WO2007122760A1 - Detection reagent and detection method for influenza infection - Google Patents

Abstract

The invention relates to a detection reagent and a detection method for influenza infection using a nuclear protein (NP) of an influenza virus as an AIV antibody detecting antigen. Preferably, the nuclear protein (NP) in which a His-tag sequence is added to a nuclear protein (NP) gene of AIV A/Duck/Aomori/478/02(H1N1) and the gene is expressed as NP-His using a baculovirus and an insect cell is used as the AIV antibody detecting antigen. According to the invention, the detection reagent and the detection method for influenza infection which enable a multi-sample treatment which is a substitute for an agar gel precipitation (AGP) test and are highly sensitive and highly specific can be obtained.

Description

 Specification

 Influenza screening reagents and methods Technical field

 [0001] The present invention relates to an influenza infection screening agent and a screening method. More specifically, the present invention relates to an influenza infection test agent and a test method using a common influenza A antigen capable of rapid, high sensitivity and multi-sample processing. Background art

 [0002] Avian influenza (Avian Influenza, hereinafter abbreviated as AI) is an important viral disease in poultry and is highly pathogenic in livestock epidemic prevention methods. avian influenza (hereinafter abbreviated as HPAI) is designated as a legal infectious disease, and low pathogenic avian influenza (hereinafter abbreviated as LPAI) is designated as a reported infectious disease. Threat. An avian influenza virus (hereinafter abbreviated as AIV) belonging to the genus Orthomyxomyces influenza A virus has an envelope, a diameter of about 80 to 120 nm, and a negative single-stranded segmental RNA. This virus is antigenically divided into three types, A, B, and C, by nucleoprotein (hereinafter abbreviated as NP) and matrix protein (hereinafter abbreviated as M). Only infects humans. Only type A viruses infect humans, horses, pigs, other mammals and a wide variety of poultry and wild birds.

[0003] Eleven types of viral proteins produced by influenza virus (Influenza virus) can be roughly classified into three types: surface proteins, internal proteins, and nonstructural proteins that are not contained in the virus particles. There are three types of surface proteins contained in virus particles: hemagglutinating protein (hereinafter abbreviated as HA), neuraminidase (hereinafter abbreviated as NA), and matrix 2 protein (hereinafter abbreviated as M2). Internal proteins include polymerase proteins (PA, PB1, PB2), NP, matrix 1 protein (hereinafter abbreviated as Ml), nonstructural protein 2 (named nonstructural, but within the virus particle) Incorporated: hereinafter abbreviated as NS2. Nonstructural protein 1 (hereinafter abbreviated as NS 1) is produced in large quantities in infected cells. It is the only protein that cannot be turned over. According to the surface proteins HA and NA, serotypes (subtypes) are classified into 15 types (H1 to H15) for HA and 9 types (N1 to N9) for NA. Recently, a new H16, which is relatively closely related to H13, has been reported. Due to the various combinations of HA and NA, there are many subtypes of influenza virus. AI is also classified into HPAI and LPAI by pathogenicity. HPAI is an acute infectious disease that causes systemic symptoms in various birds, including chickens. The symptoms vary, but the fatality rate and transmission power are extremely strong.

 [0004] Conventionally, HA and NA subtypes of AIV use antiserum prepared from 15 types of HA and 9 types of NA, respectively, and hemagglutination inhibition (hereinafter abbreviated as HI) test and It has been determined by neuraminidase inhibition (hereinafter abbreviated as NI) test. In recent years, in human influenza, reverse transcriptase polymerase chain reaction (referred to as FRT-PCR) has been used to identify serotypes of HI to H3 virus and N1 and N2 viruses. It is coming. A method for diagnosing AIV using primers H1 to H15 was reported by Lee et al. (Journal of Virological Methods. 97: 13-22.2001: Non-Patent Document 1) and adopted in Japan. PCR is said to serve as an effective and rapid method for detecting influenza A virus, which requires rapid and antisera.

 [0005] In recent years, AI has been generated one after another on a global scale, and in January 2004, HPAI was reported in Yamaguchi Prefecture for the first time in 79 years in Japan. Following the outbreak, HPAI occurred in the same year in Oita, Kyoto and Osaka prefectures, and all of these serotypes were H5N1. In addition, in 2005, the occurrence of H5N2 was observed in Ibaraki Prefecture, and the necessity of serological diagnosis important for understanding the status of AIV infection and effective control measures in the event of occurrence has increased. Therefore, it is necessary to establish an accurate and quick diagnostic method for AI.

[0006] However, AIV has 16 HA subtypes, and its serological diagnosis requires a large number of antisera (16 types and amounts) and antigens, which complicates the diagnostic method. In addition, there are many points to be improved in the current diagnostic methods in terms of sensitivity, rapidity, and specificity. Currently, the in-gel sedimentation (hereinafter abbreviated as AGP) test and the HI test are widely used for serodiagnosis techniques for detecting AIV antibodies. The AGP test uses both antigen and antibody to form sedimentation lines. Need at least 24 hours. The HI test is very laborious and costly per specimen due to the 16 HA subtype that is more sensitive and quicker than the AGP test.

 [0007] Indirect ELISA can easily detect a large number of specimens. Snyder et al. (Avian Dis. 29: 136-44.

 1985: Non-patent document 2) shows that the sensitivity and specificity are higher than those of the AGP test. It is considered that there are many nonspecific reactions, and relatively specific antigens for colorimetric determination and species-specificity for each test animal. Enzyme-linked antibodies are required. Competitive ELISA uses recombinant antigens and monoclonal antibodies (hereinafter abbreviated as MAb) to suppress non-specific reactions and increase sensitivity and specificity. Its usefulness has been proved by Zhou et al. (Avian Dis.42: 517_22.1998: Non-patent document 3) and Shafer et al. (Avian Dis. 42: 28-34.1998: Non-patent document 4). Since it does not require a bound antibody, it can be diagnosed in various animal species.

 [0008] Several usefulnesses of an influenza diagnosis system using a recombinant protein have been reported. Harmon et al. (J Med Virol. 27: 25_30.1989: Non-Patent Document 5) detected influenza antibodies in humans using NP expressed in Escherichia coli by enzyme assay (EIA), and the usefulness of the expressed NP as an antigen. Showed sex. Voeten et al. (Journal of Clinical Microbiology. 3257-3531.1998: Non-Patent Document 6) expressed influenza A NP of type A and B in Escherichia coli and used the ELISA using it to be specific for influenza A and B in humans. IgG and IgA antibody trends were examined. This experiment demonstrates the specificity and rapidity of ELISA using recombinant antigens.

[0009] Yewdell et al. (J ImmunoL 126: 1814_9.1981: Non-Patent Document 7) used MAbs to determine the expression of NP and M from influenza-infected cells by radioimmunoassay (hereinafter abbreviated as RIA), firefly. Examination by methods such as photoantibody method and agglutination test shows that NP is expressed more than M or is easily detected by these tests. In other words, it is important in the diagnosis of influenza to use MAbs against NPs that are more likely to be produced in diagnosis or are likely to be detected. In addition, this demonstrated the usefulness of diagnosis using anti-NP MAbs, and MAbs produced by Yewdell et al. Are also included in the NP-ELISA of De Boer et al. (Arch ViroL 115: 47- 61.19 90: Non-Patent Document 8). Used quickly and for various animal species It has been shown that you can.

 [0010] Competitive ELISA using baculovirus-expressed recombinant NP and anti-NP MAb has been shown to be useful by Zhou et al. (Non-patent Document 3) and Shafer et al. (Non-patent Document 4). Zhou et al. (Non-patent Document 3) used chicken, turkey, emiu, and ostrich sera to compare competitive ELISA, AGP test, and HI test, and detected competitive ELISA positive and HI positive specimens with negative AGP test. The competitive ELISA showed higher sensitivity and specificity than the AGP test, indicating that it has the same sensitivity and specificity as the HI test. Similarly, Shafer et al. (Non-Patent Document 4) also compared the competitive ELISA and AGP tests using chicken, turkey, running bird, quail, pheasant, and penguin sera. Re, correlation and competition showed high specificity and specificity of ELISA. In the competitive ELISA of Shafer et al. (Non-Patent Document 4), antigen purification is performed.

 [0011] Non-Patent Document 1 Journal of Virological Methods. 97: 13-22.2001

 Non-Patent Document 2: Avian Dis. 29: 136-44.1985

 Non-Patent Document 3: Avian Dis. 42: 517-22.1998

 Non-Patent Document 4: Avian Dis. 42: 28-34.1998

 Non-Patent Document 5:〗 Med Virol. 27: 25-30.1989

 Non-Patent Literature 6 Journal of Clinical Microbiology.3257_3531.1998

 Non-Patent Document 7: J Immunol. L26: 1814-9.1981

 Non-Patent Document 8: Arch Virol. 115: 47-61.1990

 Disclosure of the invention

 Problems to be solved by the invention

[0012] The NP of AIV that the present inventors tried to express is a protein consisting of 498 amino acid residues having several different functions throughout the life cycle of the virus. It functions mainly as a negative single-stranded RNA-binding protein and is a structural protein in ribonucleoprotein particles (hereinafter abbreviated as RNPs). In addition, NP plays an important role in the transport and transcription of RNPs between the cytoplasm and nucleus. The NP gene is l, 565 bp and is encoded in segment 5. NPs are highly conserved among viruses and have more than 95% amino acid sequence identity with other avian subtypes. Furthermore, because it exists on a different segment from the gene segment encoding HA or NA that defines the subtype, it is not affected by antigenic mutations in HA and NA. Because it is common to influenza A, it is an important protein used for diagnostic tests

[0013] Although not particularly limited, the present inventors used baculovirus-insect cells as an expression system. As the baculovirus, Autographa California nuclear polyhearosis virus (hereinafter abbreviated as AcNPV) belonging to the nuclear polyhedrosis virus (hereinafter abbreviated as NPV) was used. In addition, plasmid pAcYMl was used as a transfer vector for baculovirus. This vector is the highest in the plasmid vector containing ATG of ATG, which is the polyhydrin gene start codon (base sequence near the cloning position is AAAAAAACCTA TAAAT A CGGATCCG (SEQ ID NO: 1), underlined is the restriction enzyme BamHI recognition site) It is known that expression efficiency is obtained. The point of the baculovirus expression system lies in the promoter of the nuclear inclusion body protein called polyhydrin. Different forces depending on the gene to be expressed Many of them are at the end of virus infection, and about 50% of the total cellular protein is said to be the target protein. In this system, protein synthesis is performed in insect cells, which are eukaryotes. Therefore, unlike prokaryotes such as E. coli or lower eukaryotes such as fermenters, correct modification of the protein is possible. Be expected . In fact, modifications such as signal peptide cleavage, phosphorylation, addition of sugar chains and fatty acids, and cleavage by proteolytic enzymes have been confirmed.

 [0014] The present inventors further attempted production of MAb against NP. Compared with polyclonal antibodies, the advantages of MAb are summarized in the following three points (1) to (3).

 (1) An antibody having high titer and high specificity against the antigenic determinant of the target molecule can be obtained,

 (2) Antibody-producing hyperpridoma can be stored in liquid nitrogen like other cell lines, and antibodies can be adjusted as needed by the experimenter.

 (3) It is easy to purify MAb.

[0015] The current diagnostic methods have many points to be improved in terms of sensitivity, rapidity, specificity, etc. The AGP test, which is regarded as a diagnostic criterion for AIV, is unsuitable for multi-sample processing and has many antibodies and antigens. Need the amount of. In other words, it is important to develop a highly sensitive and specific diagnostic system that can process multiple specimens. In addition, AIV is sensitive to various mammals and birds. Therefore, it is desirable to have a method capable of monitoring these various animal species.

 Means for solving the problem

[0016] The present inventors expressed AIV NP, which is considered to be highly conserved among AIV strains, for example, in Noculus virus, and intensively studied the possibility of supplying antigens for screening. MAb was prepared, and the usefulness of competitive ELISA as a highly sensitive detection system for AIV was studied intensively. Attempts were made to establish a highly sensitive serological diagnostic system using NP, and the present invention was completed.

 That is, the present invention is an invention of an influenza infection test drug according to the following 1 to 7 and an influenza infection test method according to the following 8 to 14:

 1. An influenza infection test drug characterized by using an influenza virus nucleoprotein (NP) as an AIV (avian influenza virus) antibody detection antigen.

 2. The nucleoprotein (NP) gene of influenza virus is isolated from AIV (avian influenza virus), and the nucleoprotein (NP) expressed using a baculovirus expression system is used as an AIV antibody detection antigen as described in 1 above Influenza screening medicine.

 3. The nucleoprotein (NP) expressed as NP_His by adding the His-tag sequence to the nucleoprotein (NP) gene of AIV A / Duck / Aomori / 478/02 (HlNl) is used as the AIV antibody detection antigen. The influenza infection screening agent according to 1 or 2.

 4. The influenza infection test agent according to any one of 1 to 3 above, wherein an influenza virus nucleoprotein (NP) is used as an immobilized antigen in an enzyme-linked immunosorbent assay (ELISA).

 5. (1) Influenza virus nucleoprotein (NP) is used as an immobilized antigen in enzyme-linked immunosorbent assay (E LISA), and at least the immobilized antigen of (1) above

 (2) The influenza test agent according to any one of (1) to (4), which comprises an antinuclear protein monoclonal antibody (NP MAb) that specifically binds to a nucleoprotein (NP) of influenza virus.

 6. The influenza test agent for influenza infection according to 4 or 5 above, wherein the immobilized antigen is immobilized on an ELISA plate.

7. Antinuclear tongue immunized with AIV A / budgeriger / Aichi / l / 77 (H3N8) 6. The influenza infection test agent according to 5 above, wherein the protein monoclonal antibody NP MAb (IgGl, κ) is used.

 8. A method for detecting influenza infection, characterized by using an influenza virus nucleoprotein as an immobilized antigen in an enzyme-linked immunosorbent assay (ELISA).

 9. The nucleoprotein (ΝΡ) isolated from the AIV of the influenza virus nucleoprotein (ΝΡ) gene and expressed using the baculovirus expression system is used as an immobilized antigen in the enzyme-linked immunosorbent assay (ELISA). 9. The method for detecting influenza infection as described in 8 above.

 10. (1) Influenza virus nucleoprotein (ΝΡ) is used as an immobilized antigen in enzyme-linked immunosorbent assay (ELISA), and at least the immobilized antigen (1) and

 (2) The influenza infection test method according to 8 or 9 above, which uses an antinuclear protein monoclonal antibody (NP MAb) that specifically binds to the nucleoprotein (NP).

 11. (1) Influenza virus nucleoprotein (NP) as an immobilized antigen for enzyme-linked immunosorbent assay (ELISA),

 (2) adding test serum, reacting the anti-avian influenza (AIV) antibody with the immobilized antigen of (1),

 (3) An antinuclear protein monoclonal antibody (NP MAb) that specifically binds to the nucleoprotein (NP) of (1) above is added, and the antinuclear protein monoclonal antibody (NP M Ab) is added to the above (2). 11. The method for testing influenza infection as described in 10 above, wherein a competitive reaction with the test serum added in step 1 is carried out.

 12. (1) Influenza virus nucleoprotein (NP) was used as an immobilized antigen for enzyme-linked immunosorbent assay (ELISA),

 (2) adding test serum, reacting the anti-avian influenza (AIV) antibody with the immobilized antigen of (1),

 (3) An antinuclear protein monoclonal antibody (NP MAb) that specifically binds to the nucleoprotein (NP) of (1) above is added, and the antinuclear protein monoclonal antibody (NP M Ab) is added to the above (2). Compete with the test sera added in the next step.

(4) An enzyme-labeled antibody that reacts only with the anti-NP MAb of (3) above is added, and then (5) The influenza infection detection method according to (11), wherein color development is measured when an enzyme substrate of a labeling enzyme of the enzyme-labeled antibody of (4) is added.

 13. The method for detecting influenza infection according to any one of 10 to 12 above, using an antinuclear protein monoclonal antibody NP MAb (IgGl, K) prepared by immunizing AIV A / budgeriger / AichiZlZ77 (H3N8) .

 14. The method for detecting influenza infection according to any one of 9 to 12, wherein an ELISA plate on which an antigen is immobilized is used.

 The invention's effect

 [0018] The diagnostic system using AIV NP His according to the present invention showed high sensitivity and specificity. Among them, competitive ELISA using MAb showed high utility. There are several advantages to diagnosing type A influenza antibodies by competitive ELISA. Competitive ELISAs can save time because they can handle multiple samples. Because it uses a recombinant protein, it is necessary to use a virus. Furthermore, by immobilizing the antigen on the ELISA plate and storing it, the test can be facilitated and the time can be further shortened. There is no need to purify highly purified antigens like indirect ELISA. And monitoring in various mammals and birds could be tested in the same way.

 [0019] Preservation of the ELISA plate on which the antigen is immobilized is particularly preferable because sensitivity and specificity do not decrease, because it facilitates inspection and facilitates supply to other inspection institutions. In the present invention, AIV NP His was purified using a His-tag. When purified antigen is used, non-specific reaction of ELISA can be effectively suppressed. However, purification takes a lot of time and the amount of antigen is also significantly reduced. Non-purified competitive ELISA can handle multiple samples more efficiently. The diagnostic system according to the present invention can be widely used by conducting competitive ELISA using sera from various animal species.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0020] For cloning of the AIV NP gene, for example, the strain AZDuckZAomori Z478Z02 (H1N1), which will be described later, can be obtained by amplifying the full length of each segment by RTPCR. It ’s not limited. The NP gene-specific primer was designed to add a histidine tag to the C-terminus of NP. Insert the PCR product into pCR2.1 It was transformed into the patent cells.

 [0021] Although the expression of the AIV NP gene is not particularly limited, it is preferably carried out by a baculovirus expression system. Recombinant baculovirus is prepared by inserting AIV NP His cDNA into the baculovirus transfer vector pAcYMl, then transforming it, and preparing recombinant virus by co-transformation of pAIV NP His-pAcYMl and AcRP23-LacZ. did. This virus was plaque-cloned and named AcAI NP His. The obtained AcAI NP His was infected to Sf9 cells to express AIV NP His. Furthermore, AIV NP His was purified from AcAI NP His-infected cells using a Ni column.

 [0022] The present inventors used the expressed AIV NP His, and examined the usefulness of ELISA and AGP tests. In an indirect ELISA using purified AIV NP His, AIV immunized chicken sera showed significantly higher values than the field normal chicken sera used in the serum dilutions performed and were performed in all field normal chicken sera used. No non-specific positive reaction was observed in the serum dilution. Furthermore, in the AGP test using AIV NP His, sedimentation lines were formed only with AIV-immune chicken sera, and nonspecific positive reactions were not observed with field normal chicken sera. All of the field ostrich sera were negative. These results were the same as those using RNP antigen. From these results, it is considered that the expressed NP can be widely used as an anti-AIV antibody detection antigen in various detection methods.

[0023] For the production of anti-AIV NP MAbs, MAb 1 was obtained from mice immunized with purified AIV A / budgerigar / Aichi / l / 77 (H3 N8) (available from the Society for Animal Biological Products). 1E5 (light chain κ, heavy chain IgGl) was obtained. The MAb 11E5 was concentrated and the reactivity was examined. In the ELISA using purified AIV NP His as an antigen, the reaction was performed up to a 1.0 × 1 O ^5- fold dilution. In Western blotting using AIV AZbudgerigar / AichiZlZ77 (H3N8) and purified AI V NP His as antigens, no band was observed at any position. The following are considered to be negative for Western blotting.

[0024] In the cell, influenza NP is stable as an oligomer. In addition, the NP oligomers are non-covalently bound and are resistant to SDS resistance, a wide temperature range and the presence of salts (1M Na CI, 1 M KC1) and denaturants (8M urea). Heating above 80 ° C And dissociates into NP monomer at pH 5 or lower. In other words, the three-dimensional structure may have changed due to the effect of 100 ° C heating or ME + during the antigen preparation stage. Furthermore, when AIV A / budger igar / Aichi / 1/77 (H3N8) was used as an antigen, the band could not be confirmed in any part, and the purified AIV NP His that showed high reaction by ELISA was also used. Since it did not react at all, the NP epitope for MAb 11E5 may have changed due to the effect of electrophoresis or heating.

[0025] The present inventors examined the application of competitive ELISA using AIV NP His and MAb 11E5. In competitive ELISA, all AIV immunized chicken sera showed almost 100% inhibition, and no inhibition reaction was observed in SPF chicken sera, field normal chicken sera, and ostrich sera. In addition, 5 actual H9N2-infected chicken sera with AGP values of 2 to 8 showed inhibition rates of 100%, 34%, 100%, 91%, and 80%, respectively. Therefore, it was decided that more than 30% of inhibition was positive based on the average value and standard deviation of the inhibition rate of negative specimens and the inhibition rate of experimentally infected chicken serum.

 [0026] Furthermore, in the AIV immunized chicken serum against HI to H12, when comparing the positive limit of competitive ELISA and AGP test, the competitive ELISA positive limit / AGP test positive limit is about 1 to 10 times, compared to AGP test A competitive ELISA was found to be more sensitive.

 [0027] Specimens with different judgment results were detected by AGP test and competitive ELISA. The competitive ELISA results for 75 chicken sera that had already been diagnosed as negative by the AGP test in Yamaguchi Prefecture showed that one sample showed suppression of 30% or more, and the inhibition rate Was 4 1%. In addition, in specimens that showed a suppression of 30% or more by competitive ELISA, serum dilution could be detected up to 20-fold in the HI test using A / Duck / Hokkaido / 84/02 (H5N3). None of the samples reacted non-specifically, confirming that this reaction was H5 subtype specific. This result indicates that competitive ELISA can be used as an alternative to AGP testing as a serological diagnostic method for AIV infection. In addition, ELISA can be objectively determined as a numerical value, whereas in the AGP test, the determination becomes subjective, and there is a possibility that a positive result may be missed if a clear sediment line is not formed.

[0028] A highly sensitive and highly specific avian influenza virus (AIV) diagnostic system has been developed that can handle multiple samples in place of the gel precipitation (AGP) test. AI is highly conserved among AIV strains It expresses nucleoprotein (NP), which is considered to be useful for the diagnosis of cancer, enables the supply of antigens for screening, and further reveals the production of monoclonal antibody (MAb) against NP and the usefulness of competitive ELISA using it I made it. His-tag sequence was added to the NP gene of AIV A / Duck / Aomori / 478/02 (HI Nl) and expressed as NP-His using baculovirus and insect cells. Anti-NP MAb (IgG1, κ) was prepared by immunizing AIV A / budgeriger / Aichi / 1Z77 (H3N8). As mentioned above, the NP of NP_His combined with MAb is arbitrary.

 [0029] Expression using AIV immune serum and normal serum Indirect ELISA using NP as an antigen, AIV immune serum and expression using normal and ostrich serum AGP test using NP as an antigen, both of which significantly increased AIV immune serum No normal reaction was observed in the normal sera detected and used, and the ostrich sera were negative. Competitive ELISA with AIV immune sera, experimentally infected sera, normal and ostrich sera Significant competition was observed between AIV immune sera and experimentally infected sera that were positive in ELISA, and no competition was observed between normal and ostrich sera.

 [0030] Further, when comparing the positive limits of competitive ELISA and AGP test using AIV immune serum, it was shown that competitive ELISA was 1 to 10 times more sensitive. Therefore, competitive ELISA was performed on 75 sera subject to AI killing, and one positive sample was detected. In the hemagglutination inhibition test using this specimen, H5 subtype was specifically suppressed. From these results, the expressed NP can be widely used as an AIV antibody detection antigen for various detection methods, and competitive ELISA can detect anti-AIV antibodies in a short time and with high sensitivity, which is useful for screening. It was shown to be a high screening method.

 Example

 [0031] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited by these examples.

 [Materials and methods]

 1. Test serum

AIV (H9N2 subtype) experimentally infected chicken serum was distributed by the Institute for Animal Health (Tsukuba, Ibaraki). Eighty chicken sera subject to AI killing were distributed from the Central Animal Health Center in Yamaguchi Prefecture. Ostrich serum is from Yamagata Asahi Ostrich Industrial Center (Yamagata Pref. From Himachi). Field normal chicken serum was distributed from Aomori Portley (Hachinohe City, Aomori Prefecture). SPF chicken serum was distributed from the Institute for Chemotherapy and Serum Therapy (Akira Kumamoto, Kumamoto Prefecture). The prepared anti-AIV immunized chicken serum was used.

 [0032] 2. Avian influenza virus

 A virus containing 1 to 12 subtypes of HA and 1 to 9 subtypes of NA was used as a reference virus strain. These strains were provided by Professor Hiroshi Kita, Department of Microbiology, Graduate School of Veterinary Medicine, Hokkaido University. For the preparation of NP antigen, AZbudgerigar / Aichi / l / 77 (hereinafter abbreviated as A strain) was purchased from the Association for Animal Biological Products (Tokyo). For the NP gene clone, we used the strain A / Duck / Aomori / 478/02 isolated from soil in Aomori Prefecture.

 [0033] 3. Purification of AIV

 Strain A was phosphate buffered saline (0.14M NaCl, 2mM KC1, 3mM Na HPO, 1.5mM KH

PO, pH 7.2: hereinafter abbreviated as PBS. ) In 10- 10- ^2, 10- ^3, was inoculated in diluted to 10 ⁴ 10-day-old embryonated chicken eggs (Koiwai farm, Iwate Prefecture窄石Town). Three days later, the A strain-infected chorioallantoic fluid was collected and centrifuged at 1,100 × g for 10 minutes at 4 ° C. to recover the supernatant. The collected supernatant was centrifuged with an ultracentrifuge LE-80K (Beckman, Tokyo) and SW28Roter (Beckman) at 80,000xg for 90 minutes at 4 ° C. The supernatant was discarded, and the pellet was resuspended in 0.08% sodium azide sodium (hereinafter abbreviated as NaN) in PBS and stored at 4 ° C for 3 days. The pellet was agitated well and centrifuged at 30,000xg for 5 minutes. The supernatant was collected, sucrose was added to 10%, and 20 to 80% sucrose solution was layered on the density gradient, and centrifuged at 80,000 × g for 90 minutes at 4 ° C. Collect each fraction and save 30ml of PBS, 80,000xg, 90 minutes, 4. Centrifuged at C and discarded the supernatant. The pellet was resuspended in lml of 0.08% NaN-added PBS to obtain purified AZbudgerigar / AichiZlZ77.

 [0034] 4. AGP test method

 1) Preparation of agar gel

 The agar gel was prepared according to “Diagnosis method of chicken virus disease” (Sadaharu Horiuchi. Diagnosis method of chicken virus disease. 卯 • 595-606.1982.). Phosphate buffer (0.02M Na HPO, ImM KH PO, p

H7.4) was mixed with 8.0% NaCl and 1.0% Bacto-Agar. After that, 10% NaN was burned 1.0%. Put 25ml of agar in the petri dish, and the agar will harden and force gel puncher (hole 5m m x 7 well), agar was cut out and aspirated to make a reaction agar plate.

 2) AGP antigen

 A cultured egg culture purified strain A (hereinafter referred to as RNP antigen) was used.

 3) AGP test

 AGP antigen was placed in the center well of the agar gel, anti-AIV chicken serum was added every other well as positive sera, and test sera were placed at 30 × l / well, respectively, and observed at room temperature for 2 days. The reciprocal of the maximum serum dilution at which sedimentation lines were confirmed was taken as the AGP value.

[0035] 5 · HI test (method)

 The HI test was performed according to the “OIE MANUAL OF DIAGNOSTIC TESTS AND VA CCINES FOR TERRESTRIAL ANIMALS. HIGHLY PATHOGENIC AVIAN INFLUE NZA. OIE Terrestrial Manual. CHAPTER 2.1.14.2004”.

 1) HA test

 Using a 96-well U-type microplate (96U W / OUT LID SH MICROWELL PLATE: Nalge Nunc International, NY, USA), dilute the virus solution 25 μ1 with 0.15M physiological saline two-fold, and then add physiological saline 25 After mixing the same amount with μ1, the same amount was mixed with 50 μ1 of 0.5% chicken erythrocyte solution. After standing at room temperature for 1 hour, the reciprocal of the highest dilution factor in which phlegm was observed was determined as the evaluation. In the HI test (β method), since the aggregation inhibition value for 4 units of 単 位 antigen is standard, the 4 標準 unit antigen was determined using 1HA unit as the highest dilution virus solution at the time of positive ΗΑ determination (National Institute of Preventive Health) Society. Serum reaction. Pp. 192-201.1982. Revised 2nd edition, Virus Experimental Studies. Tokyo, Maruzen Co., Ltd., “OIE Manual”).

 2) HI test

 The test serum (25 μ1) was diluted 2-fold with physiological saline, and then mixed with an equal volume of 25 μ1 of 4ΗΑ unit antigen. After sensitizing for 1 hour at room temperature, the mixture was mixed with 50 μ 鶏 of 0.5% chicken erythrocyte solution and sensitized for 1 hour at room temperature. The reciprocal of the highest dilution factor for inhibiting aggregation was taken as the HI value.

[0036] 6. Genetic manipulation

Retired IS Ten masterpieces are TT according to the king and niece "Molecular clonmgj (SambrooK et al., Olecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harber Laboratory, NY1989.)" [0037] 7. RT-PCR

 1) RNA extraction

 Isolation A / Duck / Aomori Z478 / 02 (HlNl) strain of chorioallantoic fluid 0.25 ml was added 0.75 ml of ISOGE N-LS (Nitsubon Gene, Tokyo). Next, 200 μ 1 chlorohonome (Kanto Chemical Co., Inc., Tokyo) was stirred and centrifuged at 17,500 xg for 15 minutes at 4 ° C to recover the aqueous layer. Add 3 μ 1 Etachinmate (Nitsubon Gene Co., Ltd.), add 500 μ μ isopropanol (Kanto Chemical Co., Inc.), mix by inversion, centrifuge at 17,500 xg for 10 minutes at 4 ° C, and remove the supernatant. Abandoned. 1,000 μΐ 70% ethanol (Kanto Chemical Co., Inc.) was added to the pellet and centrifuged at 7,700 xg for 2 minutes at 4 ° C. Pellet 10 μ 1 ddH 2 O (DEPC (

 2

 Kanto Chemical Co., Ltd.) Re-suspended by treatment H 2 O) and used as an RNA extract.

 2

 [0038] 2) Primer design

 In consideration of the subsequent subcloning, the restriction enzyme BamHI site that does not exist in the original gene at both ends of the AIV NP open reading frame (hereinafter abbreviated as ORF), and the purification of the resulting protein 3 ' It was designed to add a histidine tag (hereinafter referred to as “His-tag”) sequence to the terminal side. With reference to the AIV NP gene sequence, AIV NPF: 5, -gggatccacat cATGgcgtctcaag-3 '(SEQ ID NO: 2) as a forward primer and AIV NPR His: 5, as a reverse primer-cggatcL. ό '(目 ΰ 歹') ¾ · 亏 was designed, where the start codon and stop codon are capitalized, and the BamHI site (G: GATC C) is underlined. "& 8 & § & 8 & § & 8 & § "is ^ ¾3 _ & § array part.

 [0039] 3) RT-PCR

The extracted RNA is abbreviated and RT-PCR using AI-specific primer Unil 2 (Hoffmann et al., Arch Virol. 146: 2275- 2289.2001.) Is abbreviated as complementary DNA (hereinafter referred to as cDNA). ) Was amplified. Using the GeneAmp RCA PCR core Kit (Perkin Elmer, Urayasu, Chiba), reverse transcriptase reaction was performed at 42 ° CX for 60 minutes, 99 ° CX for 5 minutes, and 30 ° CX for 1 minute. Subsequently, PCR Core Kit (Perkin-Elmer, MA, USA) was used for gene amplification. Use NP-specific primers, set denaturation at 94 ° C, primer annealing temperature at 58 ° C, cDNA synthesis at 72 ° C, and stepl (94 ° C x 4 min) for 1 cycle. , St cycle 2 (94 ° CX 20 seconds, 58 ° CX 30 seconds, 72 ° CX 7 minutes), 30 cycles, St cycle 3 (72 ° CX 7 minutes) (Journal of Virological Methods. 97: 13-22.2001).

 [0040] 8. Agarose gel electrophoresis

 For agarose gel electrophoresis, prepare SeaKem GTG Agarose (Takara Shuzo Co., Kyoto) in TBE buffer (0.1M Tris, 2mM EDTA, 85mM Boric a cid), heat-dissolve, and then abbreviate EtBr (hereinafter referred to as EtBr). ) Was added to a final concentration of 1% and lOngZml, respectively, and gelled at room temperature. Samples were run at a constant voltage of 100 V for 30 minutes to 1 hour, and nucleic acids were detected by ultraviolet irradiation. The length of DNA was estimated by comparison with lOObp DNA Ladder (Takara Shuzo Co., Ltd.).

 [0041] 9. Cloning and sequencing of AIV NP His gene

The AIV NP His gene amplified by PCR was ligated to pCR2.1, a plasmid vector, using TA Cloning Kit (Invitrogen, CA, USA). This was transduced into the host E. coli JM109 (Takara Shuzo Co., Ltd., Kyoto) using the Heat Shoe k method, and 50 g / m 1 ampicillin, 0.12% X-gal (5-Bromo-4- Chloro-3-indolyl-β—d — Galactoside (Takara Shuzo Co., Ltd.)-Added TYM agar medium (2% bacto_tryptone, 0.5% yeast ext ract, 0.1% glucose, 10 mM MgSO · 7Η0, 1.5% bacto agar) was cultured at 37 ° C. β-galatatosidase metabolizes X-gal and produces a blue pigment, but when the AIV NP His gene is inserted into the LacZ region of the vector, / 3-galatatosidase is not produced and white colonies are observed . Using this White-Blue Selection, white colonies are selected and cultured in TYM liquid medium (2% bacto-tryptone, 0.5% yeast extract, 0.1% glucose, 10 mM MgSO-7H O). PAIV NP His_pCR2.1 was obtained. The plasmid was extracted from this, digested with BamHI and confirmed by Agarose gel electrophoresis for AIV NP His gene insertion. After extraction, the BamHI digested plasmid gene and primers (AIV NPF and AIV NPR His) were used to perform a sequencing reaction by the Dideoxy Terminator method (Sanger et al., J. Mol. Biol. 94: 41.1975.). For the reaction, Dye Terminator Cycle Sequencing Kit FS (Perkin-Elmer) using labeled fluorescent dideoxynucleotide (hereinafter abbreviated as ddNTP) was used. 96 ° CX 45 seconds, 50. CX 30 seconds, 60. C4 minutes of reaction, 25 cycles, purified by spin column, ABI PRISM310 Genetic Analyzer (Per The base sequence was determined by kin-Elmer).

 [0042] 10. Expression of AIV NP His in baculovirus

 1) Preparation of transfer vector

 The pAIV NP His_pCR2.1 obtained by cloning was digested with BamHI (Takara Shuzo Co., Ltd., Kyoto) and developed by agarose gel electrophoresis, and the AIV NP His gene fragment was excised together with the gel. The AIV NP His gene was purified by ethanol precipitation after phenol treatment. The obtained fragment was ligated to BAcHI digested baculovirus transfer vector pAcYMl (Matsuura et al., J. Gen. Virol. 68: 1233-1250.1987.) Using Takara DNA Ligation Kit (Takara Shuzo Co., Ltd.) Subsequently, the host E. coli JM109 (Takara Shuzo Co., Ltd.) was transduced by the Heat Shock method. This was cultured on a TYM agar medium supplemented with 50 μg / ml ampicillin. Large-scale culture was performed from the appearing colonies in a TYM liquid medium supplemented with 50 μg / ml ampicillin to obtain pAIV NP His—pAcYMl. The plasmid was extracted, and the AIV NP His gene was confirmed by agarose gel electrophoresis after digestion with BamHI. Here, in order to confirm that the AIV NP His gene was inserted in the positive direction with respect to the polyhydrin promoter of pAcYMl, a PCR method using a plasmid containing the AIV NP His gene as a saddle type was performed. As a primer, a BaclN primer (5'-tgataacc atctcgcaaa-3 '(SEQ ID NO: 4)) with a recognition site between the polyhydrin promoter of pAcYMl and the cloning site and an AIV complementary to the 3' end of the AIV NP His gene NPR His primer was used. In this method, amplification is observed at a band of about 1,500 bp only when inserted in the forward direction with respect to the polyhydrin promoter.

 [0043] 2) Baculovirus and host cell

AcNPV was used as a baculovirus field strain, and AcRP 23_LacZ (Kitts et al., Nucleic Acid Res. 18: 5667-5672.1990.) Was used as a recombinant virus-producing parent strain. As host cells for baculovirus, we used IPLB_Sf21AE (hereinafter abbreviated as Sf21 cells) established with ovarian force of Spodoptera frugiperda, or Sf9 cells with cloned Sf21 cell force. Sf21 cells consist of TC-100 insect medium (Gibco BRL, Gaithersburg, MD, USA), hereinafter abbreviated as TC100 (0%), and fetal calf serum (hereinafter abbreviated as FCS). % Maintained at TC100 (10%) and used for cloning. It was. Sf9 cells are cultured using ESF921 medium (Expression System LLC, Woodland, CA, USA: hereinafter abbreviated as ESF921) at 28 ° C by spin culture method to produce high-titer virus. Used for expression. Baculovirus and Sf21 cells were dispensed from the NER C Institute of Virology (Oxford, UK) and Sf9 cells were purchased from Expression Systems L LC (Woodland, CA, USA).

 [0044] 3) Production of recombinant baculovirus

 pAIV NP His-pAcYMl (10 μg / ml) 1 μ1, Ac8RP23-LacZ DNA digested with Eco8 II and linearized (1 μg / ml) 1 μ1, TC I 00 (0%) Add 6 μl of the mixed solution (DNA mixed solution), TC 100 (0% FCS) medium 8 μ1, and ribofuctin (GIBCO BRL) 8 / il mixed solution (lipofectin mixed solution) for 30 minutes at room temperature. Reacted. Next, the DNA mixture and the lipofectin mixture were mixed and allowed to react at room temperature for 15 minutes, and then transferred to Sf21 cells to produce recombinant viruses by homologous recombination. After culturing at 28 ° C for 3 days, the culture broth was collected and plaque-cloned with Sf21 cells. Three days after the inoculation, plaques were stained with neutral red 300 / ig / ml and X-gal (Takara Shuzo) 75 μg / ml. Four days after the inoculation, black with no blue pigment observed under a light microscope is regarded as a plaque formed with a recombinant virus, and the plaque is collected in TC 100 (10%) medium using a Pasteur pipette and plaque cloning is performed. did. This procedure was repeated three times to obtain a recombinant baculovirus that formed only white black, and this was named AcAI NP His. This AcAI NP His was passaged several times with Sf 21 cells and used as a seed virus.

 [0045] 4) Confirmation of protein expression

Multiplicity of infection (hereinafter abbreviated as M0I) is about 5 Plaque Forming Unites (hereinafter abbreviated as PFU) in Sf9 cells (1.5 X 10 ⁶ cells) cultured in a single layer with AcAI NP His. ) Inoculated with / cell, cultured at 28 ° C using ESF921 medium, and the cells and supernatant were collected 1, 2, 3, 4 and 5 days after inoculation. In addition, a culture solution (hereinafter abbreviated as Mock) was similarly inoculated as AcNPV or negative control, and the cells and supernatant were collected after 5 days. The cells were washed with phosphate buffered saline for insect cells (0.14 M NaCl, 26.82 mM KCl, 8 mM Na HPO, 1.47 mM KH PO, pH 7.4: hereinafter abbreviated as PBS-Ac), then L

 2 4 2 4

aemmli (Laemmli, UK Nature. 277: 680-685 · 1970 ·) 4 X sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 0.02% Bromophenol blue, 5% 2_mercapto ethanol: Hereinafter abbreviated as 2Me + Buffer. ). The culture supernatant was mixed with 2Me + Buffer at a ratio of 3: 1. Each sample was boiled at 100 ° C for 5 minutes and developed by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) containing SDS. After electrophoresis, the samples were analyzed by staining with Kumashi brilliant blue staining solution (45% methanol, 10% acetic acid, 0.025% Coomassie brilliant blue) (hereinafter abbreviated as CBB staining) or Western blotting.

 [0046] 5) Western blotting analysis

 After completion of SDS-PAGE, the gel was washed with a transcription buffer (0.02M Tris, 0.15M Glycine, 0.01% SDS, 5% Methanol), and then Western blotting apparatus Holis blot (Atoichi Co., Ltd., Tokyo) was used. Nitrosenololose membranes were transferred to Hybond C Extra (Amersham, Amersham, UK) at 100 mA for 1.5 hours. After the transfer, the unbound portion of the nitrocellulose membrane was blocked with a blocking solution (1% skim milk powder, 0.1% Tween-20 dissolved in PBS). Subsequently, anti-polyhistidine mouse serum (SIGMA, Stuois, MO, USA: hereinafter abbreviated as a poly His) was used as a primary antibody and allowed to react at room temperature for 3 hours. After the reaction, it was washed 3 times with a blocking solution, and the secondary antibody was reacted at 37 ° C for 2 hours. The secondary antibody was prepared by diluting peroxidase-labeled anti-mouse IgG rabbit serum (Transduction Laboratories, Lexington, KY, USA). After the reaction, the plate was washed 3 times with PBS and developed with a BM blue POD substrate (Boehringer Mannheim, Mannheim, Germany).

 [0047] 11. Mass expression and purification of AIV NP His

AcAI NP His was inoculated into MfI 5 PFU / cell to Sf9 cells adjusted to 1.5 X 10 ⁶ cells / ml. Every 24 hours after inoculation, a part of the culture solution was collected and mixed with an equal amount of trypan blue solution, and the number of live and dead cells was counted. When the number of viable cells fell below OX 10 ⁵ cellsZml, the cells were precipitated by centrifugation at 700 × g for 15 minutes, and the pellet was collected. The pellet was purified from the Xpress System Protein Purification Kit (Invitrogen) kit, kit manual, and AIV NP His. That is, 50 ml of the cultured cell pellet was resuspended with 10 ml of Native Binding Buffer, and cells were disrupted by ultrasound using Handy Sonic model UR-20P (TOMY, Tokyo) on ice. Then centrifuge at 15,000 xg for 20 minutes at 4 ° C and The supernatant was collected and the pH was adjusted to 7.6 to 7.8 with 2M NaOH. The nickel chelate column is washed with DW, equilibrated with Native Binding Buffer, and the supernatant containing the recovered AIV NP His is placed in the nickel chelate column and shaken for 1 hour to adsorb the expression protein to which the His-tag is added. I let you. After adsorption, the column was washed with Wash Buffer, fractionated into 1 ml fractions using Imidazole adjusted to 50, 500, and 1,000 mM concentrations, and the His-tag added protein was eluted. This was developed with 12.5% SDS_PAGE, and after confirmation by CBB staining, the fraction in which the protein was confirmed was desalted through a PD10 column (Amersham Pharmacia Biotech, Tokyo) equilibrated with PBS. This was similarly developed on 12.5% SDS-PAGE, confirmed by CBB staining and Western blotting, and then sterile filtered to obtain purified AIV NP His. The protein content of purified AIV NP His was measured using the Micro BCA Protein Assay Reagent Kit (PIERCE, Rockford, IN, USA).

 [0048] 12. Indirect ELISA using NP antigen

 ELISA was performed according to the method of Kida et al. (Kida et al., Virology. 122: 38-47.1982 ·). Seed purified AIV NP His (90 μg / ml) as an antigen on an ELISA 96-well plate (SUMILON, Sumitomo Bakelite Co., Ltd., Tokyo) at 50 / l / well, 2 hours at room temperature or 24 hours at 4 ° C Left to stand. BSA10 (Bovine serum albmin fraction V (Nacalai Testa Co., Ltd., Kyoto Prefecture)) lOmgZml is added to PBS in order to recover the antigen and prevent non-specific reaction to the well portion where the antigen is not coated. .2) was added in 100 μl aliquots, allowed to stand at room temperature for 1 hour or 4 ° C for 24 hours, and washed 3 times with PBST (PBS with 0.05% Tween20, pH 7.4). Add test serum diluted 40, 160, 640, or 2,560 times with BSA5T (caroten to PBST so that BSA fraction V is 5 mg / ml) at a time, and let stand at room temperature for 1 hour. Placed. After washing 4 times with PBST, add 50 μl of peroxidase-labeled anti-chicken Ig G Usagi serum (CHEMICON International, Inc. CA, USA) diluted 1,000-fold with BSA5T, react at room temperature for 1 hour, and then color developer (PH4.0, 0.05M Citric buffer, 0.013% hydrogen peroxide, 2% 40 mM ABTS) 100 μl each, ELISA plate reader (Sanko Junyaku Co., Ltd.) after 15 and 30 minutes at room temperature Absorbance at 405 nm was measured by the company, Tokyo).

 [0049] 13. AGP test using NP antigen

AcAI NP His infected insect cells were treated with Disruption buffer (0.05M Tris-HCl, 0.5% TritonXlO 0,0.6M KC1) was treated to obtain an antigen, and the test was performed according to the AGP test.

[0050] 14. Production of monoclonal antibodies

 1) Test mouse

 One 6-week-old BALB / c female mouse (CLEA Japan, Tokyo) was used.

 2) Immune antigen

 Purified AIV A / budgerigar / Aichi ZlZ77 (H3N8) was used as an immunogen for the production of monoclonal antibodies. Purified virus at a final concentration of 0.05. /. It was inactivated with formalin and stored at 4 ° C. Freund's complete adjuvant (SIGMA) was used as an adjuvant, and an equal volume of purified virus diluted 4 times with PBS was mixed to prepare an emulsion. The prepared immunogen was inoculated into the abdominal cavity of the mouse at 200 μ1 / head. Emulsions treated in the same way were boosted several times every 2 weeks from the initial immunization in the same manner, and the mice were dissected 3 days after the final boost.

 [0051] 3) Preparation of High Pridoma

Mouse myeloma cells (J Immunol Methods. 35 (1_2): 1_21.1980: hereinafter abbreviated as FO cells) in RPMI 1640 medium (Nissi, Tokyo) plus 10% FCS (hereinafter 10% And abbreviated as RPMI 1640) at 37 ° C. and 5% CO. FO cells were also removed from the cell culture routine, and the cells were collected by centrifugation at lOOxg for 8 minutes, resuspended in 10% RPMI 1640, adjusted to 2.0 × 10 ⁷ cells / ml, and subjected to fusion. Three days after the final immunization, the mice were euthanized by blood sampling under ether anesthesia, and the blood was allowed to stand at 37 ° C, 5% C 1 hour, and further at 4 ° C for 24 hours. Serum was separated after 24 hours and stored at -20 ° C. The mouse spleen after the euthanasia treatment was removed, and the spleen cells were separated by pressing the inner cylinder of a disposable syringe and pressing the spleen on a metal mesh. Spleen cells were supplemented with 10% RPMI 1640, and spleen cells were washed twice at 100 × g for 8 minutes. Washed spleen cells were adjusted to 1.0 × 10 ⁸ cells / ml with 10% RPMI 1640.

[0052] 4) Preparation of mouse lymphokine

In order to proliferate and activate hyperidoma, mouse lymphokine was prepared (KANE, M. M. and BANKb, JN Immunoassay Making and manipulating hybndoma cells. 5.1 Facilities and media Protocol. 11 37-40.). Euthanize uninoculated BALB / c mice Thereafter, the spleen was separated by the same method as described above, and the separated spleen cells were cultured in 10% RPMI 1640 medium containing 2.5 x lZml lipopolysac charide endotoxin (SIGMA: LPS) 3 7 ° C 5% CO 4 days Cultured. After centrifuging at 390xg for 5 minutes, the supernatant is recovered and 26mm Sy

 2

rmge Filter 0.22 μ 1 (Corning Incorporated Corning, NY 4831 ermany for ermany) After thoughtful extinction, Cehumchu ¹ ~ bu (Greiner bio—one Frickenhausen 1, Germany) ί こ; ¾T / mainly at 20 ° C When used, it was diluted 20-fold with HT medium (SIGMA) and used at 100 μl / well.

 [0053] 5) Cell fusion and selection

The spleen cells of the immunized mice were adjusted to 1.0 × 10 ⁸ cell FO cells to 2 × 10 ⁷ cells and mixed with spleen cells FO cells = 5: 1. After centrifugation at 100xg for 8 minutes, the culture solution is removed, and 0.2 ml of polyethylene glycol 4000 (Kanto Chemical Co., Ltd., Saitama: hereinafter abbreviated as PEG) solution (8.75 μΜ PEG, RPMI 1640 with 15% dimethyl sulfoxide) is added. The hybridoma was made by gradually dripping to create an hybridoma.

For fusion, the cell concentration was adjusted to 10 ⁶ cells / ml with HAT medium (SIGMA), seeded in a 96-well plate (96 Wells w / Lid Flat Bottom: Greiner) at 100 μl / well, and a high-pridoma was selected. The obtained hyperidoma was treated with AIV antigen (A / Aichi / 2/68) and crude purified AIV NP provided by Professor Hiroshi Kida, Graduate School of Veterinary Medicine, Hokkaido University. Screening by ELISA, NP antibody-producing hybridoma was cloned twice by limiting dilution using HT medium supplemented with 5% lymphokine. After cloning, the isotype was examined and cultured in large quantities.

 [0054] 15. Confirmation of monoclonal antibody

 1) ELISA for detection of anti-AIV NP antibody

 ELISA was performed using crudely purified AIV NP as an antigen, hyperpridoma culture supernatant as a primary antibody, and peroxidase-labeled anti-mouse IgG rabbit serum (CHEMICON International Inc. CA USA) as a secondary antibody.

 2) Western blotting analysis

Using purified AIV A / budgerigar / Aichi / 1/77 (H3N8) diluted 100-fold with PBS (pH 7.2) as an antigen and purified AIV NP His, 12.5% SDS_PAGE electrophoresis and Western blotting were performed. As the primary antibody, MAb-producing hyperpridoma culture supernatant was used, Peroxidase-labeled anti-mouse IgG rabbit serum (Transduction Laooratones, Lexington, KY, USA) diluted 1000-fold with block solution was used as the body.

 [0055] 16. Determination of isotype

 Isotypes were determined using Immuno pure Monoclonal Antibody Isotype kit I (PIERCE) based on the product manual. Apply 50 coating antibody solutions supplied with the kit to a 96-well plate (SUMILON) for ELISA for 2 hours or 4 at room temperature. Allowed to stand at C for 24 hours. Remove the solution and add 125 μ 1 blocking solution37. C. left for 1 hour. After washing 4 times with a 125 / i 1 wash buffer, 50 μl each of the high-pridoma culture supernatant containing MAb and the positive control were added, and reacted at 37 ° C. for 1 hour. After washing 4 times with a 125 μl wash buffer, anti-mouse specific isotypes (IgG1, IgG2a, IgG3, IgM, IgA, K, E) Usagi serum and normal Usagi serum as a negative control were added dropwise. As a positive control, anti-mouse IgG was dropped and reacted at 37 ° C for 1 hour. After washing 4 times with 125 μΐ wash buffer, 50 μl of peroxidase-labeled anti-rabbit IgG goat serum was added and reacted at 37 ° C for 1 hour. After washing 4 times with 125 μ 1 wash buffer, add 100 μ 1 ABTS substrate solution, react for 30 minutes at room temperature, and measure absorbance at 405 nm using ELISA plate reader (Sanko Junyaku Co., Ltd., Tokyo) did.

 [0056] 17. Purification and concentration of MAb

The cloned MAb-producing hybridoma was cultured using a large culture flat (INTEGRA Cel ILineCL 1000) (IBS INTEGRA BIOSCIENCES, INTEGRA Biosciences AG'Switzerland). The cells were subcultured at a time when the number of cells was about 2 × 10 ⁷ to 4 × 10 ⁷ cells / ml, and the culture supernatant was collected. Mass culture flat (INTEGRA CellLineCLlOO) force MAb was purified from the collected MAb-producing hybridoma culture supernatant using Montage ™ life science kits (MILLIPORE.com, MA, USA). As a pretreatment, dust in the culture supernatant of the hybridoma produced with 0.22 mm Microless GP attached to the kit was removed. Using a binding buffer, the PROSEP-A media plug was equilibrated, and the supernatant was centrifuged at 150 xg for 20 minutes to adsorb the M Ab to the PROSEP-A media plug. Subsequently, centrifugation at 500 × g for 2 minutes was performed twice with Binding buffer to wash and remove unbound protein to the PROSEP-A media plug. Centrifuge at 500xg for 5 minutes in Elution buffer, and use PROSEP-A media plug from MA b was eluted. The obtained MAb was desalted and concentrated with Amicon Ultra 15 centrifugal Filter 30K NMWL. Then, after adding an equal amount of glycerol, it was stored at -20 ° C.

[0057] 18.Competitive ELISA

 Competitive ELISAs include Zhou et al. (Zhou et al., Avian Dis. 42: 757-761.1998.; Zhou et al., Avian Dis. 4 2: 517-22. 1998) and Shafer et al. (Shafer et al., Avian Dis. 42: 28-34.1998) The procedure was followed. AIV NP His (50 μl / well) as an antigen was placed on a 96-well plate (SUMILO N) for ELISA for 2 hours at room temperature or 4 ° C. for 24 hours. After recovering the antigen, the well was blocked with BSA 10 100 μl / well for 2 hours at room temperature or 4 ° C. for 24 hours. Wells were washed 3 times with P BST. As diluted test serum, blank, and control, BSA5T was added at 50 μl / well, respectively, and reacted at 37 ° C for 20 minutes. Without removing serum, add MAb diluted 20,000 times with PBS to the well except 50 μl / well for blank well and add 50 μl / well for BSA5T to blank we11 and react at 37 ° C for 1 hour. I let you. Wells were washed 4 times with PB ST. Peroxidase-labeled anti-mouse Ig G Usagi serum (CHEMICON International) diluted 1,000-fold with BSA5T was added at 50 μl / well and reacted at 37 ° C for 1 hour. Wells were washed 4 times with PBST. 100 μl / well of color former (pH 4.0, 0.05 M citric buffer, 0.013% hydrogen peroxide, 2% 40 mM ABTS) was added and reacted at 37 ° C. for 30 minutes. After 30 minutes, measurement was performed at 405 nm (OD) using an ELISA plate reader (Sanko Junyaku Co., Ltd.). The percent inhibition of sample sera compared to controls (MAb only) was calculated after subtracting the average of the blank values to obtain the correct value, which was subtracted from all test sera values and control values. The formula is 100- (100 X [test serum value—blank value (BSA5T) Z control value (MAb only) —blank value (BSA5T)])). A suppression rate of 30% or more was considered positive. In addition, the reciprocal of the maximum serum dilution that showed more than 30% inhibition was taken as the competitive ELISA value.

[0058] [Result]

 1. Production of AIV NPHis gene

Isolation RNA extracted from infected chorioallantoic fluid of A / Duck / Aomori / 478/02 (H1N1) strain, and using the extracted RNA as a saddle, amplify cDNA by RT-PCR using AI-specific primer Unil2 did. NP-specific primers AIV NPF and AI When V NPR His was designed and PCR was performed using these, we confirmed amplification of about 1,500 base pairs, which is considered to be the full length of NP.

 The obtained PCR product was ligated to pCR2.1 using a TA Cloning Kit and transformed into E. coli JM109, which is a recombinant cell. White colonies containing the target gene were selected using White-Blue Selection, and enrichment culture was performed. Plasmids were extracted from the resulting Escherichia coli, digested with BamHI, and developed by 1% agarose gel electrophoresis. AI compared to lOObp DNA Ladder (Takara Shuzo Co., Ltd.)

Two bands of approximately l, 500 bp presumed to be the V NP His gene and approximately 3,900 bp presumed to be the vector were observed. Furthermore, by PCR using primers (AIV NPF and AIV NPR His), the base sequence of about 500 residues from the 5 ′ end and 3 ′ end was confirmed. Sequencing of the full length NP is done.

 [0059] 2. Construction of transfer vector

 AIVNP His—pCR2.1 was digested with BamHI, and the fragment containing AIV NP His was ligated to the baculovirus transfer vector pAcYMl using the Takara DNA Ligation Kit. Furthermore, the cells were transformed into a competent cell and the plasmid was extracted from E. coli. The extracted plasmid was digested with BamHI and electrophoresed with 1% agarose gel. As a result, about 1,500 bp of inserted gene and about 10,000 bp of pAcYMl vector DNA were confirmed. In order to confirm that the AIV NP His gene was inserted in the positive direction with respect to the polyhydrin promoter, using the BaclN primer and the AIV NPR His, PCR was performed and developed by 1% agarose gel electrophoresis. The amplification of a band of about 1,500 bp was shown to be inserted in the forward direction, and one plasmid in which amplification of 1,500 bp was observed was designated as pAIV NP His-p AcYMl and used in the following expression experiments.

 [0060] 3. Production of recombinant baculovirus expressing AIV NP His

The plasmid pAIV NP His_pAcYMl and AcRP23-LacZ DNA were cotransfected, and the culture supernatant was subjected to black cloning in the presence of X-gal. Plaques that showed white color by white-blue selection were plaque-cloned three times. After several passages in Sf21 cells, high-titer virus was obtained and used as seed virus. As a result of Black Atsey, The titer of virus was 2.0 × 10 ⁸ PFU / ml. This was designated as recombinant baculovirus AcAI NP His.

 [0061] 4. Confirmation of recombinant AIV NP His expression

The obtained AcAI NP His was inoculated to 1.5 × 10 ⁶ cell / ml 300 ml of Sf 9 cells at 5 PFU / cell and collected at a time when it fell below 5.0 × 10 ⁵ cell / ml. In Western blotting using polyHis, a band of approximately 56 kDa was observed in AcAI NP His-infected cells, but no specific band was observed in AcNPV-infected cells and Mock.

 [0062] 5. Mass culture and purification of AIV NP His

 Mass culture was performed and purification was performed using a Ni column. AIV NP His adsorbed on the Ni column was eluted with Imidazole, developed by SDS-PAGE, and confirmed by CBB staining. Fractions in which a band of about 56 kDa was observed when dissolved at 500 mM were pooled, desalted through a PD10 column, and similarly subjected to CBB staining and Western blotting. Fractions in which a band of about 56 kDa was observed were pooled again and filtered through a 0.45 / im filter to obtain purified AIV NP His. As a result of protein quantification, purified AIV NP His with 90 μg / ml and total protein amount of 270 μg was obtained from 100 ml of Sf9 cell culture pellet.

 [0063] 6. ELISA using AIV NP His

 The 12 AIV immunized chicken sera showed significantly higher values than the 10 normal normal chicken sera used at the serum dilutions of 40, 160, 640, and 2560. In addition, all the normal normal chicken sera used did not show any nonspecific positive reaction in the serum dilution performed. The positive range was calculated from the mean value and standard deviation of field normal chicken sera, and a positive reaction was defined as a value equal to or greater than three times the standard deviation of the value in field normal chicken serum.

 [0064] 7. AGP test using AIV NP His

 Sedimentation lines were confirmed only in 12 samples of AIV immunized chicken sera, and no nonspecific positive reaction was observed in 164 samples of field chicken sera. All 448 field ostrich sera were negative. These results were the same as the test results using RNP antigen.

 [0065] 8. Preparation and identification of anti-AIV NP MAbs

Of mice immunized with purified AIV A / budgerigar / Aichi / 1/77 (H3N8) Ten MAb-producing hybridomas were obtained from spleen cells and myeloma cells. Of these, 8 types of culture supernatant that react with AIV NP antigen are considered as hybridomas that produce MAbs that react with NP, and 2 types that react with AIV antigen (A / AichiZ2Z68) but do not react with AIV NP antigen. Were considered to be hyperidomas that produce MAbs that respond to other proteins. These MAb isotypes were IgGl, IgG2, IgA, and IgM. IgG that reacts with the target NP was confirmed in 1E5 and 6E10. For 6E10, two types of MAb-producing hybridomas, light chain κ, heavy chain IgM and IgG2a, were cloned five times using the limiting dilution method. It was mixed. Since further separation was considered difficult in terms of time, the cells were stored frozen in liquid nitrogen. 1 1E5 was light chain κ and heavy chain IgGl.

 [0066] 9. Mass culture, purification and concentration of MAb 1 1E5

 The cloned MAb-producing hybridoma 11E5 was cultured in large quantities with INTEGRA CellLineCL 1000 to obtain 30 ml of culture supernatant. Furthermore, as a result of purifying the culture supernatant using Montage (trademark) life science kits (MILLIPORE.com, MA, USA), 400 μl of concentrated MAb 11E5 was obtained.

 [0067] 10. MAb 1 1E5 reactivity

In ELISA using purified AIV NP His as an antigen, the reaction was performed by dilution of 1.0 × 10 ³ times with MAb 1 1E5 before concentration, and dilution of 1.0 × 10 ⁵ times with MAb 1 1E5 after concentration. In Western blotting analysis using AI VA / budgerigarZAichiZlZ77 (H3N8) or purified AIV NP His as an antigen as an antigen, no band was confirmed at any position. That is, it did not react to Western blotting analysis.

 [0068] 1 1. Competitive ELISA using MAb 1 1E5

Regarding test sera, AIV immunized chicken sera were diluted 100-fold, and other sera were diluted 5-fold and used for ELISA. In the test using AIV immunized chicken serum, the blank mean value was 0.218, the control mean value was 1.311, H4N5 immune serum value was 0.201, H5N3 immune serum value was 0.23, H9N 2 immune serum value was 0.194, H1 1N6 immune serum The value was .204. The SPF chicken serum had a blank average value of 0.137, a control average value of 1.046, sample 1 of 1.035, sample 2 of 1.045, and sample 3 of 0.993. If we calculate the suppression rate using the formulas already shown, H4N5 immune serum suppression rate (%) = 100- (100 X [0.201-0.218Z1.311-0.218]) = 101.5%, H5N3 immune serum suppression rate (%) = 100- (100X [0.23-0.218Z1.311- 0.218]) = 98.857 2%, H9N2 immune serum inhibition rate (%) = 100- (100X [0.194-0.218Z1.311-0.218]) = 10 2.1%, H11N6 immune serum inhibition rate (%) = 100- (100X [0.204-0.218 / 1.311-0.218]) = 101.2%, SPF serum sample 1 inhibition rate (%) = 100- (100X [1.035-0.137 / 1.046-0.137]) = 1.174%, SPF serum sample 2 inhibition rate (% ) = 100- (100X [1.045-0.137 / 1.046-0.137]) = 0.073%, SPF serum sample 3 inhibition rate (%) = 100- (100 X [0.993-0.137 / 1.046-0.13 7]) = 5.798% The same calculation was performed for other specimens. Seven AIV immune chicken sera showed almost 100% inhibition (average inhibition rate 99.8%, standard deviation 0.432), SPF chicken serum 25 samples (average inhibition rate 1.805%, standard deviation 3.655), field normal chicken serum 43 samples In the ostrich serum 311 specimens (average inhibition rate 4.833%, standard deviation 4.683) (inhibition rate 7.462%, standard deviation 3.969), no inhibition reaction was observed. In addition, 5 actual H9N2-infected chicken sera with AGP values of 2 to 8 showed suppression rates of 100%, 34%, 100%, 91%, and 80%, respectively. Therefore, it was decided that suppression of 30% or more was positive based on the average value and standard deviation of the suppression rate of negative specimens, and the suppression rate of experimentally infected chicken blood.

 [0069] 12. Comparing the positive limit of competitive ELISA and AGP test

 In AIV immunized chicken sera against H 1 to H 12, competitive dilutions can detect serum dilutions of approximately 100-fold to 500-fold (competitive ELISA values 100-500), and AGP studies approximately 10-fold to 250-fold (AGP Values of 10 to 250) were detectable (Table 1). In other words, competitive ELIS A positive limit ZAGP test positive limit was about 1 to 10 times.

[0070] [Table 1] table 1

[0071] 13. Detection of infected sera

 In the competitive ELISA of 75 chicken sera subject to AI killing that had already been diagnosed as negative in the AGP test at the Central Animal Health Center in Yamaguchi Prefecture, one sample showed a suppression of 30% or more, and the suppression rate was 41%. One specimen showed 20-30% inhibition, the inhibition rate was 21 Q / o, 3 specimens showed 15-20% inhibition, and 9 specimens showed 10-: 15% inhibition. Specimens, 5 ~: 28 specimens showed 10% inhibition, and 33 specimens showed less than 5% inhibition (Table 2). In addition, when a HI test using A / DuckZ Hokkaido / 84/02 (H5N3) was performed on specimens showing suppression of 30% or more, H5 subtype-specific suppression was confirmed up to 20 times serum dilution.

 [0072] [Table 2]

 Table 2

 Inhibition rate Number of samples

 > 3 0% 1 (4 1%)

 2 0 to 3 0% 1

 1 5 to 2 0% 3

 1 0 to 1 5% 9

 5 to 1 0% 2 8

 <5% 3 3

Total 7 5 Brief Description of Drawings

 FIG. 1 shows setting of Cut Off value of competitive ELISA according to the present invention.

Claims

The scope of the claims

 [1] An influenza infection test drug characterized by using an influenza virus nucleoprotein (NP) as an AIV (avian influenza virus) antibody detection antigen.

 [2] The claim 1, wherein the nucleoprotein (NP) gene of influenza virus isolated from AIV (avian influenza virus) and expressed using the baculovirus expression system is used as an AIV antibody detection antigen. The influenza infection test described.

 [3] AIV A / Duck / Aomori / 478/02 (HlNl) nucleoprotein (NP) gene with a His-tag sequence and expressed as NP-His The influenza infection test agent according to claim 1 or 2, which is used as a detection antigen.

 [4] The influenza infection test agent according to any one of [1] to [3], wherein an influenza virus nucleoprotein (NP) is used as an immobilized antigen in an enzyme-linked immunoreactivity assay (ELIS A).

 [5] (1) An influenza virus nucleoprotein (NP) is used as an immobilized antigen in an enzyme-linked immunosorbent assay (ELIS A), and at least the immobilized antigen of (1) above

 (2) The influenza infection test agent according to any one of claims 1 to 4, which comprises an antinuclear protein monoclonal antibody (NPMAb) that specifically binds to a nucleoprotein (NP) of influenza virus.

 [6] The influenza infection test agent according to claim 4 or 5, wherein the immobilized antigen is immobilized on an ELISA plate.

 [7] The test for influenza infection according to claim 5, wherein the antinuclear protein monoclonal antibody NPMAb (IgGl, κ) prepared by immunization with AIV AZbudgerigerZAichi / lZ77 (H3N8) is used.

 [8] A method for testing influenza infection, comprising using an influenza virus nucleoprotein as an immobilized antigen in an enzyme-linked immunosorbent assay (ELISA).

[9] The nucleoprotein (ΝΡ) gene of influenza virus nucleoprotein (ΝΡ) isolated from AIV and expressed using the baculovirus expression system was used as a solid-phased antigen in enzyme-linked immunosorbent assay (ELISA). The method for inspecting influenza infection according to claim 8 to be used

[10] (1) An influenza virus nucleoprotein (NP) is used as an immobilized antigen in an enzyme-linked immunosorbent assay (ELIS A), and at least the immobilized antigen (1) and

 (2) The influenza infection screening method according to claim 8 or 9, wherein an antinuclear protein monoclonal antibody (NPMAb) that specifically binds to the nucleoprotein (NP) is used.

 [11] (1) Influenza virus nucleoprotein (NP) as an immobilized antigen for enzyme-linked immunoreactivity assay (ELIS A),

 (2) adding test serum, reacting the anti-avian influenza (AIV) antibody with the immobilized antigen of (1),

 (3) An antinuclear protein monoclonal antibody (NP MAb) that specifically binds to the nucleoprotein (NP) of (1) above is added, and the antinuclear protein monoclonal antibody (NP M Ab) is added to the above (2). 11. The method for testing influenza infection according to claim 10, wherein the test is performed by a competitive reaction with the test serum added in step 10.

 [12] (1) Influenza virus nucleoprotein (NP) is used as an immobilized antigen for enzyme-linked immunosorbent assay (ELIS A),

 (2) adding test serum, reacting the anti-avian influenza (AIV) antibody with the immobilized antigen of (1),

 (3) An antinuclear protein monoclonal antibody (NPMAb) that specifically binds to the nucleoprotein (NP) of (1) above is added, and the antinuclear protein monoclonal antibody (NPMAb) is added according to (2) above. Compete with the added test serum, and at the next stage,

 (4) Add an enzyme-labeled antibody that reacts only with the anti-NPMAb of (3) above, and in the next step,

 (5) The method for detecting influenza infection according to claim 11, wherein color development is measured when an enzyme substrate of a labeling enzyme of the enzyme-labeled antibody of (4) is added.

 [13] The influenza infection detection method according to any one of claims 10 to 12, wherein an antinuclear protein monoclonal antibody NPMAb (IgGl, κ) prepared by immunizing AIV AZbudgerigerZAichi / lZ77 (H3N8) is used.

 [14] The influenza infection screening method according to any one of [9] to [12], wherein an ELISA plate on which an antigen is immobilized is used.