and back in the totally 18 routine confirmed cases.Since 2003, the H5 virus strain is broken out in succession whole East Asia and country in Southeast Asia, and WHO and influenza scholarly forecast H5 avian influenza virus will most possibly become the epidemic strain that human next time big influenza is broken out.At the beginning of 2004, H5 type high pathogenic avian influenza is also successively broken out in tens provinces of China, and in the Hong-Kong, Thailand, Holland etc. find the incident that the bird flu of H5 type infects multiple animals such as chicken, duck, heron, tiger, cat.More alarmingly be, doubtful people occurred in Thailand and infected people's incident that also there is the many cases report in Malaysia.Enter 2005, European countries such as Romania, Russia, Turkey find that in succession bird infects the deadly incident of H5 type avian influenza virus, the expert thinks that this is the result with malicious migratory bird migration, and this makes the further diffusion of the highly pathogenic H5 type avian influenza virus of control propagate and becomes more difficult.Scholarly forecast, propagation along with transient, H5 type avian influenza virus might be by African country Eurasian, that the Asia and Africa land bridge further propagates into the sanitary condition extreme difference, make chance and the time that H5 type avian influenza virus obtains and other human influenza virus fully recombinates, to form a kind of brand-new influenza virus highly fatal to the mankind when the time comes probably, its various losses that bring to the mankind will be difficult to estimate.According to the WHO statistics, by on January 19th, 2006, the whole world reached 80 examples because of the human case that infects H5N1 virus death, brought great test for global public health security.

[0004] current research result (Li, K.S.et al., 2004, Nature) show, south China aquatic bird (tame duck) is the main carrier and the circulator of H5 type avian influenza virus, and the outburst of H5 type bird flu has obvious seasonal, and is accompanied by the differentiation of biological multiformity (several genes type).Yet molecule epidemic disease-ology research shows, the duck group plants nearly 30% the positive and infects and there is no any symptom at present, and Ji Qunzhong has also that to reach 10% popular and asymptomatic band malicious.These infected animals can constantly make the people be subjected to new infection again, and human beings'health is constituted huge threat.The relevant expert is consistent to be thought and will control H5 type Highly Pathogenic Avian Influenza Virus (HPAIV) popular in whole East Asia, South East Asia and European various countries well, and early diagnosis is a prerequisite, just can accomplish then early to isolate, early handle, and the people is accomplished early treatment.

[0005] adopt traditional virus separation and serum diagnosis method diagnosis avian influenza virus to take 4-5 days, and most people and animal disease control system experimentation chamber lack the three-grade biological safety laboratory at present, so the diagnosis that break out H5 in Southeast Asian countries and area obviously lags behind.Common situation is that a large amount of chickens are dead does not have the breadboard report of making a definite diagnosis with slaughtering after action is finished yet, brings very big inconvenience for breaking out of control virus.In addition, because of minority bird (particularly aquatic bird, as tame duck) exists the malicious situation of asymptomatic band, the quarantine system does not have effective detection means again, and the sustainable development of this situation has caused and should virus break out repeatedly in a plurality of countries and regions, delays continuous situation.

[0006] simultaneously, because H5 type avian influenza virus (wherein Goose/Guangdong/1/96 is a representative strains) belongs to highly pathogenic virus, animal model commonly used at present all there is lethality, and the hemagglutinin (HA) that adopts gene engineering method to express can not completely be obtained antigenicity, and the monoclonal antibody that a plurality of in the world famous laboratories successively attempt preparing at this virus stock is not all succeeded.At present this virus antigenicity analysis is had to adopt specificity and the reactive monoclonal antibody by A/chicken/Pennsylvania/1370/83 (H5N2) and A/chicken/Pennsylvania/8125/83 (H5N2) preparation that does not all obviously reach the diagnostic reagent requirement.

[0007] in view of said circumstances, at present urgent expectation can have a kind of convenient, fast, real-time diagnostic method and means, thereby in time the first-generation being striden ethnic infectious patient isolates for treatment, prevent that virus from developing to human-to-human transmission, before virus does not adapt to the mankind, just interrupt it and propagate chain, thereby fundamentally eliminate of the big influenza threat of this virus the mankind.

[0008] China is domestic, and relevant anti-avian influenza virus H5 hypotype detects the existing bibliographical information of research.(Qin Aijian such as likes to build in the Qin of institute of animal husbandry and veterinary medicine of Yangzhou University, Shao Hongxia, Qian Kun etc., China's Preventive Veterinary Medicine newspaper, 2003,03 phase) developed anti-avian influenza virus H5 and H9 hypotype hemagglutinin monoclonal antibody specific, used these monoclonal antibodies and carry out indirect immunofluorescence assay and be proved to be and in 24 hours, detect corresponding avian influenza virus rapidly.Beijing Administration for Entry-Exit Inspection and Quarantine utilizes fluorescence RT-PCR rapid detection highly pathogenic avian influenza virus H5 hypotype, shortens to 4 hours detection time, on December 10th, 2005 by clinical verification.Guo Yuanji has summarized H 5 hypotype strain antibody tests and has needed with microneutralization experiment or the high ELISA (Guo Yuanji of specificity in " human and bird fluenza present Research " literary composition, China's experiment and clinical virology magazine, 2004, but the relevant ELISA of utilization detects the research document of H5 hypotype does not see relevant report 03 phase).

[0009] the existing report of the external relevant research that utilizes ELISA to detect H5N1 antibody.Rowe etc. have reported and have used the reorganization hemagglutinin as antigen coated, and utilize indirect ELISA to detect H5N1 antibody, the sensitivity of its ELISA is 80%, and specific degree is that 62% (April 1999 for Rowe T.et al., J.Clin.Microbiol.; 37 (4): 937-43), but the document is not the monoclonal antibody at H5N1 hypotype hemagglutinin HA gene specific.(Zhou such as Zhou, E.M.et al., Avian Dis., 1998,42 (4): 757-61), (Shafer such as Shafer, A.L.et al., Avian Dis., 1998,42 (1): 28-34) adopt the competitive ELISA method to detect the anti-core protein antibody of avian influenza virus, but detected object is the NP protein antibodies of all H1-H16 hypotypes of A type bird flu, can not determine hypotype.Lu has reported that the Dot-ELISA based on monoclonal antibody detects avian influenza virus (AIV), and this method directly detects AIV antigen, and its specificity is can cross reaction (Lu H., Avian Dis., 2003,47 (2): 361-9) not take place with other bird virus.Though Sala etc. have set up the ELISA based on the special monoclonal antibody of H7 hypotype surface glycoprotein, its hypotype is H7; Monoclonal antibody is the special monoclonal antibody of surface glycoprotein, is not monoclonal antibody (Sala G, Cordioli P, Moreno-Martinet al.Avian Dis.2003,47 (3Suppl): 1057-9) of H5 hypotype hemagglutinin HA gene specific.

[0010] regrettably, the monoclonal antibody major part that existing avian influenza virus immunology diagnosis means are used at be nucleoprotein (NP albumen), thereby its detection is A type (also claiming the first type) influenza virus, but in fact A type influenza virus comprises H1～H16 totally 16 hypotypes, the equal no pathogenicity of wherein sizable hypotype or low pathogenicity is arranged has only the H5 subtype avian influenza virus to be the maximum Highly Pathogenic Avian Influenza Virus (HPAIV) of harm.Thereby prior art far can not satisfy the needs of clinical detection.

[0011] basic goal of the present invention is to overcome the defective of existing avian influenza virus immunoassay technology, the monoclonal antibody that is adopted at be the HA albumen of H5 hypotype, thereby can special detection have highly pathogenic H5 subtype avian influenza virus.

Summary of the invention

[0012] but the invention provides the monoclonal antibody of specificity in conjunction with H5 subtype avian influenza virus hemagglutinin (HA) albumen, and the monoclonal antibody of capable of blocking at least 50% monoclonal antibody and H5 subtype avian influenza virus hemagglutinin (HA) protein binding activity. the present invention also provides relevant hybridoma cell strain, isolated nucleic acid molecule and small peptide, with and contain pharmaceutical composition and the medical diagnositc equipment and the test kit of this monoclonal antibody.The present invention also provides and has utilized this monoclonal antibody to survey, diagnosis, prevention and treatment avian influenza virus, the particularly method of H5 subtype avian influenza virus.

Description of drawings

[0013] Fig. 1 is the measurement result of golden mark method H5 subtype influenza virus HA antigen detection kit, and wherein two red line appear in " a ", regard as the positive; Nature controlling line only appears in " b ", regards as feminine gender; Red line does not appear in " c ", and it is invalid then to regard as.

[0014] Fig. 2 is the measurement result of golden mark method H5 subtype influenza virus anti-HA antibody assay kit, and wherein nature controlling line only appears in " a ", regards as the positive; Two red line appear in " b ", regard as feminine gender; Red line does not appear in " c ", and it is invalid then to regard as.

[0015] Fig. 3 is the measurement result of H5 subtype influenza virus HA antigen detection kit, and wherein, oval color development area appears in "+" expression, regards as the positive; Oval color development area does not appear in "-" expression, regards as feminine gender.

[0016] Fig. 4 is the expression plasmid figure of 6 kinds of chimeric antibodies: pcDNA3.1-Ak8H5, pcDNA3.1-AH8H5, pcDNA3.1-Ak10F7, pcDNA3.1-AH 10F7, pcDNA3.1-Ak4D1 and pcDNA3.1-AH4D1.

[0017] Fig. 5 is the blood clotting inhibition experiments of three kinds of chimeric antibodies to the Ck/HK/Yu22/02 strain, wherein, and the 1st, 2,3 row: PBS contrast; The 4th and 5 row: 10F7cAb; The 6th row: 10F7mAb; The 7th and 8 row: 4D1cAb; The 9th row: 4D1mAb; The 10th and 11 row: 8H5cAb; The 12nd row: 8H5mAb.

[0018] Fig. 6 is chimeric antibody and the immunofluorescence test experience result who expresses the cell response of H5 hemagglutinin, and wherein A is cAb 4D1 (DAPI); B is cAb 4D1 (FITC); C is cAb 10F7 (DAPI); D is cAb 10F7 (FITC); E is anti--HBV cAb (DAPI); F is anti--HBV cAb (FITC).

[0019] Fig. 7 is the histogram of the ELISA detected value OD (450/620) of phage polypeptide.

[0020] Fig. 8 is the plasmid synoptic diagram of pTO-T7 and pTO-T7-239-123.

[0021] Fig. 9 is the plasmid synoptic diagram of pTO-T7 and pTO-T7-239-125.

[0022] Figure 10 is the SDS-PAGE electrophorogram of fusion rotein 239-123 purification of samples, wherein, and 1: the molecular wt label; 2: the whole lysates of E.coli of expressing 239-123; The supernatant of the ultrasonic lysate of 3:239-123; 4: the 239-123 among the damping fluid I; 239-123 purified fusion protein in the 5:2M urea; The purified fusion protein of 239-123 in the 6:4M urea; The purified fusion protein of 239-123 in the 7:8M urea.

[0023] Figure 11 is the SDS-PAGE electrophorogram of fusion rotein 239-125 purification of samples, wherein, and 1: the molecular wt label; 2: the whole lysates of E.coli of expressing 239-125; 3: the centrifugation supernatant of whole lysates; 4: the 239-125 among the damping fluid I; The purified fusion protein of 239-125 in the 5:2M urea; The purified fusion protein of 239-125 in the 6:4M urea; The purified fusion protein of 239-125 in the 7:8M urea.

[0024] Figure 12 has shown the binding characteristic of 239-123 fusion rotein: the colouring intensity histogram of its ELISA detected value OD (450/620), wherein, the 239-123 fusion rotein is attached on the various strains that transverse axis identifies.

[0025] Figure 13 has shown the binding characteristic of 239-125 fusion rotein: the colouring intensity histogram of its ELISA detected value OD (450/620), wherein, the 239-125 fusion rotein is attached on the various strains that transverse axis identifies.

[0026] Figure 14 is that the 239-123 fusion rotein is attached to the colouring intensity of the ELISA detected value OD (450/620) of 8H5mAb (trilateral dotted line) or 8C11mAb (square dotted line) under various mAb dilutions.

[0027] Figure 15 is the plasmid figure of pC149-mut and pC149-mut-123.

[0028] Figure 16 is the plasmid figure of pC149-mut and pC149-mut-125.

[0029] Figure 17 is the SDS-PAGE electrophorogram of full born of the same parents' lysate of the recombinant protein of expressing in a small amount, wherein, and 1:D123; 2:T123; 3:F123; 4:Q123; 5:D125; 6:T125; 7:F125; 8:Q125.

[0030] Figure 18 is the SDS-PAGE electrophorogram of purification of recombinant proteins, wherein, and 1:D123; 2:T123; 3:F123; 4:Q123; 5:D125; 6:T125; 7:F125; 8:Q125.

[0031] Figure 19 is the Electronic Speculum figure of viruslike particle, and this viruslike particle is that the polypeptide by segmental fusion rotein of HBV cAg and binding antibody assembles.

[0032] Figure 20 is for showing the colouring intensity histogram of the ELISA detected value OD (450/620) of binding affinity between fusion rotein HBc-123/125 and the monoclonal antibody 8H5.

[0033] Figure 21 is for showing the colouring intensity histogram of the ELISA detected value OD (450/620) of binding affinity between fusion rotein HBc-Q123 or HBc-D125 and the various monoclonal antibody; The result shows that fusion rotein HBc-Q123 and HBc-D125T are specifically in conjunction with monoclonal antibody 8H5.

[0034] Figure 22 has shown that ELISA detects HBc-123 fusion protein immunization mouse serum antibody titer upcurve, and wherein, the time is 0-4 week, and antibody titers detects by ELISA.

[0035] Figure 23 has shown that ELISA detects HBc-125 fusion protein immunization mouse serum antibody titer upcurve, and wherein, the time is 0-4 week, and antibody titers detects by ELISA.

[0036] Figure 24 has shown that immunofluorescence detects the proteic reaction of expressing in immune mouse serum and the SF21 cell of HA.

[0037] Figure 25 is the Electronic Speculum figure of the viruslike particle of HBc-122, HBc-124, HBc-128 and the assembling of HBc-129 recombinant protein.

[0038] Figure 26 is the colouring intensity histogram of the ELISA detected value OD (450/620) of the binding affinity between demonstration fusion rotein HBc-122, HBc-124, HBc-128 and HBc-129 and the various monoclonal antibody; The result shows that fusion rotein HBc-122, HBc-124, HBc-128 and HBc-129 are attached to monoclonal antibody 8H5 specifically.

[0039] Figure 27 shown by 12 peptide fusion proteins assemblings viruslike particle compete the result who combines the 8H5 enzyme labelled antibody with H5N1 virus, wherein, the longitudinal axis is colouring intensity (with OD (a 450/620) value representation), and transverse axis is used various types of virion and a PBS contrast in the experiment.

Embodiment

[0040] relational term that relates in the present patent application is defined as follows:

[0041] " hemagglutinin " speech among the present invention refers to the envelope glycoprotein of avian influenza virus.Hemagglutinin mediation influenza virus is at the absorption of host cell and enter.The hemagglutinin of avian influenza virus has 16 serology hypotypes, and HA1-HA16 corresponds respectively to 16 virus subtypes, i.e. H1-H16.

[0042] " antibody " speech among the present invention refers to any one immunoglobulin (Ig), comprise can binding specificity antigenic monoclonal antibody, many anti-, dual specifics or multi-specificity antibody.A complete antibody comprises two heavy chains and two light chains.Every heavy chain contains a variable region and three constant regions of first, second, third, etc.; Every light chain comprises a variable region and a constant region.Antibody is " Y " type, and the neck of " Y " type structure contains the second and the 3rd constant region of two heavy chains, and it forms by disulfide-bonded.Every arm of " Y " type structure contains wherein first constant region of a heavy chain and the variable region and the constant region of a variable region and a light chain.The variable region of light chain and heavy chain determines antigenic combination; Three hypervariable regions are all contained in the variable region of every chain, claim that (CDR of light chain (L) comprises LCDR1, LCDR2, LCDR3 to complementary determining region (CDR), the CDR of heavy chain (H) comprises HCDR1, HCDR2, (it is named by people such as Kabat HCDR3, see Sequencesof Proteins of Immunological Interest, Fifth Edition (1991), the 1-3 volume, NIHPublication 91-3242, Bethesda Md).Wherein, three CDR are spaced apart by framework region (FR).Framework region is more conservative and form a shelf shape support structure hypervariable region than the CDR district.The constant region of heavy chain and light chain combines irrelevant with antigen, but has multiple effector function.Antibody can be divided into several classes according to the aminoacid sequence of CH, mainly is: IgA, IgD, IgE, IgG and IgM, wherein some class also further is divided into subclass, as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 etc.

[0043] " antibody " speech among the present invention, ultrawhite except that refering in particular to complete immune globulin, the fragment (as being an immunocompetence section of immunoglobulin molecules at least) that also refers to immunoglobulin (Ig) is as Fab, Fab ', F (ab ') 2, Fv fragment, single-chain antibody molecule or the multi-specificity antibody that formed by any fragment of the immunoglobulin molecules that contains one or more CDR district.In addition, the antibody that the present invention relates to also can be the antibody that is formed in conjunction with the framework region of one or more different human normal immunoglobulins by the one or more CDR district in the specific human normal immunoglobulin.

[0044] " Fab " fragment that antibody is relevant is meant the part of the antibody molecule that the variable region of the variable region of containing a light chain and a constant region and a heavy chain and constant region are got up through disulfide-bonded.

[0045] " Fab ' " fragment is meant the Fab fragment that has comprised the part hinge area.

[0046] F (ab ') 2 refers to the dimer of Fab '.

[0047] part of the antibody that becomes through disulfide-bonded of second, third constant region of " Fc " of antibody second, third constant region of referring to first heavy chain and second heavy chain.The Fc section of antibody has multiple different function, but does not participate in antigenic combination.

[0048] refer to can be in conjunction with the minimal segment of the antibody of complete antigen binding site for " Fv " of antibody section.A Fv fragment comprises that the variable region of a light chain is attached to the variable region of a heavy chain.

[0049] " single-chain antibody " among the present invention or " scFv " refer to the engineered antibody (Houston 1988) that is directly linked to each other with variable region of heavy chain or be formed by connecting by a peptide chain by variable region of light chain

[0050] " the single-chain antibody Fv-Fc " among the present invention or " scFv-Fc " comprise that also scFv connects the engineered antibody of the Fc section formation of antibody.

[0051] " antigenic determinant " among the present invention (or claim epi-position) refers in the antigen molecule that part of amino acid or the atomic radical with antibodies.

[0052] " monoclonal antibody " speech among the present invention refers to a segment from antibody in a group height homologous antibody molecule or antibody, also promptly except that the spontaneous mutation that only under a few cases, may occur, and the identical antibody molecule of a group.Monoclonal antibody has high specific to the single epi-position on the antigen.Monoclonal antibody is with how anti-different, and resist is the antibody molecule that has comprised the different epi-positions on the identification antigen more.Though traditional monoclonal antibody is by the hybridoma excretory, the monoclonal antibody that the present invention relates to is not limited in this preparation method.As, the monoclonal antibody that the present invention relates to can adopt the hybridoma technology of reported first such as Kohler to obtain (Nature, 256:495,1975), also can adopt recombinant DNA technology to obtain (as referring to U.S.P 4,816,567).

[0053] " chimeric antibody " speech among the present invention refers to light chain of antibody or/and the part of heavy chain is the identical or homologous antibody of sequence that is derived from a certain specific species or belongs to a certain specific antibodies class or subclass, and light chain of antibody is or/and another part of heavy chain is the identical or homologous antibody of sequence that is derived from another species or belongs to another antibody class or subclass.In any case, this antibody fragment still kept to target antigen (U.S.P 4,816,567to Cabilly et al. in conjunction with active; Morrison et al., Proc.Natl.Acad.Sci.USA, 81:68516855 (1984)).

[0054] among the application, the all or part of CDR district that " humanized antibody " speech refers to people source immunoglobulin (Ig) (receptor antibody) obtains antibody or antibody fragment after being replaced by the CDR district of a non-human antibody (donor antibody), and donor antibody wherein can be that expection specificity, affinity and reactive mouse, rat or rabbit antibody are arranged.In addition, the aminoacid sequence of the framework region of people source immunoglobulin (Ig) (FR) also can be replaced by the aminoacid sequence of corresponding non-human antibody.And the amino-acid residue of humanized antibody also can be neither derive from receptor antibody, also non-CDR district or the framework region sequence that derives from donor antibody.These manually modified purposes are further to improve or optimization antibody performance.In a word, humanized antibody be meant contain at least one, two almost complete variable regions normally, wherein Dui Ying all or nearly all CDR district are from non-human antibody, FR district all or almost all wherein is from human antibody.The ideal humanized antibody contains the part in the Fc district of immunoglobulin (Ig) at least, normally the Fc district of people source immunoglobulin (Ig).More detailed contents see also: Jones et al., Nature, 321:522525 (1986); Reichmann et al., Nature, 332:323329 (1988); Presta, Curr.Op.Struct.Biol., 2:593596 (1992); And Clark, Immunol.Today 21:397402 (2000).

[0055] " separated " speech of relating to of the application refers to and obtains through artificial means under the native state.If the material or the composition of a certain " separated " appear in occurring in nature, may be that its residing natural surroundings has taken place to change or separate out this material under natural surroundings so, or the two situation all have generation.Such as naturally occurring certain not separated polynucleotide or polypeptide in, a certain living animal body, and highly purified identical polynucleotide or the polypeptide separated under this native state promptly are referred to as separated.Here " separated " do not got rid of and is mixed with artificial or the synthetic material, and not getting rid of existence does not influence active other impurity of material yet.

[0056] " carrier " speech refers among the present invention, a kind of nucleic acid launch vehicle that certain proteic polynucleotide of coding can be inserted wherein and the albumen acquisition is expressed.Carrier can make its genetic material element that carries be expressed at host cell inner expression by conversion, transduction or transfection host cell.For instance, carrier comprises: plasmid; Phagemid; Coemid; The artificial chromosome (PAC) in artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 source; Phage such as lambda particles phage or M13 phage and animal virus etc.Animal virus kind as carrier has retrovirus (comprising slow virus), adenovirus, adeno-associated virus, simplexvirus (as hsv), poxvirus, baculovirus, papilloma virus, papova viruses (as SV40).A kind of carrier may contain the element that various control is expressed, and comprises promoter sequence, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene.In addition, carrier also can contain replication origin.Carrier also might include assists it to enter the composition of cell, as virion, liposome or protein coat, but not only has only these materials.

[0057] " host cell " speech refers to the cell that imports carrier among the present invention, comprise following many cell types, as prokaryotic cell prokaryocytes such as intestinal bacteria or withered grass bacterium, as fungal cells such as yeast cell or aspergillus tubigensis, as insect cells such as S2 drosophila cell or Sf9, perhaps as fibroblast, Chinese hamster ovary celI, COS cell, the NSO cell, the HeLa cell, bhk cell, the zooblast of HEK 293 cells or people's cell.

[0058] " neutralizing antibody " speech refers to and can remove or significantly reduce antibody or the antibody fragment of target viral antigen in conjunction with virulence.

[0059] " sequence same percentage " speech refer to nucleic acid in the candidate sequence or amino acid respectively with the corresponding nucleic acid or the per-cent of peptide sequence amplifying nucleic acid or amino acid homogeny.Here relate to nucleotide sequence or polypeptide preface to relevant term " sequence similarity per-cent " be defined as candidate nucleic acid sequence or amino acid residue sequence respectively to the similar per-cent of purpose nucleotide sequence or aminoacid sequence.For some sequences, it and aim sequence are compared, can skip the sudden change breach in case of necessity, reaching the maximum similar per-cent of gene, and do not go to consider any conservative property sudden change of similar sequences.The multiple comparison method of this area can be used for determining the similarity of nucleic acid or aminoacid sequence, comprises BLAST as the available computer software, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) etc.The staff of skilled understands to the suitable measuring parameter of comparison setting, is included as and uses some algorithms of maximum comparability to reach and the comparison of full length sequence.

[0060] " specificity in conjunction with " speech refers to, and refers to two intermolecular nonrandom association reactions, as antibody with produce reaction between the antigen of this antibody.Herein, in conjunction with first kind of antigenic antibody to second kind of antigenic binding affinity be detect less than or very weak.In some embodiments, certain antigen-specific antibodies is meant with avidity (KD)≤10 ^-5M is (as 10 ^-6M, 10 ^-7M, 10 ^-8M, 10 ^-9M, 10 ^-10M etc.) in conjunction with this antigen, wherein KD refers to the ratio (koff/kon) of dissociation yield and combination rate, and its method that can adopt those skilled in the art to be familiar with is measured.

[0061] antibody

[0062] monoclonal antibody of the present invention can specificity in conjunction with the H5 subtype avian influenza virus.One aspect of the present invention relates to monoclonal antibody and the corresponding antigen binding fragment thereof of energy specificity in conjunction with H5 subtype avian influenza virus hemagglutinin.

[0063] anti-H5 monoclonal antibody of the present invention is by mouse hybridoma cell strain 8H5,3C8, and 10F7,4D1,3G4 and 2F2 are secreted.The title of these monoclonal antibodies is named with its corresponding hybridoma cell strain.Just, these anti-H5 monoclonal antibodies are produced by hybridoma cell strain 8H5,3C8,10F7,4D1,3G4 and 2F2 respectively, and difference called after 8H5,3C8,10F7,4D1,3G4 and 2F2.Monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2 energy specificity are in conjunction with H5 subtype avian influenza virus hemagglutinin.Mouse hybridoma cell strain 8H5,3C8,10F7,4D1,3G4 and 2F2 on January 17th, 2006 in China typical culture collection center (CCTCC, Wuhan University, Wuhan, China) carry out preservation, preserving number is CCTCC-C200607 (hybridoma cell strain 8H5), CCTCC-C200605 (hybridoma cell strain 3C8), CCTCC-C200608 (hybridoma cell strain 10F7), CCTCC-C200606 (hybridoma cell strain 4D1), CCTCC-C200604 (hybridoma cell strain 3G4) and CCTCC-C200424 (hybridoma cell strain 2F2).

[0064] the present invention relates to monoclonal antibody and also comprise and can block monoclonal antibody 8H5,3C8,10F7,4D1,3G4 or 2F2 are in conjunction with the monoclonal antibody of H5 subtype avian influenza virus hemagglutinin.Epi-position on these monoclonal antibody bonded hemagglutinin can be identical with the epi-position that monoclonal antibody 8H5,3C8,10F7,4D1,3G4 or 2F2 are discerned.The epi-position that these monoclonal antibodies are discerned also can spatially have overlapping with the epi-position that monoclonal antibody 8H5,3C8,10F7,4D1,3G4 or 2F2 are discerned.Such monoclonal antibody can reduce the bonding force at least 50% of monoclonal antibody 8H5,3C8,10F7,4D1,3G4 or 2F2 and H5 subtype avian influenza virus hemagglutinin, or at least 60%, or preferably at least 70%, or more preferably at least 75%, or more preferably at least 80%, or more preferably at least 85%, or more preferably at least 90%, or more preferably 95%, or most preferably 99%.

[0065] can adopt ordinary method such as Antibodies:A Laboratory Manual, ColdSpring Harbor Laboratory, the method of describing among the Ed Harlow and David Lane (1988) is measured a certain unknown monoclonal antibody and is reduced the ability of a certain known monoclonal antibody in conjunction with the H5 hemagglutinin.For example, earlier be coated on antigen on the microwell plate in advance, hatching in the microwell plate behind the above-mentioned pre-bag quilt of the common adding of the known monoclonal antibody behind the mark of the antibody unlabelled to be measured of serial dilution and specific concentrations, known antibodies is attached to the quantity on the plate under the different dilution antibody to be measured of washing back mensuration then.The ability of antibody competition known antibodies conjugated antigen to be measured is strong more, and the ability of known antibodies conjugated antigen is just weak more.Usually, antigen is to be coated in advance on the 96 hole microwell plates, and utilizes radio-labeled method or enzyme labelling method to measure unmarked monoclonal antibody and block the ability of mark monoclonal antibody.

[0066] can adopt the hybridoma preparation method of report in Nature 256:495 (1975) such as Kohler to prepare monoclonal antibody.At first with immunogen (adding adjuvant in the time of necessity) immunization mouse or other appropriate host animal.The injection system of immunogen or adjuvant is generally subcutaneous multi-point injection or abdominal injection.Immunogen lotus root in advance is linked to some known protein, on serum albumin or soybean one's mother's sister enzyme inhibitors, may help the immunogenicity of enhancement antigen in the host.Adjuvant can utilize freund adjuvant or MPL-TDM etc.After animal was subjected to immunity, the lymphocyte that has the former antibody of secretion specificity binding immunoassay in the body produced.In addition, lymphocyte also can utilize external immunity to obtain.Collect purpose lymphocyte and myeloma cell also with suitable fusogen,, merge to obtain hybridoma (Goding as PEG, MonoclonalAntibodies:Principles and Practice, pp.59-103, Academic Press, 1996).

[0067] hybridoma of above-mentioned preparation can be inoculated in the suitable nutrient solution and grow, and preferably contains material that one or more can suppress not merge, parent myeloma cell growth in the nutrient solution.For example, to lacking the parent myeloma cell of xanthoglobulin guanine monophosphate transferring enzyme (HGPRT or HPRT), in nutrient solution, add the growth that xanthoglobulin, aminopterin-induced syndrome and thymus pyrimidine materials such as (HAT substratum) can suppress the HGPRT-deficient cells.

[0068] preferred myeloma cell should have the fusion rate height, and the antibody-secreting ability is stable, to abilities such as HAT nutrient solution sensitivities.Wherein, the first-selected mouse of myeloma cell source myelomatosis, as MOP-21 and MC-11 mouse tumor strain (the THE Salk Institute Cell Distribution Center that derives, San Diego, Calif.USA), with SP-2/0 or X63-Ag8-653 cell strain (American Type Culture Collection, Rockville, Md.USA).Also there is the research report to utilize human myeloma and people mouse allos myeloma cell strain to prepare people's monoclonal antibody (Kozbor, J.Immunol., 133:3001 (1984) in addition; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).

[0069] nutrient solution of hybridoma growth is used to detect the generation at the monoclonal antibody of specific antigen.The binding specificity of measuring the monoclonal antibody of hybridoma generation has immunoprecipitation or external in conjunction with test, as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA).For example, the Scatchard analytical method of utilizing Munson etc. to describe at Anal.Biochem.107:220 (1980) can be used to measure the avidity of monoclonal antibody.

[0070] after specificity, avidity and the reactivity of the antibody that hybridoma produces are determined, the purpose cell strain can pass through (Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103, Academic Press, 1996) limiting dilution assay of described standard carries out subcloning.Suitable nutrient solution can be DMEM or RPMI-1640 etc.In addition, the form that hybridoma can also ascitic tumor is grown in animal body.

[0071] utilizes traditional immunoglobulin purification method,, the monoclonal antibody of subclone emiocytosis can be separated from cell culture fluid, ascites or serum as albumin A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography etc.

[0072] monoclonal antibody of the present invention can also be to obtain by the genetically engineered recombinant technology.Utilize specificity to carry out pcr amplification, can from hybridoma, separate the dna molecular of obtain encoding monoclonal antibody heavy chain and light chain gene in conjunction with the nucleic acid primer of monoclonal antibody heavy chain and light chain gene.The gained dna molecular inserts in the expression vector, and transfection host cell does not then produce the myeloma cell of immunoglobulin (Ig) as E.coli cell, ape and monkey COS, Chinese hamster ovary celI or other.Target antibody is cultivated and expressed to host cell after the transfection under given conditions.

[0073] combination has high specific and high-affinity to antibody of the present invention to the H5 hemagglutinin.These antibody may have weak cross reactivity to the hemagglutinin of other hypotype, and preferably, these antibody do not have cross reactivity fully to the hemagglutinin of other hypotype.On the one hand, the KD value of antibodies H5 hemagglutinin of the present invention is less than 1 * 10 ^-5M; Preferably, the KD value is less than 1 * 10 ^-6M; More preferably, the KD value is less than 1 * 10 ^-7M, most preferably, the KD value is less than 1 * 10 ^-8M.

[0074] monoclonal antibody of the present invention can be the antibody that comprises traditional " Y " type structure shape of two heavy chains and two light chains.In addition, described antibody also can be Fab fragment, Fab ', the F (ab) that has kept on the antibody of traditional " Y " type structure shape of hemagglutinin avidity ₂, Fv or other type the part fragment, its avidity in conjunction with hemagglutinin can be higher or lower than the antibody of traditional " Y " type structure shape.

[0075] antibody fragment of the present invention can utilize the complete antibody molecule of hydrolysis to obtain (referring to Morimoto et al., J.Biochem.Biophys.Methods 24:107-117 (1992) andBrennan et al., Science 229:81 (1985)).In addition, these antibody fragments also can directly produce (reviewed in Hudson, Curr.Opin.Immunol.11:548-557 (1999) by recombinant host cell; Little et al., Immunol.Today, 21:364-370 (2000)).Such as, Fab ' fragment can directly obtain from the E.coli cell or chemical lotus root connection forms F (ab ') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).For another example, F (ab ') ₂Fragment can connect acquisition with leucine zipper GCN4.In addition, Fv, Fab or F (ab ') ₂Fragment also can be directly directly separated from the recombinant host cell nutrient solution and is obtained.Those of ordinary skill in the art knows other technology of preparation antibody fragment fully.

[0076] antibody nucleotide sequence

[0077] the present invention relates to specificity in conjunction with the antibody of H5 hemagglutinin or the coding nucleic acid molecule of antibody fragment.The nucleic acid molecule of encoding antibody can separate from hybridoma and obtains.Those of ordinary skill in the art knows the nucleotide sequence that utilizes routine techniques can measure these molecules fully.The antibody nucleic acid molecule that the present invention relates to also can utilize traditional genetically engineered recombinant technology or chemical synthesis process to obtain.The sequence of the antibody nucleic acid molecule that the present invention relates on the one hand, has comprised the variable region of heavy chain of anti-H5 antibody or the part nucleotide sequence of antibody molecule.The sequence of the antibody nucleic acid molecule that the present invention relates on the other hand, also comprises the variable region of light chain of anti-H5 antibody or the part nucleotide sequence of antibody molecule.The sequence of the antibody nucleic acid molecule that the present invention relates on the other hand, also comprises the CDR sequence of heavy chain or variable region of light chain.

[0078] an aspect of of the present present invention relates to the nucleic acid molecule of coding monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2 heavy chain and light chain variable region sequence.The nucleic acid molecule of monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2 weight chain variabl area sequence corresponds respectively to SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:24.The nucleic acid molecule of monoclonal antibody 8H5,3C8,10F7,4D1 and 2F2 light chain variable region sequence corresponds respectively to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:18 and SEQ ID NO:26.The invention still further relates to the nucleic acid molecule varient or the analog that have comprised monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2 heavy chain and light chain variable region sequence.

[0079] on the other hand, the invention still further relates to the varient of various isolated nucleic acid molecule, the identical SEQ ID of its sequence NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:24 or SEQ ID NO:26 with following nucleotide sequence.Particularly, the homogeny of the sequence of nucleic acid varient and following nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:24 or SEQ ID NO:26 reaches 70% at least, preferably reaches 75% at least, more preferably reaches 80% at least, more preferably reaches 85% at least, more preferably reaches 90% at least, most preferably reaches 95% at least.

[0080] the present invention also provides the nucleic acid molecule of energy specificity in conjunction with the encoding sequence of the antibody fragment of H5 subtype avian influenza virus.

[0081] the present invention further also relate to encoding antibody weight chain variable region amino acid sequence by SEQID NOs:28-30, SEQ ID NOs:34-36, SEQ ID NOs:40-42, SEQ ID NOs:46-48, SEQ ID NOs:52-54 and SEQ ID NOs:58-60 the separated nucleic acid molecule of correspondence.The invention still further relates to encoding antibody light chain variable region amino acid sequence is the pairing nucleic acid molecule of SEQ ID NOs:31-33, SEQID NOs:37-39, SEQ ID NOs:43-45, SEQ ID NOs:49-51 and SEQ ID NOs:61-63.

[0082] the present invention relates to contain the recombinant expression vector of described nucleic acid molecule, also relate to the host cell that has transformed these molecules.And, the invention still further relates to and utilize the host cell comprised described nucleic acid molecule to cultivate under given conditions and separate the method that obtains inventing described antibody.

[0083] antibody polypeptides sequence

[0084] monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2 heavy chain and light chain variable region amino acid sequence can be derived from the nucleotide sequence of correspondence and be obtained.Monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2 weight chain variable region amino acid sequence are respectively SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:25.Monoclonal antibody 8H5,3C8,10F7,4D1 and 2F2 light chain variable region amino acid sequence are respectively SEQ ID NO:4, SEQ ID NO:8, SEQID NO:12, SEQ ID NO:19 and SEQ ID NO:27.On the one hand, the weight chain variable region amino acid sequence that comprises of anti-H5 monoclonal antibody provided by the invention is SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:25.On the other hand, the light chain variable region amino acid sequence that comprises of anti-H5 monoclonal antibody provided by the invention is SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:19 and SEQ ID NO:27.

[0085] on the other hand, the sequence similarity of the aminoacid sequence of the variable region of heavy chain of antibody provided by the invention and SEQID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:21 or SEQ ID NO:25 reaches 70% at least, preferably at least 75%, preferably at least 80%, preferably 85%, again preferably at least 90%, best is at least 95%.

[0086] on the other hand, the sequence similarity of the aminoacid sequence of the variable region of light chain of antibody provided by the invention and SEQID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:19 or SEQ ID NO:27 reaches 70% at least, preferably at least 75%, preferably at least 80%, preferably 85%, again preferably at least 90%, best is at least 95%.

[0087] aminoacid sequence of the CDR of the heavy chain of monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2 and variable region of light chain is determined as follows:

CDR1, the CDR2 of monoclonal antibody 8H5 heavy chain and the aminoacid sequence of CDR3 are respectively SEQ IDNos:28-30.CDR1, the CDR2 of monoclonal antibody 8H5 light chain and the aminoacid sequence of CDR3 are respectively SEQ ID Nos:31-33.

CDR1, the CDR2 of monoclonal antibody 3C8 heavy chain and the aminoacid sequence of CDR3 are respectively SEQ IDNos:34-36.CDR1, the CDR2 of monoclonal antibody 3C8 light chain and the aminoacid sequence of CDR3 are respectively SEQ ID Nos:37-39.

CDR1, the CDR2 of monoclonal antibody 10F7 heavy chain and the aminoacid sequence of CDR3 are respectively SEQ IDNos:40-42.CDR1, the CDR2 of monoclonal antibody 10F7 light chain and the aminoacid sequence of CDR3 are respectively SEQ ID Nos:43-45.

CDR1, the CDR2 of monoclonal antibody 4D1 heavy chain and the aminoacid sequence of CDR3 are respectively SEQ IDNos:46-48.CDR1, the CDR2 of monoclonal antibody 4D1 light chain and the aminoacid sequence of CDR3 are respectively SEQ ID Nos:49-51.

CDR1, the CDR2 of monoclonal antibody 3G4 heavy chain and the aminoacid sequence of CDR3 are respectively SEQ IDNos:52-54.

CDR1, the CDR2 of monoclonal antibody 2F2 heavy chain and the aminoacid sequence of CDR3 are respectively SEQ IDNos:58-60.CDR1, the CDR2 of monoclonal antibody 2F2 light chain and the aminoacid sequence of CDR3 are respectively SEQ ID Nos:61-63.

[0088] on the other hand, the invention provides anti-H5 monoclonal antibody heavy chain or fragment, it contains following CDR:(i) from one or more CDR of SEQ ID NOs:28-30; (ii) from one or more CDR of SEQ ID NOs:34-36; (iii) from one or more CDR of SEQ ID NOs:40-42; (iv) from one or more CDR of SEQ ID NOs:46-48; (v) from one or more CDR of SEQ ID NOs:52-54; Or (vi) from one or more CDR of SEQ ID NOs:58-60.In one embodiment, anti-H5 monoclonal antibody heavy chain or the fragment CDR that to contain three aminoacid sequences be SEQ ID NOs:28-30.In another embodiment, anti-H5 monoclonal antibody heavy chain or the fragment CDR that to contain three aminoacid sequences be SEQ ID NOs:34-36.In another embodiment, the CDR that it is SEQ ID NOs:40-42 that anti-H5 monoclonal antibody heavy chain or fragment contain three aminoacid sequences.In another embodiment, the CDR that it is SEQ ID NOs:46-48 that anti-H5 monoclonal antibody heavy chain or fragment contain three aminoacid sequences.In another embodiment, the CDR that it is SEQ ID NOs:52-54 that anti-H5 monoclonal antibody heavy chain or fragment contain three aminoacid sequences.In another embodiment, the CDR that it is SEQ ID NOs:58-60 that anti-H5 monoclonal antibody heavy chain or fragment contain three its aminoacid sequences.

[0089] on the other hand, the aminoacid sequence that contains of the heavy chain of anti-H5 monoclonal antibody or segmental CDR may be one or more amino acid whose sudden changes to occur or increase or lack on SEQ ID NOs:28-30,34-36,40-42,46-48,52-54 and 58-60.Preferably, the amino acid of sudden change or interpolation or disappearance is no more than 3 amino acid.More preferably, the amino acid of sudden change or interpolation or disappearance is no more than 2 amino acid.Most preferably, the amino acid of sudden change or interpolation or disappearance is no more than 1 amino acid.

[0090] on the other hand, the invention provides anti-H5 monoclonal antibody light chain or fragment and contain following CDR:(i) from one or more CDR of SEQ ID NOs:31-33; (ii) from one or more CDR of SEQ ID NOs:37-39; (iii) from one or more CDR of SEQ ID NOs:43-45; (iv) from one or more CDR of SEQ ID NOs:49-51; (v) from one or more CDR of SEQ ID NOs:55-57; Or (vi) from one or more CDR of SEQ ID NOs:61-63.In one embodiment, anti-H5 monoclonal antibody light chain or the fragment CDR that to contain three its aminoacid sequences be SEQ ID NOs:31-33.In another embodiment, anti-H5 monoclonal antibody light chain or the fragment CDR that to contain three its aminoacid sequences be SEQ ID NOs:37-39.In another embodiment, the CDR that it is SEQ ID NOs:43-45 that anti-H5 monoclonal antibody light chain or fragment contain three its aminoacid sequences.In another embodiment, the CDR that it is SEQ ID NOs:49-51 that anti-H5 monoclonal antibody light chain or fragment contain three its aminoacid sequences.In another embodiment, the CDR that it is SEQ ID NOs:55-57 that anti-H5 monoclonal antibody light chain or fragment contain three its aminoacid sequences.In another embodiment, the CDR that it is SEQ ID NOs:61-63 that anti-H5 monoclonal antibody light chain or fragment contain three its aminoacid sequences.

[0091] on the other hand, the aminoacid sequence that the light chain of anti-H5 monoclonal antibody or segmental CDR contain may be at SEQ ID NOs:31-33,37-39, and 43-45,49-51 and 61-63 go up and one or more amino acid whose sudden changes occur, increase or lack.The amino acid of preferably, sudden change, interpolation or disappearance is no more than 3 amino acid.The amino acid of more preferably, sudden change, interpolation or disappearance is no more than 2 amino acid.The amino acid of most preferably, sudden change, interpolation or disappearance is no more than 1 amino acid.

[0092] varient after the amino acid of the variable region of above-mentioned antibody or CDR is undergone mutation, added or lacks still keeps the ability of specificity in conjunction with the H5 subtype avian influenza virus.The present invention also comprises the varient of such Fab.

[0093] monoclonal antibody varient of the present invention can obtain by traditional gene engineering method.Those skilled in the art knows the method for utilizing nucleic acid mutation to transform dna molecular fully.In addition, the nucleic acid molecule of encoding heavy chain and light chain varient also can obtain by chemosynthesis.

[0094] chimeric antibody, humanized antibody close fusion rotein

[0095] on the other hand, the present invention also provides chimeric antibody, and it is by mouse source monoclonal antibody 8H5,3C8, and 10F7,4D1, the variable region heavy chain of 3G4 or 2F2 or its varient and/or intact light chain or part is formed in conjunction with people source monoclonal antibody constant region.And the present invention has also comprised humanized antibody, and they are by mouse source monoclonal antibody 8H5,3C8,10F7,4D1,3G4 or 2F2 or its varient one or more CDR graftings form to the framework of human antibody.

[0096] on the other hand, the present invention also provides lotus root to join a kind of fusion rotein that contains monoclonal antibody of the present invention wholly or in part of certain molecule.

[0097] chimeric antibody, humanized antibody and fusion rotein can utilize traditional genetic engineering technique to obtain.As, the DNA of coding monoclonal antibody can be transformed into (Morrison to the mouse source sequence that the sequence of the constant region of the heavy chain of human antibody and light chain replaces to homology by the method for sudden change, et al., or obtain chimeric or humanized antibody or fusion rotein Proc.Nat.Acad.Sci.81:6851 (1984)), by whole or partial immunity sphaeroprotein encoding sequence and NIg encoding sequence are carried out covalency lotus root connection.

[0098] neutralizing antibody

[0099] on the other hand, the invention provides can in and the anti-H5 antibody of the virus activity of H5 subtype avian influenza virus.In one embodiment, this neutralizing antibody can in and the virus activity of H5 subtype avian influenza virus at least 60%, or at least 70%, or preferably at least 75%, or preferably at least 80%, or preferably at least 85%, or preferably at least 90%, more preferably at least 95%, most preferably at least 99%.

[0100] those of ordinary skills know fully and utilize traditional technological method can measure in the antibody and the virus activity of H5 subtype avian influenza virus.The neutralization activity that promptly can be used for measuring the some specific H5 monoclonal antibody among the present invention as the method for the neutralization test described in the embodiments of the invention 1.

Small peptide

The present invention also provides a kind of small peptide of simulating monoclonal antibody identification epi-position.

Ten screened come out that contain 7 amino acid whose small peptides because it combines with monoclonal antibody 8H5,3C8. wherein, 6 with 8H5 monoclonal antibody bonded small peptide aminoacid sequence SEQ ID NOS:64-69 is arranged, 4 with 3C8 monoclonal antibody bonded small peptide aminoacid sequence SEQ ID NOS:70-73. is arranged

It is very good that seven peptide 8H5A (SEQ ID NO:64) and 8H5E (SEQ ID NO:68) have shown that reactive behavior .8H5A seven peptides and the combination of 8H5 monoclonal antibody get, but get relative relatively poor with other monoclonal antibodies in conjunction with .8H5E seven peptides a little less than getting and the combination of 8H5 monoclonal antibody.

Further, the present invention also provide 13 be combined with monoclonal antibody 8H5 and characteristic contain 12 amino acid whose small peptides really (table 16, SEQ ID NOS:74-97).

This 12 peptide 123 or 125 can be used for making fusion rotein 239-123 and 239-125, this fusion rotein is fine in conjunction with getting with 8C11 and 8H5 monoclonal antibody respectively. and this 12 peptide 123 or 125 can be used for making fusion rotein with HBVcAg, this fusion rotein and 8H5 monoclonal antibody but not other antibodies have characteristic. fusion rotein HBc-122, HBc-124, HBc-128, HBc-129 and 8H5 monoclonal antibody but not other antibodies have characteristic. fusion rotein HBc-122, HBc-124, HBc-128, HBc-129 also can simulate monoclonal antibody identification epi-position and form the assembling of similar virion.

[0101] detection method

[0102] the present invention also provides a kind of and utilizes monoclonal antibody of the present invention to detect the antigen in the H5 type avian influenza virus sample and/or the method for antibody.

[0103] on the one hand, the invention provides the method that detects the H5 subtype avian influenza virus, comprise following step: (i) with viral be combined into antibody virus in a certain strain monoclonal antibody of the present invention or its certain fragment and the above-mentioned sample or antibody fragment virus mixture; (ii) detect this sample composites to determine in the sample whether virus being arranged.

[0104] on the other hand, the invention provides the method for H5 subtype avian influenza virus in a kind of test sample, comprise following step: (i) first antibody is adsorbed onto on the solid support; (ii) in above-mentioned upholder, add the suspicious testing sample that may contain the H5 subtype avian influenza virus; (iii) in above-mentioned upholder, add the second antibody that has marker; Thereby the existence that (iv) detects this marker judges whether the H5 subtype avian influenza virus exists.

[0105] another aspect of the present invention provides the detection method of H5 subtype avian influenza virus in a kind of test sample, comprises following a few step: (i) antibody is adsorbed onto a certain solid support; The sample that may contain the H5 subtype avian influenza virus that (ii) in this solid support, adds pre-mixing H5 hemagglutinin marker; (iii) detect and whether have H5 hemagglutinin marker.

[0106] detection method can be used enzyme linked immunological absorption (ELISA), enzyme immunodetection, chemiluminescence immunoassay detection, radioimmunity detection, fluorescence immunoassay detection, immunochromatography, competition law and similar detection method.Utilize competition law or sandwich assay mode, above-mentioned detection method can be used to detect target antigen or antibody.

[0107] competition law is the quantitative relation of the labelled antigen competition of antigen and a kind of known quantity in the comparative sample in conjunction with monoclonal antibody of the present invention.Carry out immunology detection based on competition law and be the sample that will contain the target antigen of unknown number join the known physics of prior usefulness or chemical process monoclonal antibody bag of the present invention by to solid support and carried out.Target antigen behind the quantitative in advance mark of adding reacts simultaneously.After hatching, the flushing solid support detects the activity that is attached to the marker on this upholder.

[0108] in sandwich assay, the target antigen in the sample is sandwiched between the anti-and mark monoclonal antibody of Sheet, and then adds the substrate of marker such as enzyme, and the variation by substrate colors detects and judges antigenic existence.Carry out immunology detection, for example, earlier the sample that contains a kind of unknown number target antigen is joined to wrap in advance with physics or chemical process and reacted on the solid support of monoclonal antibody described in the present invention based on sandwich assay.Then, adding mark monoclonal antibody of the present invention reacts.After hatching, wash this upholder, again the activity that is attached to the marker on this upholder is detected.Marker can be substrate, luminophore such as different luminol,3-aminophthalic acid cyclic hydrazide and acridinium ester, fluorescent substance such as fluorescein and rhodamine, vitamin H and coloring matter such as latex particle and the Radioactive colloidal gold etc. of radio isotope as 125 iodine, enzyme, enzyme.The enzyme that mark is used can be peroxidase (as horseradish peroxidase HRP), alkaline phosphatase, beta galactosidase enzyme and glucose oxidase.Have 2 for suitable substrate in these reactions; 2 '-azino-two (3-ethyl benzo thiophene pyrroline-6 sulfonic acid), luminol,3-aminophthalic acid cyclic hydrazide-hydrogen peroxide, O-Phenylene Diamine-hydrogen peroxide (at peroxidase), para-nitro-pheneye phosphate, 4-methyl acid phosphate umbellate form ketone, 3-(2 '-the spiral diamantane)-4-methoxyl group-4-(3 " phosphoryl) phenyl-1,2-diethoxy alkane (at alkaline phosphatase), p-nitrophenyl-β-D-semi-lactosi and methyl umbelliferone-β-D-semi-lactosi (at beta galactosidase enzyme).Other mark comprises quantum dot-labeled, chromophore's mark, enzyme labelling, the affinity ligand mark, the electromagnetism spin labeling, the heavy atom mark, be marked with the probe of nanoparticle scattering of light mark or other nanoparticle, fluorescein isothiocyanate (FITC), TRITC, rhodamine, the tetramethyl-rhodamine, the R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas is red, Phar-Red, different phycoerythrin (APC), epi-position mark such as FLAG or HA epi-position, and enzyme labelling such as alkaline phosphatase, horseradish peroxidase, I ²-tilactase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase and hapten conjugation thing such as digoxigenin or dinitrophenol(DNP), maybe can form title complex in conjunction with pairing as streptavidin/vitamin H, avidin/biotin or antigen/antibody title complex as comprising rabbit igg and resisting-rabbit igg; Fluorophor such as umbelliferose (umbelliferone), fluorescein, fluorescein isothiocyanate, rhodamine, the tetramethyl-rhodamine, Yihong, green fluorescent protein, algae is red, tonka bean camphor, methylcoumarin, pyrene, Victoria Green WPB, toluylene, fluorescent yellow, Cascade indigo plant, dichlorotriazine base fluorescein, dansyl chloride, phycoerythrin, the fluoresce lanthanide complex compound is as comprising europium and terbium, Cy3, Cy5, molecular beacon (molecular beacons) and its fluorescent derivative, luminescent material such as luminol,3-aminophthalic acid cyclic hydrazide; Scattering of light or plasmone group resonance material is as gold or silver-colored particle or quantum mottle (quantum dot): or radio active material as ¹⁴C, ¹²³I, ¹²⁴I, ¹³¹I, Tc99m, ³⁵S or ³H; Or ball (spherical shell), and be marked with the probe that any other signal known in the art produces marker.For example, detectable molecule includes but not limited to fluorophor and noted earlier other is known, as the Principles of Fluorescence Spectroscopy that is compiled at Joseph R.Lakowicz (Editor), Plenum Pub Corp, the Molecular Probes Handbook of the sixth version of second edition (July1999) and Richard P.Hoagland is described.In some embodiments, marker comprises semiconductor nano crystallite such as quantum mottle (being Qdots), referring to U.S.P 6,207,392.Qdots can buy from Quantum Dot Corporation.Be used for semiconductor nano crystallite of the present invention and comprise the semi-conductive nano microcrystalline of Group II-V such as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe and its mixture and the semi-conductive nano microcrystalline of Group III-V such as GaAs, InGaAs, InP, InAs and its mixture.The use of Group IV semi-conductor such as germanium or silicon, or organic semi-conductor use may be convenient feasible under certain conditions.The semiconductor nano crystallite also can comprise alloy, and it contains two or more semi-conductors that is selected from Group III-V compound, Group II-VI compound, Group IV element and its composition.

[0109] in some embodiments, the fluorescent energy acceptor is connected to the detection probes thing that serves as a mark.In one embodiment, the fluorescent energy acceptor can form fluorescent chemicals by the reaction of compound and singlet oxygen and form, or reacts and be translated into fluorescent chemicals with an ancillary compound by compound and form.This compounds can be included in the damping fluid in apparatus of the present invention.In other embodiment, the fluorescent energy acceptor can be a part that comprises the compound of chemoluminescence agent or group, and for example, the fluorescent energy acceptor can comprise metal complexs such as rare earth metal such as europium, samarium, tellurium.To have a sharp-pointed photoluminescent band attractive especially because of it for these materials; And group of the lanthanides marker such as europium (III) can provide effectively signal emission for a long time, are difficult for photobleaching simultaneously, thereby can place longer for some time under the situation of needs so that contain the test set of processing/response sample.In various allos and homoimmune assay method, used long-life fluorescence europium (III) complex compound nanoparticle thing that serves as a mark, for example can be referring to Huhtinen et al.Clin.Chem.2004Oct; 50 (10): 1935-6.But when the nanoparticle of these inner markers is used with the time resolved fluorescence detection, measure performance and can improve.In allos was measured, the dynamicrange that lower concentration is measured down can be expanded; And, detecting the high specific activity nanoparticle marker that antibody applies by using, rather than use the detection antibody of conventional mark, the dynamics of mensuration also can improve.In homology was measured, for FRET (fluorescence resonance energy transfer), europium (III) nanoparticle was effectively donor, thereby can carry out simply, screen fast and efficiently.In one embodiment, the fluorescent marker of marker as disclose herein comprises and biomolecules link coupled nanoparticle marker.In other words, nanoparticle can be as detecting or capturing probe.For example, among the present invention, can utilize the nanoparticle of europium (the III)-mark that is connected to monoclonal antibody or streptavidin (SA) to come analyte specific in the test sample, as the immunoassay of nanoparticle base.Nanoparticle can be used as the substrate that adheres to the particular combination agent, and these particular combination agent are at analyte and detection (as marker) or catch composition.The example of associated mark thing can be referring to U.S.P4, and 695,554; 4,863,875; 4,373,932; With 4,366,241.U.S.P4 has then disclosed colloidal metal and dyed particles in 313,734 and 4,373,932.And U.S.P4 has then disclosed in 954,452 and how to prepare and use nonmetallic colloid; U.S.P4 has disclosed the organic polymer latex particle as marker in 252,459.

[0110] marker is attached to method on antigen or the antibody, can pass through maleimide method (J.Biochem. (1976), 79,233), vitamin H activation method (J.Am.Chem.Soc. (1978), 100,3585), hydrophobic combined techniques, ester activation method or isocyanic ester method (" Enzyme immunoassay techniques ", published in 1987 by Igaku Shoin).

[0111], then need make radiation-resistant work top or the liquid-state protective equipment made good use of if above-mentioned marker is a radio isotope.If above-mentioned marker is an enzyme, need to add substrate, the activity of enzyme is measured by colorimetry or photofluorometer.If above-mentioned marker is fluorescent substance, luminophore or coloring matter, measuring method can use method well known in the art to measure accordingly.

[0112] among the present invention, the sample that is used for detecting the H5 subtype avian influenza virus includes but not limited to the totivirus of animal or patient's movement, oral cavity and nasal secretion, chicken embryo culture or lytic virus etc.

[0113] proofing unit and test kit

[0114] the invention still further relates to the diagnostic kit of H5 subtype avian influenza virus antigen in a kind of detection H5 subtype avian influenza virus diagnosis of infection test kit, the especially test sample or antibody.Diagnostic kit of the present invention comprises at least a monoclonal antibody of the present invention.The employed monoclonal antibody of the present invention of diagnostic reagent of the present invention is not done special restriction, it can be the antigenic monoclonal antibody of any strain identification H5 hemagglutinin, also can be the antibody fragment of arbitrary monoclonal antibody of tool antigen-specific of the present invention, as F (ab ') ₂, Fab ', Fab etc.

[0115] on the one hand, the present invention relates to two kinds of test kits that detect the H5 subtype avian influenza virus, these reagent comprise a kind of monoclonal antibody or their active fragments or mutant at least.Preferably, test kit of the present invention also comprises the detection reagent that is fit to detect antigen-antibody reaction.

[0116] on the other hand, the present invention relates to a kind of test kit that detects anti-H5 subtype avian influenza virus antibody, it comprises at least a monoclonal antibody of the present invention or its active fragments or its mutant.Preferably, test kit described in the present invention comprises the detection reagent that is fit to detect antigen-antibody reaction.

[0117] solid support that relates in the diagnostic kit of the present invention or solid support include but not limited to microwell plate, magnetic particle, immune chromatograph filter paper, polymer for example polystyrene, granulated glass sphere, glass filter and other insoluble carrier.In one embodiment, one comprise a plurality of compartments or the zone solid support in, have at least a compartment to coat with antibody of the present invention.Preferably, at least one compartment (or first compartment) is coated with antibody of the present invention, and at least one remaining compartment (or second compartment) with can with hypotype (for example, the H1 of avian influenza virus except that H5, H2, H3, H4, H6, H7, H9, H10, H11, H12, H13, H13, H14, H15orH16) bonded antibody is coated specifically, preferably hypotype H1, H3, H7, H9.

[0118] diagnostic reagent of the present invention also comprises some other compositions, includes but not limited to enzyme, corresponding substrate, radio isotope, reflective material, fluorescent substance, coloring matter, damping fluid and lath and above-mentioned suchlike material that mark is used.

[0119] in the diagnostic reagent of the present invention, employed invention monoclonal antibody must be coated on the solid support in advance.One preferred embodiment in, to the bag quilt monoclonal antibody direction of travel location can help to improve the antigen-antibody joint efficiency.(Proteomics 5,416-419 (2005)) such as TaeWoon Cha have confirmed to control the conformation of pre-coating protein molecule and design solid support surface ideal chemical environment and will help to keep and improve the proteic reactive behavior of immobilization and tire.Document had reported that several different methods can be combined in antibody on the solid support by predetermined conformation direction.(Proteomics 2,48-57 (2002)) such as Shawn Weng reported once that utilizing nucleic acid to connect proteic method made protein molecular be combined on certain surface with identical spatial positioning.Soellner, M etc. (J.AM.CHEM.SOC.125,11790-11791 (2003)) have reported and a kind ofly can be attached to certain lip-deep method to the albumen that comprises antibody and antigen by the Staudinger ligation in a kind of identical mode.In the Staudinger reaction, triazo-compound and the reaction of phosphorus thioesters form a kind of aminocompound.(Anal.Chem such as Hairong Zhang, 78,609-616 (2006)) reported a kind of method that antibody on the gold-plated magnetic bead is navigated to particle surface by the free thiol reactant on monoclonal antibody ' fragment, according to this, the antigen binding site on all antibody all is positioned on the ideal conformation.(J.Phys.Chem.B, 110,1907-1914 (2006)) such as Hai Xu once reported the method that antibody is adsorbed onto hydrophilic Si oxide/water surface.(Proteomics, 3,2176-2189 (2003)) such as Seung-yong Seong summarized directionally with proteopexy to certain surperficial method and in these methods used protein molecular.All these reference all intactly are summarized in here.

[0120] in the diagnostic kit of the present invention, employed monoclonal antibody or antigen must be in advance with the above-mentioned marker marks of mentioning.

[0121] but the invention still further relates to the automatization proofing unit of the avian influenza virus in a kind of automatization test sample.

[0122] the various devices that whether have analyte in the immunochemistry detection of biological liquid sample that utilize is disclosed in the prior art.This class device can utilize so-called " sandwich assay " to measure, and for example, target analytes such as antigen are clipped in the antibody of mark and are fixed between the antibody of solid support.Whether exist the antibody complex of bonded antigenic mark and/or its quantity to measure by observing.This class device also can adopt the competition law immunoassay, wherein, the antibody that is incorporated into solid phase surface is contacted with the sample that contains unknown number antigen analysis thing and with the labelled antigen of same-type.Then, measure the antigenic quantity of the mark be incorporated into solid phase surface, thereby provide indirect measurement for the quantity of antigen analysis thing in the sample.Different mensuration can be utilized the device that is suitable for measuring different analytes, for example, different antibody or antigen is incorporated into design district or the addressable area (as suction or non-absorbent membrane) of testing substrate.Because these methods and other the method for discussing below both can detect antibody, also can detect antigen, their so-called immunochemistry ligand-receptors are measured or are called for short immunoassay.

[0123] no matter solid-phase immunoassay device is sandwich assay type or competition law type, can provide sensitive to detect the analyte in biological fluid sample such as blood or the urine.The solid-phase immunoassay device comprises solid support, wherein, a member of ligand-receptor centering, normally antibody, antigen or haptens are incorporated on this solid support.Usually, the form of early stage solid support is flat board, test tube or polystyrene bead, and this knows in radioimmunoassay and enzyme immunoassay.Recently, people adopt various porous materials such as nylon, soluble cotton, cellulose acetate ester, glass fibre and other porous polymer as solid support.People also disclose many self the band immunoassay test kit, it adopts the solid phase carrier of porous material as immunochemical component such as antigen, haptens or antibody.Usually these test kits can adopt Mierocrystalline cellulose test paper, circulation or or migration in design.Can adopt among the present invention any routine, known devices finishes immunoassay or specificity in conjunction with mensuration, to detect influenza.

[0124] in some aspects, the present invention includes the device of the infection that detection causes by various influenza viruses or its hypotype.In some embodiments, will contain in the sample feeder of one or more influenza viruses or resisiting influenza virus antibody the main body that whether is infected with working sample by one or more influenza viruses or its hypotype.The device that comprises solid support can comprise resisiting influenza virus antibody or influenza antigen placed on it, thereby can measure the sample that suspection contains influenza virus, influenza virus protein or resisiting influenza virus antibody.In many embodiments, employed antibody includes but not limited in apparatus of the present invention: the arbitrary combination of many anti-, monoclonal antibody (MAb) or its conservative property or functional variant, chimeric antibody, distortion antibody, humanized antibody, its bioactive fragment or these antibody; Functional antibodies or its fragment just are meant antibody on the whole herein.Antibody of the present invention can adapt to any device, to detect influenza virus.For example, H5 avian influenza virus can be detected by H5 albumen in the target random sample product or anti-H5 hypotype antibody.In one embodiment, H5 is from avian influenza virus (AIV).

[0125] many devices of buying on the market can be installed (or additional) antibody or antigen disclosed herein at an easy rate.These devices can comprise employed solid phase substrate in the detection method, include but not limited to filter paper, polymkeric substance such as polystyrene, granulated glass sphere, glass filter and other insoluble carrier that microwell plate, magnetic bead, immune chromatograph are used.Substrate can be usually but be not limited to following shape: banded, tabular, chip, spherical, pearl, poroid as the hole on the titer plate or any other suitable shape.And what cooperate partner's (being antigen or antibody) in conjunction with it can be Any shape, for example, and droplet price fixing, test tube, Mierocrystalline cellulose test paper, micro-centrifuge tube, pearl, from capstan or the like.Suitable material comprises glass, plastics (as polyethylene, PVC, polypropylene, polystyrene etc.), albumen, paper, carbohydrate and other solid support.Other operable material comprises pottery, metal, metalloids, semiconductor material, cement etc.In some embodiments, immunoassay (as ELISA) titer plate comprises the form in 96 holes, 384 orifice plates or 1536 holes, or more hole, as other commercial plate.

[0126] some operable devices comprise Mierocrystalline cellulose test paper, lateral flow devices, cartridge, multiple device, titer plate, microfluidic devices, plate or array or high-throughout platform, as disclosed in following United States Patent (USP): the U.S. patent No.: 6,448,001; 4,943,522; 6,485,982; 6,656,744; 6,811,971; 5,073,484; 5,716,778; 5,798,273; 6,565,808; 5,078,968; 5,415,994; 6,235,539; 6,267,722; 6,297,060; 7,098,040; 6,375,896; 7,083,912; 5,225,322; 6,780,582; 5,763,262; 6,306,642; 7,109,042; 5,952,173 and 5,914,241.For example, microfluidic devices can be U.S.P 5,707,799 and WO 2004/029221 in disclosed.

[0127] Mierocrystalline cellulose test paper

[0128] in some very usual Mierocrystalline cellulose test paper were measured, in home pregnancy and ovulation tests test kit, immunochemical component such as antibody were to be combined on the solid phase.Proofing unit dips in suspection contains the sample of unknown antigen analyte, and insulation is cultivated then.Perhaps also a spot of sample can be placed the sample region of acceptance.The antibody that adds mark then, and certification mark thing, the indication that whether exists as interested analyte.In some cases, marker is an enzyme, like this, needs to add the antibody of enzyme labelling, and it can add simultaneously or cultivate the back in insulation and add.Then, washing unit, and it is inserted in second solution of the substrate that contains enzyme again.If there is enzyme labelling, it can interact with substrate, makes the product that produces the band look, this product or deposit on the solid phase as throw out, or the variation of generation visible light in substrate solution.Baxter etc. disclose the immunoassay .Kali et of so sandwich shape Mierocrystalline cellulose test paper etc. and disclose in EP-A 0 282 192 and a kind ofly can be used for the Mierocrystalline cellulose test paper device that competition law is measured in EP-A 0125118.Material, form and the marker of T Mierocrystalline cellulose test paper are known, and can be used for influenza test.The example of Mierocrystalline cellulose test paper device comprises U.S.P4,235,601,5,559,041,5,712,172 and 6,790, described in 611.In some embodiments, antibody of the present invention can place on the Mierocrystalline cellulose test paper device.For example, can use solid support Mierocrystalline cellulose test paper, the anti-H5 hypotype AIV antibody in the test sample, wherein can be at a place or many places matrix (matrix) some position this test paper is installed.One place's matrix can be attached with antibody or its functional fragment of non-specific control.These matrix points position can be protein binding position and/or antigen in conjunction with the position, and normally make, but also can use any suitable medium known in the art, as some nylon and Polyvinylidene class by soluble cotton.In some embodiments, can adhere to many mesostromas at solid support, each matrix contains at the antigen of multiple subtype influenza virus or antibody.

[0129] Continuous Flow

[0130] design of flow pattern immunoassay apparatus can be avoided needed a large amount of cultivation and cleaning in the Mierocrystalline cellulose test paper mensuration continuously.Valkirs etc. are at U.S.P4, disclose a kind of device that comprises antibody (the target antigen analyte is had specificity) in 632,901, and this antibodies and adds liquid sample in this device in porous membrane or filter.Because liquid flow warp or Continuous Flow are crossed in the film, target analytes is attached on the antibody.After adding sample, can add the antibody of mark again.The visual detection of traget antibody can point out whether there is the target antigen analyte in the sample.Korom et etc. discloses a kind of improvement on the Continuous Flow device in EP-A 0 299 359, wherein, the antibody appendix of mark is in a film, and this film plays the effect that reagent transmits system.Such device can comprise the layer that the composition in the sample is played the strainer effect, and comprises employed reagent in the detection.When sample when one deck flows to another layer, it contacts with specific binding reagents and reacts, in some cases, the component in the mark system can provide the indication that whether has target analytes.

[0131] immunofiltration device

[0132] the immunofiltration device can buy from the market that (as Pierce, Rockford IL), and can transform at an easy rate, with appendix antibody of the present invention.In enzyme linked immunological flow assay method (E LIFA), between 96 hole sample panel and vacuum chamber, use nitrocellulose membrane.Reagent is added on the sample panel, and vacuum makes reagent pass through this nitrocellulose membrane.Sleeve pipe will not have the bonded product to transfer in the collecting chamber.In order to detect, before adding enzyme substrates, in collecting chamber, place a microtest plate (microwell plate).Vacuum makes the product of band look transfer in the hole of microtest plate, so that analyze on the microtest plate reading device of automatization.The ELIFA system comprises the Polymethylmethacrylate of clean cut, and has seal washer, thereby can provide from a hole to constant flow rate another hole.Sleeve pipe can accurately be transferred to the product of band look in the hole of microtest plate, so that analyze.Capture antibodies of the present invention is that point is on substrate (as titer plate, film or chip) basically.Apply and suspect and to have the biological sample of influenza virus or influenza antigen and hatch, make capture antibodies combine with it.Subsequently, add detection antibody.U.S. patent application 2003/0108949 discloses a kind of high-throughout immunofiltration device.Such device can comprise the layer of strainer effect and/or comprise reagent used in the detection.When sample and specific binding reagents react, in some cases, the component in the mark system just can provide the indication that whether has a certain analyte.

[0133] lateral flow devices

[0134] in lateral flow assays, utilize some or all detection to come impregnated membranes with reagent.The detection of analytes district is provided, and distinguishes the analyte of certification mark at this.For example, can be referring to the U.S.P4 of Tom etc., 366,241 and the EP-A 0 143 574 of Zuk.Knownly can carry out many improvement for the effluent proofing unit.This class device can comprise some specificity in conjunction with the reagent in detecting (sample before being applied to the effluent band can with some reagent react, perhaps can in turn on the effluent band, add extra reagent), perhaps the effluent band can comprise and be used for specificity in conjunction with all necessary reagent that detect.Lateral flow devices often comprises reagent, and these reagent can be attached on the colour-coded, thereby can observe directly detected result, and does not need further to add other material.For example, can be referring to people's such as the people's such as U.S.P 4,770,853, May of Bernstein WO 88/08534 and Ching EP-A 0299428.This class device so makes up usually, makes it comprise position, reagent area and the detection zone that applies sample.This type of device is normally made by water-absorbing material, like this can so that sample from sample apply the district through reagent or reaction zone Continuous Flow to detection zone.Although some is reflected at sample and is applied to and may just takes place before being with, in some embodiments, reaction zone comprises the reagent that is used for immunoassay.A kind of specificity combinating reagent such as antibody, it can be attached to diffustivity sample and apply district or being with of reaction zone, and makes that it can be in conjunction with the antigen in the sample, and flows along band with sample.At detection zone, can be directly with another specificity of antigen or antibody in conjunction with the partner, capture antigen-antibody complex, perhaps, also can as avidin or streptavidin and vitamin H, be caught indirectly with other specificity in conjunction with the partner.Similarly, marker can be directly or indirectly attached on antigen or the antibody.The example of lateral flow devices can be referring to U.S.P4,818,677,4,943,522,5,096,837 (RE 35,306), 5,096,837,5,118,428,5,118,630,5,221,616,5,223,220,5,225,328,5,415,994,5,434,057,5,521,102,5,536,646,5,541,069,5,686,315,5,763,262,5,766,961,5,770,460,5,773,234,5,786,220,5,804,452,5,814,455,5939,331 and 6,306,642.And U.S.P 4,703,017,6,187,598,6,352,862,6,485,982,6,534,320 and 6,767 then disclose other lateral flow devices in 714, and it can improve, for use in a plurality of analytes in the tracer liquid sample.

[0135] in the routine techniques, can in a single calibration tape, set up independent detection zone, thereby can detect a plurality of analytes in the sample for each analyte.By using different markers or detecting same marker, can distinguish different analytes at different detection zones.Can adopt the device of any routine, finish mensuration a plurality of analytes.

[0136] immunoassay utilize the mechanism of immune system, and wherein, when having morbific antigen or external foreign matter, human body will be reacted, and produce antibody.These antibody and antigen also are immunoreactant, can be bonded to each other, thereby produce a kind of reaction mechanism of high specific, this mechanism can be used for determining whether existing in the specimen specific antigen with and concentration.

[0137] such lateral flow devices generally includes a porous membrane, and it randomly is carried on the rigid material.Usually, this porous membrane can be made by many materials, and it can allow fluid to pass through to get final product.For example, the material that is used to form porous membrane includes but not limited to natural materials, synthetic materials or exists naturally but through the material of synthetic modification such as polysaccharide (as cellulose materials such as paper, derivatived cellulose such as cellulose acetate ester, soluble cotton); Polyethersulfone; Polyethylene; Nylon; PVDF; Polyester; Polypropylene; Silicon-dioxide; Inorganic materials such as deactivation aluminum oxide, diatomite, magnesium sulfite, or other inorganic fine material, these materials are distributed in the porous polymer matrix equably, polymkeric substance such as vinylchlorid, ethene propenyl chloride multipolymer, ethene vinylchlorid acetate copolymers, fabric of Sheng Chenging (as cotton) or synthetic (as nylon or regenerated fiber) naturally; Porous infiltration colloid, as silica gel, agarose, dextran and gel; Polymer thin film is as polyacrylamide or its analogue.In a special embodiment, described porous water-permeable membrane has soluble cotton and/or polyether sulfone materials to form.It should be understood that described term " soluble cotton " refers to cellulose nitrate, it can be the miscellany of soluble cotton or it and nitric ether or other acid esters, as has the aliphatic carboxylic acid of 1-7 carbon atom.

[0138] such equipment also can comprise one and contain the band that absorbs liner, and this band is in the upstream or the downstream of described test/detection or check plot.As well known to the skilled person, described absorption liner can help to promote capillary force formula liquid flow by described film.In some embodiments, absorb liner and can contain movably immunoassay reagent (as, antibody).Certainly, it will be appreciated that described removable or fixed immunoassay reagent also can be processed at any position of the upstream of described test/detection or check plot, just as in the independent component of detection system.

[0139] in many embodiments, some suitable material can be used to form described sample liner, includes but not limited to soluble cotton, Mierocrystalline cellulose, porous polyethylene liner, fiberglass test paper.If desired, the sample liner also can comprise one or more pretreated analytical reagents, they or covalently or non-covalently be attached on the liner.Described testing sample from the sample liner transfer to be communicated with sample liner one end in conjunction with on the liner.Describedly form by a kind of material that can circulate for liquid in conjunction with liner.For example, in one embodiment, described have fiberglass to form in conjunction with liner.Should be understood that other also can be used for the present invention in conjunction with liner.Selectively, in some embodiments, binding substances or other immunoreagents can be included in the component, described component be applied to detect band before with sample mix.

[0140] whether detects existence analyzed in the testing sample for convenient, can use multiple detection probes in conjunction with liner described.When these detection probes in conjunction with liner, when assay when the sample liner flows in conjunction with liner, these probes still can supply the assay combination.Any is attached to assay, and the existence that detection probes is used to discern assay subsequently whether.Detection that detection probes can be used for analyzing or standard.But in another embodiment, independent standard probe can be applied in conjunction with on the liner, to combine with described detection probes so that normalise simultaneously and detect, so can eliminate the mistake that the routine analysis modular system causes usually.

[0141] in some cases, may need to modify described detection probes in some way so that their easier being attached on the assay.In this case, described detection probes can be modified by specific specificity binding member, and described specificity binding member is adhered on the detection probes to form bonding probes.The specificity binding member refers generally to specificity in conjunction with element of paired, as, two kinds of different molecules are incorporated on another molecule to one of them molecular chemistry and/or physical property.For example, immunoreactivity specific combination element can comprise antigen, haptens, ligand, antibody (primary or secondary) and their corrupt and evil goods, comprises the material that those are formed by recombinant DNA method or synthetic peptide method.Antibody can be monoclonal antibody or polyclonal antibody, recombinant protein or their miscellany or segment, or antibody is in the miscellany of other specificity binding members.Prepare the details of such antibody and they are open at this as the well-formedness that the specificity binding member uses.Other common specificitys include but not limited in conjunction with pairing, vitamin H and avidin (or derivatives thereof), vitamin H and strep antibiont albumen, carbohydrate and hemagglutinin, the nucleotide sequence that replenishes (comprising probe and the capture nucleic acid sequence of in the DNA hybridization assays that detects the target nucleic acid sequence, using), the peptide sequence that replenishes, comprise the peptide sequence that those are formed by recombination method, effector molecule and acceptor molecule, hormone and hormone are conjugated protein, enzyme cofactor and enzyme, enzyme inhibitor and enzyme, or the like.And specificity comprises the element similar to the original specific element in conjunction with pairing.For example, the derivative of assay or segment, for example, the assay analogue all can be employed, as long as it contains at least one epi-position identical with assay.

[0142] for example, in one embodiment, the fluid that contains test sample is transported to gold mark pad, mixes to form analyte complex at gold mark pad place's analyte with by a kind of detection probes of special combination element modified.Because it is unimpeded to flow between gold mark pad and the porous water-permeable membrane, so described mixture can be got the detection zone that is transferred on the porous water-permeable membrane discard from the gold mark.Perhaps, can utilize a plurality of surveyed areas by the specific antibody of integrating not synantigen (as, different influenza viruses or from the virus antigens of different influenza viruses).Described surveyed area can contain fixing agent, and described fixing agent generally can form chemistry with analyte and/or analyte complex (as, the mixture of analyte and detection probes) or material resources are connected.In some embodiments, described reagent can be a kind of biological reagent, as antibody disclosed herein.Other biological reagent is known by those skilled in the art, and they include but not limited to, antigen, haptens, antibody, a-protein or G, avidin, streptavidin or their mixture.In some situation, people wish that these biological reagents have the mixture bonded ability with analyte and/or analyte and detection probes.

[0143] these reagent are used for the secure bond site of detection probes/analyte complex.In some cases, analyte as antibody, antigen etc., contains two binding sites.In case the arrival detection zone, one of them binding site is occupied by the specific combination element of combined probe.But, the site that is not occupied of analyte can with the fixing agent combination.One and and the fixing agent combination, combined probe just forms a kind of new triple sandwich mixtures.

[0144] detects or the test zone generally can provide the different surveyed areas of any amount, so that the existence that the user can determine specific analyte in the testing sample better whether.Each zone is all contained identical reagent or is contained the fixedly different reagent of multiple analytes.For example, detection zone may comprise two or more different surveyed areas (as, wire, point-like etc.).Surveyed area may be processed into the form of wire, and its direction is vertical in fact with the direction that testing sample flows through analytical equipment.Equally, in some embodiments, surveyed area may be processed into the form of wire, and its direction is parallel in fact with the direction that detected sample flows through analytical equipment.

[0145] in some cases, described film also can be determined a check plot (not shown), and it can be used to the signal that normally carries out to analysis of user.For example, described check plot (not shown) can contain a kind of fixing agent, described fixing agent generally have with probe or be fixed on the probe reagent chemistry and/ability of physical bond.This reagent includes but not limited to, for example, and antigen, haptens, antibody, a-protein or G, avidin, streptavidin, secondary antibody or their mixture.In addition, also may wish to use various non-biological material to distinguish reagent in contrast.For example, in some embodiments, described check plot reagent may comprise a kind of polyelectrolyte, and is aforesaid, can be incorporated on the loose probe.Because the reagent of described check plot is specific to probe only, whether analyte exists all can have signal to form.Described check plot can be placed on any position along film, but is preferably placed at the upstream of detection zone.

Whether [0146] use this analytical equipment to detect certain analyte exists and can use various form.For example, in the above-described embodiment, utilized a kind of sandwich mode.Other utilize example that this sandwich measures referring to No. the 4th, 168,146, people's such as Grubb United States Patent (USP), and No. the 4th, 366,241, the United States Patent (USP) of Tom, and these documents are quoted by drink fully at this.In addition, also can use other modes, as competitive mode.In competitive mode was analyzed, label probe matched with the same or analogous molecule of analyte with one usually.So, label probe just and test analyte compete utilizable reagent.Usually measure with competitive mode when check and analysis thing such as haptens, each unit price haptens is only in conjunction with an antibody molecule.The example of competitive mode immunoassay is referring to No. the 4th, 235,601, people's United States Patent (USP)s such as Deutsch, and the 4th, 442, No. 204 and the 5th, 208, No. 535 patents of Buechler of Liotta are quoted fully at these these documents.Other forms of equipment is referring to the 6th, 194, No. 220 patents of people such as people's such as the 5th, 395, No. 754 patents of people such as Lambofte, Jou the 5th, 670, No. 381 patents and Malick.These patent documentations are quoted fully at this.

[0147] microfluidic control device

[0148] of the present invention aspect some, antibody disclosed herein can be integrated in the micro-fluidic device.This equipment is a microfluidic flow system, can be in conjunction with one or more analytes.Can directly separate on analysis or the slave unit on the equipment in conjunction with last analyte, for example do further to analyze or handle.Selectable, the analyte that is not attached on the equipment can be collected, and for example, further processes or analyzes.

[0149] exemplary equipment is a kind of flowing-type instrument that has the dull and stereotyped passage that can supply the sample circulation.This kind equipment is referring to United States Patent (USP) the 5th, 837, No. 115.Sample can be transferred in this equipment by gravity, kapillary or a kind of active force, as by infusion pump with a kind of sample such as blood perfusion in micro-fluidic device.Other known carrying methods also can use.The micro-fluidic device at random row of one in dependence equipment structure fixes analyte.Described structure can be by the several different methods manufacturing, include but not limited to laser radiation, embossing, x optical micro-image (LIGA), plating, electroforming, photolithography, active-ion-etch, ion beam processing method, compression molding, casting, power injection molding, jet molding method, micromachining material.Need be appreciated that it is not crucial making the employed method of present device, as long as method therefor causes a large amount of same structure and equipment.And this method must cause the high surface area of described structure and arrange closely to produce a narrow passage mutually.Described passage allow analyte in liquid, disperses with raising in fixing site the fixing efficient of analyte and/or labelled reagent.

[0150] structure of described production in enormous quantities is preferably made by the polymeric material of any amount.These materials comprise, but be not limited to, polyolefins such as polypropylene, polyethylene, polyester such as polyethylene terephthalate, the vinylbenzene such as the polystyrene that contain polymkeric substance, styrene-acrylonitrile, the acrylonitrile butadiene estyrene, polycarbonate, acrylate copolymer such as polymethylmethacrylate and polyacrylonitriles contain the muriate of polymkeric substance such as the muriate and the polyvinylidene dichloride of ethylene polymerization thing, acetal homogeneity polymkeric substance and multipolymer, Mierocrystalline cellulose and ester class thereof, cellulose nitrite contains the fluorochemical such as the poly(vinylidene fluoride) of polymkeric substance, tetrafluoroethylene, polymeric amide, imide, polyether-ether-ketone contains sulfide such as the polyphenylene sulfides and the polyether sulfides of polymkeric substance, Polyurethanes contains the silicide such as the polydialkysiloxane of polymkeric substance.In addition, described structure can be made by the mixture of multipolymer, above-mentioned materials and/or the metal that rolls the film of thing, tinsel such as aluminium foil, metal treatment or be deposited on above-mentioned materials, also can be made by glass or stupalith.In a kind of method therein, a kind of laser such as excimer laser can be used to the irradiates light mask, so that the light that passes from photomask melts primer to form passage (Sercel in material basic unit, J., et al., SPIE Proceedings, Vol.998, September, 1988).

[0151] common, micro-fluidic device can comprise an inlet that supplies testing sample to enter.Common described passage is a kapillary, be used for shifting described testing sample from the import to equipment in, comprise that also row's structure is used to provide fixedly site, and port is as the outlet of emission gases.Can also increase reactive tank (chambers) and extra kapillary when in addition, customizing this equipment.Usually, testing sample relies on capillary force to move through equipment.In addition, can use one or more kapillaries drive testing samples from the import to the passage in.In addition, can use the structure area (structuresarea) of one or more kapillary separation instrumentations.But, also can use different pressures to drive liquid and in equipment, flow, replace or increase kapillary power with this.

[0152] for liquid the mobile passage by producing between the adjacent structure body.Design is important for the contacting of surface and fluid molecule of optimizing structure to passage with structure.Typically, the degree of depth of passage approximately from 1 μ m to 1mm.The width average of passage generally approximately from 0.02 μ m to 20 μ m.Passage can comprise the structure of different shape, comprises rhombus, hexagon, annular or square, its highly generally approximately from 1mu.m to 1mm, width average approximately from 1 μ m to 1mm.

[0153] fixing agent can be as the surface that covalently or non-covalently is attached to structure in kapillary and/or reactive tank.Described reagent can be the release reagent of prescribing a time limit, spatial separation reagent, or coated and dry in the surface.Placing fixing agent is known by those skilled in the art to the technology on described surface.In one embodiment, described fixing agent be disclosed herein be the antibody of target with influenza antigen (as H5 AIV).

[0154] uses the method for present device to comprise and utilize the specificity binding member.The method that detects comprises that going into fluorescent dye with coloured marker obtains combining of coloured particle.Selectively, detecting step can comprise and the combining of a kind of enzyme that produces coloured product.

[0155] can use one or more displaced flowpathss in the equipment of the present invention.Kapillary is branched off into different paths behind import transportation testing sample: lead to the major avenues of approach of structure and replace path.Replace path can allow the existence in a plurality of fixedly sites, and whether or the quantity of a plurality of analytes the existence that allows to realize simultaneously analyte on single test site.In a preferred implementation, a plurality of analytes in test set (different influenza subtype) are determined.

[0156] alternative path can comprise the zone of mixing described test kit testing sample.For example, reactive tank can be used as the zone of adding reagent.In addition, can comprise also in this device channels that trapping apparatus (trapping device) is in order to remove the fluid composition more than the certain size.For example, in equipment of the present invention, can comprise a separator, for example from whole blood, isolate blood plasma or serum.For example, a kind of adhesive substance of hydrophobic sintered porous seepage material (matrix) can make a kind of erythrocyte adhesive application to its surface.Described adhesive substance can be placed on the front of structure described in the equipment.Erythrocyte in the whole blood sample is trapped in the space of described adhesive substance, and substantial hemocyte freely blood plasma and serum pass described adhesive substance, and be transported on the structure in the equipment by capillary force.Quote United States Patent (USP) herein totally No. 4933092.

[0157] automatization

[0158] antibody of the present invention is easy to adapt to automatization immunochemical analyses instrument.For making things convenient for the automatization of the inventive method, and reduce fringe time, a sessile antibody in the immunoassay of the present invention needs and the magnetic-particle combination.

[0159] by obtainable business method antibody is combined with such magnetic beads, as [the Lake Success of Dynal company, N.Y. (USA)] the Dynabeads of M-280 goat anti-rabbit igg bag quilt and the rabbit antibody of target protein, or the M-450 Tosylactivated Dynabeads of use Dynal company, the state is in conjunction with a last associated antibodies.Selectively, reagent such as glutaraldehyde can be used to make target antibody to be covalently bound on the solid support, preferred magnetic beads.Representational binding reagents can include organic compounds such as thioester, carbonization imide, succinimide ester, vulcabond, glutaraldehyde, diazobenzene, hexamethylene-diamine.

[0160] preferred automatization/immunoassay system is ACS:180.RTM. robotics luminescent system [USA New York Ta Lidun and a Massachusetts horse Field Beyer Co., Ltd; Comprise ACS:180 PLUS system; ACS:180SE system and ACS:CENTAUR.RTM. system].ACS:180.RTM. active immunity is measured system referring to document Dudley, B.S., J.Clin.immunoassay, 14 (2): 77 (Summer 1991).This system utilize chemiluminescent labels as tracer agent and paramagnetic particle (PMP) as solid-phase reagent.The ACS:180 system provides competitive mode combination and sandwich in conjunction with two kinds of mensuration modes, and wherein each step all is automatization.The ACS:180 system uses the micron order paramagnetic particle so that the maximization of available surface-area, and provides a kind of the need centrifugally just can separate the method for bonded tracer agent by quick magnetic from unconjugated tracer agent.Reagent can be added simultaneously or be added after a while.Other markers as enzyme mark thing, also can replace using as chemiluminescent labels such as acridinium esters.Preferably detect luminous signal with photometer.The preferred immune 1.TM. immunoassay system that uses Beyer Co., Ltd.Other exemplary automatic equipments that can adapt to the operation immunoassay of using antibody of the present invention comprise United States Patent (USP) the 5th, 807, No. 522 and the 6th, 907, and No. 722.

[0161] in another embodiment, anti influenza antibody of the present invention can be integrated into automatic porous plate so that use method of immunity.Porous is analyzed mould (as flat board) and can be adapted to analyze inductive in one or more holes of mould or the reactive tank (as, the hole of porous analysis plates) based on chemiluminescent mensuration in porous.The porous analysis plates can comprise several elements, for example comprises dull and stereotyped top, dull and stereotyped bottom, hole, working electrode calculates electrode, reference electrode, insulating material, the contact surface of electrical connection, be electrically connected conduction through hole, tackiness agent, analytical reagent, identification marking or the mark of described electrode and contact surface.Hole on the flat board can be the opening at dull and stereotyped top, and the inwall of opening can be a hole wall.(can be directly bonding or bonding by other compositions) bottom as the hole can be bonded together with the top bottom dull and stereotyped.

[0162] described porous analysis mould (as flat board) can contain any amount, arbitrary shape or size, reach hole and/or the reactive tank of being made up of multiple differing materials with arbitrary shape or conformational array.In a preferred real-time mode of the present invention, described porous analysis plates is the porous plate that utilizes industrial standards to produce, and has the flat board of standard form and quantity, size, shape and the conformation in hole.The standard pattern example comprises 96-, 384-, and 1536-and 9600-orifice plate, described hole is with two-dimensional arrangements.Other styles comprise single hole, diplopore, 6-hole and 24-hole and 6144-orifice plate.Preferably, described hole and/or reactive tank have first electrode that is incorporated into wherein at least, more preferably, comprise at least one second electrode.According to preferred real-time mode, described hole and/or reactive tank contain a working electrode that is incorporated into wherein at least, more preferably also comprise at least one calculating electrode.According to a particularly preferred embodiment, working electrode, calculate electrode and randomly, reference electrode all is integrated in hole and/or the reactive tank.Described analysis is dull and stereotyped preferred smooth, but also can be crooked (uneven).

[0163] and, in the hole, in the reactive tank and/or analyze that (for example, in the hole of porous analysis plates) can comprise one or more analytical reagents in the analyzed area of mould.For example, can use the analytical reagent of the different phenotype antibody that contain different Antibody of Influenza or a kind of influenza virus polypeptide in the different zones of droplet plate.These analytical reagents can be fixed or be placed on the surface of one or more holes and/or reactive tank (preferably on the surface of electrode, most preferably on the surface of working electrode), and (for example can be fixed in or be placed in one or more analyzed areas, be fixed in or be placed on the surface of one or more holes and/or reactive tank with the reagent of certain style arrangement, preferably on the working electrode and/or the surface of calculating electrode, most preferably on the surface of working electrode).Described analytical reagent also can be by the profile of described hole and/or reactive tank involved or location within it.For example, the insulating material of certain style can limit or locate fluid.

[0164] in one embodiment, instrument of the present invention can be used to impel and measure luminous in analyzing mould, preferably porous analysis plates.What can combine has, for example, and one or more photon detectors; Light tight shell; The electric connector that is used for the contact analysis mould; Transmit porous and analyze the mechanism of mould turnover instrument (more specifically, being the mechanism of the light tight shell of turnover); Associating and location porous are analyzed the mechanism of mould and photon detector and electric connector.Observe and distinguish mould device (as, one or more bar code scanners (as, a side of bar code scanner scanning flat board or mould, the opposite side of another scanning flat board or mould)); Aspect sensor; Make the mechanical means that electric connector is arranged with mould, one or more guiding luminous power supply in mould; With suitable electronic installation and software.

[0165] described instrument also can comprise storage, stacking, move and/or the mechanical means of one or more analysis moulds that distribute (as, porous plate stack).Described instrument can use photon detector array (for example, photodiode array) or image-forming photon detector array (for example, CCD photographic camera) luminous to measure easily.The light that these detectors allow these apparatus measures to send from porous (and/or reactive tank), simultaneously also/or make the light intensity and the spatial distribution imaging of from single hole (and/or reactive tank), sending.

[0166] preferably, described instrument can be measured the light that sends from the one or more districts that analyze mould, and preferably analyzing mould is the porous analysis plates.In some embodiments, a district comprises a group hole (and/or reactive tank), and quantity is analyzed the total hole of mould (and/or reactive tank) number (for example, the delegation of a porous plate mesopore, row or a two-dimentional submatrix) from one to being less than.One preferred embodiment in, a district comprises the hole of the 4%-50% of porous plate hole count.In a particularly preferred embodiment, the porous analysis plates is divided into cylindrical portion face (for example, there are a delegation or a row hole in district) or square department face (for example, standard-sized porous plate cup is divided into the square department face of six equidimensions).In some embodiments, a district comprises one or more holes and more than one fluid containment zone is arranged in the hole.Described instrument, preferably, can in a given mould (preferred dull and stereotyped), progressively induce ECL also/or progressively measure from described district and the ECL that comes.

[0167] described instrument also can be integrated microprocessor and computer and comes some function in the controling appliance, and helps storage, analyzes and display data.These microprocessors and computer can be positioned within the instrument, or be positioned at a distance and and instrument continuous (for example being connected) by network.

[0168] film/surface

[0169] in all many-sides of the present invention, the equipment of having integrated influenza antigen or resisiting influenza virus antibody comprises a surface or film.Kinds of surface or film can provide a surface for various common immunoassay device, and antibody or antigen are fixed or are processed thereon.Like this, film can provide and comprise surveyed area and use immunoreagent to make the visual control zone of detected result (for example, no matter sample contains in one or multiple virus).In numerous embodiments, containing influenza antigen or handling thereon has the film of resisiting influenza virus antibody to be handled (for example lateral flow or Mierocrystalline cellulose test paper device) on the solid support successively.

[0170] antigen/antibody can be incorporated on it film or the surface form by a kind of material, this material includes but not limited to Mierocrystalline cellulose, nitrocotton, nylon, has the positively charged ion nylon of a tetravalence amino (Zata probe), be converted into the aminophenyl thioether (aminophenylthioether of DPT, APT) test paper, azido derivant (not being colored when using with enzyme certification mark thing) or hydrophilic polyvinylidene fluorochemical (PVDF) (can be by Mass., Billerica, Millipore obtains).Term " digestion fiber " refers to any cellulosic nitric ether.So suitable material can comprise and cellulosic carboxylicesters bonded nitrocotton.Can there be very big variation in the aperture of nitrocellulose membrane, but normally about the 5-20 micron, preferably about 8-15 micron.But those skilled in the art can expect that other materials known also can be used.In some embodiments, described test zone comprises that the quilt of being produced by Millipore overstocks the netted assembly of nitrocotton that rolls into to the nitrocotton of the polyester film backing of a light.In another embodiment, this contains antigen/antibody zone (or " test zone ") and is made by nylon.In another embodiment, the particle of the reagent of another specificity bound analyte can be carried, therefore a test zone, for example Ya Zhi nylon powder, or glass fibre can be determined but described test zone comprises a kind of fixedly rubber or other.In some embodiments, described test zone comprises a kind of opaque and in the dampness material transparent in drying regime.

[0171] test and check plot

[0172] equipment can comprise film or the surface of containing test and check plot, and test zone can be made of any of above-mentioned materials.Usually the composition of test zone can be determined in test zone and check plot.In one embodiment, described test and check plot comprise and the test zone identical materials.Usually, term " test zone " this be used for explaining in equipment/on comprise the zone of at least one test and check plot.In some embodiment, equipment uses water-absorbing material and in some embodiments, and for non-absorptive fluid is provided, these materials will be terminated liquid and handle and cause the absorptive reactive force of absorbent membrane to interrupt.Suitable stop buffer comprises bovine serum albumin, methylated bovine serum albumin, and the whole serum of animal, casein, skim-milk, a large amount of sanitising agents and polymkeric substance, for example, PEG, PVA and analogue thereof.In some embodiments, the interference site on untreated absorbent membrane is because the existence of stop buffer and completely destroy has allowed expense water-absorbent fluid to pass through.As pointing out that herein testing apparatus of the present invention is an equipment that has a plurality of tests and check plot.

[0173] surveyed area generally comprises one or more check plots, and whether flow for verification sample like this is being very useful of expection.Each check plot all comprises different zone on the space, a specificity is often arranged in conjunction with the paired retaining element in this zone, and this element can react with the contrast agents of mark.In certain embodiment, the check plot on the program comprises the reliable sample of a test analyte, or a segment of sample.In this embodiment, used a kind of labelled reagent, described liquid sample carries labelled reagent and arrives test and check plot; The labelled reagent that is not incorporated on the assay just will be attached on the reliable sample of the assay to be measured that is positioned at the check plot.In another embodiment, control line contains antibody, and this antibody is specific to labelled reagent or provides for the fixed labelled reagent.In operation, labelled reagent is limited in each one or a plurality of check plot, even any one or all interested analytes do not exist in sample to be tested.

[0174] in some embodiments, solid support comprises the zone of certain style, and described zone comprises antigen/antibody in conjunction with the adhesive substance field, and this field can be designed to any shape of wanting (for example, square, ellipse, circle, vertical line or sea line).For example, antigen is processed on the plain test paper of solid Muller's fibers in conjunction with adhesive substance (matrix) field, and described Mierocrystalline cellulose test paper can be made by plastics or polyester film material.The application of the invention, by integrating each contains different specific antigenss on the different positions of single calibration tape or single solid support Mierocrystalline cellulose test paper a plurality of square adhesive substance, might in an independent test, be measured to a plurality of anti-H5AIV antibody subtypes.

[0175] in various embodiments, contains antibody of the present invention and the device that is applied in the immunoassay can be included in the test kit.Use for the immunoassay of special form, this test kit has comprised necessary reaction reagent.This test kit comprises a Mierocrystalline cellulose test paper and the independent reagent with its application, has fixed the lateral flow device of analyzing the required antibody of the multiple hypotype of influenza on it, perhaps any conventional equipment that has necessary reagent.For example, process by the frequent a series of reagent that in test kit, provides of dipping of Mierocrystalline cellulose test paper, being with or without specific anti--subtype influenza virus (for example, H5AIV antibody) or influenza antigen (for example, H5 antigen) in sample can be determined quickly and easily.This test kit is fit to expert or personnel's use in the industry.Utilize the influenza virus that test kit of the present invention can carry out obtaining from body fluid fast (for example, serum diagnosis AIV), body fluid can be blood, urine, saliva, seminal fluid, ight soil, saliva, bile, cerebrospinal fluid, nasal mucus, genitourinary/urogenital liquid, nose are exhaled, spinal fluid, or the like.

[0176] in another embodiment, plain test paper of the Muller's fibers of solid phase or lateral flow device are placed in a test tube or the similar container, this testing tube or similar containers have added influenza virus (for example, patient AIV) or the animal specimen of having infected under a cloud.Sample and the antigen/antibody that is combined in the Mierocrystalline cellulose test paper react.Take out Mierocrystalline cellulose test paper and cleaning lightly then.The plain test paper of solid phase Muller's fibers is taken out from scavenging solution and is placed in another test tube, this test tube contains high dilution, immunoglobulin (Ig) through affine separation and purification, this immunoglobulin (Ig) is to being specific from the species that wherein obtain sample, and can with alkaline phosphatase or other suitable enzyme combinations.Then the Mierocrystalline cellulose test paper takes out and puts into a container that has scavenging solution from second antibody solution.In case taking out from scavenging solution, the Mierocrystalline cellulose test paper just is placed in the final test tube that has developer or other suitable substrate solutions.Positive reaction can evaluate by the comparison of simple observation reference standard (top line) and positive line (following line).If positive line color is darker than standard lines, detected result is exactly positive so.Covalently bound enzyme and the substrate reactions that arrives through the immunoglobulin (Ig) of affinity purification, described substrate produces a colored reaction product when the end of enzyme-substrate reactions.The immunoglobulin (Ig) of the closely-related purifying of Lian Jieing just can detect easily in this way, and this detection is presented in the sample and exists the specific antibody of influenza virus (as, H5 antibody).This technology has been integrated known elisa technique, and exposure is also arranged herein.

[0177] required being understood that selects the technology of suitable enzyme and substrate and proper reaction conditions to be known by those skilled in the art.These enzymes with immunoglobulin (Ig) in conjunction with after activity will still be arranged.Each enzyme-substrate paired chemical reaction all will obtain a coloured product.In addition, the matching method that has plurality of optional to select, wherein enzyme and substrate can with the immunoglobulin (Ig) combination through affinity purification, but only after the immunoglobulin (Ig) of purifying has been connected to sample antibody, the reaction of enzyme and substrate could produce coloured reaction product.

[0178] certain, by using different antibody/antigen, sample can filter out a different set of virus and different hypotypes easily.Those skilled in the art should be understood that by immunoassay described herein need analyze different groups.Referring to CURRENT PROTOCOLS INIMMUNOLOGY (Coligan, John E.et.al., eds.1999).

[0179] array

[0180] antibody of the present invention can be used in the equipment easily, method with this Equipment Inspection analyte is a kind of method for quick, this method comprises that one or more influenza virus proteins of detecting in the sample (for example, H5) or one or more anti--Antibody of Influenza.This method comprises the embodiment that antibody is showed with the form of the array that contains other antibody, described other antibody its with multiple different virus such as other influenza virus sub-strain (as, AIV) be target.In other embodiment, antibody can be different epitope combination on target or specific and the set polypeptide with identical antigen.

[0181] in the further embodiment of the present invention, the array of antibody comprises substrate, the small segment (patch) of a large amount of discrete arrangements, on the known region of substrate surface part, wherein (i) each fragment comprises the antibody that is fixed on the substrate, antibody can be in conjunction with specific expressing viral product in the wherein said set fragment, the segment of product, perhaps host protein, antiviral antibody for example, (ii) array comprises a lot of different antibody, each antibody can be in conjunction with different expressing viral products, the viral product segment, perhaps host protein is as antiviral antibody.

[0182] preferably, antibody covalently is fixed on the segment of array, and can directly fix also can indirect securement.As a rule, array comprises 10 segments at least.In a preferred embodiment, array comprises 50 fragments at least.In a particularly preferred embodiment, array comprises at least 100 fragments.At one optionally in the preferred embodiment, antibody array can comprise and surpasses 10 ³, 10 ⁴, perhaps 10 ⁵Individual fragment.

[0183] the substrate surface zone that is covered by each fragment preferably is no more than 0.25mm ²Preferably, the substrate surface zone of each fragment covering is at about 1 μ m ²To 1000 μ m ²In a particularly preferred embodiment, the substrate surface zone of each fragment covering is at about 1 μ m ²To 2500 μ m ²In an alternate embodiments, the area that a fragment on array can cover substrate surface has 2500nm ²So little, even so little fragment generally there is no need in the middle of the application of array.

[0184] fragment in the array can be any geometrical shape.For example, fragment can be an orthogonal, circular.Fragment in the array also can be irregular shape.Fragment also can be random begin to increase from lower floor's substrate intermediate layout (median plan).

[0185] the segmental distance that separates on the array can be different.Preferably, the intersegmental about 1 μ m of distance between adjacent of the sheet on the array is to 500 μ m.Typically, if segmental diameter greater than 10 μ m, separates on the array segmental apart from proportional with segmental diameter or side length approximately.If fragment is smaller, generally all the diameter than fragment itself is big at this moment to separate segmental distance.

[0186] in a special embodiment of array, all fragments of array all are comprised in about 1cm on the substrate surface ²Or littler zone.So in the embodiment of a preferred array, the about 1cm of array total area on substrate surface ²Or 100 or multi-disc section have more been comprised in the littler zone.Selectively, the about 1cm of particularly preferred array total area on substrate surface ²Or comprised 10 in the littler zone ³Individual or more fragment.The preferred about 1cm of array total area on substrate surface ²Or in the littler zone, even can arbitrarily comprise 10 ⁴Perhaps 10 ⁵Perhaps more fragment.In other embodiments of the invention, the fragment of all arrays is included in that area is approximately 1mm on the surface of substrate ²Or in the littler zone.

[0187] typically, on a fragment of array, has only an antibody.If more than one antibody on a fragment, all antibody on that fragment must a shared common binding partner so.For example, fragment can comprise the antibody (though antibody potentially can be in conjunction with the different epitopes on the set influenza virus) of a variety of influenza virus proteins.In a preferred embodiment, described influenza virus protein/antigen is that H5 and influenza virus are AIV.

[0188] array among the present invention can contain the different antibody of day with quantity.Typically, described array comprises at least 10 kinds of different antibody.Preferably, described array comprises at least 50 kinds of different antibodies.More preferably, described array comprises at least 100 kinds of antibody.Selectable preferred array comprises above 10 ³Plant different antibodies or surpass 10 ⁴Plant different antibodies.Described array even can arbitrarily comprise and surpass 10 ⁵Plant different antibodies.

[0189] in an embodiment of array, each fragment of array comprises a kind of different antibody.For example, an array comprises that about 100 fragments can comprise about 100 different antibody.Similarly, one contains about 10000 segmental arrays and suddenly comprises about 10000 kinds of different antibodies.Yet in an alternate embodiments, each different antibody is fixed on a plurality of fragments of array.What for example, each different antibody can be random occurs in 2-6 different fragment.Therefore, an array of the present invention can comprise about 3000 antibody fragments, but only comprises about 1000 kinds of different antibody, because each different antibody occurs in 3 different fragments.

[0190] typically, can be had at least 10 by the different proteic quantity of a large amount of different antibodies bonded on the array.Yet preferably, a large amount of different antibody on array can be used in conjunction with the different albumen of a large amount of quantity, for example, and about at least 50 or about at least 100.In further preferred embodiment, a large amount of different antibodies can be in conjunction with about at least 10 on array ³Individual albumen.

[0191] utilize the antibody array in the present embodiment can arbitrarily be included in the flowing reactive groove with every 25mm ²The form of surface-area 1-10uL liquid volume is placed two-dimensional array.The case that covers on the array in the flowing reactive groove can be transparent or semitransparent.In a real-time mode, described case can comprise thermal glass or silica glass.In other embodiment, described case may be the part of detection system, is fixed on antibody and solution on the array with monitoring, as the reaction between the protein in the extraction liquid of cell.Described flowing reactive groove should keep containing suitable liquor with protection antibody.Salinity, temperature and other condition optimizations keep similar to normal physiological conditions.Sample in fluid solution may infiltrate in the above-mentioned flowing reactive groove, and they are affirmed and the fixed antibody response.Need provide time enough to allow antibody and binding partner generation combination thereof.This required time is depended on the avidity of antibody to its binding partner.Fluid is transported to does not need special miniflow pump, valve or hybrid technology in the array.

[0192] detection method

[0193] uses in the suitable any device of antibody of the present invention, can obtain the existence that being detected as of a lot of classes assigns to detect binding partner.Detection can be quantitative also can be qualitative.Array of the present invention can share with absorption, chemoluminescence and the fluorescence (comprising validity period, polarization, fluorescence correlation spectroscopy (FCS), FRET (fluorescence resonance energy transfer) method (FRET)) of optical detecting method such as visible light or UV-light.And, other detecting patterns, as based on light wave, referring to PCT open text (WO 96/26432 and United States Patent (USP) the 5th, 677, No. 196), based on surface plasmon resonance analytical technology (surface plasmonresonance), based on the surface charge transmitter, based on the surface action force transducer can with the many embodiments of the present invention compatibility.Selectively, based on Brewster angle microscopy (Brewster Angle microscopy) (Schaaf et al., Langmuir (BAM), 3:1131-1135 (1987)) and ellipsometry (U.S.Pat.Nos.5,141,311and 5,116,121; Kim, Macromolecules, 22:2682-2685 (1984)) science and technology also can be employed.Quartzy little poising action (microbalances) and desorption process (referring to No. the 5th, 719,060, United States Patent (USP) for example) also provide the detection method of other suitable some embodiments of array of the present invention.Referring to United States Patent (USP) the 5th, 313, No. 264 visible a kind of optical biosensor system can be compatible mutually with matrixes more of the present invention, also can be compatible mutually with multiple unlabelled detection method such as surface plasmon resonance analytical technology, internal reflection fluorescence microscope (TIRF), Brewster angle microscopy, light wave light-emitting mode spectrography (OWLS).

[0194] in some embodiments, the described equipment of having integrated antibody of the present invention can be integrated in the system, described system comprises reader (reader), particularly be arranged on the reader in the computer, as reader based on reflection and/or fluorescence, and comprise a data processing software that uses reduction of data (data reduction) and curve fitting algorithm, preferably neural network (trained neuralnetwork) logotype of being trained with quilt is to determine whether existing and concentration of in biological sample analyte exactly.Reader as used herein is meant the instrument of a kind of detection and/or quantitative data, on calibration tape (test strips) in being included in the testing apparatus of using antibody of the present invention.Data should be Glassless, but do not need so.Described method is included in the step of carrying out immunoassay on the patient samples, uses the step based on the reader reading of data of reflection and/or fluorescence, uses and utilizes the data processing software of reduction of data to handle the gained data.Preferred software comprises curve fitting algorithm, can be at random and the neural network logotype of being trained, and to determine whether existing and concentration of in testing sample assay.The data that obtain from reader can be handled output data so that the diagnosis of risk assessment or medical condition to be provided by medical diagnostic system further.Can select in the embodiment, described output data can be used to be input to follow-up conclusion supporting system, and as neural network, it can be by training to assess these data.

[0195] in how various embodiments, described reader can be the reflection reader, the emission reader, the fluorescence reader, the luminous reader of chemical-biological, magnetic force reader or current measurement reader (or two or more combination), this depends on wants the signal that detects in the slave unit.And some detection method generally is used for traditional immunoassay, and this method needs the applying marking thing, and these detection methods may be used in the array of the present invention.These technology comprise non-competing formula immunoassay, competitive mode immunoassay, double-tagging thing method, ratio immunoassay.These specific technology mainly are useful in when having the number very little (approximately less than 100) of homospecific different antibodies not and antibody array when using together.In described competitive mode method, occupying of binding site is to determine indirectly.In this method, the antibody of array is exposed in the development reagent of mark, and described development reagent normally has analyte or its analogue of mark.Described development reagent and analyte competition are combined in the site on the antibody.Development reagent is attached to can be formed on occupying on a small quantity of antibody on the different segments on the single pulsating antibody.

[0196] in non-competing formula method, occupying of binding site is directly to determine.In this method, the segment of described array is exposed in the developing solution of mark, described developing solution can be incorporated on the combined analyte or in proteopexy reagent with by on the binding site that occupies.For example, described developing solution can be and be occupied the directly corresponding antibody that is labeled (for example, " sandwich analysis ") of site.Selectively, double label method, when described antibody by a kind of marker mark, adopt ratioing technigue (Ekins, et al., Clinica Chimica Acta., 194:91-114,1990) when developing solution is carried on the back another marker mark.In aforesaid technology, can use a lot of different marking methods, comprise radioisotope method, enzyme process, chemoluminescence method and fluorescent method.In some embodiments, preferred fluorescence detection.The method that detects includes but not limited to the change of the change of color, photoabsorption or light transmission, the change of PH, conductive change, epipolic change, the variation of physics phase or similar approach.

[0197] testing sample should provide the detected composition of detection system or this composition to be added into.Described composition widely different, this depends on the character of detection system.Wherein a kind of detection method comprises the use particle, and particle is used to provide astigmatism or changes velocity of flow.Described particle can be, but be not limited to cell, the polyparticle that can not be blended in liquid system, rubber grain, ceramic particle, nucleic acid particle, bonding particle or its analogue.Particulate selects to depend on detection method, when change of fluid dispersive distribution situation and stability, activity, participations etc. are like that.Fixed bit point analysis thing combine with the specificity binding member can be by test set in the pressure of testing sample arbitrarily detect.For example, the pressure detector that is connected with testing sample enters or can detect when withdrawing from passage pressure to be reduced, and it is because the combination of analyte that described pressure reduces, and it can cause, and flow velocity slows down in the passage.

[0198] for example, for determining that (for example, antibodies) amount reaches the amount that antigen exists to already present detectable marker, can use people's such as Hazelgrove method and instrument (referring to Anal.Biochem150:449-456,1985).This method is based on the television camera that is connected with computer.Described mark is presented on the luminous box by the television camera imaging, and by a kind of digitizer tablet (Techmar company) digitizing.After the digitizing, computer can read out position, width, height and the relative area of each mark.Photo densitometry also is used to measure proteinic absolute concentration.

[0199] in another embodiment, will comprise that the device that Dot-ELISA tests is used to detect direct target protein from any sample.Thereby, the method that antibody of the present invention can be used for comprising the following steps:

(a) provide a solid support, so that carry out the mensuration of monoclonal antibody;

(b) sample that suspection is had an influenza virus is applied on this solid support;

(c) solution that will contain organic acid such as citric acid or lactic acid is applied on the solid support;

(d) solution that will contain molten mucus agent (mucolytic agent) and stain remover is applied on this solid support;

(e) this solid support being contacted with elementary monoclonal antibody (primary Mab), chimeric monoclonal antibody, its variant or fragment is the enough time, make elementary monoclonal antibody, chimeric monoclonal antibody, its variant or fragment and H5AIV protein binding be in the same place, form the elementary monoclonal antibody of conjugated antigen;

(f) contact time enough, make the elementary monoclonal antibody of this conjugated antigen be convenient to be attached on this conjugate with the anti--monoclonal antibody conjugate (conjugate) of enzyme labelling above-mentioned; And

(g) colouring reagents is applied on this solid support, wherein, this colouring reagents is formed the sign of band look by enzyme catalysis, thereby makes it possible to whether exist in the visual detection sample H5AIV albumen.

A kind of Dot-ELISA method is disclosed in the U.S. Patent application 2006/0246429, herein with its integral body document as a comparison.

[0200] in some embodiments, device of the present invention or test kit can be used for nursing (caresetting) field, wherein, can utilize the color developing detection system as adopting the system of alkaline phosphatase.For example, isolate some bottles, its fowl has the streptavidin-alkaline phosphatase conjugate in damping fluid, NBT or 5-bromo-4-chloro-3-indyl phosphoric acid ester (BCIP), and it is designed to reach desired color successively, thereby makes it can be used as the component of test kit.Utilize the color developing detection system, its result can detect by an unaided eye and without any need for the help of equipment.In one embodiment, proofing unit can utilize alkaline phosphatase quantitatively to determine the AIV amount that exists.When using with photodensitometer, this device that comprises alkaline phosphatase can provide quantitative result accurately.

[0201] in one embodiment, the test kit of detection H5AIV albumen or anti-H5 hypotype AIV antibody can comprise: the certification mark thing is as combination streptavidin-alkaline phosphatase conjugate thereon; The reagent that contains NBT or 5-bromine 4-chloro-3-indyl phosphoric acid ester (BCIP); And reference standard.

[0202] in arbitrary embodiment that the application describes, specimen may be from but be not limited to the physiology source.The specimen that can be used for apparatus of the present invention comprises suspects the sample that has antigen or antibody, and these samples can be from inhuman animal body or human body; The specimen that can be used for apparatus of the present invention includes but not limited to: physiological fluid such as blood, serum, blood plasma, saliva, eye discharge, cerebrospinal fluid, purulence, transudate, milk, sweat, tears, ear effluent, phlegm, lymph liquid, urine, ight soil, mouth and nose secretory product; Tissue is as lung, spleen and kidney; Totivirus in the chicken embryo culture or the liquid of lytic virus; And other suspection contains the sample of influenza virus protein or resisiting influenza virus antibody, and having only it is solublely maybe can be suspended in the suitable fluid.Specimen can be through anticipating, as extraction, add, separate, dilution, concentrate, filter, distillation, dialysis or the like.Except physiological liquid, also can use other liquid testing sample, and interested composition both can be liquid, also can be solid, as long as this solid can dissolvedly maybe can be suspended in the liquid medium.In one embodiment, sample is taken from nasal cavity.Device of the present invention has the surface that can adhere to one or more antigens or antibody usually.

[0203] methods of treatment and pharmaceutical composition

[0204] method that the invention provides a kind of prevention and treat avian influenza virus related viral infections patient comprises the patient is intervened a certain amount of pharmaceutical cpd that pharmaceutical activity is arranged that has comprised one or more monoclonal antibodies of the present invention.The present invention also provides a kind of salt medicine that contains the pharmaceutical cpd of one or more monoclonal antibodies of the present invention or obtain on this basis.

[0205] means of intervention of pharmaceutical cpd of the present invention can be traditional intervention approach, comprise in oral, oral cavity, hypogloeeis, eyeball, part, parenteral, rectum, the leaf sheath, in the endoplasm net groove, inguinal region, intravesical, part (as, pulvis, ointment or drops), or nasal, but not only be confined to this.

[0206] pharmaceutical cpd of suitable parenteral route injection may contain and meet sterilized water or non-aqueous solution, aerosol, suspension or the emulsion that medication preparation requires, can be at the sterile powder that faces injectable solution of resuspended one-tenth of time spent or aerosol.As water-based and the non-aqueous carrier that is fit to, instrument and various diluent such as water, ethanol, polyol (as propyleneglycoles, macrogol, glycerol and analogue thereof), suitable mixture, rape oil (as sweet oil), with the alicyclic organic that can be used for injecting, as ethane oleic acid.As use the Yelkin TTS capsid to keep the proper flow of medicine.As use aerosol, tensio-active agent to keep suitable particle size.

[0207] pharmaceutical composition of the present invention can contain also that some play protectiveness, preserve moisture, the adjuvant of emulsification and aerosolization; also can contain the instant composition that prophylaxis of microbial is polluted; as various antibacterium reagent, antifungal agents; as parabens; chlorobutanol; phenol, Sorbic Acid and analogue.Also can comprise the reagent of keeping osmotic pressure, as sugar, NaCl and analogue thereof.Can use the reagent that prolongs absorption to prolong injectable drug composition adsorption time, as Monostearate and gel etc.

[0208] oral solid phase formulation comprises capsule, tablet, pulvis, granule etc.Activeconstituents in these solid phase formulations is mixed with a kind of traditional inert pharmaceutical vehicle (or carrier) at least as Trisodium Citrate, calcium phosphate, or (a) weighting agent or additive such as starch, lactose, sucrose, seminose and silicic acid; (b) tackiness agent is as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Sudan Gum-arabic; (C) wetting agent is as glycerine; (d) cracked dose, as agar, lime carbonate, potato powder or Tapioca Starch; (e) retardant is as paraffin; (f) short absorption agent is as the tetramino mixture; (g) wetting Agent for Printing Inks is as the pure and mild glyceryl monostearate of hexadecyl; (h) sorbent material is as kaolin and bentonite; (i) lubricant, as talcum, calcium stearate, Magnesium Stearate, solid polyethylene glycol, sulfuric acid Lauryil Sodium, or the mixture of its above-mentioned substance.In tablet and capsule formulation, may also contain buffer reagent.

[0209] the solid phase formulation can discharge or pulsed release dosage form by making improvement, be in the above-mentioned various direct release excipient of mentioning, to add some can change the vehicle of drug release rate and form, can be included in the form that also can make coat in the formulation.Speed discharges the transformation agent and comprises the carboxylic propyl methocel, methylcellulose gum, carboxymethyl cellulose sodium, Mierocrystalline cellulose ethane, cellulose acetate, polyethylene oxide, the xanthan glycocoll, different vinylformic acid ammonia multipolymer, hydrogenation flavor oil, carnauba wax, paraffin, the phthalic acid cellulose acetate, phthalic acid carboxylic propyl methocel, the mixture of Sipacril 2739OF or above-mentioned substance.Improvement discharges and pulsed release dosage form may contain a kind of or one group of vehicle with improvement rate of release.

[0210] pharmaceutical cpd of the present invention also can be made up of propellant or the solvent that disappears (FDDFs) fast, comprises following composition: aspartyl-phenylalanine methyl ester, sulfanilamide (SN) potassium, citric acid, croscarmellosesodium, crospovidone, xitix, the ethyl group acrylate, ethyl group Mierocrystalline cellulose, gelatin, the hydroxy propyl methocel, Magnesium Stearate, N.F,USP MANNITOL, methyl second butenoate, the seasoning peppermint, polyoxyethylene glycol, silica aerogel, silicon-dioxide, Vivastar P 5000, stearic acid fumaric acid sodium, sorbyl alcohol, Xylitol.Here being used to describe " atomize and disappear molten " speech of FDDFs, depending on the solvability of used medicine, is insoluble as medicine, can be made into nebulizer formulation fast, is soluble as medicine, then can be made into solvent-borne type fast.

[0211] the solid phase composition of similar type also uses the filling formulation of making soft gelatin or glutoid such as lactose or caramel or other high-molecular weight polyoxyethylene glycol and similar vehicle.

[0212] solid dosage such as tablet, sugar-coat agent, capsule and granule etc. can be made by the mode of the outsourcing capsid all known such as casing or other this area ordinary person.Also can be contain opacifying agent, also can be contain can rise slowly, postpone, similar composition that the control active medicine discharges.Also can use compositions such as polymer and paraffin to carry out embedding.If suitable, the formulation of the form of micro-capsule made activeconstituents by also available above-mentioned one or more vehicle.

[0213] is used for oral liquid dosage form, comprises emulsus agent, solution, suspension, syrup and the elixir etc. that meet the medicine requirement.Except activeconstituents, liquid dosage form also can contain this area some inertia solution commonly used, as water or other solvent, soluble reagents and emulsifying agent, as ethyl group alcohol, isopropyl alcohol, ethyl group carbonate, phenyl benzoate, third rare ethylene glycol, 1,3-methyltrimethylene glycol, oil, particularly, Oleum Gossypii semen, Peanut oil, Semen Maydis oil, sweet oil, flavor oil and sesame oil, glycerine, tetrahydrofurfuryl alcohol, polyoxyethylene glycol and fatty acid sorbitol ester, and the mixture of above-mentioned substance or similar material.

[0214] except these inert diluents, pharmaceutical cpd also can comprise adjuvants such as wetting Agent for Printing Inks, emulsifying agent, suspension agent, saccharifying agent, seasonings and flavouring agent.In addition, pharmaceutical cpd also can comprise ethoxylation homopolymerization ethanol, polyxyethylated sorbyl alcohol and sorbitanic fat, Microcrystalline Cellulose, an aluminium hydroxide, wilkinite, agar polymkeric substance and tragacanth, or the suspension agent of the mixture of these materials and so on.

[0215] pharmaceutical cpd of the present invention is also made the mixture that is fit to animals for treating, or meet salt for animals, or meet solvent for animals or first patent medicine, and make a kind of dosage of the most suitable certain particular animals and the dosage forms of approach medicine according to common animal doctor and animal doctor practitioner's requirement.

[0216] one or more monoclonal antibody of the present invention can be used for prevention and or treatment H5 avian influenza virus infection relative disease in conjunction with other Anti-virus agent.Monoclonal antibody can with these Anti-virus agents simultaneously, separately or successive administration.Other Anti-virus agent comprises ribavirin, diamantane, and the carboxyl urea, IL-2, IL-12 and five carboxylic chain born of the same parents acid, but be not limited only to these.

[0217] polypeptide of polypeptide screening method and antibody recognition and vaccine

[0218] the invention provides a kind of detection method of screening the mimic epitopes polypeptide of monoclonal antibody identification of the present invention.And the present invention also provides the epitope mimic peptide of monoclonal antibody identification of the present invention.On the one hand, the small peptide of the present invention's announcement contains aminoacid sequence SEQ ID Nos:64-68,70-73,74,76,78,80,82,84,86,88,90,92,94 and 96.These polypeptide can be in conjunction with monoclonal antibody of the present invention.Therefore, these polypeptide have the antigen-specific identical with the H5 hemagglutinin.These polypeptide also can be used for preparing H5 subtype avian influenza vaccine, also can be used to diagnose the existence of anti-H5 hemagglutinin antibody.

[0219] on the other hand, screening method of the present invention comprises the steps: that (i) cultivates the polypeptide display libraries of a suitable expression of polypeptides under given conditions; (ii) culture solution and monoclonal antibody of the present invention are mixed; (iii) screen the phage clone of specificity in conjunction with above-mentioned monoclonal antibody.The monoclonal antibody that is used to screen is not limited only to monoclonal antibody 8H5,3C8,10F7,4D1,3G4 and 2F2.Describe a kind of phage-displayed polypeptides library that utilizes among the embodiment of the present application 11-13 in detail and successfully screen small peptide detection method in conjunction with monoclonal antibody of the present invention.

Embodiment

[0220] below in conjunction with specific embodiment and accompanying drawing, the present invention is further described, but these descriptions is not construed as limiting the invention.

[0221] embodiment 1: the preparation of anti-H5 subtype avian influenza virus HA gene monoclonal antibody

[0222] antigenic preparation:

[0223] with virus strain Ck/HK/Yu22/02 (H5N1) (being called for short Yu22), inoculates the chicken embryo of being fertilized 9 day age, after 30 are hatched 2 days, collect the chicken blastochyle, the Yu22 virus after obtaining increasing.Collect live virus, under 4 with 0.03% Formalin formalin deactivation, inactivation of viruses detects through HA, (annotate: the concrete grammar that HA titer determination and HI detect is referring to the WHO operational guidance to determine the titre of inactivation of viruses liquid, we select HA=1024, and this virus strain is provided by Hong Kong University's department of microbiology).

[0224] small white mouse

[0225] 6 age in week, female Balb/c mouse was available from the anticancer center of Xiamen University, and at this center raising experiment.

[0226] preparation of hybridoma:

[0227] we use the interior immunization ways of body and the PEG fusion method of standard to obtain monoclonal antibody, detailed method is referring to Ed Harlow et al., " Antibodies A Laboratory Manual ", ColdSpring Harbor Laboratory 1988. concise and to the point processes are as follows:

[0228] mouse immune: with above-mentioned pretreated viral liquid and Fu Shi Freund's complete adjuvant (CFA) equal-volume mixing and emulsifying, through the limb muscle multi-point injection, every per injection 300ul.15d and 29d after the first immunisation add Freund's incomplete adjuvant (IFA) with the viral liquid of same dosage respectively and carry out booster immunization.Second strengthens the inhibition that the back blood sampling detects HI tires, reach 1: 640 when tiring after, get mouse spleen and do fusion.72hr booster immunization once more before merging, through tail vein injection virus liquid 1 time, 50ul/ only.Prepare 10 and merge plate.

[0229] merges: get the highest mouse spleen cell of serum HI titre and merge mutually with murine myeloma cell, earlier spleen is ground and obtain splenocyte suspension, the SP2/0 murine myeloma cell that is in logarithmic phase with low ten times of cell count mixes then, through PEG1500 effect 1min together, then fused cell liquid 100ml is divided to install in 10 96 orifice plates and cultivate two kinds of cytogamy.Merging substratum is the complete screening culture medium of RPMI1640 that contains HAT and 20%FBS.The antigen-specific sex clone suppresses (HI) experiment screening by blood clotting, after 3 time cloningizations, obtains stable cell strain of monoclonal antibody.

[0230] screening of hybridoma: merge the back cell after cultivating 10 days on the 96 porocyte plates, draw cell conditioned medium and do blood clotting inhibition (HI) detection, cloning is continued in positive hole, till the secreted antibody of cell strain can be stablized inhibition Yu22 strain virus and chicken blood generation aggegation.

[0231] The selection result: obtain six strain monoclonal antibody 2F2,3G4,3C8,4D1,8H5,10F7.

[0232] cultivation of hybridoma: stable authentic monoclonal antibody cell strain is amplification cultivation in CO2gas incubator earlier, is transferred to 24 holes through 96 holes, transfers to 50ml cell bottle through amplification cultivation.Injection cell in the collecting cell bottle is drawn ascites after 7-10 days from mouse peritoneal in mouse peritoneal then.

[0233] Purification of Monoclonal Antibodies:

[0234] earlier with 50% sulphur ammonium precipitation process, then to PBS, pH7.2's ascites dialyses, and uses DEAE post purifying under HPLC afterwards, obtains the monoclonal antibody behind the purifying, the monoclonal antibody purity after the SDS-PAGE purification Identification.

[0235] blood clotting of monoclonal antibody suppresses the active checking of experiment:

[0236] select for use 34 strains to derive from (the Chen et al. that belongs to the Different Variation subbreed on ground such as Vietnam, Indonesia, Malaysia, Thailand, Hong Kong, China and Europe, PNAS, 103:2845,2006) H5N1 virus and the non-H5 virus of 14 strains (H1～H13, chicken NDV), utilize hemagglutination-inhibition test method (HI) to identify described monoclonal antibody and viral reactivity, result such as table 1 and 2, as seen 5 strain monoclonal antibodies all have excellent specificity, and are all reactionless with non-H5 virus strain; And to the reaction of H5 virus strain, different monoclonal antibody strains there are differences the reactivity of different virus strain, except 3G4 response spectrum minimum, the response spectrum of remaining four strain monoclonal antibody and virus substantially all near or reach 100%.

[0237] table 1 monoclonal antibody is to the hemagglutination-inhibition test positive reaction ratio of H5 and non-H5 virus

[0238] table 2 monoclonal antibody suppresses titre to the blood clotting of 34 strain H5 C-type virus Cs

Annotate:＜, titre is less than 100.

[0239] monoclonal antibody is tested the neutralization of virus:

[0240] utilizes in the micropore and experiment detects neutralization activity (the Hulse-Post et al.PNAS of described monoclonal antibody to H5N 1 virus, 102:10682-7 (, 2005)), the results are shown in Table 3, it is active that visible monoclonal antibody 8H5 all demonstrates good neutralization to all H5N1 virus strains.

[0241] table 3 monoclonal antibody is to the neutralization experiment titre of H5N1 virus

Annotate :/, No data;＜, titre is less than 100

[0242] embodiment 2:H5 subtype influenza virus HA antigen detection kit (euzymelinked immunosorbent assay (ELISA), assembling ELISA)

[0243] this test kit uses double antibody sandwich method to detect the HA antigen of the H5 subtype influenza virus in the sample.At first on the polyethylene micropore lath of test kit, wrap in advance by the monoclonal antibody of anti-H5 type influenza virus HA gene, after the H5 type influenza virus HA antigen after the cracking is added to micropore, the monoclonal anti physical efficiency of pre-bag quilt is caught it, also combination with it of the monoclonal antibody linked with peroxidase of Jia Ruing subsequently is then by substrate for enzymatic activity colour developing degree judged result.When not containing influenza antigen in the sample or be not H5 type influenza virus, substrate can not develop the color.Detectable sample comprises the intact virus of movement, mouth and nose solinocrine thing, chicken embryo culture or lytic virus etc.

[0244] preparation of enzyme plate:

[0245] pre-bag is by the grand antibody of the monoclonal antibody of anti-H5 type influenza virus HA gene on the polyethylene micropore lath of test kit, and the monoclonal antibody bag is used the 10mM phosphate buffered saline buffer, and (PB pH7.4), wraps under 37 and spent the night; Use PBST (10mM PBS+0.05%Tween 20) washing once then, button adds confining liquid (10mMPBS+2% gelatin) after doing again, and 37 seal 2hr, detains the dry doubling vacuum again and drains and be packaged into finished product test kit enzyme plate (8 * 12 hole).

[0246] configuration of other composition of test kit:

[0247] employing virus cracking liquid is formed:

Lysate A (LB-A): 6%CHAPS+2%Tween-20+1%Tween-80.

Lysate B (LB-B): 100mM PMSF, use the Virahol dissolving, the work final concentration is 2mM.

Lysate C (LB-C): 10mM PBS, pH7.4

[0248] enzyme marking reagent:, obtain the suitable enzyme marking reagent of extent of dilution with the grand antibody of the monoclonal antibody of the anti-H5 type of HRP mark influenza virus HA gene

[0249] positive control: the H5N1-Yu22 strain inactivation of viruses that uses suitable titre is as positive control

[0250] negative control: with the negative contrast of lysate A.

[0251] developer A liquid: 13.4g/L Na ₂HPO ₄.12H ₂O+4.2g/L citric acid .H ₂The O+0.3g/L Urea Peroxide

[0252] developer B liquid: 0.2mM/ L tetramethyl benzidine 3,3 ', 5,5 '-Tetramethylbenzidine (TMB)+20mM/L dimethyl formamide

[0253] stop buffer: the 2M vitriol oil

[0254] concentrated cleaning solution: 20x PBST

[0255] shrouding film: 2

[0256] valve bag: 1

[0257] specification sheets: 1 part

[0258] testing process:

[0259] dosing: it is standby that 50ml concentrated solution (20 *) is diluted to 1000ml with distilled water or deionized water

[0260] numbering: the corresponding microwell plate of sample is numbered according to the order of sequence, and every plate should be established negative control 3 holes, positive control 2 holes and blank 1 hole (the blank hole does not add sample and enzyme marking reagent, and all the other respectively go on foot identical).

[0261] sample preparation and application of sample:

[0262] when being liquid, sample (comprises type specimen, chicken embryo culture sample, cell cultures sample): need the prewired an amount of LB-A of 100ul LB-A+4ul LB-B to mix with mixed solution and the vibration of LB-B by every hole.Every hole adds 100ul sample to be measured earlier on micropore then, adds the above-mentioned lysate that 100ul prepares again and reacts.

[0263] when sample is dried swab type specimen: after getting 1ml LB-A+40ul LB-B+1ml PBS and mixing, adds in the specimen tube, sample is dissolved in vibration, after room temperature leaves standstill 30min, shake again outstanding after, the centrifugal 5min of 6000rpm, the absorption supernatant detects, and every hole adds 100ul and reacts.

[0264] when sample be argol just during type specimen: after getting 1ml LB-A+40ul LB-B+1ml PBS and mixing, add in the specimen tube, argol just is configured to the excrement suspension sample of 10% (w/v), vibration dissolving sample, after room temperature left standstill 30min, it was outstanding to shake again, the centrifugal 5min of 6000rpm, the absorption supernatant detects, and every hole adds 100ul and reacts.

[0265] each detection all needs to be provided with the yin and yang attribute control wells, and every hole adds 100ul contrast liquid.

[0266] hatches: with sealing moderate speed's room temperature (25～28) concussion 60min on the rearmounted micro oscillator of film shrouding.

[0267] washing: carefully take the shrouding film off, wash 5 times with washing the plate machine washing, last button is as far as possible done.

[0268] enzyme-added: as in respective aperture, to add enzyme marking reagent 100ul respectively.

[0269] hatches: after sealing the film shrouding, put 37 degree incubations 30 minutes.

[0270] repeating step 6

[0271] colour developing: every hole adds developer A, each 50ul of B liquid, the mixing that vibrates gently, 37 lucifuges colour developing 30 minutes.

[0272] measure: every hole adds 1 of stop buffer (50ul), and the mixing that vibrates is gently measured each hole OD value with single wavelength 450nm (need establish the blank hole) of microplate reader or dual wavelength 450nm/630nm.

[0273] result judges:

[0274] a) normal range of negative control: under the normal circumstances, (negative control hole OD value is if should give up greater than 0.1, if all negative control hole OD values are answered repeated experiments all greater than 0.1 in negative control hole≤0.1.If negative control hole less than 0.03, then calculates by 0.03).

[0275] normal range of positive control b): under the normal circumstances, positive control hole OD value 〉=0.50.

[0276] c) threshold value (CUTOFF) is calculated: negative control hole OD average+0.15.

[0277] positive judgement d): sample OD value 〉=threshold value (CUTOFF) person is the avian influenza virus H5 type HA antigen-reactive positive.

[0278] negative judgement e): sample OD value＜threshold value (CUTOFF) person is an avian influenza virus H5 type HA antigen-reactive feminine gender.

[0279] clinical samples determination experiment:

[0280] utilize this test kit to detect various H5 and non-H5 virus sample, the results are shown in Table 4, this test kit has good detection sensitivity and specificity as can be seen.

[0281] table 4 utilizes the H5 antigen ELISA detecting kit to detect H5 and non-H5 virus sample

Annotate :-, feminine gender; A detects quantity; B detects sample size; HA, HA unit.

[0282] embodiment 3:H5 subtype influenza virus anti-HA antibody assay kit (euzymelinked immunosorbent assay (ELISA), assembling ELISA)

[0283] this test kit adopts the H5 type influenza virus HA antigen-specific antibodies in the competition law detection serum specimen.At first once wrap on the micropore lath in test kit by the monoclonal antibody of anti-H5 subtype influenza virus HA, the secondary bag is by the recombinant expressed antigen of H5 subtype influenza virus HA gene.After adding serum specimen and monoclonal antibody linked with peroxidase, antigen on specific antibody in the sample and the monoclonal antibody linked with peroxidase competition desmoenzyme target, if the serum specimen that adds can obviously suppress monoclonal antibody linked with peroxidase and combine with antigenic, then show to contain the antigenic specific antibody of influenza virus H5 type HA in the sample.In sample, do not contain Antibody of Influenza or be not H5 type Antibody of Influenza, can not suppress monoclonal antibody linked with peroxidase and antigenic reaction.

[0284] preparation of enzyme plate:

[0285] once pre-bag is by the grand antibody of the monoclonal antibody of anti-H5 type influenza virus HA gene on the polyethylene micropore lath of test kit, and the monoclonal antibody bag is used the 10mM phosphate buffered saline buffer, and (PB pH7.4), wraps under 37 and spent the night; Use PBST (10mM PBS+0.05%Tween 20) washing then once, button adds confining liquid (10mMPBS+2% gelatin) after doing again, 37 ℃ of sealing 2hr.Button carries out secondary bag quilt after doing, use 10mM PBS, join on the microwell plate that once wraps after processed by every hole 100ul behind the recombinant expressed antigen of pH7.4 solution dilution, 37 ℃ of bags are by 2hr, 37 sealing 2hr again after the washing once detains the dry doubling vacuum at last and drains and be packaged into finished product test kit enzyme plate (8 * 12 hole).

[0286] configuration of other composition of test kit:

[0287] a) enzyme marking reagent: the monoclonal antibody with the anti-H5 type of HRP mark influenza virus HA gene obtains the suitable enzyme marking reagent of extent of dilution.

[0288] positive control b): the monoclonal antibody of the anti-H5 type influenza virus HA gene of use suitable concn is as positive control.

[0289] negative control c): use 100% calf serum (NBS) as negative control.

[0290] developer A liquid d): 13.4g/L Na ₂HPO ₄.12H ₂O+4.2g/L citric acid .H ₂The O+0.3g/L Urea Peroxide

[0291] developer B liquid: 0.2mM/ L tetramethyl benzidine 3,3 ', 5,5 '-Tetramethylbenzidine (TMB)+20mM/L dimethyl formamide

[0292] stop buffer: the 2M vitriol oil

[0293] concentrated cleaning solution g): 20x PBST

[0294] shrouding film h): 2

[0295] valve bag i): 1

[0296] specification sheets j): 1 part

[0297] testing process:

[0298] a) dosing: it is standby that 50ml concentrated solution (20 *) is diluted to 1000ml with distilled water or deionized water.

[0299] numbering b): the corresponding microwell plate of sample is numbered according to the order of sequence, and every plate should be established negative control 3 holes, positive control 2 holes and blank 1 hole (the blank hole does not add sample and enzyme marking reagent, and all the other respectively go on foot identical).

[0300] application of sample c): in respective aperture, add testing sample or feminine gender, positive control 50ul respectively.

[0301] d) enzyme-added: as in respective aperture, to add enzyme marking reagent 50ul.

[0302] e) hatches: with sealing the film shrouding, put 37 degree incubations 60 minutes behind the mixing.

[0303] washing f): carefully take the shrouding film off, wash 5 times with washing the plate machine washing, last button is as far as possible done.

[0304] colour developing g): every hole adds developer A, each 50ul of B liquid, the mixing that vibrates gently, 37 lucifuges colour developing 15 minutes.

[0305] h) measure: every hole adds 1 of stop buffer (50ul), and the mixing that vibrates is gently measured each hole OD value with single wavelength 450nm (need establish the blank hole) of microplate reader or dual wavelength 450nm/630nm.

[0306] result judges:

[0307] a) normal range of negative control: under the normal circumstances, negative control hole 〉=0.1.

[0308] normal range of positive control b): under the normal circumstances, positive control hole OD value≤0.1.

[0309] c) threshold value (Cutoff value) is calculated: Cutoff value=negative control hole OD average/2.

[0310] positive judgement d): sample OD value＜Cutoff value is declared the H5 type influenza virus HA antigen-specific antibodies positive.

[0311] negative judgement e): sample OD value 〉=Cutoff value is declared H5 type influenza virus HA antigen-specific antibodies feminine gender.

[0312] clinical samples determination experiment:

[0313] utilizes this H5 antibody kit to detect human serum and chicken serum sample, the results are shown in Table 5, illustrate that this test kit has good sensitivity and specificity.

[0314] table 5 utilizes H5 antibody ELISA kit measurement serum specimen

Annotate :-, feminine gender; A detects quantity; B detects sample size.

[0315] assembling of embodiment 4:H5 subtype influenza virus HA antigen detection kit (golden mark method)

[0316] this test strip is to adopt the diagnostic reagent of new generation of colloidal gold immunochromatographimethod technology development, and detectable sample comprises the intact virus of movement, mouth and nose solinocrine thing, chicken embryo culture or lytic virus etc.This product deft design, disposable use, easy and simple to handle, safe, reliable, pollution-free, carry the Quality Control contrast, do not need any additive reagent, display result is clear and definite, is swift in response, and the entire operation time only needs 30 minutes.

[0317] the detection zone bag is by the monoclonal antibody of anti-HA on nitrocellulose filter respectively for this test strip, and wrap by sheep anti-mouse igg the check plot.During detection in the sample anti-HA monoclonal antibody (Ab--Au) of H5 type influenza virus and mark Radioactive colloidal gold form mixture (Ag-Ab-Au), because chromatography effect mixture along the film Tape movement, can form the double-antibody sandwich immunocomplex with the monoclonal antibody of the anti-HA that is coated on detection zone.As positive sample, then can form a red line at detection zone and each aggegation of check plot respectively; As negative sample, then only form a red line in the check plot.

[0318] preparation of test kit test strip: same ordinary method.

[0319] test kit is formed: test strip, lysate, specification sheets.

[0320] operating process:

[0321] a) sample preparation and application of sample:

(comprise type specimen, chicken embryo culture sample, cell cultures sample) when [0322] the i. sample is liquid:

[0323] need the prewired an amount of LB-A of 100ul LB-A+4ul LB-B to mix by every hole with mixed solution and the vibration of LB-B.Every hole adds 100ul sample to be measured earlier on micropore then, adds the above-mentioned lysate that 70ul prepares again and reacts.

When [0324] the ii. sample is dried swab type specimen:

[0325] get 1ml LB-A+40ul LB-B+1ml PBS and mix after, adds in the specimen tube, vibration dissolving sample, after room temperature leaves standstill 30min, shake again outstanding after, the centrifugal 5min of 6000rpm draws the supernatant detection, every hole adds 70 μ l and reacts.

[0326] the iii. sample is an argol just during type specimen:

After getting 1ml LB-A+40ul LB-B+1ml PBS mixing, add in the specimen tube, argol just is configured to the excrement suspension sample of 10% (w/v), vibration dissolving sample, after room temperature left standstill 30min, it was outstanding to shake again, the centrifugal 5min of 6000rpm draws supernatant and detects, and every hole adds 70ul and reacts.

[0327] b) the slow application of sample 70ul at the application of sample place is flat on room temperature.

[0328] result judges: observations in 30 minutes.

[0329] two red line occur, regard as the positive; Nature controlling line only occurs, regard as feminine gender; Red line do not occur, it is invalid then to regard as, and the result is referring to Fig. 1.

[0330] embodiment 5:H5 subtype influenza virus anti--assembling of HA antibody assay kit (golden mark method)

[0331] this test strip is to adopt the diagnostic reagent of new generation of colloidal gold immunochromatographimethod technology development, can detect the H5 type Antibody of Influenza in the serum specimen.This product deft design, disposable use, easy and simple to handle, safe, reliable, pollution-free, carry the Quality Control contrast, do not need any additive reagent, display result is clear and definite, is swift in response, and the entire operation time only needs 30 minutes.Be applicable to the primary dcreening operation of H5 type HA antibody.The detection zone bag is by the monoclonal antibody of anti-HA on nitrocellulose filter respectively for this test strip, and wrap by sheep anti-mouse igg the check plot.Freeze-drying has anti-HA labeling of monoclonal antibodies Radioactive colloidal gold and H5 type influenza virus reorganization HA antigen expressed on the glass fibre, and the employing competition law detects the H5 type influenza virus anti-HA antibody in the testing sample.Thereby then competing the formation of blocking mixture with anti-HA labeling of monoclonal antibodies Radioactive colloidal gold as the antibody that contains anti-H5 in the sample can not develop the color; If negative, then form the mixture colour developing.

[0332] preparation of test kit test strip: same ordinary method

[0333] test kit is formed: test strip, specification sheets

[0334] testing process:

[0335] sealing bag is opened, taken out required test strip, slowly add serum sample 70 microlitres at the application of sample place, be flat on room temperature, observations in 30 minutes exceeds 30 minutes, and the result is invalid.

[0336] result judges:

Nature controlling line only occurs, regard as the positive; Two red line occur, regard as feminine gender; Red line do not occur, it is invalid then to regard as, referring to Fig. 2.

[00337] assembling of embodiment 6:H5 subtype influenza virus HA antigen detection kit

[0338] this test kit utilizes the enzyme of new generation of immunity percolation technology development to exempt from diagnostic reagent, can detect the HA antigen of the H5 subtype influenza virus in the sample, detectable sample comprises the intact virus of movement, mouth and nose solinocrine thing, chicken embryo culture or lytic virus etc.It is disposable use that the oozing of this test kit considered proofing unit, easy and simple to handle, safe, reliable, pollution-free, can carry out rapid detection, and display result is clear and definite, be swift in response, and the entire operation time needs about 45 minutes approximately.This test kit wraps by sheep anti-mouse igg by the monoclonal antibody of anti-H5 (HA) with in the check plot at the pre-bag in sample detection district that oozes on the nitrocellulose filter of considering proofing unit.Adding is after cracked contains H5 subtype influenza virus HA sample, the monoclonal antibody body of pre-bag quilt is caught it, form immune complex (Ab-Ag), adding enzyme mark monoclonal antibody (Ab-HRP) subsequently combines with immune complex, final antibody-antigen-the hrp-antibody complex (Ab-Ag-Ab-HRP) that forms then carries out the result by the substrate for enzymatic activity colour developing and judges.When not containing H5 subtype influenza virus in the sample, detection zone does not develop the color, and only forms a colour developing point in the check plot.

[0339] preparation of leak detection apparatus:

[0340] nitrocellulose filter and absorbent filter place a flat bottom to support.The suitable lid of shape covers on the support of bottom, and an opening is afterwards arranged in the middle part of the lid.The flow control unit of a shape appropriate lid intermediate openings inserts opening.This flow control unit has two holes to be respectively load sample and contrast.The bottom of flow control unit is made it to remain on nitrocellulose membrane and the filter paper with the restriction sample flow by on supporting in the bottom tightly.Anti--H5 (HA) monoclonal antibody is painted on the test zone of flow control unit, and goat anti-mouse IgG is painted on the check plot of flow control unit.Test paper in air air-dry 1 hour, being then packed in vacuum becomes leak detection apparatus.

[0341] test kit is formed:

[0342] a) oozes the worry proofing unit

[0343] sample processing device b): a bottle is tightened by the strainer block; Strainer block middle part comprises strainer to make solution to separate and filters and pass through.

[0344] enzyme marking reagent c):, obtain the suitable enzyme marking reagent of extent of dilution with the grand antibody of the monoclonal antibody of the anti-H5 type of HRP mark influenza virus HA gene.

[0345] lysate d): 3%NP40+1%Triton X-100+40mM PBS, pH7.4.

[0346] washings e): 2%Triton X-100+20mM EDTA+0.25%Tween 20+0.1%Proclin 300+150mM NaCl+5mM PBS, pH7.4.

[0347] colour developing liquid f): 3,3 ', 5,5 '-Tetramethylbenzidine (TMB) Liquid substrate System for Membranes.

[0348] stop buffer g): 50mM aqueous citric acid solution

[0349] specification sheets h)

[0350] testing process:

[0351] a) sample preparation and application of sample:

Add 200 μ l testing samples in each sample processing device, drip 8 lysis buffers subsequently, fully mixing. then, all lysate sample are pressed to pack into by the strainer block in sample processor detects the hole.Treat that sample absorbs back (about 25 minutes) fully, removes the b of flow control unit [0352]) washing: Dropwise 5 drips washing lotion, allows washing lotion absorb fully;

[0352] washing b): add 5 lavation buffer solutions, make it to absorb fully.

[0353] c) enzyme-added: as to drip 4 enzyme marking reagents, treat that enzyme oozed the clean afterreaction of worry 2 minutes fully;

[0354] d) wash: divide 2 washings, splash into 8 washingss for the first time, washing lotion splashes into 5 washingss after oozing fully and considering totally again, allows washings ooze and considers totally;

[0355] colour developing e): drip 2 colour developing liquid, liquid to be developed the color blots and carried out result's judgement in back 2 minutes, and the result who surpasses 5 minutes does not have clinical meaning, and the result judges that synoptic diagram is referring to Fig. 3.

[0356] clinical samples determination experiment: utilize this H5 quick detection kit to measure clinical samples, obtain the data results of table 6, show that this test kit has good sensitivity and specificity.

[0357] table 6 utilizes the H5 antigen immune to ooze the mensuration of worry method to clinical viral sample

Annotate :-, feminine gender; A detects quantity; B detects sample size.

[0358] embodiment 7: the monoclonal antibody light chain gene separates with the heavy chain gene variable region

[0359] partly pastes an ancient piece of jade, round, flat and with a hole in its centre and cultivate 10 ⁷Individual hybridoma, blowpipe blows afloat attached cell and makes suspension, transfers in the new 4ml centrifuge tube, the centrifugal 3min of 1500rpm, the cell of collecting precipitation is resuspended among the aseptic PBS of 100ul (pH7.45), transfers in the new 1.5ml centrifuge tube.(Roche Germany), puts upside down mixing gently, leaves standstill 10min to add 800ul Trizol.Add the 200ul chloroform, thermal agitation 15s leaves standstill 10min, and 4 ℃ of centrifugal 15min of 12000rpm shift in the new 1.5ml centrifuge tube of supernatant liquid to, add isopyknic Virahol, and mixing leaves standstill 10min.4 ℃ of centrifugal 10min of 12000rpm abandon supernatant, add 600ul 75% washing with alcohol, and 4 ℃ of centrifugal 5min of 12000rpm abandon supernatant, are deposited in 60 vacuum and drain 5min.Transparent precipitation is dissolved in 70ul DEPC H ₂Among the O, be distributed into two pipes.Every pipe adds 1ul reverse transcription primer, wherein the reverse transcription primer of a pipe adding is MVJkR (5 '-CCg TTT (T/g) AT (T/C) TC CAg CTT ggT (g/C) CC-3 '), be used to the chain variable region gene that increases, the reverse transcription primer that another pipe adds is MVDJhR (5 '-C ggT gAC Cg (T/A) ggT (C/g/T) CCTTg (g/A) CC CCA-3 '), is used to the heavy chain variable region gene that increases.Every pipe adds 1ul dNTP (worker is given birth in Shanghai) again, puts 72 ℃ of water-bath 10min, is put into the mid-5min of ice bath immediately, add 10ul 5x reverse transcription damping fluid, and 1ul AMV (10u/ul, Pormega), (40u/ul Promega), becomes cDNA in 42 with the RNA reverse transcription behind the mixing to 1ul Rnasin.

[0360] polymerase chain reaction (PCR) method is adopted in the separation of antibody gene variable region, use is according to the Ig-Prime kits synthetic primer sets of Novagen company and design synthetic two downstream primer MVJkR, MVDJhR (Bo Ya company in Shanghai is synthetic) in addition, MVJkR is the downstream primer of chain variable region gene amplification, and MVDJhR is the downstream primer of heavy chain variable region gene amplification.Template is two kinds of cDNA of above synthetic.The PCR condition is: 94 ℃ of 5min, 72 ℃ of 50s 35cycles of 53 ℃ of 1min of 94 ℃ of 40s, 72 ℃ of 15min.Reclaim the purpose fragment and also be cloned into pMD 18-T carrier, deliver to the order-checking of Shanghai Bo Ya company, sequence is through the definite antibody variable region sequence in blast comparison back, and infers and amino acid sequence corresponding.

[0361] from 6 strain bird flu monoclonal antibody hybridoma cell strains, clones its antibody variable gene according to aforesaid method, and infer and amino acid sequence corresponding.Table 7 is depicted as used upstream primer sequence, the numbering of 6 strain variable region of mab nucleic acids and aminoacid sequence shown in the table 8.(complementary determinant region CDR) determines (table 9) by I MGT/V-QUEST (http://imgt.cines.fr/textes/vquest/) to complementary determining region.

[0362] the upstream primer sequence of table 7 amplification bird flu monoclonal antibody variable region gene

Table 86 strain variable region of mab nucleic acids and aminoacid sequence numbering

Table 96 strain monoclonal antibody cdr amino acid sequences

[0363] expression of embodiment 88H5 single-chain antibody and active the detection

[0364] heavy chain and the variable region of light chain with the 8H5 antibody gene connects into the single-chain antibody dna fragmentation by (GGGGS) 3 small peptides.With 8H5 HF1/8H5 HR1 is primer to amplifying 8H5 variable region of heavy chain fragment, with 8H5 KF1/8H5 KR1 be primer to amplifying 8H5 variable region of light chain fragment, primer sequence sees Table 10.Reclaim two fragments respectively, primer and template are carried out overlapping extension in new PCR system each other with these two fragments again, obtain a small amount of complete single chain antibody fragments, be template with complete fragment then, with 8H5 HF1/8H5 KR1 is that primer increases in a large number, reclaim single chain antibody fragments, receive single chain antibody fragments, be cloned in the pTO-T7 prokaryotic expression carrier that same enzyme cuts with the switchback of BamHI/Sal I enzyme.As expression strain, adopt ordinary method to express with the ER2566 intestinal bacteria, the protein of results expression is to exist with insoluble inclusion body form.With ordinary method washing purifying inclusion body, single-chain antibody mainly is dissolved in the 8M urea as a result.To be dissolved in single chain antibody protein in the 8M urea progressively to 1 * PBS dialysis renaturation, 12000rpm removed precipitation in centrifugal 10 minutes, and the single-chain antibody solution of the preliminary purification that finally obtains is carried out determination of activity.

[0365] table 10 single-chain antibody and chimeric antibody clone uses primer

[0366] adopt the competitive ELISA method to detect the activity of the 8H5 single-chain antibody that preliminary purification goes out.Bag is by the bird flu polyclonal antibody in the hole of polystyrene batten, and seal with BSA, treat to add in the gaging hole above-mentioned single-chain antibody solution of 50 μ l and 50 μ l bird flu H5 viruses, then add 50 μ l1 * PBS solution and 50 μ l bird flu H5 viruses in the negative control hole, add 50 μ l bird flu polyclonal antibodies and 50 μ l bird flu H5 viruses in the positive control hole, repeat in three holes.Each hole slightly behind the mixing in 37 ℃ of reactions after 1 hour, be two anti-ly to react half an hour again with the bird flu polyclonal antibody of HRP mark, add A, B colour developing liquid in 37 ℃ of colour developings 15 minutes, use the microplate reader reading after the termination reaction.The negative control mean values is 1.871 as a result, and the positive control mean values is 0.089, treats that the gaging hole mean values is 0.597, illustrates that the 8H5 single chain antibody protein that preliminary purification goes out has higher activity.

[0367] selects 26 strain H5N, 1 virus for use, utilize hemagglutination-inhibition test method (HI) to identify described 8H5 single-chain antibody and viral reactivity.In 96 hole blood-coagulation-boards, every hole adds 25 μ L PBS, adds 25ul 8H5 single-chain antibody solution (0.08mg/ml) and mixing in first hole, gets 25 μ L to second hole, doubling dilution so backward.Get 25 μ l virus and add in the single-chain antibody, incubated at room 30min adds 0.5% chicken red blood cell, 50 μ L again, and incubated at room 30min observes red cell agglutination.The 8H5 single-chain antibody all has HI activity (table 11) to wherein 16 strain virus as a result.

[0368] expression of embodiment 9 10F7,4D1 single-chain antibody and active the detection

[0369] method is as described in the embodiment 8, and the heavy chain and the variable region of light chain of antibody gene connected into the single-chain antibody dna fragmentation by (GGGGS) 3 small peptides.With 10F7VHF/10F7VHR be primer to amplifying 10F7 variable region of heavy chain fragment, be that primer is to amplifying 10F7 variable region of light chain fragment with 10F7VKF/10F7VKR.With 4D1VHF/4D1VHR be primer to amplifying 4D1 variable region of heavy chain fragment, be that primer is to amplifying 4D1 variable region of light chain fragment with 4D1VKF/4D1VKR.

[0370] being the primer eclipsed 10F7 single chain antibody fragments that increases in a large number with 10F7VHF/10F7VKR, is the primer eclipsed 4D1 single chain antibody fragments that increases in a large number with 4D1VHF/4D1VKR.Receive single chain antibody fragments with the switchback of BamH I/SalI enzyme, be cloned in the pT0-T7 prokaryotic expression carrier that same enzyme cuts.Equally with the ER2566 intestinal bacteria as expression strain, the expression process is the same, expressed protein exists with insoluble inclusion body form.Ultrasound precipitation is through identical purge process, and single-chain antibody mainly is dissolved in the 8M urea as a result.To be dissolved in single chain antibody protein in the 8M urea progressively to 1 * PBS dialysis renaturation, 12000rpm removed precipitation in centrifugal 10 minutes, and the single-chain antibody solution of the preliminary purification that finally obtains is carried out determination of activity.

[0371] selects 26 strain H5N1 viruss for use, utilize hemagglutination-inhibition test method (HI) to identify the 10F7 of described preliminary purification, the activity of 4D1 single-chain antibody.Method is the same, and the concentration of single-chain antibody 10F7 is 1.06mg/ml, and the concentration of 4D1 is 0.34mg/ml.The 4D1 single-chain antibody all has the HI activity to wherein 23 strain virus as a result, and the 10F7 single-chain antibody has HI activity (table 11) to 14 strain virus wherein.

[0372] three kinds of single-chain antibodies of table 11 are to the HI result of 25 strain H5N1 avian influenza virus

[0373] annotate: the HI titre is the n power of dilution 2, and n is numerical value in the table

[0374] adopt in and the activity of the 10F7 single-chain antibody of measuring preliminary purification.Choose 7 strains during 2002 to 2006, in Hong Kong, ground such as Indonesia, Qinghai, the strain that from chicken, duck and several wild birds, is separated to, utilize blood clotting to suppress the described 10F7 single-chain antibody of experimental identification in the reactivity of virus, this single-chain antibody has all shown neutralization preferably active (seeing Table 12) to 5 strain virus wherein as a result.At the Ck/HK/Yu22/02 strain, scFv still can suppress virus infected cell after 64 times of dilutions.

[0375] among table 12 10F7scFv and experimental result

[0377] expression of embodiment 10 chimeric antibodies and active the detection

[0378] with the variable region of heavy chain and the chain variable region gene of 4D1,10F7 and 8H5 monoclonal antibody, adds signal peptide sequence respectively, be cloned into again in the eukaryon expression plasmid that contains people gamma 1 heavy chain and kappa constant region of light chain sequence.Wherein pcDNA3.1-AH contains people gamma 1 CH sequence, and pcDNA3.1-Ak contains people's light chain kappa constant region sequence.

[0379] be that primer is to amplifying 8H5 part heavy chain signal peptide sequence and variable region fragment with 8H58CHF1/8H5VHR, with this PCR product is template, 8H58CHF2/8H5VHR is that primer is right, amplify the 8H5 weight chain variabl area sequence of band complete signal peptide, be cloned into the pcDNA3.1-AH plasmid of Bam HI/Xho I double digestion, thereby obtain the expression plasmid pcDNA3.1-AH8H5 of expressing human mouse chimeric heavy chain.With 8H58CKF1/8H5VKR1 is that primer is to amplifying 8H5 part light chain signal peptide sequence and variable region fragment, with this PCR product is template, 8H58CKF2/8H5VKR1 is that primer is to amplifying the 8H5 variable region of light chain fragment of band complete signal peptide sequence, be cloned into the pcDNA3.1-Ak plasmid of EcoR I/Xho I double digestion, thereby obtain the expression plasmid pcDNA3.1-Ak8H5 of expressing human mouse chimeric light chain.

[0380] be that primer is to amplifying 10F7 part heavy chain signal peptide sequence and variable region fragment with 10F78CHF1/10F7VHR, with this PCR product is template, 10F78CHF2/10F7VHR is that primer is right, amplify the 10F7 weight chain variabl area sequence of band complete signal peptide, be cloned into the pcDNA3.1-AH plasmid of Bam HI/Xho I double digestion, thereby obtain the expression plasmid pcDNA3.1-AH10F7 of expressing human mouse chimeric heavy chain.With 10F78CKF1/10F7VKR is that primer is to amplifying 10F7 part light chain signal peptide sequence and variable region fragment, with this PCR product is template, 10F78CKF2/10F7VKR is that primer is to amplifying the 10F7 light chain variable region sequence of band complete signal peptide sequence, be cloned into the pcDNA3.1-Ak plasmid of EcoR I/Xho I double digestion, thereby obtain the expression plasmid pcDNA3.1-Ak10F7 of expressing human mouse chimeric light chain.

[0381] be that primer is to amplifying 4D1 part heavy chain signal peptide sequence and variable region fragment with 4D1VHF1/4D1VHR, with this PCR product is template, 4D1VHF2/4D1VHR is that primer is right, amplify the 4D1 weight chain variabl area sequence of band complete signal peptide, be cloned into the pcDNA3.1-AH plasmid of Bam HI/Xho I double digestion, obtain the expression plasmid pcDNA3.1-AH4D1 of expressing human mouse chimeric heavy chain.With 4D1VKF/4D1VKR be primer to amplifying 4D1 light chain signal peptide sequence and variable region fragment, be cloned into the pcDNA3.1-Ak plasmid of EcoR I/Xho I double digestion, thereby obtain the expression plasmid pcDNA3.1-Ak4D1 of expressing human mouse chimeric light chain.

[0382] Fig. 4 is the expression plasmid figure of 3 kinds of chimeric antibodies.

[0383] will contain two plasmid co-transfection 293FT cells of chimeric heavy chain and chimeric light chain by calcium phosphate transfection method, collect culture supernatant, obtain the chimeric antibody (cAb) of preliminary purification through saturated sulphur ammonium precipitation.The starting point concentration of adjusting chimeric antibody and mouse monoclonal antibody (mAb) is to 0.7ug/ml, carries out blood clotting with the Ck/HK/Yu22/02 strain and suppresses that HI is active to be detected, and the result shows, the HI activity of three kinds of chimeric antibodies all with corresponding mouse monoclonal antibody consistent (Fig. 5).

[0384] select 23 strain H5N1 viruss for use, utilize hemagglutination-inhibition test method (HI) to identify the 10F7 of described preliminary purification, the activity of 4D1 chimeric antibody, method is the same.Two kinds of chimeric antibodies of result all have HI activity (table 13) to this 23 strain virus.

[0385] two kinds of chimeric antibodies of table 13 are to the HI result of 23 strain H5N1 avian influenza virus

[0387] further adopt immunofluorescence method to measure the activity of cAb.In 24 porocyte culture plates, place cover glass, cultivate insect cell SF21 therein,, in the SF21 cell, express the HA albumen of avian influenza virus by insect cell-baculovirus expression system.Cell after the expression is through the PBS washing, and 4% Paraformaldehyde 96 is fixed, after the sealing of goat polyvalent antibody, with 4D1 or 10F7 chimeric antibody incubated at room 1 hour, with the negative contrast of the special human mouse chimeric antibody of HBV.With fluorescently-labeled goat anti-human antibody (MO is two anti-USA) for Sigma, St.Louis, room temperature reaction half an hour, get DAPI (Sigma, St.Louis, MO, USA) transfect cell nuclear, room temperature 10 minutes, film-making is in just putting observation fluorescent microscope (Nikon) under then.By the result of Fig. 6 as can be known, 4D1,10F7 chimeric antibody all can be specifically with the SF21 cell in express the HA protein binding of avian influenza virus.

[0388] embodiment 11 screens the small peptide of simulation monoclonal antibody identification epi-position from phage 7 peptide storehouses

[0389] selects for use the 7 peptide phage display libraries (ph.D.C7C peptide library) of New England Biolabs company to screen and monoclonal antibody 8H5,3C8 bonded 7 peptides, undertaken by process specifications.As follows substantially:

[0390] draws 50 μ l Protein A-agarose media (50% aqueous suspensions) in Eppendorf tube, add 1ml TBS+0.1%Tween (TBST) solution.Flick tube wall or the resuspended medium of gentle concussion.Low-speed centrifugal 30 seconds, precipitation medium, the careful suction removed supernatant.Medium is resuspended in 1ml and blockades in the damping fluid, 4 ℃ of effects 60 minutes, mixing once in a while.During this period, use the TBS damping fluid with 2 * 10 ¹¹Individual bacteriophage particles (the original library that is equivalent to 10 μ l) and 300ng antibody dilution are to final volume 200 μ l, and the antibody final concentration is 10nM.Room temperature effect 20 minutes.Blockade after the reaction, the low-speed centrifugal precipitation medium, and wash 4 times with 1mlTBS, all need precipitation medium at every turn.Phage-mixtures of antibodies is added in the medium of washing gentle mixing, room temperature effect 15 minutes and mixing frequently.The low-speed centrifugal precipitation medium is abandoned supernatant, washes 10 times with 1mlTBTS.Resuspended precipitation medium is in the Glycine-HCI of 1ml 0.2M (pH2.2), among the 1mg/mlBSA, and room temperature effect 10 minutes, the phage of elution of bound.Centrifugal wash-out mixed solution 1 minute carefully changes supernatant in another new Eppendorf tube over to.Use 150 μ l 1M Tris-HCI immediately, in the damping fluid of pH9.1 and elutriant.Take out about 1 μ L titration phage titre.Remaining phage solution is added in the 20mL logarithmic growth ER2738 host bacterium liquid in early stage, and 4.5h is cultivated in 37 ℃ of concussions.Bacterium liquid is moved in the 50mL centrifuge tube the centrifugal 10min of 10000rpm.Draw top 80% supernatant, add PEG/NaCl (20%PEG-8000, the 2.5M NaCI) solution of 1/6 volume, 4 ℃ of standing over night.4 ℃ of centrifugal 15min of 10000rpm.Outwell supernatant, with 1mL PBS dissolution precipitation phage.4 ℃ of centrifugal 5min draw supernatant, add the PEG/NaCI solution of 1/6 volume, and 4 ℃ leave standstill 1h.4 ℃ of centrifugal 15min of 10000rpm.Outwell supernatant, with 200 μ L PBS dissolution precipitation phages, 4 ℃ of preservations.Repeat above-mentioned steps, carry out a screening of taking turns again.

[0391] with the ER2738 host bacterium of incubated overnight according to 1: 10 dilution proportion in the LB substratum, and bacterium liquid branch installed to (1mL/ pipe) in the culture tube.10 of every kind of monoclonal antibody pickings are inoculated in the above-mentioned bacterium liquid through the 3 plaque mono-clonals of taking turns blue look on the LB/IPTG/Xgal culture plate of screening.4.5h is cultivated in 37 ℃ of concussions.Bacterium liquid is moved on in the 1.5mL centrifuge tube, after 10000g is centrifugal, gets the 200ul supernatant and preserve, remaining extracts ssDNA according to a small amount of M13 extracting and purifying test kit (Shanghai China Shun biotechnology company limited) operational manual.Through the order-checking of Shanghai Bo Ya Bioisystech Co., Ltd, know 7 peptide sequence such as tables 14 of insertion.

[0392] table 14 and monoclonal antibody 8H5,3C8 bonded 7 peptide sequences

[0393] activity of embodiment 12 phages 7 peptides detects

[0394] three kinds of phages that will contain 8H5A, 8H5E, 3C8A 7 peptides are increased in a large number, through the PEG post precipitation, are dissolved in PBS, and measuring titre is 10 ¹¹-10 ¹²Between.Wrap by 4 kinds of mouse monoclonal antibody 8H5,4A1,9N7,4,D11 5% skimmed milk PBS sealing respectively with 5ug/ml concentration.With adding three kinds of phages carrying out behind the gradient dilution respectively, react after 1 hour, wash 5 times, adds 1: 5000 goat-anti M13 enzyme labelled antibody, reaction developed the color 15 minutes after half an hour, reading after the termination.The result is as shown in Table 15, and the reaction of 8H5A and 8H5 monoclonal antibody has specificity preferably as can be known, and very weak with the reactivity of other three kinds of monoclonal antibodies.8H5E is relatively poor to the atopy of 8H5 monoclonal antibody.

[0395] the specific binding activity detected result of table 15 phage 7 peptides

[0396] embodiment 13 screens the small peptide of simulation 8H5 identification epi-position from phage 12 peptide storehouses

[0397] selects for use the 12 peptide phage display libraries (ph.D.12peptide library) of New England Biolabs company to screen and monoclonal antibody 8H5 bonded 12 peptides, screen by process specifications.Concrete steps are with reference to embodiment 11.

[0398] after the third round screening, takes out the phage that about 1ul elutes and carry out titration.The single phage plaque of picking is cultivated 4.5～5h for 37 ℃ to the ER2738 bacterium of logarithmic phase, centrifugal collection phage supernatant carries out ELISA and detects.Bag is by the mouse monoclonal antibody 8H5 of 10ug/ml concentration, with the phage supernatant is one anti-, Anti-M 13/HRP antibody (Amersham Phmarcia Biotech, UK) be two anti-with dilution in 1: 5000, getting the proteic uncorrelated antibody 8C11mAb of bird flu associated antibodies 4D1mAb, 10F7mAb and anti-HEV E2 is the contrast of mouse monoclonal antibody.Fig. 7 shows wherein the detected result of 12 phage polypeptides preferably.By result among the figure as can be known, most of phage display peptide is higher than control antibodies more than 3 times at the value of reading of target antibody 8H5, and specificity is preferably arranged.

[0399] according to phage ssDNA extraction agent box (Omega, USA) routine operation extracting DNA is with this dna profiling check order (Invitrogen, Shanghai, China), obtain the Nucleotide and the aminoacid sequence (seeing Table 16) of above 12 strain phage displays, 12 peptides.

[0400] table 16 and grand antibody 8H5 bonded 12 peptide sequences of monoclonal antibody

[0401] embodiment 14.12 peptides 123 and 125 and 239 protein fusion expressions and active the detection

[0402] structure of 239-123 and 239-125 fusion expression vector

[0403] this laboratory at expression in escherichia coli the fragment (a.a.368-606) of HEV ORF2, the recombinant protein 239 of acquisition can be assembled into and is viruslike particle, has good immunogenicity.We are by PCR method, and the C end that constructs 239 sequences merges the prokaryotic expression carrier pTO-T7-239-123 (Fig. 8) and the pTO-T7-239-125 (Fig. 9) of 12 peptide sequences.At first, at 239 sequences and 12 peptide sequences design primer (table 17), be template with 239 genes, use primer 2 39-123F/239-123R1 and 239-125F/239-125R1 and carry out first round pcr amplification.As template, application primer 2 39-123F/239-123R2 and 239-125F/239-125R2 carry out second and take turns pcr amplification, amplify 239-123 and 239-125 fragment behind product recovery, the purifying.Then, reclaim PCR product 239-123 and 239-125 respectively, Ndel and EcoRI double digestion rear clone are gone among the carrier pTO-T7.Connector transformed into escherichia coli ER2566 expresses in a small amount and plasmid enzyme restriction identifies that positive colony is recombinant prokaryotic expression vector pTO-T7-239-123 and pTO-T7-239-125.

[0404] table 17 239-123 and 239-125 clone primer sequence

[0405] 239-123 and 239-125 Expression of Fusion Protein and purifying

[0406] picking transforms the single bacterium colony of E R2566 of pTO-T7-239-123 or pTO-T7-239-125 plasmid, contain in the LB substratum of Kn resistance in 2mL, 37 ℃ of concussions are cultured to OD600 about 0.5, transfer enlarged culturing to 500mL LB (containing the Kn resistance) according to 1: 1000 ratio then, be cultured to OD600 at about 1.0 o'clock, add IPTG 500 μ L, induce 4h for 37 ℃.Collect thalline, the centrifugal 10min of 8000rpm down for 4 ℃.Abandon supernatant, bacterial sediment is resuspended with the lysate of 20mL/ bottle.Ice-water bath adopts Ultrasonic Cell Disruptor to handle smudge cells.Ultrasound condition is as follows: the working hour: 10min; Pulse: beat 2sec, stop 5sec; Output rating: 70%.The centrifugal 10min of 12000rpm keeps supernatant, and precipitation is resuspended with 20mL 2%Triton, concussion 30min.The centrifugal 10min of 12000rpm keeps supernatant, and precipitation is resuspended with 20mL Bufferl, concussion 30min.Repeating Triton/Bufferl handles once.The centrifugal 10min of 12000rpm keeps supernatant, and precipitation is resuspended with 20mL 2M Urea/Bufferl, concussion 30min.The centrifugal 10min of 12000rpm keeps supernatant, and precipitation is resuspended with 20mL 4M Urea/Bufferl, concussion 30min.The centrifugal 10min of 12000rpm keeps supernatant, and precipitation is resuspended with 20mL 8MUrea/Bufferl, concussion 30min.The centrifugal 10min of 12000rpm keeps supernatant.Each keeps supernatant and makes on the albumen all product and analyze (Figure 10, Figure 11) to carry out SDS-PAGE.Learn that by the SDS-PAGE analysis albumen mainly is dissolved among the 8M Urea, purity can reach 90%.With the albumen gradient among the 8M Urea dialyse to PBS (8M Urea-4M Urea-2M Urea-PBS).

[0407] activity of 239-123 and 239-125 fusion rotein detects

[0408] directly the ELISA method detects

[0409] 239-123 of preliminary purification and 239-125 fusion rotein with the concentration bag of 10 μ g/mL by 96 orifice plates, 37 ℃ of 2h.Wash plate 1 time, ED confining liquid sealing nonspecific binding site, 4 ℃ are spent the night behind 37 ℃ of 2h.Behind the deblocking liquid, every hole adds the different mouse monoclonal antibody of 100 μ L, comprising: 8C11,7H8,3C8,8H5,1A6,13E1,1D8,1G2,3G41,13A2,11H8,4D1,10HD4,14H12,6CF3,7D1,7E8,10DE2,16A12,3FC1,8E2,3D2,10D122,13E7 be totally 24 strain monoclonal antibodies.Wherein 8C11 is the special monoclonal antibodies of 239 albumen, and 8H5 is the used monoclonal antibodies of 12 peptide screenings, and all the other 22 strain monoclonal antibodies are to go up blood clotting albumen (HA) monoclonal antibody at avian influenza virus (AIV).Hatch 1h for 37 ℃.PBST washes plate 5 times, and every hole adds GAM-HRP (1: 10000) 100 μ L, hatches 30min for 37 ℃.PBST washes plate 5 times, adds colour developing liquid colour developing 10min, and stop buffer stops, the microplate reader value of reading.By Figure 12, Figure 13 result as can be known, 239-123 and 239-125 fusion rotein only react with antibody 8C11 and 8H5, and be all reactionless with other monoclonal antibody.The atopic that 239-123 and 239-125 fusion rotein are described is very good.

The 239-123 of preliminary purification and 239-125 fusion rotein respectively with the concentration bag of 10 μ g/mL by 96 orifice plates, 37 ℃ of 2h.Wash plate 1 time, ED confining liquid sealing nonspecific binding site, 4 ℃ are spent the night behind 37 ℃ of 2h.Every hole adds the 8H5 antibody (having positive control 8C11 antibody simultaneously) of 100 μ L doubling dilutions, hatches 1h for 37 ℃.PBST washes plate 5 times, and every hole adds GAM-HRP (1: 10000) 100 μ L, hatches 30min for 37 ℃.PBST washes plate 5 times, adds colour developing liquid colour developing 10min, and stop buffer stops, the microplate reader value of reading.The result of Figure 14 shows that along with the reduction of 8H5 antibody concentration, 8H5 also decreases with the activity that combines of 239-125 fusion rotein.This with positive control antibody 8C11 with 239 proteic to combine activity change consistent.239-123 fusion rotein detected result is consistent therewith.Further specifying these two kinds of fusion roteins of 239-123 and 239-125 is specific reaction with combining of antibody 8H5.

[0410] embodiment 1512 peptides 123 and 125 and HBV cAg protein fusion expression

[0411] structure of fusion expression vector

[0412] utilize the a.a.1-149 of HBcAg can be in intestinal bacteria with the character of viruslike particle formal representation, this laboratory is with among these 149 aminoacid insertion coli expression carrier pTO-T7, and substitute the 79th, 80 two amino acid of HBcAg B cell advantage epi-position section with connexon, made up mutant HBc expression plasmid pC149-mut.HBcAg has very strong T cell immunogenicity, and (mayor immunodominant region, fusion a.a.78-83) can not change its particulate poly characteristic to allogenic polypeptide, and can make foreign epitope be exposed to particle surface at the inner MIR of HBcAg.

[0413] copies the 79th and 80 amino acids of inserting HBcAg with 1 to 5 respectively with 123 and 125, obtain serial fusion rotein, be referred to as HBc-123, HBc-125 respectively.According to 12 peptide sequences, and the carrier sequence of pC149-mut, design contains the forward primer of 12 peptide sequences and general reverse primer 149MRP (table 18, underscore represent insert peptide sequence) respectively.With pC149-mut is template, uses primer HBc123F2/HBcR and HBc123F2/HBcR and carries out first round pcr amplification.Product reclaims, behind the purifying as template, use primer HBc123F1/HBcR and HBc123F1/HBcR and carry out second and take turns pcr amplification, amplify the C149a.a.81-149 that is connected with 12 peptide sequences, cut back recovery purifying through Bgl II and EcoR I enzyme and obtain fragment.With BamH I and EcoR I double digestion carrier pC149-MUT, be connected with fragment behind the recovery carrier simultaneously.Connector transformed into escherichia coli ER2566 expresses and identifies and the plasmid enzyme restriction evaluation, identifies that correct plasmid has promptly inserted one 12 peptide, respectively called after pC149-mut-123 and pC149-mut-125.This plasmid gets carrier with BamH I and EcoR I double digestion again, with be connected with the segment that contains 12 peptide sequences that EcoR I enzyme is cut through Bgl II before, positive colony is the reorganization prokaryotic expression plasmid that contains 2 12 peptide copy numbers, respectively called after pC149-mut-D123 and pC149-mut-D125.Use similar approach, construct recombinant prokaryotic expression vector pC149-mut-T123, pC149-mut-F123, pC149-mut-Q123 and the pC149-mut-T125, pC149-mut-F125, the pC149-mut-Q125 that contain 3 copies, 4 copies and 5 copy numbers respectively.Make up plasmid figure such as Figure 15, (being example only) shown in Figure 16 with pC149-mut-123 and pC149-mut-125.

[0414] table 18123,125 and clone's primer sequence of HBV cAg expressing fusion protein

(underscore is 12 peptide sequences)

[0416] Expression of Fusion Protein and purifying

[0417] fusion rotein that pC149-mut-D123, pC149-mut-T123, pC149-mut-F123, pC149-mut-Q123, pC149-mut-D125, pC149-mut-T125, pC149-mut-F125, pC149-mut-Q125 expression plasmid are expressed is called D123, T123, F123, Q123, D125, T125, F125, Q125.The ER2566 bacterial classification that will contain eight kinds of expression plasmids respectively is inoculated in 2mL and contains in the LB substratum of Kn resistance, and it is about 0.5 that 37 ℃ of concussions are cultured to OD600, then according to 1: 1000 ratio switching enlarged culturing to 500mL LB (containing the Kn resistance), be cultured to OD ₆₀₀About 0.8 o'clock, add IPTG 500 μ L, induce 20h for 18 ℃.Collect thalline, the centrifugal 10min of 8000rpm down for 4 ℃.Abandon supernatant, bacterial sediment is resuspended with the lysate of 20mL/ bottle.Ice-water bath adopts Ultrasonic Cell Disruptor to handle smudge cells.Ultrasound condition is as follows: the working hour: 10min; Pulse: beat 2sec, stop 5sec; Output rating: 70%.The centrifugal 10min of 12000rpm keeps supernatant.Identify that through SDS-PAGE all fusion roteins mainly are expressed in (Figure 17) in the supernatant.

[0418] because the supernatant expressing protein is more assorted, so need carry out purifying to it.And this proteinoid can spontaneous assembling form particle under suitable condition, so when albumen carried out purifying, should consider that its spontaneous assembling forms the particulate condition.We adopt following method that albumen is carried out purifying and promote the spontaneous assembling of albumen to form particle: the amount adding saturated ammonium sulphate with cumulative volume 20% carries out albumen precipitation, ice bath reaction 30min.The centrifugal 10min of 12000rpm abandons supernatant.Precipitation blows afloat with the CBBuffer that contains 5% beta-mercaptoethanol, 37 ℃ of concussion 30min, and the centrifugal 10min of 12000rpm, supernatant dialyse to the PB5.8 buffer solution system of (containing 300mM NaCl and 50mM EDTA).Every 8h changes liquid once, collects dialyzate after changing liquid 6 times, and the centrifugal 10min of 12000rpm, supernatant sample carry out SDS-PAGE and identify purity.In this method, earlier albumen is carried out preliminary purification with 20% saturated ammonium sulphate, impelling albumen to form the particle assembling with the CB Buffer that contains 5% beta-mercaptoethanol again causes dimer, promotes the spontaneous assembling of albumen to form particle then under the condition of low pH value and high salt.Under this condition, target protein can obtain further purifying (Figure 18) simultaneously.

[0419] above 8 kinds of fusion roteins of expressing are all with behind the aforesaid method preliminary purification, and sample directly carries out electron microscopic observation (Figure 19) with 2% phosphoric acid tungsten negative staining.Assemble successful particle and be uniform hollow ball shape (the part particle is solid shape), two kinds of sizes are arranged, diameter is about 35nm and 20nm respectively.

[0420] embodiment 16ELISA detects HBc-123 and HBc-125 fusion rotein activity

[0421] with eight kinds of fusion roteins respectively with the concentration bag of 10 μ g/mL by 96 orifice plates, 37 ℃ of 2h.Wash plate 1 time, ED confining liquid sealing nonspecific binding site, 4 ℃ are spent the night behind 37 ℃ of 2h.Every hole adds 100 μ L 8H5 antibody, hatches 1h for 37 ℃.PBST washes plate 5 times, and every hole adds GAM-HRP (1: 10000) 100 μ L, hatches 30min for 37 ℃.PBST washes plate 5 times, adds colour developing liquid colour developing 10min, and stop buffer stops, the microplate reader value of reading.By the result of Figure 20 as can be known, eight kinds of fusion roteins all have the activity with the 8H5 antibodies.

[0422] is example with Q123 and D125 albumen, carries out the detection of antibodies specific.The protein concentration bag of 10 μ g/mL is by 96 orifice plates, 37 ℃ of 2h.Wash plate 1 time, ED confining liquid sealing nonspecific binding site, 4 ℃ are spent the night behind 37 ℃ of 2h.Every hole adds 100 μ L different antibodies, comprising: 8H5,8G9,3C8,4D1,10F7,1G2,3D2,3CF1,7D1,6CF3,7H8,10DE2,13E7,16A12 be totally 14 strain monoclonal antibodies.Wherein 8H5 is the used monoclonal antibodies of 12 peptide screenings, and all the other 13 strain monoclonal antibodies are to go up blood clotting albumen (HA) monoclonal antibody at avian influenza virus (AIV), hatch 1h for 37 ℃.PBST washes plate 5 times, and every hole adds GAM-HRP (1: 10000) 100 μ L, hatches 30min for 37 ℃.PBST washes plate 5 times, adds colour developing liquid colour developing 10min, and stop buffer stops, the microplate reader value of reading.To E LISA interpretation of result (Figure 21), Q123 and D125 fusion rotein only react with antibody 8H5, and be all reactionless with other bird flu monoclonal antibody.The atopic that Q123 and D125 fusion rotein are described is very good.

[0423] the immunogenicity analysis of embodiment 17HBc-123 and HBc-125 fusion rotein

[0424] above-mentioned purified eight kinds of HBc-123 and HBc-125 fusion rotein are mixed with isopyknic freund adjuvant respectively and (mix with complete freund adjuvant during initial immunity, mix with incomplete freund adjuvant during booster immunization), mode with the muscle multi-point injection, the immunity BALB/c mouse, the immunity amount of every mouse is 100 μ g albumen, and 3-4 mouse is one group.Behind the initial immunity, booster immunization is once taked eyeball blood simultaneously week about.Carrying out antiserum(antisera) separates: the blood suspension is put 37 ℃ of 2hr, move into 4 ℃ again and spend the night, make hemagglutination.The centrifugal 10min of 4000g draws supernatant, promptly obtains antiserum(antisera).Deposit in-20 ℃ standby.

[0425] wraps by 239-123 and 239-125 fusion rotein with 1 μ g/ hole, the sheep anti-mouse igg of HRP mark is as ELIAS secondary antibody, set up the indirect method ELISA that anti-12 peptides 123 or 125 specific antibodies detect, be used for detecting 123 peptides or 125 peptide specific antibodies and 12 reactive polypeptides and the antibody generation titre situation of animal antiserum.After each fusion protein immunization serum of HBc-123 diluted with 1: 1000, ELISA detected result such as Figure 22.After each fusion protein immunization serum of HBc-125 diluted with 1: 2000, ELISA detected result such as Figure 23.

[0426] immunofluorescence of embodiment 18 immune serums detects

[0427] in 24 porocyte culture plates, places cover glass, cultivate insect cell SF21 therein,, in the SF21 cell, express the HA albumen of avian influenza virus by insect cell-baculovirus expression system.Cell after the expression is through the PBS washing, and 4% Paraformaldehyde 96 is fixed, after the sealing of goat polyvalent antibody, with the T123 of dilution in 1: 20 and F125 immune mouse serum incubated at room 1 hour, with the negative contrast of HBc immune mouse serum.With fluorescently-labeled sheep anti-mouse antibody (MO is two anti-USA) for Sigma, St.Louis, room temperature reaction half an hour, get DAPI (Sigma, St.Louis, MO, USA) transfect cell nuclear, room temperature 10 minutes, film-making is in just putting observation fluorescent microscope (Nikon) under then.By the result of Figure 24 as can be known, T123 and F125 immune mouse serum all can be specifically with the SF21 cell in express the HA protein binding of avian influenza virus.Show that further 123 and 125 have simulated the epitope of HA preferably.

[0428] embodiment 1912 peptides 122,124,128,129 and HBV cAg protein fusion expression and active the detection

[0429] 4 kind of 12 peptide 122,124,128,129 inserts in the HBV cAg albumen with two copies respectively, and the fusion rotein that obtains is called HBc-122, HBc-124, HBc-128 and HBc-129.

[0430] construction process of HBc-122, HBc-124, HBc-128 and HBc-129 fusion protein expression vector is identical with the construction process of HBc-123, HBc-125 fusion protein expression vector, the primer such as table 19.The upstream primer of the first round is F3, and second upstream primer of taking turns is F2, and the upstream primer of third round is F1, and downstream primer is HBcR.Take turns PCR by 3, purpose 12 peptide segments are linked to each other with C 149-mut fragment, and, be connected, be built into expression plasmid (concrete grammar sees embodiment 15 for details) with the pC 149-mut carrier of BamH I and EcoR I double digestion with behind Bgl II and the EcoR I double digestion.

[0431] table 19 HBc-122, HBc-124, HBc-128 and HBc-129 fusion rotein clone primer sequence

[0432] HBc-122, HBC-124, HBc-128 and HBc-129 expressing fusion protein and purifying and above-mentioned HBc-123, HBc-125 Expression of Fusion Protein, purification process identical (concrete grammar sees embodiment 15 for details).The expression of HBc and 12 peptide fusion proteins and particle assembling situation are shown in table 20.Particulate electron microscopic observation picture such as Figure 25.

[0433] expression of table 20HBc and 12 peptide fusion proteins and VLP form

[0434] indirect ELISA detects HBc-122, HBc-124, HBc-128 and HBc-129 fusion rotein activity

[0435] HBc-122, HBc-124, HBc-128 and HBc-129 fusion rotein with the concentration bag of 10 μ g/mL by 96 orifice plates, 37 ℃ of 2h.Wash plate 1 time, ED confining liquid sealing nonspecific binding site, 4 ℃ are spent the night behind 37 ℃ of 2h.Every hole adds 100 μ L different antibodies, comprising: 8H5,8G9,3C8,1G2,3D2,3CF1,7D1,6CF3,7H8,10DE2,13E7,16A12 be totally 12 strain monoclonal antibodies.Wherein 8H5 is the used monoclonal antibodies of 12 peptide screenings, and all the other 11 strain monoclonal antibodies are to go up blood clotting albumen (HA) monoclonal antibody at avian influenza virus (AIV), hatch 1h for 37 ℃.PBST washes plate 5 times, and every hole adds GAM-HRP (1: 10000) 100 μ L, hatches 30min for 37 ℃.PBST washes plate 5 times, adds colour developing liquid colour developing 10min, and stop buffer stops, the microplate reader value of reading.By the result of Figure 26 as can be known, HBc-122, HBc-124, HBc-128 and HBc-129 fusion rotein only react with antibody 8H5, all reactionless with other bird flu monoclonal antibody, illustrate that the atopic of HBc-122, HBc-124, HBc-128 and HBc-129 fusion rotein and 8H5 monoclonal antibody is very good.

[0436] embodiment 20 shows the competitive ELISA experiment of the viruslike particle and the avian influenza virus of 12 peptides

[0437] get the special mouse monoclonal antibody of avian influenza virus H 5 N 1 2F2 with the 10ug/ml wrapper sheet, virus strain Ck/HK/Yu22/02 adds in the orifice plate with 1: 40 dilution back hatches, and 37 ℃, 1h; Discard virus in the hole, the viruslike particle that 10ug is just pure and the 8H5/HRP of dilution in 1: 1000 add in the orifice plate jointly hatches, and 37 ℃, 30min.126 and 127 do not combine with the H5 monoclonal antibody, go up as negative control but be showed in VLP equally, and be the negative control that does not add VLP with PBS again in addition.The result as shown in figure 27, above-mentioned 5 viruslike particles to be measured all can combine the 8H5 enzyme labelled antibody with virus competition, further point out these 5 12 peptides all might simulate the segment space structure of 8H5 epitope.

Sequence table

SEQ?ID?NO：1(8H5?Vh?Nucleotide?sequence)

CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACTTTCAGTAACTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGCGATAGAACAAACTACAATGGGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCCACATGCAACTCAGTAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAATAGATACGACGGGTATTATTTTGGTTTGGATTACTGGGGTCAAGGAACCTCAGTCGCCGTCTCCTCAGCC

SEQ?ID?NO：2(8H5?Vh?Amino?Acid?sequence)

QVQLQQSGAELMKPGASVKISCKATGYTFSNYWIEWIKQRPGHGLEWIGEILPGSDRTNYNGKFKGKATFTADTSSNTAHMQLSSLTSEDSAVYYCANRYDGYYFGLDYWGQGTSVAVSSA

SEQ?ID?NO：3(8H5?Vk?Nucleotide?sequence)

GAAATCGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATTTCGTTTACTGGTACCAGCAGAGGTCAGATGCCTCCCCCAAACTATTGATTTACTATTCATCCAACCTGGCTCCTGGAGTCCCACCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCGGCTTGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCACTTTACTAGTTCCCCGTACACGTTCGGAGGGGGGACCAACCTGGAAATAAAACGG

SEQ?ID?NO：4(8H5?Vk?Amino?Acid?sequence)

EIVLTQSPAIMSASLGEKVTMSCRASSSVNFVYWYQQRSDASPKLLIYYSSNLAPGVPPRFSGSGSGNSYSLTISGLEGEDAATYYCQHFTSSPYTFGGGTNLEIKR

SEQ?ID?NO：5(3C8?Vh?Nucleotide?sequence)

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCCTCTGGGTACAGCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTCTAAAGTGGATGGGCTGGATAAACACCTACACCGGAGAGCCAGCCTATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTCTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGATGGAATAGAGATGCTATGGACTACTGGGGTCAAGGAACCTCGGTCACCGTATCTAGC

SEQ?ID?NO：6(3C8?Vh?Amino?Acid?sequence)

QIQLVQSGPELKKPGETVKISCKASGYSFTNYGMNWVKQAPGKGLKWMGWINTYTGEPAYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARWNRDAMDYWGQGTSVTVSS

SEQ?ID?NO：7(3C8?VK?Nucleotide?sequence)

GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTTGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTCTGACAATAGTCTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTATTGGGGATCCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG

SEQ?ID?NO：8(3C8VK?Amino?Acid?sequence)

DIVLTQSPASLAVSLGQRATISCRASESVDSSDNSLMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSIGDPPYTFGGGTKLEIKR

SEQ?ID?NO：9(10F7?Vh?Nucleotide?sequence)

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGATTCTTATACTAACTACAATCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGGGGGGTACAGGAGACTTTCACTATGCTATGGACTACTGGGGTCAAGGCACCTCGGTCACCGTATCATCG

SEQ?ID?NO：10(10F7?Vh?Amino?Acid?sequence)

QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGGTGDFHYAMDYWGQGTSVTVSS

SEQ?ID?NO：11(10F7?VK?Nucleotide?sequence)

GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCATTAGCAGTAATATAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCAGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGTTCAGTTCCCGTACACGTTCGGAGGGGGCACCAAGCTGGAAATCAAACGG

SEQ?ID?NO：12(10F7?VK?Amino?Acid?sequence)

DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYVQFPYTFGGGTKLEIKR

SEQ?ID?No：13

CATGGGATGCTGCCGGTGTAT

SEQ?ID?No：14

AATTCTGGGCCTTGGCTGACG

SEQ?ID?No：15

TGGCCGCCTCTGTCGAAGAAG

SEQ?ID?NO：16(4D1?VH?Nucleotide?sequence)

CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCT

TCAGTGAACCTGTCCTGTAAGGCTTCTGGCTACACCTTCACCAGCTACT

GGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATC

GGAGAGATTGATCCTTCTGATAGTTTTACTACCTACAATCAAAACTTCA

AAGACAGGGCCACATTGACTGTAGACAAATCATCCAGCACAGCCTAC

ATGCAGCTCAGAAGTCTGACATCTGAGGACTCTGCGGTCTATTACTGT

GCCAGGGGGGGTCCAGGAGACTTTCGCTATGCTATGGATTACTGGGGC

CAAGGCACCTCGGTCACCGTCTCCTCA

SEQ?ID?NO：17(4D1?VH?Amino?Acid?sequence)

QVQLQQPGAELVKPGASVNLSCKASGYTFTSYWMHWVKQRPGQGLEWI

GEIDPSDSFTTYNQNFKDRATLTVDKSSSTAYMQLRSLTSEDSAVYYCAR

GGPGDFRYAMDYWGQGTSVTVSS

SEQ?ID?NO：18(4D1?VK?Nucleotide?sequence)

GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAG

ACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCATTAGCAGTAATA

TAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCAGATTATTCTCTCACCATCAGCAGCCTGGAATCCGAAGACTTTGCAGACTATTACTGTGTACAGTATGTTCAGTTTCCCTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT

SEQ?ID?NO：19(4D1?Vk?Amino?Acid?sequence)

DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYVQFPYTFGGGTKLEIKRA

SEQ?ID?NO：20(3G4VH?Nucleotide?sequence)

CAGGTCCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCTGGCTACACATTCACTGATTATGCTATGCATTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGACTTATTAATACTGACTATGGTGATACTACTTACAACCAGAAGTTCAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAACACAGCCTATATGGAACTTGCCAGACTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGATCGGACTATGATTACTATTTCTGTGGTATGGACTACTGGGGTCAAGGAACCACGGTCACCGAATCTCTA

SEQ?ID?NO：21(3G4?VH?Amino?Acid?sequence)

QVQLQQSGAELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGLINTDYGDTTYNQKFKGKATMTVDKSSNTAYMELARLTSEDSAIYYCARSDYDYYFCGMDYWGQGTTVTESL

SEQ?ID?NO：22

SEQ?ID?NO：23

SEQ?ID?NO：24(2F2?VH?Nucleotide?sequence)

CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGCGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTACACTGGATTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGGCTGAGGGAAGAACCGACTATAATTCAGTTCTCAAATCCAGACTGAGCATCAATAAGGACAATTCCAGGAGCCAAGTTTTCTTAGAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGAGGTGATTACTACGGAAGCCTGGTACTTCGATGTCTGGGGCCAAGGAACCTCGGTCACCGAATCT

SEQ?ID?NO：25(2F2?VH?Amino?Acid?sequence)

QVQLKESGPGLVAPSQRLSITCTVSGFSLTGYGVHWIRQSPGKGLEWLGMIWAEGRTDYNSVLKSRLSINKDNSRSQVFLEMNSLQTDDTARYYCAREVITTEAWYFDVWGQGTSVTES

SEQ?ID?NO：26(2F2?VK?Nucleotide?sequence)

GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTATACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATATGCTTCCCAATCCATCTCTGGGATCCCCTCCAGATTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCACTATCAACAGTGTGGAACCTGAAGATGTTGGAATGTATTACTGTCAAAATGGTCACACCTTTCCGCTCACGTTCGGTGCTGGCACCAAGCTGGAAATCAAACGG

SEQ?ID?NO：27(2F2?VK?Amino?Acid?sequence)

DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLTINSVEPEDVGMYYCQNGHTFPLTFGAGTKLEIKR

SEQ?ID?NO：28(8H5?Vh?CDR1?Amino?Acid?sequence)

GYTFSNYW

SEQ?ID?NO：29(8H5?Vh?CDR2?Amino?Acid?sequence)

ILPGSDRT

SEQ?ID?NO：30(8H5?Vh?CDR3?Amino?Acid?sequence)

ANRYDGYYFGLDY

SEQ?ID?NO：31(8H5?Vk?CDR1?Amino?Acid?sequence)

SSVNF

SEQ?ID?NO：32(8H5?Vk?CDR2?Amino?Acid?sequence)

YSS

SEQ?ID?NO：33(8H5?Vk?CDR3?Amino?Acid?sequence)

QHFTSSPYT

SEQ?ID?NO：34(3C8?Vh?CDR1?Amino?Acid?sequence)

GYSFTNYG

SEQ?ID?NO：35(3C8?Vh?CDR2?Amino?Acid?sequence)

INTHTGEP

SEQ?ID?NO：36(3C8?Vh?CDR3?Amino?Acid?sequence)

ARWNRDAMDY

SEQ?ID?NO：37(3C8?Vk?CDR1?Amino?Acid?sequence)

ESVDSSDNSL

SEQ?ID?NO：38(3C8?Vk?CDR2?Amino?Acid?sequence)

RAS

SEQ?ID?NO：39(3C8?Vk?CDR3?Amino?Acid?sequence)

QQSIGDPPYT

SEQ?ID?NO：40(10F7?Vh?CDR1?Amino?Acid?sequence)

GYTFTSYW

SEQ?ID?NO：41(10F7?Vh?CDR2?Amino?Acid?sequence)

IDPSDSYT

SEQ?ID?NO：42(10F7?Vh?CDR3?Amino?Acid?sequence)

ARGGTGDFHYAMDY

SEQ?ID?NO：43(10F7?Vk?CDR1?Amino?Acid?sequence)

QGISSN

SEQ?ID?NO：44(10F7?Vk?CDR2?Amino?Acid?sequence)

HGT

SEQ?ID?NO：45(10F7?Vk?CDR3?Amino?Acid?sequence)

QYVQFPYT

SEQ?ID?NO：46(4D1?Vh?CDR1?Amino?Acid?sequence)

GYTFTSYW

SEQ?ID?NO：47(4D1?Vh?CDR2?Amino?Acid?sequence)

IDPSDSFT

SEQ?ID?NO：48(4D1?Vh?CDR3?Amino?Acid?sequence)

ARGGPGDFRYAMDY

SEQ?ID?NO：49(4D1?Vk?CDR1?Amino?Acid?sequence)

QGISSN

SEQ?ID?NO：50(4D1?Vk?CDR2?Amino?Acid?sequence)

HGT

SEQ?ID?NO：51(4D1?Vk?CDR3?Amino?Acid?sequence)

VQYVQFPYT

SEQ?ID?NO：52(3G4?Vh?CDR1?Amino?Acid?sequence)

GYTFTDYA

SEQ?ID?NO：53(3G4?Vh?CDR2?Amino?Acid?sequence)

INTDYGDT

SEQ?ID?NO：54(3G4?Vh?CDR3?Amino?Acid?sequence)

ARSDYDYYFCGMDY

SEQ?ID?NO：55(3G4?Vk?CDR1?Amino?Acid?sequence)

SEQ?ID?NO：56(3G4?Vk?CDR2?Amino?Acid?sequence)

SEQ?ID?NO：57(3G4?Vk?CDR3?Amino?Acid?sequence)

SEQ?ID?NO：58(2F2?Vh?CDR1?Amino?Acid?sequence)

GFSLTGYG

SEQ?ID?NO：59(2F2?Vh?CDR2?Amino?Acid?sequence)

IWAEGRT

SEQ?ID?NO：60(2F2?Vh?CDR3?Amino?Acid?sequence)

AREVITTEAWYFDV

SEQ?ID?NO：61(2F2?Vk?CDR1?Amino?Acid?sequence)

QSISDY

SEQ?ID?NO：62(2F2?Vk?CDR2?Amino?Acid?sequence)

YAS

SEQ?ID?NO：63(2F2?Vk?CDR3?Amino?Acid?sequence)

QNGHTFPLT

SEQ?ID?NO：64(Antibody-binding?peptide-8H5)

HGMLPVY

SEQ?ID?NO：65(Antibody-binding?peptide-8H5)

PPSNYGR

SEQ?ID?NO：66(Antibody-binding?peptide-8H5)

PPSNFGK

SEQ?ID?NO：67(Antibody-binding?peptide-8H5)

GDPWFTS

SEQ?ID?NO：68(Antibody-binding?peptide-8H5)

NSGPWLT

SEQ?ID?NO：69(Antibody-binding?peptide-8H5)

SEQ?ID?NO：70(Antibody-binding?peptide-3C8)

WPPLSKK

SEQ?ID?NO：71(Antibody-binding?peptide-3C8)

NTFRTPI

SEQ?ID?NO：72(Antibody-binding?peptide-3C8)

NTFRDPN

SEQ?ID?NO：73(Antibody-binding?peptide-3C8)

NPIWTKL

SEQ?ID?NO：74(Antibody-binding?peptide)

MEPVKKYPTRSP

SEQ?ID?NO：75(Antibody-binding?peptide)

ATGGAGCCGGTGAAGAAGTATCCGACGCGTTCTCCT

SEQ?ID?NO：76(Antibody-binding?peptide)

ETQLTTAGLRLL

SEQ?ID?NO：77(Antibody-binding?peptide)

GAGACTCAGCTGACTACGGCGGGTCTTCGGCTGCTT

SEQ?ID?NO：78(Antibody-binding?peptide)

ETPLTETALKWH

SEQ?ID?NO：79(Antibody-binding?peptide)

GAGACGCCTCTTACGGAGACGGCTTTGAAGTGGCAT

SEQ?ID?NO：80(Antibody-binding?peptide)

QTPLTMAALELF

SEQ?ID?NO：81(Antibody-binding?peptide)

CAGACGCCGCTGACTATGGCTGCTCTTGAGCTTTTT

SEQ?ID?NO：82(Antibody-binding?peptide)

DTPLTTAALRLV

SEQ?ID?NO：83(Antibody-binding?peptide)

GATACTCCGCTGACGACGGCGGCTCTTCGGCTGGTT

SEQ?ID?NO：84(Antibody-binding?peptide)

TPLTLWALSGLR

SEQ?ID?NO：85(Antibody-binding?peptide)

ACGCCGCTTACGCTTTGGGCTCTTTCTGGGCTGAGG

SEQ?ID?NO：86(Antibody-binding?peptide)

QTPLTETALKWH

SEQ?ID?NO：87(Antibody-binding?peptide)

CAGACGCCTCTTACGGAGACGGCTTTGAAGTGGCAT

SEQ?ID?NO：88(Antibody-binding?peptide)

QTPLTMAALELL

SEQ?ID?NO：89(Antibody-binding?peptide)

CAGACGCCTCTGACTATGGCGGCTCTTGAGCTTCTT

SEQ?ID?NO：90(Antibody-binding?peptide)

HLQDGSPPSSPH

SEQ?ID?NO：91(Antibody-binding?peptide)

CAGACGCCTCTGACTATGGCGGCTCTTGAGCTTCTT

SEQ?ID?NO：92(Antibody-binding?peptide)

GHVTTLSLLSLR

SEQ?ID?NO：93(Antibody-binding?peptide)

GGGCATGTGACGACTCTTTCTCTTCTGTCGCTGCGG

SEQ?ID?NO：94(Antibody-binding?peptide)

FPNFDWPLSPWT

SEQ?ID?NO：95(Antibody-binding?peptide)

TTTCCGAATTTTGATTGGCCTCTGTCTCCGTGGACG

SEQ?ID?NO：96(Antibody-binding?peptide)

ETPLTEPAFKRH

SEQ?ID?NO：97(Antibody-binding?peptide)

GAGACGCCTCTTACGGAGCCGGCTTTTAAGCGGCAT

Claims (61)

1. an energy specificity is in conjunction with the monoclonal antibody of H5 subtype avian influenza virus hemagglutinin, and wherein, described monoclonal antibody comprises the variable heavy chain that is selected from as next group:

(i) comprising one or more, aminoacid sequence is the variable heavy chain of the CDR of SEQ ID NOs:28-30;

(ii) comprising one or more, aminoacid sequence is the variable heavy chain of the CDR of SEQ ID NOs:34-36;

(iii) comprising one or more, aminoacid sequence is the variable heavy chain of the CDR of SEQ ID NOs:40-42;

(iv) comprising one or more, aminoacid sequence is the variable heavy chain of the CDR of SEQ ID NOs:46-48;

(v) comprising one or more, aminoacid sequence is the variable heavy chain of the CDR of SEQ ID NOs:52-54; And

(vi) comprising one or more, aminoacid sequence is the variable heavy chain of the CDR of SEQ ID NOs:58-60.

2. monoclonal antibody as claimed in claim 1, wherein, described variable heavy chain comprises the aminoacid sequence that is selected from as next group:

SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ IDNO:21 and SEQ ID NO:25.

3. monoclonal antibody as claimed in claim 1, it further comprises the variable light chain that is selected from as next group:

(i) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:31-33;

(ii) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:37-39;

(iii) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:43-45;

(iv) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:49-51;

(v) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:55-57; And

(vi) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:61-63.

4. monoclonal antibody as claimed in claim 3, wherein, described variable light chain comprises the aminoacid sequence that is selected from as next group:

SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:19 and SEQ IDNO:27.

5. monoclonal antibody as claimed in claim 2, it further comprises the variable light chain that is selected from as next group:

(i) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:31-33;

(ii) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:37-39;

(iii) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:43-45;

(iv) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:49-51;

(v) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:55-57; And

(vi) comprising one or more, aminoacid sequence is the variable light chain of the CDR of SEQ ID NOs:61-63.

6. monoclonal antibody as claimed in claim 5, wherein, described variable light chain comprises the aminoacid sequence that is selected from as next group:

SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:19 and SEQ IDNO:27.

7. monoclonal antibody as claimed in claim 6, wherein, described monoclonal antibody is Fab, Fab ', F (ab) ₂Or Fv.

8. monoclonal antibody as claimed in claim 1, wherein, described monoclonal antibody is Fab, Fab ', F (ab) ₂Or Fv.

9. monoclonal antibody as claimed in claim 1, wherein, described monoclonal antibody in conjunction with the KD value of H5 subtype avian influenza virus hemagglutinin less than 1 * 10 ^-5M.

10. monoclonal antibody as claimed in claim 9, wherein, described monoclonal antibody in conjunction with the KD value of H5 subtype avian influenza virus hemagglutinin less than 1 * 10 ^-6M.

11. monoclonal antibody as claimed in claim 1, wherein, described monoclonal antibody comprises non--CDR district, this non--CDR district is from the species that are not muroid.

12. monoclonal antibody as claimed in claim 11, wherein, described non--the CDR district is the antibody from the mankind.

13. monoclonal antibody as claimed in claim 12, wherein, the described mankind are non--and the CDR district has one or more substituted amino acids from murine antibody.

14. an energy specificity is in conjunction with the monoclonal antibody of H5 subtype avian influenza virus hemagglutinin, wherein, described monoclonal antibody comprises the monoclonal antibody that is selected from as next group:

(i) monoclonal antibody that produced of hybridoma cell strain 8H5, wherein, the preserving number of hybridoma cell strain 8H5 is CCTCC-C200607;

The (ii) monoclonal antibody that produced of hybridoma cell strain 3C8, wherein, the preserving number of hybridoma cell strain 3C8 is CCTCC-C200605;

The (iii) monoclonal antibody that produced of hybridoma cell strain 10F7, wherein, the preserving number of hybridoma cell strain 10F7 is CCTCC-C200608;

The (iv) monoclonal antibody that produced of hybridoma cell strain 4D1, wherein, the preserving number of hybridoma cell strain 4D1 is CCTCC-C200606;

(the v) monoclonal antibody that produced of hybridoma cell strain 3G4, wherein, the preserving number of hybridoma cell strain 3G4 is CCTCC-C200604;

(the vi) monoclonal antibody that produced of hybridoma cell strain 2F2, wherein, the preserving number of hybridoma cell strain 2F2 is CCTCC-C200424.

15. a hybridoma cell strain, this hybridoma cell strain is selected from the hybridoma cell strain as next group:

(i) preserving number is the hybridoma cell strain 8H5 of CCTCC-C200607;

(ii) preserving number is the hybridoma cell strain 3C8 of CCTCC-C200605;

(iii) preserving number is the hybridoma cell strain 10F7 of CCTCC-C200608;

(iv) preserving number is the hybridoma cell strain 4D1 of CCTCC-C200606;

(v) preserving number is the hybridoma cell strain 3G4 of CCTCC-C200604; And

(vi) preserving number is the hybridoma cell strain 2F2 of CCTCC-C200424.

16. an energy specificity is in conjunction with the monoclonal antibody of H5 subtype avian influenza virus hemagglutinin, wherein, described monoclonal antibody can seal described hemagglutinin be selected from as next group monoclonal antibody combine active at least 50%:

(i) monoclonal antibody that produced of hybridoma cell strain 8H5, wherein, the preserving number of hybridoma cell strain 8H5 is CCTCC-C200607;

The (ii) monoclonal antibody that produced of hybridoma cell strain 3C8, wherein, the preserving number of hybridoma cell strain 3C8 is CCTCC-C200605;

The (iii) monoclonal antibody that produced of hybridoma cell strain 10F7, wherein, the preserving number of hybridoma cell strain 10F7 is CCTCC-C200608;

The (iv) monoclonal antibody that produced of hybridoma cell strain 4D1, wherein, the preserving number of hybridoma cell strain 4D1 is CCTCC-C200606;

(the v) monoclonal antibody that produced of hybridoma cell strain 3G4, wherein, the preserving number of hybridoma cell strain 3G4 is CCTCC-C200604;

(the vi) monoclonal antibody that produced of hybridoma cell strain 2F2, wherein, the preserving number of hybridoma cell strain 2F2 is CCTCC-C200424.

17. monoclonal antibody as claimed in claim 16, wherein, described monoclonal antibody is sealed described hemagglutinin in conjunction with active at least 70%.

18. monoclonal antibody as claimed in claim 17, wherein, described monoclonal antibody is sealed described hemagglutinin in conjunction with active at least 90%.

19. an isolated nucleic acid molecule, its comprise can the encoding antibody variable region of heavy chain nucleotide sequence, described antibody heavy chain variable region then comprises the aminoacid sequence that is selected from as next group:

(i)SEQ?ID?NOs：28-30；

(ii)SEQ?ID?NOs：34-36；

(iii)SEQ?ID?NOs：40-42；

(iv)SEQ?ID?NOs：46-48；

(v) SEQ ID NOs:52-54; And

(vi)SEQ?ID?NOs：58-60。

20. isolated nucleic acid molecule as claimed in claim 19, wherein, described variable region of heavy chain comprises the aminoacid sequence that is selected from as next group:

SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ IDNO:21 and SEQ ID NO:25.

21. isolated nucleic acid molecule as claimed in claim 20, wherein, described nucleic acid molecule comprises the nucleotide sequence that is selected from as next group:

SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:16, SEQ IDNO:20 and SEQ ID NO:24.

22. expression vector that comprises nucleic acid molecule as claimed in claim 19.

23. host cell that comprises expression vector as claimed in claim 22.

24. an isolated nucleic acid molecule, its comprise can the encoding antibody variable region of light chain nucleotide sequence, described antibody chain variable region then comprises the aminoacid sequence that is selected from as next group:

(i)SEQ?ID?NOs：31-33；

(ii)SEQ?ID?NOs：37-39；

(iii)SEQ?ID?NOs：43-45；

(iv)SEQ?ID?NOs：49-51；

(v) SEQ ID NOs:55-57; And

(vi)SEQ?ID?NOs：61-63。

25. isolated nucleic acid molecule as claimed in claim 24, wherein, described variable region of light chain comprises the aminoacid sequence that is selected from as next group:

SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:19 and SEQ IDNO:27.

26. isolated nucleic acid molecule as claimed in claim 25, wherein, described nucleic acid molecule comprises the nucleotide sequence that is selected from as next group:

SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:18 and SEQ IDNO:26.

27. expression vector that comprises nucleic acid molecule as claimed in claim 26.

28. host cell that comprises expression vector as claimed in claim 27.

29. the method for H5 subtype avian influenza virus in the test sample, it comprises the steps:

A) described sample is contacted with monoclonal antibody in the claim 1;

B) reaction of described monoclonal antibody of detection and described virus.

30. method as claimed in claim 29, wherein, described monoclonal antibody is attached on the solid phase.

31. method as claimed in claim 30, wherein, described solid phase is selected from as next group: titer plate, magnetic particle, latex particle and nitrocellulose membrane.

32. method as claimed in claim 30, wherein, described monoclonal antibody be by direction like this attached on the described solid phase, it can increase the joint efficiency of described monoclonal antibody and described sample.

33. method as claimed in claim 32, wherein, described monoclonal antibody by its constant zone attached on the described solid phase.

34. method as claimed in claim 29, wherein, described reaction is measured by the enzyme colour developing.

35. method as claimed in claim 29, wherein, described reaction is measured by fluorescence.

36. method as claimed in claim 29, wherein, described reaction is measured by chemoluminescence.

37. method as claimed in claim 29, wherein, described monoclonal antibody is Fab, Fab ', F (ab) ₂Or Fv.

38. method as claimed in claim 29, wherein, described sample comes from the birds or the mankind.

39. a pharmaceutical composition, it comprises according to claim 1 monoclonal antibody at pharmacy acceptable salt.

40. a pharmaceutical composition, its comprise one or more be selected from as next the group monoclonal antibody, its pharmacy acceptable salt:

(i) monoclonal antibody that produced of hybridoma cell strain 8H5, wherein, the preserving number of hybridoma cell strain 8H5 is CCTCC-C200607;

The (ii) monoclonal antibody that produced of hybridoma cell strain 3C8, wherein, the preserving number of hybridoma cell strain 3C8 is CCTCC-C200605;

The (iii) monoclonal antibody that produced of hybridoma cell strain 10F7, wherein, the preserving number of hybridoma cell strain 10F7 is CCTCC-C200608;

The (iv) monoclonal antibody that produced of hybridoma cell strain 4D1, wherein, the preserving number of hybridoma cell strain 4D1 is CCTCC-C200606;

(the v) monoclonal antibody that produced of hybridoma cell strain 3G4, wherein, the preserving number of hybridoma cell strain 3G4 is CCTCC-C200604;

(the vi) monoclonal antibody that produced of hybridoma cell strain 2F2, wherein, the preserving number of hybridoma cell strain 2F2 is CCTCC-C200424.

41. pharmaceutical composition as claimed in claim 40, it comprises that further other antiviral ingredients is at pharmacy acceptable salt.

42. pharmaceutical composition as claimed in claim 41, wherein, described monoclonal antibody is Fab, Fab ', F (ab) ₂Or Fv.

43. a method that prevents or treat the disease that on a main body, causes by avian influenza, it comprise to described main body administration effective dose, the described pharmaceutical composition of claim 39.

44. a composition that is used for the test sample avian influenza virus, it comprise appendix on the solid phase substrate, the described monoclonal antibody of claim 1.

45. composition as claimed in claim 44, wherein, described monoclonal antibody is Fab, Fab ', F (ab) ₂Or Fv.

46. composition as claimed in claim 44, wherein, described monoclonal antibody comprises variable heavy chain, and this variable heavy chain contains the peptide sequence that is selected from as next group:

SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ IDNO:21 and SEQ ID NO:25.

47. as the composition that claim 46 is stated, wherein, described monoclonal antibody further comprises and can lighten, this can lighten and contain the peptide sequence that is selected from as next group:

SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:19 and SEQ IDNO:27.

48. composition as claimed in claim 44, wherein, described solid phase substrate is selected from as next group: titer plate, magnetic particle, latex particle and nitrocellulose membrane.

49. composition as claimed in claim 44, wherein, described solid phase substrate is a calibration tape.

50. composition as claimed in claim 49, wherein, described calibration tape has an at least one test zone and a control region.

51. composition as claimed in claim 44, wherein, described monoclonal antibody be by direction like this attached on the described solid phase substrate, it can increase the joint efficiency of described monoclonal antibody and described sample.

52. a device that is used for the test sample avian influenza virus, it comprises the solid phase substrate, and this solid phase substrate comprises between a plurality of marker spaces, wherein, is coated with monoclonal antibody as claimed in claim 1 between one or more described marker spaces.

53. device as claimed in claim 52, wherein, be coated with between one or more described marker spaces be different from can specificity in conjunction with the described monoclonal antibody of H5 subtype avian influenza virus hemagglutinin, binding reagents.

54. device as claimed in claim 53, wherein, described binding reagents be can specificity in conjunction with H1, H3, H7, or the antibody of H9 subtype avian influenza virus.

55. device as claimed in claim 52, it further comprises the automatization proofing unit, and this automatization proofing unit can detect combining between described monoclonal antibody and the H5 subtype avian influenza virus hemagglutinin.

56. the test kit of avian influenza virus in the test sample, it comprise as claimed in claim 1, be attached to monoclonal antibody and second monoclonal antibody detectable, that be labeled on the solid phase substrate.

57. test kit as claimed in claim 56, wherein, described second monoclonal antibody can be specifically in conjunction with avian influenza virus.

58. test kit as claimed in claim 56, wherein, described second monoclonal antibody can be specifically in conjunction with avian flu virus hemagglutinin protein.

59. test kit as claimed in claim 56, it further comprises control criterion.

60. it is SEQID NOs:64-69 that an energy specificity comprises aminoacid sequence in conjunction with the small peptide of 8H5 monoclonal antibody, 74,76,78,80,82,84,86,88,90,92,94, or 96, or by SEQ IDNOS:75,77,79,81,83,85,87,89,91,93,95, or 97 nucleic acid sequence encoding.

61. it is SEQID NOs:70-73. that an energy specificity comprises aminoacid sequence in conjunction with the small peptide of 3C8 monoclonal antibody

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