KR20120024313A - Diagnostic kit for h9 type avian influenza virus using rapid immunochromatography and the method for diagnosing h9 type avian influenza by using the same - Google Patents

Abstract

PURPOSE: An H9 type avian influenza virus diagnosis kit by quick immunochromatography is provided to ensure quick and simple diagnosis without special equipment. CONSTITUTION: A kit for diagnosing H9 type avian influenza virus contains monoclonal antibodies. The monoclonal antibody is prepared from hybridoma cells of deposit number 11753BP, KCTC 11754BP, and KCTC 11755BP. A H9 type avian influenza diagnosis strip contains the monoclonal antibody. The diagnosis strip comprises a sample pad(1), a gold pad(2), nitrocellulose membrane(3), and moisture absorption pad(4). The gold pad contains a gold conjugate in which gold particles are conjugated with a monoclonal antibody produced from hybridoma cells of deposit number KCTC 11755BP.

Description

Diagnostic kit for H9 type avian influenza virus using rapid immunochromatography and the method for diagnosing H9 type avian influenza by using the H9 type avian influenza virus diagnostic kit by rapid immunochromatography method and method for diagnosing H9 type avian influenza using the same same}

The present invention is a diagnosis capable of quickly and accurately diagnosing H9 type avian influenza infection in chicken total excretory cavity (fecal) swab samples by a rapid immunochromatography method using an antibody specific for hemagglutinin antigen of H9 type avian influenza. Kits and methods for diagnosing H9 type avian influenza using the same.

Avian Influenza is an acute viral epidemic that infects several types of birds, including chickens, turkeys and wild birds, and mainly damages poultry in chickens and turkeys. Avian influenza is a disease spreading rapidly and various pathogenicity, and the World Animal Health Organization (OIE) classifies it as high and low pathogenic avian influenza.

The causative agent of avian influenza is the Influenza virus type A of the Orthomyxoviridae family. The serotypes of influenza A are two proteins, hemagglutinin and neuraminidase. There are two H serotypes and N serotypes. The serotypes of avian influenza virus are expressed as H serotypes and N serotypes (e.g., H9N2), and there are 16 H serotypes and 9 N serotypes. There are 144 serotypes. The serotype of avian influenza, which has been popular in Korea since its first occurrence in 1996, is a low pathogenic avian influenza belonging to the H9N2 type.

Avian influenza virus can be transmitted by droplets or water (latency 3-14 days), and the most important transmission method is direct contact with feces. Viruses in feces can survive for at least 35 days at 4 ° C, and 1 g of virus contaminated feces can infect about 1 million chickens. Avian influenza virus spreads directly on other chickens because of feces on human feet, feed cars, appliances, equipment, or egg surfaces. Egg-flood transmission through eggs does not occur, but can spread through egg-contaminated feces, so that eggs and chickens produced in diseased breeders can spread the disease. The clinical symptoms of H9N2 type low-pathogenic avian influenza, which are currently popular in Korea, vary according to the type of virus, and they show cyanosis in the head area such as respiratory symptoms, diarrhea, rapid decrease in spawning rate or crest. A variety of observations up to 10-50% are observed.

Diagnosis of avian influenza virus infection includes genetic testing, isolation of virus and serological tests. Genetic testing can extract the genes from infected chicken feces, organs, cecal tonsils and other parenchymal samples, and then identify the virus and its serotypes by RT-PCR. Virus isolation is performed by inoculating infected samples into SPF (Specific Pathogen Free) eggs to identify and isolate hemagglutinin virus, and by using H and N serotype specific antibodies, HI (hemagglutination inhibition) and NI (neurami) Suppression of isolated virus can be identified through a nidase inhibitory response. Serological tests include blood hemagglutination suppression (HI), agar sedimentation (AGID), and enzyme-linked immunosorbent assay (ELISA) to determine whether the chickens are infected. I use it by a method. Genetic testing, virus isolation, and serological testing should be done in a laboratory with the appropriate reagents, equipment, and expertise. Diagnostics can be as early as three to five days.

Recently, human influenza virus antigen rapid test kits using immunochromatography have been developed and widely used for diagnosing human influenza virus infection mainly in domestic and overseas hospitals, and these test kits all depend on imports. Rapid animal immunoassay kits have been developed and commercially available for the common avian influenza virus and H5 type highly pathogenic avian influenza virus. Development did not take place. Thus, there is a need for a diagnostic kit of commercially available H9 type low pathogenic avian influenza.

Therefore, the present inventors quickly and accurately discriminate whether H9 type avian influenza is infected by rapid immunochromatography in the total excretory cavity (fecal) swab sample when hemagglutinin antigen is prepared and used for antibodies specific for H9 type avian influenza. It has been found that the present invention has been completed.

Accordingly, it is an object of the present invention to provide a diagnostic kit for diagnosing avian influenza infection in a short time without special testing equipment in the field, and a method for diagnosing avian influenza using the same.

In order to achieve the above object, in the present invention, H9 type avian influenza produced from hybridoma cells of Accession Nos. KCTC 11753BP, KCTC 11754BP and KCTC 11755BP, and the hemagglutinin antigen is a monoclonal antibody specific for H9 avian influenza. Provide a diagnostic kit.

The present invention also provides a method for diagnosing avian influenza using the diagnostic kit.

H9 type avian influenza diagnostic kit according to the present invention was able to easily and quickly diagnose the infection of H9 type avian influenza in the field using a total excretory cavity (fecal) swab sample as a sample.

Figure 1a is a cross-sectional view of the H9 type avian influenza diagnostic strip according to the present invention (1) sample pad, 2) gold pad, 3) membrane (T: inspector position, C: control line position), 4) hygroscopic pad, 5) plastic Card], Figure 1B is a schematic diagram of the antibody-antigen response of the diagnostic strip.
2a to 2c show the measurement range and detection limit of the diagnostic strip according to the invention.
3A and 3B show the cross reactivity of the diagnostic strip according to the present invention.
Figures 4a to 4d are the results of reactivity investigation on the standard strain and domestic isolated H9 type avian influenza of the diagnostic strip according to the present invention.
5a and 5b show the results of comparing the timing and the detection rate through the larynx and total excretory cavity swab after H9 type avian influenza virus artificial infection using the diagnostic strip according to the present invention.
6 is a test result of the sensitivity of the diagnostic strip according to the present invention.

The present invention is a diagnostic that can differentially detect H9 type avian influenza virus in total excretory cavity (fecal) swab samples by rapid immunochromatography using antibodies specific for hemagglutinin antigen specific to H9 type avian influenza virus. Relates to a kit.

The diagnostic kit according to the invention comprises monoclonal antibodies produced from hybridoma cells of Accession Nos. KCTC 11753BP, KCTC 11754BP and KCTC 11755BP and whose hemagglutinin antigens are specific for H9 type avian influenza.

In one embodiment of the present invention, the diagnostic kit may be manufactured in the form of a diagnostic strip having the configuration shown in FIG. 1A, but is not limited thereto. In FIG. 1A, the diagnostic strip consists of 1) sample pad, 2) gold pad, 3) membrane (T: inspector position, C: control line position), 4) hygroscopic pad, and 5) plastic card.

In one embodiment of the present invention, hemagglutinin antigens can be diagnosed with H9 type avian influenza virus by immunochromatography using the diagnostic strip. In more detail, a specific antibody capable of detecting H9 type avian influenza virus on a membrane is adsorbed to an adsorption pad, and the gold conjugate to which another antibody is conjugated to gold particles is dried on a gold pad. H9 type avian influenza diagnosis is placed in a plastic device equipped with a sample application hole and a result display window by superimposing the gold pad including the dried gold conjugate and the sample pad applying the sample on the membrane and attaching the hygroscopic pad to the opposite position. After the kit is prepared, approximately 120-150 μl (4-5 drops) of the sample solution from the chicken swab (fecal) sample is placed in the sample application hole and the avian influenza virus present in the sample reacts with each antibody attached to the gold particles. Is developed on the membrane by capillary action.

Figure 1b is a schematic diagram of the antibody-antigen reaction of the diagnostic strip, the H9-type specific antibody (# 17) and the antigen-antibody complex (virus- # 17) to which gold particles are attached when there is a certain amount of H9 type avian influenza virus in fecal samples Complex) to begin development. The H9 specific antibodies (2G9, 5D9) are then attached to the test line so that when the previous antigen-antibody complex (virus- # 17 complex) is developed, the antigen-antibody complex (2G9,5D9-virus- # 17 complex) is added. ) Is formed, and gold particles accumulate on the inspection line, and become purple.

In this case, the monoclonal antibodies (# 17, 2G9, 5D9)) is produced in the mouse. In addition, mouse immunoglobulin (IgG) was purified and then inoculated into goats to produce antibodies to immunoglobulins, which were then attached to the control line. Mouse antiserum with gold particles reacted with the test line, and the remaining antibodies must be used in the control line. It forms a complex with an immunoglobulin antibody, making it purple. In the absence of H9 type avian influenza virus, no response should be observed on the test line, and the control line should have a purple response.

Membrane that can be used for the preparation of the H9 type avian influenza diagnostic kit according to the present invention can be used as a material of a conventional diagnostic strip, for example, various synthesis of nitrocellulose, cellulose, cellulose acetate or polyethylene, etc. Polymers may be used, but are not limited thereto.

The membrane has a hemagglutinin antigen adsorbed to a specific position specific to the H9 type avian influenza, and when the virus antigen is present in the sample, each virus antigen in response to the antibody conjugated to the gold particles is attached to the membrane. Reacts with the adsorbed antibody to form a purple band of gold particle color at that location. Anti-mouse IgG is adsorbed at the control line position so that the gold particles to which mouse IgG is adsorbed will always react and show a purple band regardless of the presence of viral antigen in the sample. The unreacted content penetrates into the hygroscopic pad so that the membrane of the inspection window appears clean white to easily read the formed band. That is, when a certain amount of H9 type low pathogenic avian influenza virus is present in the sample for hemagglutinin antigen, purple bands are simultaneously formed at the position where the monoclonal antibody specific for the virus is adsorbed and the control line. In addition, if avian influenza virus other than H9 type is present in the sample or there is no virus, a violet band is formed only at the control line.

In one embodiment of the present invention, the monoclonal antibody (# 17 antibody) of Accession No. KCTC 11755BP forms a conjugate with a gold particle and is adsorbed onto the membrane, and the monoclonal antibody produced from the hybridoma cells of Accession Nos. KCTC 11753BP and KCTC 11754BP. 2G9 and 5D9 antibodies) are adsorbed on the test line.

The present invention also relates to a method for diagnosing H type avian influenza using the H9 type avian influenza diagnostic kit.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Example 1 Preparation of monoclonal antibody for diagnosing avian influenza

1) Purification of avian influenza antigen

It was prepared from H9N2 type avian influenza virus [A / Ck / Kor / 301/01 (H9N2)] which is used as diagnostic antigen for H9N2 and antigen for vaccine production in Korea. In other words, avian influenza virus incubated for 2 to 3 days at 9 days of age SPF eggs at 37.0 ℃ inactivated with a final concentration of 0.2% formalin, concentrated and purified. The inactivated avian influenza virus was first centrifuged at 4,000xg for 30 minutes, and the supernatant was secondly ultracentrifuged at 70,000xg for 3 hours (L8-70M, Beckman, USA). Resuspend the centrifuged pellet (1/25 times the amount of sucrose) to sucrose density gradient ultracentrifugation (100,000 xg, 90 minutes), and isolate the avian influenza virus fraction to isolate the avian influenza monoclonal It was used as an antigen for antibody production.

The antigen purified by the above method was used as an immunogen to immunize Balb / c mice. Afterwards, splenocytes were isolated, conjugated with myeloma, and hybrid cell lines secreting monoclonal antibodies against avian influenza virus antigens were selected by ELISA, and several mononuclear cell groups were established by limiting dilution. These cell lines were cultured in bulk and inoculated into the abdominal cavity of Balb / c mice to obtain ascites containing a large amount of antibody, and IgG antibodies were purified using protein A / G gels.

The purified antibodies were used to select antibodies that specifically react to H9 type avian influenza virus through hemagglutination inhibition (HI). HI positive reaction was observed in 72 clones of the obtained antibodies, the three most reactive clones (2G9, 5D9 and # 17) were selected and shown in Table 1 below. In addition, the present inventors deposited the hybridoma cells producing the three clones with the Korea Biotechnology Research Institute Gene Bank on August 25, 2010, respectively, and received KCTC 11753BP, KCTC 11754BP, and KCTC 11755BP.

Evaluation of the efficacy of selected antibodies by HI reaction Clone number Hybridoma cells
Deposit number Clonal Antigens HI test 2G9 KCTC 11753BP A / Ck / Kor / 301/01 (H9N2) 2 ⁴ log ₂ 5D9 KCTC 11754BP 2 ⁶ log ₂ # 17 KCTC 11755BP 2 ⁹ log ₂

Example 2 Preparation of Diagnostic Strip

Using the 2G9, 5D9 and # 17 antibodies prepared in Example 1, H9 type avian influenza diagnostic strips were prepared as follows.

1) membrane coating

Among the antibodies against the purified H9 type avian influenza virus antigen, 2G9 and 5D9 antibodies prepared in Example 1 were mixed at the same concentration, diluted in PBS at a concentration of 0.58 µg / strip, and nitrocellulose membrane test line (T: H9 type). Adsorbed at the location of detecting avian influenza virus) was used as a coating solution. In addition, the control line (C) position was coated with a goat anti mouse antibody 0.75 μg / strip.

2) gold pad manufacturer

The # 17 antibody prepared in Example 1 was combined with a gold colloid. In other words, by reducing the gold chloride to sodium citrate solution to prepare a plain gold (plain gold) after the addition of each antibody, 40 nm antibody bound gold particles at 532 nm so that the absorbance at 10 + / -1 Prepared. PEG solution was treated to stabilize the antibody bound thereto. Each gold conjugate to which the gold particles and the antibody were combined was mixed, soaked in polyester or glass fibers to 8.3 μl / strip, and dried to prepare a gold pad.

3) Hygroscopic pad and sample pad manufacture

The moisture absorption pad and the specimen pad were prepared by drying so that the reaction solution could be absorbed well.

4) Device assembly

As shown in FIG. 1, 1) sample pad, 2) gold pad, 3) membrane (T: inspector position, C: control line position), 4) hygroscopic pad, and 5) plastic card, respectively, were prepared. Combined and cut to 4.45 mm / strip size. The strip thus cut was placed in the bottom plate of the final device and covered with the top plate.

5) Final product composition

Sampling swab, diluent (50 mM Tris, 0.02% Sodium azide, 0.1% Triton X-100, pH 8.0, 1 ml for one test) in the device described above Use) to make the final diagnostic strip.

6) Test principle of H9 type avian influenza diagnostic strip according to the present invention

When using the H9 type avian influenza diagnostic strip according to the present invention, the total excretory cavity (fecal) swab sample of the chicken is diluted with a sample dilution and applied to the sample application hole. At this time, if a certain amount of H9 type avian influenza virus is present in the sample, the H1 type avian influenza virus is first bound to the # 17 antibody conjugated to the gold particles of the gold pad, and then the conjugate is moved along the membrane by immunochromatography principle. Two violet bands are shown along with the control band, forming an antibody-antigen-antibody complex, secondary binding to 2G9 and 5D9 antibodies at the test line position (T) of (FIG. 1B).

[Test Example 1] Detection limit investigation

In order to determine the measurement range and detection limit of the diagnostic strip prepared in Example 2, it was carried out using H9 type avian influenza virus different from the isolate from the hemagglutination titer and EID titer.

Hemagglutination Titer was prepared by adding 25 μl of PBS (pH 7.2) to all wells of a V-bottom 96 well plate and adding 25 μl of H9 type avian influenza virus from different strains to each well. Dilutions were then made. Thereafter, 25 µl of 1% chicken erythrocyte suspension was added, shaken well, and allowed to stand at room temperature for 40 minutes. Hemagglutination titer was determined as the highest dilution factor that aggregates red blood cells.

Egg Infectious Dose (EID ₅₀ ) was used to inoculate 10 ml of H9 type avian influenza virus from 9-11 days old SPF embryonated eggs and inoculate 0.1 ml into the intestinal ureter for 3 days at 37.0 ° C. After incubation, the intestinal urea was mixed with 5% chicken erythrocyte suspension and plate hemagglutination was performed to determine whether the virus was infected. The titer was calculated by the Reed & Munch method, and the results are shown in Tables 2 to 4 and FIGS. 2A to 2C.

Chicken-derived H9 type avian influenza virus, 1HAU = 10 ^5.1 EID ₅₀ /0.1ml Dilution factor 2 in ¹ 2 ² 2 ³ 2 ⁴ 2 ⁵ 2 ⁶ 2 ⁷ 2 ⁸ 2 ⁹ 2 ¹⁰ Hemagglutination + + + + + + + - - - HAU 64 32 16 8 4 2 One Kit Reactivity + + + + + + + - - -

Duck-derived H9 type avian influenza virus, 1HAU = 10 ^4.2 EID ₅₀ /0.1ml Dilution factor 2 in ¹ 2 ² 2 ³ 2 ⁴ 2 ⁵ 2 ⁶ 2 ⁷ 2 ⁸ Hemagglutination + + + + + - - - HAU 16 8 4 2 One Kit Reactivity + + + + + - - -

Wild bird-derived H9 type avian influenza virus, 2HAU = 10 ^5.4 EID ₅₀ /0.1ml Dilution factor 2 in ¹ 2 ² 2 ³ 2 ⁴ 2 ⁵ 2 ⁶ 2 ⁷ 2 ⁸ 2 ⁹ Hemagglutination + + + + + + + - - HAU 64 32 16 8 4 2 One Kit Reactivity + + + + + + - - -

In the results of Tables 2-4 and FIGS. 2A-2C, high reactivity was observed in the diagnostic strip of Example 2 comprising three monoclonal antibodies (2G9, 5D9 and # 17) according to the present invention. More specifically, there was a difference according to the isolate derived from the test line (T). For chicken-derived H9 type avian influenza virus, up to 1 HAU (10 ^5.1 EID ₅₀ /0.1ml), duck-derived H9 type algae Up to 1 HAU (10 ^4.2 EID ₅₀ /0.1 ml) of influenza viruses and up to 2 HAU (10 ^5.4 EID ₅₀ /0.1 ml) of wild bird-derived H9 avian influenza viruses were detected.

Test Example 2 Cross-Reactivity

In order to investigate the cross-reactivity of the diagnostic strip prepared in Example 2, samples infected with H1 to H15 type avian influenza virus and various other viruses with pathogenicity to birds were applied to the diagnostic strip. And 6, and FIGS. 3A and 3B.

Crossreactivity of Avian Influenza HA Serum Serotype H1N1 H2N2 H3N8 H4N6 H5N3 H6N5 H7N1 H8N4 H9N2 H10N7 H11N6 H12N5 H13N6 H14N5 H15N6 Kit Reactivity - - - - - - - - + - - - - - -

Investigate cross-reactivity with algae-derived viruses virus Bird-derived virus H9N2 avian influenza virus APV IBV NDV IBDV 310/01 Q30 / 04 164/04 136/04 116/04 ms96 / 96 Kit Reactivity - - - - + + + + + +

※ APV: Pneumovirus, TRTV strain, IBV: Infectious bronchitis virus, KS96 strain, NDV: Newcastle disease virus, Correction strain strain, IBDV: Gamboro virus, Winterfield 2510 strain

In the results of Table 5, Table 6, Figure 3a and 3b, the diagnostic strip prepared in Example 2 was specifically positive only to H9 type avian influenza virus. On the other hand, they were negative for blood serotypes other than H9, namely H1-8 and H10-15 avian influenza viruses and other avian-derived viruses (pneumovirus, infectious bronchitis virus, Newcastle disease virus and infectious F cystic virus). .

[Test Example 3] Reactivity of A9 influenza virus derived from H9 type strains, chickens, ducks, and wild birds

In order to investigate the responsiveness to the H9 type avian influenza virus of the diagnostic strip prepared in Example 2, the diagnosis was performed on various H9 type avian influenza viruses isolated from H9 type US standard strains and domestic chickens, ducks, and wild birds. The reactivity was examined by applying a strip, and the results are shown in Tables 7 to 10 and FIGS. 4A to 4D.

Virus-derived H9 type American standard stock (Wisconsin1 / 66) Kit Reactivity +

Chicken origin H9 type avian influenza virus GJ180 / 08 GJ92 / 07 GJ91 / 07 GJ89 / 07 YS61 / 07 AS51 / 06 DY65 / 04 164/04 136/04 Q30 / 04 310/01 ms96 / 96 Kit Reactivity + + + + + + + + + + + +

Duck origin H9 type avian influenza virus GJ77 / 07 H102-60 / 07 GJ113 / 07 GJ84 / 07 GJ83 / 07 Kit Reactivity + + + + +

Derived from wild birds H9 type avian influenza virus PJ24 / 07 PSC29 / 05 KH21 / 05 UP59 / 04 ES12 / 04 Kit Reactivity + + + + +

As a result, the diagnostic strip prepared in Example 2 responded positively to both the US standard strain of H9 type avian influenza virus as well as to various domestic isolated avian influenza viruses.

[Test Example 4] Comparison of detection timing and detection rate through total excretory steel swap

In order to investigate the detection ability of the diagnostic strip prepared in Example 2 in the actual infected animals, H9N2 type low pathogenic avian influenza virus (A / Ck / Kor / 310/01, H9N2) in 6-week-old SPF chicken 10 ^6.5 EID ₅₀ / After infecting 0.1 ml of the nasal cavity, the presence of virus was examined by the date of inoculation, and the presence or absence of responsiveness of the diagnostic strip was confirmed. The results are shown in FIGS. 5A and 5B.

In the results of FIG. 5A, it was confirmed that avian influenza virus was excreted up to 7 days after infection as a result of virus isolation through the inoculation method in the chicken throat. On the other hand, as a result of investigating the reactivity of the diagnostic strip of Example 2 according to the present invention, a positive reaction was detected only in one individual 4 days after infection, and 10% of kit positive reactions compared to virus isolation were confirmed.

On the contrary, when the virus was isolated from the total excretory river swap of chickens through the inoculation method, it was confirmed that the avian influenza virus was excreted until 11 days, and inoculated when the diagnostic strip of Example 2 according to the present invention was applied to the virus inoculation population. 50-57% of kit positives were observed for 3-6 days later (FIG. 5B). On the other hand, when the virus was isolated during the same period (3-6 days post infection), it was confirmed that a relatively high kit positive rate of 57-83% was shown in the diagnostic strip of Example 2.

These results show that the infection is effective when applied to 5-10 individuals whose clinical symptoms are observed in order to confirm the infection of H9 type avian influenza virus using the diagnostic strip of Example 2 according to the present invention in an outdoor chicken farm. It was judged that it could be confirmed. In addition, it was judged appropriate to test the detection samples for infection using total excretory cavity swabs rather than laryngeal swabs.

Test Example 5 Sensitivity Investigation

It was difficult to collect feces of avian influenza infection cases in an outdoor farm, and the sensitivity of the diagnostic strip was investigated by spiking a minimum amount of virus in the feces in the diagnostic strip prepared in Example 2. Was first fecal 0.1 g, 10 ^6.1 EID ₅₀ / of 100㎕ virus (310/01, H9N2) 100 ㎕, and a mixture of the diluent 800 ㎕ of the diagnostic strip, the supernatant was allowed to react 100 ㎕ to diagnostic strip of which, The results are shown in Table 11 and FIG. 6.

In the results of Table 11 and FIG. 6, 79% positive, 15% positive and 6% negative were found, and the diagnostic strip of Example 2 according to the present invention was found to be effective against the minimum amount of virus detected in feces. It was judged that there was.

Test Example 6 Specificity Investigation

500 total excretory river swabs were collected from six farms where no clinical symptoms were observed in Gyeonggi-do, Chungbuk and Chungnam, and virus specificity test was performed by the inoculation method and specificity test using the diagnostic strip prepared in Example 2 above. And the results are shown in Table 12 below.

Farm address kind copper Week sample water Test result virus
detach Kit
Responsive A farm Dongsak-dong, Pyeongtaek-si, Gyeonggi-do Laying hen 2 buildings 45 weeks old 20 points voice voice 3 buildings 45 weeks old 20 points voice voice 4 buildings 45 weeks old 20 points voice voice B farm Dongsak-dong, Pyeongtaek-si, Gyeonggi-do Laying hen 2 buildings 17 weeks old 20 points voice voice 1 building 49 weeks old 20 points voice voice C farm Godeok-myeon, Pyeongtaek-si, Gyeonggi-do Laying hen Beef 14 weeks old 25 points voice voice 1 building 66 weeks old 25 points voice voice 2 buildings 32 weeks old 25 points voice voice 4 buildings 49 weeks old 25 points voice voice D farm Entrance Area, Seobuk-gu, Cheonan-si, Chungnam Laying hen 1 building 53 weeks old 20 points voice voice 2 buildings 47 weeks old 20 points voice voice 3 buildings 66 weeks old 20 points voice voice 4 buildings 81 weeks old 20 points voice voice 5 buildings 30 weeks old 20 points voice voice E farm Chuchi-ri, Sotae-myeon, Chungju-si, Chungbuk Laying hen 4 buildings 17 weeks old 25 points voice voice 5 buildings 66 weeks old 25 points voice voice 3 buildings 31 weeks old 25 points voice voice 6 buildings 38 weeks old 25 points voice voice F farm Gunup-ri, Silchon-myeon, Gwangju-si, Gyeonggi-do Laying hen A-1 ridge 54 weeks old 25 points voice voice A-2 ridge 36 weeks old 25 points voice voice B-1 ridge 13 weeks old 25 points voice voice B-2 38 weeks old 25 points voice voice

In the results of Table 12, in all total excretory steel swab samples that were determined to be avian influenza negative, the specificity of the diagnostic strip prepared in Example 2 was all negative was confirmed that the specificity is 100%.

Korea Research Institute of Bioscience and Biotechnology KCTC11753BP 20100825 Korea Research Institute of Bioscience and Biotechnology KCTC11754BP 20100825 Korea Research Institute of Bioscience and Biotechnology KCTC11755BP 20100825

Claims (6)

H9 type avian influenza diagnostic kit comprising a monoclonal antibody specific for avian influenza produced from hybridoma cells of Accession Nos. KCTC 11753BP, KCTC 11754BP and KCTC 11755BP and whose hemagglutinin antigen is H9. Comprising the monoclonal antibody according to claim 1,
H9 type avian influenza diagnostic strip consisting of sample pad, gold pad, nitrocellulose membrane and hygroscopic pad. The method of claim 2,
H9 type avian influenza diagnostic strips in which each monoclonal antibody produced from hybridoma cells of Accession Nos. KCTC 11753BP and KCTC 11754BP was adsorbed onto a nitrocellulose membrane. The method of claim 2,
Gold pad is a H9 type avian influenza diagnostic strips comprising a gold conjugate combining the gold particles and monoclonal antibodies produced from hybridoma cells of Accession No. KCTC 11755BP. A method for diagnosing H9 type avian influenza using the H9 type avian influenza diagnostic kit according to claim 1. A method of diagnosing H9 avian influenza using the H9 type avian influenza diagnostic strip according to any one of claims 2 to 4.

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