KR20090044763A - Diagnostic kit for avian influenza virus antibody using competetive elisa and diagnostic method using the same - Google Patents

Abstract

The present invention relates to a diagnostic kit for avian influenza virus antibodies and a diagnostic method using the same. More specifically, the present invention relates to an influenza antigen adsorption plate in which a recombinant nuclear protein antigen is bound to a microplate on which a monoclonal antibody against an avian influenza virus is adsorbed, and a monoclonal antibody and an enzyme against a nucleoprotein of an avian influenza virus. The present invention relates to a diagnostic kit for avian influenza virus antibodies comprising an enzyme-labeled antibody conjugated thereto, and a diagnostic method for measuring avian influenza virus antibodies in a test sample using the same.

Avian influenza, nuclear protein, monoclonal antibody, enzyme-labeled antibody

Description

Diagnostic kit for avian influenza virus antibody using competetive ELISA and diagnostic method using the same}

The present invention relates to a diagnostic kit for avian influenza virus antibodies using a competitive enzyme immunoassay and a diagnostic method using the same. More specifically, the present invention relates to an influenza antigen adsorption plate in which a recombinant nuclear protein antigen is bound to a microplate on which a monoclonal antibody against an avian influenza virus is adsorbed, and a monoclonal antibody and an enzyme against a nucleoprotein of an avian influenza virus. The present invention relates to a diagnostic kit for avian influenza virus antibodies comprising an enzyme-labeled antibody conjugated thereto, and a diagnostic method for measuring avian influenza virus antibodies in a test sample using the same.

Avian influenza spreads rapidly and is highly pathogenic, and infects various kinds of birds such as chickens, turkeys and wild birds, and is an acute viral infectious disease mainly affecting chickens and turkeys. In particular, duck is a disease that does not show any clinical symptoms even if it is infected. Among them, highly pathogenic avian influenza (HPAI) is required to be reported by the OIE. Species are classified as livestock epidemics. In Korea, two highly pathogenic avian influenza (H5N1) cases have occurred twice (December 2003 to March 2004, November 2006 to July 2013), and low pathogenic avian influenza (H9N2) has been present since 1996. It is a situation that is commercialized.

The causative agent of avian influenza is the Influenza virus type A of Orthomyxoviridae . The serotypes of influenza A are classified according to the two types of proteins, hemagglutinin and neuraminidase, and there are H serotypes and N serotypes. The serotypes of influenza virus are indicated by their respective H and N serotypes (e.g., H9N2). Avian influenza viruses have 16 H serotypes and 9 N serotypes. There are 144 serotypes of. To date, highly pathogenic avian influenza belongs to the H5 and H7 serotypes.

Currently, the diagnosis of avian influenza virus infection includes the isolation of the virus and serological tests. Among them, serological tests include hemagglutination inhibition (HI), agar sedimentation reaction (AGP), enzyme immunoassay (ELISA), etc., and they are used as mass monitoring test methods to determine whether the chickens are infected or vaccinated. Doing. Serological tests are performed in two steps. The first step is to detect a common antibody against the AI virus, and the second step is to perform a test to determine the subtype if the antibody is positive in the first step. The relevant OIE provisions are as follows. The first step is to test for group antigens. This test is a method for detecting a common antibody against group A virus, which is an antibody test for nucleoprotein (NP) or matrix (M) protein. This antigen is present in all types of viruses, regardless of serotype (H or N). Positive responses to this reaction indicate infection with the influenza A virus but do not give information about the subtype.

As a test for detecting a common antibody against avian influenza, the AGP test (agar gel sedimentation method) is representative. This method is very simple and easy to test in chicken and turkey serum is a test that can be done with only basic equipment. It is very specific but has a disadvantage of low sensitivity. For this reason, it should be used for flocks rather than individual testing. However, aquatic algae should not be used because they do not produce sedimentation antibodies and are not yet fully validated for many species of algae.

The following is an ELISA (Enzyme Immunoassay) method, which has the disadvantage of having laboratory equipment such as a spectrophotometer. It is very sensitive but has a disadvantage of low specificity. Most of the commercially available ELISA kits for detecting avian influenza antibodies are indirect ELISA. In this case, the second antibody conjugate must be used differently depending on the animal species to be tested. Serum testing of animals other than chickens and pigs is not possible.

The second stage serum test is a subtype specific antibody test, and haemagglutination inhibition (HI) is typical. When performing the HI method, there is a disadvantage in that different antigens must be used depending on the H serotype.

Therefore, the present inventors are studying a method for diagnosing avian influenza antibodies that can be commonly used in various kinds of animals, and using competitive enzyme immunoassay using monoclonal antibodies against nuclear proteins, which are common antigens of avian influenza viruses, and recombinant nuclear protein antigens ( The present invention was completed by confirming that avian influenza virus antibodies can be detected effectively and quickly in various species by Competitive ELISA.

Accordingly, an object of the present invention is an influenza antigen adsorption plate in which a recombinant nucleoprotein antigen is bound to a microplate in which a monoclonal antibody is adsorbed against a nucleoprotein of avian influenza virus; And it provides a diagnostic kit of avian influenza virus antibodies comprising an enzyme-labeled antibody conjugated with a monoclonal antibody and an enzyme conjugate to the avian influenza virus nuclear protein.

Another object of the present invention to provide a method for diagnosing avian influenza virus antibodies using the diagnostic kit.

In order to achieve the object of the present invention as described above, the present invention is an influenza antigen adsorption plate in which a recombinant nucleoprotein antigen is bound to a microplate in which a monoclonal antibody against a nucleoprotein of avian influenza virus is adsorbed; And it provides a diagnostic kit of avian influenza virus antibody comprising a monoclonal antibody to the nucleoprotein of the avian influenza virus and an enzyme-labeled antibody conjugated to the enzyme.

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

Hereinafter, the present invention will be described in detail.

Diagnosis kit of avian influenza virus antibody and a diagnostic method using the same according to the present invention by adsorbing a monoclonal antibody against avian influenza virus (Avian Influenza Virus, Nucleoprotein: NP) in a microplate first, the nucleoprotein antigen is only the microplate After pretreatment for adsorption, the enzyme conjugate reacts with the antibody in the sample by reacting the recombinant nucleoprotein antigen and simultaneously reacting the test sample to be tested and the enzyme conjugate conjugated to the monoclonal antibody against the nuclear protein. It is based on the principle of competitive enzyme immunoassay (see FIG. 1). More specifically, the test sample and the enzyme conjugate are added to the microplate prepared in the present invention at the same time. If the test sample contains an antibody to the nuclear protein, the reaction reacts competitively with the antibody of the enzyme conjugate. In this case, only the antibody of the enzyme conjugate reacted with the antigen, and after the washing process, if the coloring agent was added, the color was hardly developed when the antibody was present in the test sample, and when the antibody was not present, the color was strongly reacted with the enzyme conjugate. . In other words, if the color development hardly occurs, the antibody was determined to be positive, and if the color became strong, the principle of determining to be antibody negative was used.

Diagnostic Kit

Preferably, the diagnostic kit of the avian influenza virus antibody of the present invention comprises an influenza antigen adsorption plate in which a recombinant nucleoprotein antigen is bound to a microplate in which a monoclonal antibody is adsorbed against a nucleoprotein of avian influenza virus; And an enzyme-labeled antibody in which a monoclonal antibody and an enzyme are conjugated to the nucleoprotein of the avian influenza virus.

(a) Influenza antigen adsorption plate

Influenza antigen adsorption plate of the present invention is a high purity purified monoclonal antibody against the nuclear protein of avian influenza virus adsorbed to the microplate wells preferably 0.095 ~ 0.105ug / well, more preferably 0.1ug / well concentration, It can be prepared by reacting avian influenza virus nucleoprotein expressed in the baculovirus and then blocking with 1% bovine serum albumin or the like and drying the dried plate in a silver foil with dried silica gel.

Monoclonal antibodies against the avian influenza virus nucleoprotein can be prepared using conventional cloning and cell fusion techniques using the avian influenza virus nuclear protein as an immunogen (antigen). The avian influenza virus used as an immunogen of the monoclonal antibody is not particularly limited, but may be preferably a highly pathogenic avian influenza (A / CK / Korea / ES / 03 (H5N1)) isolated in Korea. In the embodiment, the immunogen for the preparation of the monoclonal antibody by inactivating the avian influenza (A / CK / Korea / ES / 03 (H5N1), concentrate and purify, and isolate and recover the fraction showing hemagglutination through centrifugation. (See Example <1-1>).

The method for producing monoclonal antibody using the prepared immunogen is known fusion method (Kohler and Milstein, European J. Immnunol . 6: 511-519 1976), recombinant DNA method (US Patent No. 4816567) and phage Antibody libraries (Clackson et al., Nature , 352, 624-628, 1991; Marks et al., J. Mol. Biol . 222, 58: 1-597, 1991). For example, an immunogen (antigen) of interest can be administered to wild type or breeding mice (eg BALB / c) to produce natural or human monoclonal antibodies. Such antigens can be administered alone, in admixture with an adjuvant, can be expressed from a vector and can elicit an immune response as a DNA or fusion protein. Fusion proteins include carrier proteins coupled with peptides intended for an immune response, such as β-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH), and bovine serum albumin, Carrier proteins are not limited thereto. In this case the peptide acts as a hapten for the carrier protein. The method for producing monoclonal antibody is briefly described as follows. After boosting the animal, the spleen is removed and the spleen cells are extracted by methods known in the art [Kohler and Milstein, Nature 256: 495-497 (1975); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)]. The hybrid cells obtained are cloned by conventional methods, for example by limiting dilution, and the resulting clones are cultured to produce the desired monoclonal antibody. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and because they are synthesized by hybridoma culture, they also have another advantage that they are not contaminated by other immunoglobulins. Have

Meanwhile, the monoclonal antibody according to the present invention is prepared by using a nucleoprotein, which is a common antigen of avian influenza, as an immunogen, and can adsorb all kinds of avian influenza viruses having H1 to H15 serotypes (Example <1-2). And and FIG. 2).

The recombinant nucleoprotein antigen, which is one of the components of the diagnostic kit of the present invention, may be prepared according to a method for preparing a recombinant protein known in the art. For example, a DNA sequence encoding a nucleoprotein of avian influenza is inserted into a vector, the host is transformed with a recombinant expression vector formed therefrom, and the resulting transformant is a medium suitable for causing the DNA sequence to be expressed and Incubated under conditions, and recovering substantially pure recombinant protein from the culture.

Preferably, the recombinant nucleoprotein can be prepared by introducing a nucleoprotein gene cloned from avian influenza virus (A / CK / Korea / MS96 / 96 (H9N2)) into baculovirus and then inoculating it into insect cells.

In one embodiment of the present invention, RNA extraction of avian influenza virus (A / CK / Korea / MS96 / 96 (H9N2)) and RT-PCR were performed to clone the nuclear protein gene and introduce it into baculovirus to introduce recombinant baculovirus. After inoculating the recombinant baculovirus into Sf9 cells, which are insect cells, the cell supernatant was removed about 4-5 days, and cell lyolysis was repeated three times, followed by centrifugation to remove cell debris. The supernatant was then set as a nuclear protein (see Example 2.) The nuclear protein gene cloned in the present invention has a nucleotide sequence represented by SEQ ID NO: 3.

(b) enzyme-labeled antibodies

Enzyme-labeled antibodies can be prepared by conjugating enzymes to monoclonal antibodies against the nucleoprotein of avian influenza virus.

Monoclonal antibodies against the avian influenza virus nucleoprotein can be prepared in the same manner as the monoclonal antibodies used for the preparation of influenza antigen adsorption plates.

The enzyme is a labeling agent for qualitatively or quantitatively measuring the formation of an antigen-antibody complex, for example, but not limited to, β-glucuronidase, β-D-glucosidase, urease, fur Oxidase, alkaline phosphatase, acetylcholinesterase, glycos oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase and the like can be used. Preferably peroxidase can be used.

Methods for conjugating enzymes to the monoclonal antibodies are known in the art, and in one embodiment of the present invention, enzyme-labeled antibodies were prepared by conjugating HRPO (horseradish peroxidase) to a monoclonal antibody of the present invention by a known Nakane method ( See Example 3).

(c) other

The diagnostic kit of the present invention may further include reagents known in the art used for competitive enzyme immunoassay in addition to the influenza antigen conjugate plate and the enzyme-labeled antibody.

For example, such reagents may include negative control serum, positive control serum, wash solution, substrate solution capable of measuring enzyme activity, and reaction stop solution.

The negative control serum influenza antigen and antibody can be prepared by screening normal chicken serum negative and adding calcium to form and remove fibrin.

The positive control serum is selected by removing chicken serum positive for influenza antibodies and forming fibrin by adding calcium. Concentrated by saturated ammonium sulphate can be prepared by appropriate dilution with normal chicken plasma treated with calcium.

The wash solution may be prepared in phosphate physiological salt buffer (pH 7.2) to which 0.2% of polysorbate 20 is added.

The substrate solution may vary depending on the type of enzyme used. In one embodiment of the present invention, tetramethylbenzidine (8 mg / ml) and hydrogen peroxide (20-32%, 0.331 ml) are mixed as a substrate solution for HRPO. Prepared.

The reaction stop solution may be 1N hydrochloric acid.

Diagnosis method

In the method for diagnosing avian influenza virus antibodies using the diagnostic kit of the present invention, a test sample and an enzyme-labeled antibody are simultaneously added to the avian influenza antigen adsorption plate so that the antibody and the enzyme-labeled antibody in the test sample react competitively. The presence or absence of the antibody in the test sample is characterized in that the addition.

Preferably, the diagnostic method of the present invention comprises the steps of: (a) dispensing the test sample, negative control serum and positive control serum to the influenza antigen adsorption plate of the present invention, respectively, and simultaneously reacting and treating the enzyme-labeled antibody;

(b) washing the plate in which the reaction is completed in step (a) and treating the substrate solution; And

(c) treating the reaction stopper solution on the plate of step (b) and measuring the absorbance.

The "test sample" may be serum derived from an animal to measure the presence and extent of avian influenza virus antibody. For example, the animal may be any one selected from the group consisting of duck, turkey, pig, chicken, goose, guinea pole, quail, gray partridge, red partridge, pheasant, swan and horse. .

Most preferably, the diagnostic method of the present invention can be performed as follows.

Approximately 30 minutes before starting the test, the reagent components are left at room temperature, gently shaken before use, mixed well, and the plate is opened after use at room temperature.

The plate is made in strip form to determine the number of wells required for inspection, and the remaining wells are sealed with silica gel in their own silver foil so that they can be stored at 2 to 8 ° C.

Dispense the negative control solution (serum), the positive control solution (serum), and the sample to be tested into each well of the aliquot-plate of the sample, and simultaneously dispense concentrated enzyme-labeled antibody solution into each well. Shake carefully to prevent splashing and seal the plate with the attached sealing tape.

The sealed plate is preferably reacted at 37 ° C. for 30 minutes.

After the reaction, the contents of each well are aspirated and washed with the washing solution.

The substrate solution is dispensed into all wells, sealed with the attached sealing tape, and allowed to react for 10 minutes at room temperature.

Add the stop solution to all wells and mix well so that blue turns completely yellow.

Air blank is used to measure the negative control, the positive control, and the absorbance of each sample. In this case, the measurement wavelength of absorbance is 450 nm, and when a dual wavelength reader is used, the reference wavelength is 620 nm, and the absorbance value is measured within 30 minutes after adding the reaction stopper.

The diagnosis result according to the above method may be determined by the following method.

Negative control (serum) should be tested using 3 wells, average absorbance value should be at least 1.8000, positive control (serum) should be tested using 2 wells, average absorbance value should be less than 0.2000.

The PI value is calculated from the measured absorbance of the test sample using the following formula, and is positive if the calculated PI value (Percent Inhibition: PI) is greater than or equal to the determination reference value, and negative if less.

PI value = [1- (sample absorbance / negative control mean absorbance)] X 100

According to the diagnostic method of the present invention, in case of chicken, duck, quail, goose, guinea pole, gray partridge, red partridge, pheasant, swan, horse, and pig, a PI value of 50 or more is positive, and a negative value is negative. can do. In the case of turkey, it can be judged as positive if PI value is 85 or more, and negative when it is less.

The PI value can be established by the following method. Using the diagnostic kit according to the present invention, the sensitivity and specificity are plotted by the TG-ROC method based on the antibody titer tested for each animal and the antibody titer tested by the conventional HI method. The PI value is established when the sensitivity and specificity are not high either.

In one embodiment of the present invention, the efficacy of the diagnostic kit of the present invention was tested on various standard antisera of AIV (see Example <7-1>). As a result, the diagnostic kit of the present invention is specifically designed for all types (H1N1, H2N3, H3N8, H4N8, H5N1, H5N2, H5N3, H5N9, H6N2, H7N1, H7N3, H7N7, H8N4, H9N7, H10N1, H11N6, H11N9). , H13N6, H14N5, H15N6) was able to detect nuclear protein antibodies against AIV (see Table 5), cross-reactivity with antiserum against EDS-76, Mycoplasma, Newcastle Disease It was confirmed that there was no.

In addition, in another embodiment of the present invention using the indirect enzyme immunoassay principle of the conventional commercial kit and diagnostic kit according to the AIV antiserum detection ability of the test according to the comparison result of the chicken serum (Example <7- 2), it was confirmed that the diagnostic kit of the present invention can detect the common antibody against avian influenza more effectively than commercially available kits (see Table 6).

In another embodiment of the present invention, H9N2 virus was inoculated into SPF chickens and tested for the presence of antibodies using the diagnostic kit of the present invention (see Example <7-3>). At this time, it was compared with the existing HI method. As a result, from the fourth day of challenge inoculation it was possible to observe the antibody positive phenomenon with the diagnostic kit of the present invention, it was confirmed that the diagnostic kit of the present invention can detect the antibody similar to the existing HI method (Fig. 3 and See Table 7).

In another embodiment of the present invention, chicken, goose and ducks are inoculated with H5N1 vaccine and the antibody is changed to the diagnostic kit of the present invention or the existing commercially available kit (Synbiotics Inc. kit, Avivac Inc. kit, all of which are indirect enzyme immunity). Diagnostic tests using a measurement ELISA kit) and HI testing for H5 were performed (see Example <7-4>). As a result, all of the chickens, geese and ducks vaccinated with the H5N1 vaccine using the diagnostic kit of the present invention were able to detect the antibodies (see Table 9). On the other hand, the existing commercial ELISA kits can not only test the goose and duck serum because only the chicken serum can be used, the diagnostic kit of the present invention was available for all livestock breeding.

Furthermore, in one embodiment of the present invention, to confirm whether the diagnostic kit according to the present invention is applicable to all animal species, the sensitivity and specificity of each animal species were confirmed (see Example <7-5>). As a result, it was confirmed that the diagnostic kit of the present invention can be used in 12 animals (see Table 10).

Therefore, the diagnostic kit of avian influenza antibody and the diagnostic method using the same according to the present invention can be commonly used in various kinds of animals by using nuclear protein, which is a common antigen of avian influenza, and can be diagnosed to a degree similar to that of the HI standard, which is a conventional standard method. It can be seen.

As described above, the method of the present invention is applicable to all livestock breeds, and in particular, it is possible to effectively detect viral antibodies in ducks and wild birds that do not show clinical symptoms even when infected, and use different antigens according to H serotypes. It is simpler and easier than the conventional HI method which should be used. Therefore, virus antibodies can be diagnosed in a larger amount and more quickly, which is very useful for disease monitoring in animals.

In addition, by using a nuclear protein derived from domestically isolated avian influenza virus in the production of diagnostic kits, in particular, it is possible to detect a more sensitive virus infection antibodies that are popular in Korea.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

< Example  1>

Avian Influenza Diagnostics Monoclonal  Antibody manufacturing

<1-1> Avian Influenza Antigen Purification

Avian influenza virus used for monoclonal antibody production was prepared from highly pathogenic avian influenza (A / CK / Korea / ES / 03 (H5N1)) occurring in Korea. That is, inoculated with 10 days-old SPF eggs at 37.6 ℃ incubated for 2 to 3 days and collected, and then inactivated with 0.2% formalin final concentration, and concentrated and purified. 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). After centrifugation, the fish pellet was resuspended (1/25 times the amount of sucrose) to perform sucrose density gradient ultracentrifugation (100,000 xg, 90 minutes) and hemagglutination activity. Visible fractions were separated and used as antigens for the production of avian influenza monoclonal antibodies.

<1-2> Monoclonal  Antibody production

Balb / c mice were immunized using the antigen purified by the method of Example <1-1> as an immunogen. Then, splenocytes were isolated and conjugated with myeloma cells to select hybrid cell lines that secrete monoclonal antibodies against avian influenza virus antigens by ELISA, and established several mononuclear cell groups by limiting dilution. These cell lines were mass cultured and inoculated to Balb / c mice intraperitoneally to obtain ascites containing a large amount of antibody, and IgG antibodies were purified using protein A / G gel.

<1-3> Avian influenza antigen diagnosis Monoclonal  Specificity Test of Antibodies

Antibodies that specifically react to the general avian influenza virus were selected by performing dot immunoassay using the antibodies purified in Example <1-2>.

As a result, the clone No. AIV NP-1 antibody (deposited with KCLRF-BP-00166 on Sept. 11, 2007 to the Korea Cell Line Research Foundation) was assigned to the AIV NP-2 antibody (2007 to Korea Cell Line Research Foundation) for microplate adsorption. Accession No. KCLRF-BP-00167 dated September 11, 2008 was selected as an antibody for the enzyme conjugate. The antibody was shown to be adsorbed avian influenza virus having a H1 to H15 serotype (Fig. 2).

< Example  2>

Baculovirus  Production of Avian Influenza Antigen Recombinant Protein

Recombinant nuclear protein was produced using low pathogenic A / CK / Korea / MS96 / 96 (H9N2) strain first isolated in 1996 in Korea.

RNA extraction of avian influenza virus and RT-PCR were performed as a first step for expression in baculovirus. That is, total RNA was isolated and templated using TRIzol reagent (Invitrogen, USA), and RT-PCR was performed with a one-step RT-PCR kit (Qiagen, Cat No. 2102212) using the following primer.

Forward primer: SEQ ID NO: 1

5'-AGCAAAAGCAGGGTTAATAATCAC-3 '

Reverse primer: SEQ ID NO: 2

5'-AGTAGAAACAAGGGTATTTTTCCTC-3 '

At this time, RT-PCR conditions were as follows (Table 1).

RT-PCR Condition cycle reaction Temperature time One  Reverse transcription reaction 45 ℃ 30 minutes 40  RTase Inactivation 94 ℃ 5 minutes  denaturalization 94 ℃ 30 seconds  Annealing 50 ℃ 30 seconds  kidney 72 ℃ 40 seconds One  Final height 72 ℃ 5 minutes

The 1,565 bp nucleoprotein gene obtained after PCR was electrophoresed on an agarose gel and extracted from agarose gel using a gel extraction kit (Qiagen). Nucleoprotein genes were cloned using a TA cloning kit (Invitrogen, USA), and the sequences were checked by an external professional company (SEQ ID NO: 3).

The cloned nuclear protein gene was inserted into pFastBac (Invitrogen, USA) donor vector and translocated to bacmid to obtain recombinant bacmid DNA. The recombinant bacmid DNA was transformed into Sf9 cells (Invitrogen) using a cellfectin reagent (Invitrogen, USA) and further cultured for 72 hours to prepare a recombinant baculovirus. Virus infectivity titer was performed using a plaque assay.

Sf9 cells were used as cells for culturing the recombinant baculovirus expressing the nuclear protein. That is, the recombinant baculovirus was inoculated in Sf9 cells with an MOI of 0.4 PFU / cell, and further cultured for 72 hours, and the cells were recovered by centrifugation at 3,000xg for 10 minutes. At this time, the composition of the medium was 90% insect cell culture media (insect cell culture media) and 10% FBS. The recovered cells were subjected to 12% SDS-PAGE, followed by Western blot using coomasie blue staining and rabbit antiserum to confirm nuclear proteins of approximately 57.4 KD in size. The rabbit antiserum was obtained by the following method. Avian influenza virus was incubated in embryonated eggs for 3-4 days to collect allantoic fluid and inactivated by 0.4% paraformaldehyde treatment. The inactivated virus was subjected to ultrafast centrifugation using a sucrose gradient to isolate the virus and immunize the rabbit to obtain rabbit antiserum.

The present inventors inoculated the recombinant baculovirus to Sf9 cells, and then removed the cell supernatant around 4-5 days and repeated freeze-thawing three times, followed by centrifugation to remove cell debris and the supernatant. Was set as a nuclear protein

< Example  3>

For nuclear proteins Monoclonal  Preparation of enzyme-labeled antibody conjugated with an enzyme

An enzyme conjugate was prepared by conjugating the monoclonal antibody AIV NP-2 against the nuclear protein prepared in Example <1> with a horseradish peroxidase (HRPO) by a known Nakane method. That is, 5 mg of HRP is dissolved in 1.2 ml of distilled water, and 0.3 ml of sodium phosphate (pH 7.0) in which 0.1 M of sodium-m-periodate (Sigma, S-1878) is dissolved. Added. This was reacted for 20 minutes at room temperature and dialyzed overnight with 1 mM sodium acetate (pH 4.0). Monoclonal antibody # 16 was prepared in 20mM carbonate (carbonate, pH9.5) at a concentration of 10 mg / ml, and 0.5 ml of the HRP solution dialyzed above was reacted at room temperature for 2 hours. Then, 100ul of sodium borohydride (sodium borohydride, 4mg / ml in water) was added and reacted at 4 ° C for 2 hours. It was dialyzed again with PBS (pH 7.2).

< Example  4>

According to the invention Diagnostic Kit  Produce

1) Influenza antigen adsorption plate: monoclonal antibody AIV NP-1 against avian influenza virus nuclear protein prepared in Example <1-3> was purified with high purity and adsorbed to the microplate well at a concentration of 0.1 ug / well, The avian influenza virus nucleoprotein expressed in the baculovirus of Example 2 was reacted. Thereafter, the resultant was blocked by 1% bovine serum albumin or the like and dried, and the dried plate was sealed with silver silica gel with dried silica gel cloth and stored until use.

2) Negative Control Serum: Prepared by screening normal chicken serum with negative influenza antigen and antibody and removing calcium by adding fibrin.

3) Positive control serum: Select chicken serum positive for influenza antibody and remove it by forming fibrin by adding calcium. Concentrated by treatment with saturated ammonium sulphate and prepared by appropriately diluting with normal chicken plasma treated with calcium.

4) Washing solution: Polysorbate 20 was prepared in phosphate physiological salt buffer (pH 7.2) to which 0.2% was added.

5) Substrate solution: Tetramethylbenzidine (8mg / ml) and hydrogen peroxide (20-32%, 0.331ml) were prepared by mixing.

6) The reaction stop solution was made with 1N hydrochloric acid.

< Example  5>

According to the invention Kit  Diagnostic method

The kit according to the present invention was used to diagnose the presence of avian influenza antibodies by the following method.

1) Leave the reagent components at room temperature for about 30 minutes before starting the test, shake well before use, mix well, and use the plate after opening at room temperature.

2) The plate was made in the form of a strip to determine the number of wells required for the test, and the remaining wells were sealed with silica gel in their own silver foil to be stored at 2 to 8 ° C.

3) Dispense 50 μl each of the negative control solution (serum), positive control solution (serum), and the sample to be tested into each well of the aliquot-plate of the sample. After the aliquots were aliquoted, the samples were carefully shaken to prevent the samples from splashing out of the well for about 10 seconds, and then the plate was sealed with the attached sealing tape.

4) The sealed plate was reacted at 37 ° C. for 30 minutes.

5) After the reaction, the contents of each well were aspirated and washed six times with the washing solution as follows. First, the contents of the wells were removed by the suction device, and then the washing solution was completely filled with the wells (about 350 μl per well) and the solution was aspirated six times.

6) 100 μl of the substrate solution was dispensed into all wells, sealed with the attached sealing tape, and reacted at room temperature for 10 minutes.

7) 100 μl of each solution was added to each well of each well, and the mixture was mixed well so that the blue color changed to yellow completely.

8) The air blank was measured for the negative control, the positive control and the absorbance of each sample. At this time, the measurement wavelength of absorbance was 450 nm, and when a dual wavelength reader was used, the reference wavelength was 620 nm, and the absorbance value was measured within 30 minutes after adding the reaction stopper.

< Example  6>

Determination of Diagnostic Results According to the Method of the Invention

The diagnosis of the diagnostic result using the kit of the present invention was performed as follows.

1) Quality Control

(1) Negative control solution (serum) is tested using 3 wells, and the average absorbance value is 1.8000 or more, and when about one value is out of range, the average value of the remaining two values is calculated. If more than one value is out of the above range, the test should be repeated.

(2) The positive control solution (serum) is tested using 2 wells, and the average absorbance value should not be more than 0.2000. If the average absorbance value is out of the above range, there is a problem with the test procedure or the reagent. Check the cause and retest.

2) Calculation of Percent Inhibition (PI)

(1) Calculation of negative control (serum) average value

According to the test method of the present invention, the absorbance of the negative control solution (serum) is obtained, and then the average value of the three values is calculated.

Example of calculating negative control mean Negative control number Absorbance (450 nm, reference wavelength 620 nm) One 2.520 2 2.522 3 2.521 Sum 7.563

* Average absorbance of negative control (serum): NCx = 7.563 / 3 = 2.521

(2) Calculation of positive control (serum) average value

According to the test method of the present invention, the absorbance of the positive control (serum) is obtained, and then the average value of the two values is calculated.

Example of calculating positive control mean Positive Control Number Absorbance (450 nm, reference wavelength 620 nm) One 0.058 2 0.060 Sum 0.118

Average absorbance of positive control (serum): PCx = 0.118 / 2 = 0.059

(3) Calculation of PI Value Average

PI value = [1- (sample absorbance / negative control mean absorbance)] X 100

Example) Sample Absorbance = 2.420, Average Absorbance of Negative Control = 2.521

PI value = [1- (2.420 / 2.521)] X 100 = 4

Result judgment: negative

3) Judgment of the result

(1) Negative: Specimens showing PI values below the determination criterion shall be judged negative.

(2) Positive: Specimens showing PI values above the criterion value are considered positive.

(3) Determination of Positive Test in Primary Test A sample shall be retested for at least 2 wells, and if it is positive for at least 1 well from the retest result, it is determined as final positive.

(4) The final positive sample should be comprehensively diagnosed by a professional veterinarian using other clinical or experimental results.

(5) judgment standard value

Judgment thresholds for various animals animal chicken duck Turkey Quail goose Guinea Falls Gray partridge Red partridge pheasant Swan Words pig Positive PI value 50 or more 50 or more 85 or more 50 or more 50 or more 50 or more 50 or more 50 or more 50 or more 50 or more 50 or more 50 or more Negative PI value Less than 50 Less than 50 Less than 85 Less than 50 Less than 50 Less than 50 Less than 50 Less than 50 Less than 50 Less than 50 Less than 50 Less than 50

< Example  7>

According to the invention Diagnostic Kit  Efficacy test

<7-1> AIV  Antiserum Test

The efficacy of the diagnostic kit of the present invention was tested by the method of Example 5 above and determined by the method of Example 6 on standard antiserum conducted in Italian IZS laboratory and Russian FGI (all OIE reference laboratories).

As a result, the diagnostic kit of the present invention is H1N1, H2N3, H3N8, H4N8, H5N1, H5N2, H5N3, H5N9, H6N2, H7N1, H7N3, H7N7, H8N4, H9N7, H10N1, H11N6, H11N9, H5, H5 It was confirmed that it is reactive with standard antiserum against, and no cross-reactivity with antiserum against EDS-76, Mycoplasma, Newcastle Disease.

Therefore, it was confirmed that the diagnostic kit of the present invention can specifically detect nucleoprotein antibodies against all types of AIV (Table 5).

Diagnostic Results for AIV Antisera Using the Diagnostic Kit of the Invention Serum number AI type HI titer AGID Results Diagnostic result Absorbance PI value result Standard training Standard voice Batch 2/05 SPF negative serum neg neg 1.334 14.0 Neg 0.061 1.552 Batch 1/01 H1N1 1: 512 pos 0.069 96.9 Pos 0.075 2.206 Batch 1/05 H2N3 1: 2048 pos 0.058 97.4 Pos 0.075 2.206 Batch 1/02 H3N8 1:32 pos 0.075 96.6 Pos 0.075 2.206 Batch 1/04 H4N8 1: 512 pos 0.050 97.7 Pos 0.075 2.206 Batch 1/05 H5N1 1: 512 pos 0.048 97.8 Pos 0.075 2.206 Batch 1/01 H8N4 1: 256 pos 0.062 97.2 Pos 0.075 2.206 Batch 1/05 H9N7 1: 4096 pos 0.092 95.8 Pos 0.075 2.206 Batch 1/01 H10N1 1:64 pos 0.110 95.0 Pos 0.075 2.206 Batch 1/01 H11N6 1: 128 pos 0.050 97.7 Pos 0.075 2.206 Batch 1/01 H12N5 1: 512 pos 0.053 97.6 Pos 0.075 2.206 Batch 1/01 H13N6 1: 512 pos 0.049 97.8 Pos 0.075 2.206 Batch 1/01 H14N5 1: 256 pos 0.052 97.6 Pos 0.075 2.206 Batch 1/04 H15N6 1: 256 pos 0.264 88.0 Pos 0.075 2.206 Batch 2/01 NDV 1: 256 neg 1.698 23.0 Neg 0.075 2.206 H7N1 1: 128 pos 0.045 98.6 Pos 0.105 3.179 H7N3 1: 256 pos 0.069 97.8 Pos 0.105 3.179 H6N2 1: 1024 pos 0.060 98.1 Pos 0.105 3.179

 neg: negative, pos: positive

<7-2> of the present invention Diagnostic Kit  Existing Commercialized With the kit  Comparison of efficacy

The ability to detect AIV antiserum by the conventional kit using the indirect enzyme immunoassay principle and the diagnostic kit according to the present invention was compared and tested in chicken serum.

As a result, it was confirmed that the diagnostic kit of the present invention can detect a common antibody against avian influenza more effectively than commercially available kits (Table 6).

Comparative test results of AIV antiserum detection efficacy of the diagnostic kit according to the present invention and the existing commercially available kit Serum number Diagnostic kit Pos≥51 of the present invention Synbiotics, Pos≥338 EU, Avivac, Pos≥15 H5 HI titer, Pos≥1: 16 H1N1 (Italy) * 94 4151 97 <1: 2 H2N3 (Italy) 82 9401 121 1: 8 H3N8 (Italy) 93 11263 258 <1: 2 H4N8 (Italy) 97 Not tested Not tested <1: 2 H5N1 95 7775 87 1: 512 H5N2 (Italy) 96 11424 264 1: 256 H5N3 (Italy) 95 8727 128 1: 1024 H5N9 (Italy) 97 9713 92 1: 256 H7N1 (Italy) 97 11673 227 <1: 2 H7N3 (Italy) 94 Not tested Not tested <1: 2 H7N7 (Italy) 92 11741 239 <1: 2 H8N4 (Italy) 95 12273 259 <1: 2 H10N1 (Italy) 94 Not tested Not tested   1: 8 H11N9 (Italy) 97 Not tested Not tested <1: 2 H12N5 (Italy) 89 11196 293 <1: 2 H13N6 (Italy) 92 8937 120 <1: 2 H14N5 (Italy) 96 Not tested Not tested <1: 2 H15N6 (Italy) 91 Not tested Not tested <1: 2 EDS-76 10 101 9 <1: 2 Mycoplasma gallisepticum 42 170 10 <1: 2 NDV 24 114 9 <1: 2

* Standard serum (Institute of Zooprophylactic, Viennese, Italy)

<7-3> H9N2  Detection timing test after virus challenge

The test was conducted twice. The first test was inoculated with 10 ml of H9N2 virus in 10 male 6-week-old SPF chickens by 0.1 ml of 10 ^8.1 EID _{50 /} /0.1 ml, and the blood was collected by different individuals at 20-day intervals. The presence of the antibody was detected by the kit method. At this time, the presence of the antibody was detected by the HI method, which is an existing standard method, and compared with the method of the present invention.

The second test was inoculated intranasally with 6 ml of 6-week-old SPF chickens using 10 ^4.5 EID _{50 /} /0.1 ml of virus titer lower than that of the first test, and then collected on 4, 7, 11 and 14 days. The presence of the antibody was detected using the diagnostic kit of the present invention. At this time, the presence of the antibody was detected by the HI method, which is an existing standard method, and compared with the method of the present invention. As a comparative test, the HI method proceeded according to the procedure of OIE. Dilute 25 ul of each serum to a V buttom plate, add 4 HAU of H9N2 virus in the same amount, and react for 30 minutes at room temperature. Add 25 ul of 1% chicken erythrocytes and leave for 40 minutes at room temperature. The highest dilution factor that did not occur was calculated by titer.

As a result of the experiment, both the groups inoculated with 10 ^8.1 EID _{50 /} 0.1ml and 10 ^4.5 EID _{50 /} /0.1ml by nasal challenge were able to observe the antibody positive phenomena with the diagnostic kit of the present invention from the 4th day of inoculation. Therefore, the diagnostic kit of the present invention was able to confirm that the antibody can be detected similar to the existing HI method (Fig. 3 and Table 7).

Antibody changes after H9N2 challenge Diagnostic Results According to the Invention HI Days after vaccination Absorbance PI value NC 2.511 2.388 PC 0.007 0.010 day 0-1 2.091 15 0 day 0-2 2.140 13 0 day 0-3 1.542 37 0 day 4-1 1.099 55 2 day 4-2 0.865 65 3 day 4-3 1.648 33 2 day 7-1 0.140 94 4 day 7-2 0.019 99 7 day 7-3 0.019 99 5 day 11-1 0.178 93 10 day 11-2 0.040 98 10 day 11-3 0.003 100 10 day 14-1 0.022 99 10 day 14-2 -0.018 101 10 day 14-3 0.515 79 10 blank 2.291 6

-Inoculation: 10 ^4.5 EID ₅₀ /0.1ml, 0.1ml, / IN

<7-4> H9N2  vaccine After inoculation  Antibodies Test for Change

H9N2 Vaccine SPF Chicken After inoculation at 12 water, the change in antibody was measured using the diagnostic kit of the present invention, and the control group was placed at 3 water. At this time, the antibody was also measured by the HI method, which is the existing standard method, and compared with the diagnostic kit of the present invention.

The vaccine disclosed in the trial was a vaccine in which an inactivated H9N2 virus (2 (10) HAU) was mixed with an AlOH ₃ gel, which was inoculated into the muscle 0.5 ml. Two weeks later, a booster was given. Blood collection for antibody titration was performed before and 14 days after the first inoculation and two weeks after the second inoculation.

As a result of the experiment, antibodies were detected from 14 days after the first inoculation after inoculation with the H9N2 vaccine (Table 8). However, although it is expected to be able to detect enough before that, since the specimens were obtained from 14 days after inoculation, the antibody positiveness before the test could not be confirmed.

 Antibody after H9N2 vaccination results of a measurement Serum number Diagnostic Kit of the Invention HI (2 ⁿ ) Absorbance Judgment Titer Judgment Before vaccination-1 2.665 voice One voice Before vaccination-2 2.660 voice 2 voice Before vaccination-3 2.578 voice One voice Before inoculation-4 2.348 voice 2 voice Pre-inoculation-5 2.553 voice 2 voice 2 weeks after the first vaccination 0.053 positivity 7 positivity 2 weeks after the first vaccination-2 0.055 positivity 10 positivity 2 weeks after the first vaccination 0.029 positivity 9 positivity 2 weeks after the first dose-4 0.025 positivity 9 positivity Two weeks after the first shot-5 0.022 positivity 8 positivity Two weeks after the first vaccination-6 0.279 positivity 9 positivity 2 weeks after the first vaccination-7 0.039 positivity 5 positivity 2 weeks after the first dose-8 0.026 positivity 9 positivity 2 weeks after the first vaccination-9 0.029 positivity 8 positivity Two weeks after the first vaccination-10 0.022 positivity 9 positivity Two weeks after the first vaccination-11 0.058 positivity 7 positivity Two weeks after the first dose-12 0.026 positivity 8 positivity Two weeks after the first vaccination-control-1 2.615 voice 2 voice Two weeks after the first vaccination-control-2 2.625 voice One voice Two weeks after the first vaccination-control-3 2.586 voice 2 voice 2 weeks after the 2nd vaccination 0.035 positivity 10 positivity 2 weeks after the 2nd vaccination-2 0.023 positivity 9 positivity 2 weeks after the 2nd vaccination 0.053 positivity 7 positivity 2 weeks after the 2nd vaccination-4 0.040 positivity 10 positivity 2 weeks after the 2nd vaccination-5 0.013 positivity 12 positivity 2 weeks after the 2nd vaccination-6 0.055 positivity 9 positivity 2 weeks after the 2nd vaccination-7 0.020 positivity 11 positivity 2 weeks after the 2nd vaccination-8 0.036 positivity 10 positivity 2 weeks after the 2nd vaccination-9 0.013 positivity 10 positivity 2 weeks after the 2nd vaccination-10 0.021 positivity 9 positivity 2 weeks after the 2nd vaccination-11 0.044 positivity 11 positivity 2 weeks after the 2nd vaccination-control-1 2.728 voice 0 voice 2 weeks after the 2nd vaccination-control-2 2.845 voice One voice 2 weeks after the 2nd vaccination-control-3 2.690 voice 2 voice

<7-4> H5N1  Testing for Changes in Antibodies After Vaccination

Intramuscularly H5N1 vaccine on chicken, 5 ducks and 8 geese 0.5 ml each was inoculated. The chickens were then inoculated at 10 and 28 days of inoculation, and the geese and ducks were collected at 30 days and the diagnostic kit of the present invention or the existing commercial kit (Synbiotics kit, Avivac kit, all indirect enzyme immunoassay ELISA) Kit) and HI test for H5.

As a result, all of the chickens, goose, and ducks inoculated with the H5N1 vaccine were able to detect antibodies. The chickens were able to detect the goose and the ducks on the 30th day after 10 days (Table 9). Of course, the chicken 10 days before, geese and ducks 30 days before it was determined that it can be detected, but there was no specimen disclosed in the test could not be confirmed. On the other hand, the existing commercial ELISA kits could not be tested because only the chicken serum can be used, the diagnostic kit of the present invention was available for all livestock and the results also appeared similar to the existing HI method.

Antibody results after H5N1 vaccination No. Serum History Invention Pos≥51 Synbiotics, Pos≥338 EU, Avivac, Pos≥15 H5 HI titer, Pos≥1: 16 One Chickens, 10 days after vaccination 63 2242 36 1: 8 2 Chickens, 10 days after vaccination 64 810 37 1: 8 3 Chickens, 10 days after vaccination 68 3358 10 1: 8 4 Chickens, 10 days after vaccination 71 1049 25 1:32 5 Chickens, 10 days after vaccination 64 1659 38 1: 8 6 Chickens, 28 days after vaccination 73 8475 72 1: 128 7 Chickens, 28 days after vaccination 90 7427 59 1: 128 8 Chickens, 28 days after vaccination 91 5622 57 1: 256 9 Chickens, 28 days after vaccination 92 4199 28 1: 256 10 Chickens, 28 days after vaccination 93 1355 18 1: 256 11 Goose, 30 days after vaccination 80 Not tested Not tested 1: 8 12 Goose, 30 days after vaccination 84 Not tested Not tested 1:32 13 Goose, 30 days after vaccination 82 Not tested Not tested 1:16 14 Goose, 30 days after vaccination 85 Not tested Not tested <1: 2 15 Goose, 30 days after vaccination 82 Not tested Not tested 1: 8 16 Goose, 30 days after vaccination 83 Not tested Not tested 1: 8 17 Goose, 30 days after vaccination 67 Not tested Not tested 1:16 18 Goose, 30 days after vaccination 81 Not tested Not tested 1:64 19 Duck, 30 days after vaccination 12 Not tested Not tested 1: 8 20 Duck, 30 days after vaccination 84 Not tested Not tested <1: 2 21 Duck, 30 days after vaccination 87 Not tested Not tested 1: 8 22 Duck, 30 days after vaccination 86 Not tested Not tested 1:16 23 Duck, 30 days after vaccination 88 Not tested Not tested 1:16

<7-5> By breeder  Sensitivity and Specificity Measurements

In order to confirm whether the diagnostic kit according to the present invention is applicable to all animal species, sensitivity and specificity of each animal species were checked. At this time, the diagnostic kit of the present invention was compared with the ELISA and HI method of the Italian IZS. Using the diagnostic kit according to the present invention, the sensitivity and specificity are plotted by the TG-ROC method based on the antibody titer tested for each animal and the antibody titer tested by the conventional HI method. PI values were established when the sensitivity and specificity were not high either.

On the other hand, in general, the sensitivity and specificity of the 95% or more is very good, it is judged that the utilization is more than 80%, the sensitivity and specificity of the diagnostic kit of the present invention appeared to be mostly excellent level for each animal 12 It can be confirmed that the species can be used in animals (Table 10).

Sensitivity and specificity of each diagnostic kit according to the present invention Breeder PI value * Test HI contrast IZS ELISA responsiveness(%) % Specificity responsiveness(%) % Specificity duck 50 1,031 95.4 98.5 Not tested Not tested Turkey 85 213 97.1 100 97.1 100 pig 50 266 95.2 97.4 Not tested Not tested chicken 50 1,613 98.2 97.3 Not tested Not tested goose 50 25 100 86.7 100 100 Guinea Falls 50 19 87.5 100 87.5 100 Quail 50 46 100 97 100 78 Gray Partridge 50 38 75 94.1 100 100 Red Partridge 50 5 Not tested 100 Not tested 100 pheasant 50 18 100 66.7 100 75 Swan 50 4 50 Not tested 100 100 Words 500 63 Not testd Not testd 100 Not testd

* If PI value is 50 standard, if PI value is 50 or more, positive, if less than 50,

Figure 1 illustrates the diagnostic method of the present invention based on the principle of competitive enzyme immunoassay.

2 is a result of testing the specificity of the monoclonal antibody for diagnosing avian influenza antigen according to the present invention using a dot immunoassay (Dot immunoassay).

Figure 3 is a graph showing the results of measuring the change in antibody titer after H9N2 challenge inoculation using the diagnostic kit according to the present invention.

Inoculation amount: 10 ^8.1 EID ₅₀ /0.1ml, 0.1ml, / IN

<110> National Veterinary Research and Quarantine Service (NVRQS) <120> Diagnostic kit for avian influenza virus antibody using          competetive ELISA and diagnostic method using the same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 1 agcaaaagca gggttaataa tcac 24 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 2 agtagaaaca agggtatttt tcctc 25 <210> 3 <211> 1565 <212> DNA <213> Avian Influenza Virus (A / CK / Korea / MS96 / 96 (H9N2)) <220> <221> gene (222) (1) .. (1565) <223> Nucleoprotein <400> 3 agcaaaagca gggttaataa tcactcactg agtgacatca aaatcatggc gtcccaaggc 60 accaaacggt cttatgaaca gatggaaact gatggggatc gccagaatgc aactgagatt 120 agggcatccg tcgggaagat gattgatgga attgggagat tctacatcca aatgtgcact 180 gaacttaaac tcagtgatca tgaagggcgg ttgatccaga acagcttgac aatagagaaa 240 atggtgctct ctgcttttga tgaaagaagg aataaatacc tggaagaaca ccccagcgcg 300 gggaaagatc ccaagaaaac tggggggccc atatacagga gagtagatgg aaaatggatg 360 agggaactcg tcctttatga caaagaagaa ataaggcgaa tctggcgcca ggccaacaat 420 ggtgaggatg cgacagctgg tctaactcac ataatgatct ggcattccaa tttgaatgat 480 gcaacatacc agaggacaag agctcttgtt cgaactggaa tggatcccag aatgtgctct 540 ctgatgcagg gctcgactct ccctagaagg tccggagctg caggtgctgc agtcaaagga 600 atcgggacaa tggtgatgga actgatcaga atggtcaaac gggggatcaa cgatcgaaat 660 ttctggagag gtgagaatgg gcggaaaaca agaagtgctt atgagagaat gtgcaacatt 720 cttaaaggaa aatttcaaac agctgcacaa agagctatgg tggatcaagt gagagaaagt 780 cggaacccag gaaatgctga gatcgaagat ctcatatttt tggcaagatc tgcattgata 840 ttgagagggt cagttgctca caaatcttgc ctacctgcct gtgcgtatgg acctgcagta 900 tccagtgggt acgacttcga aaaagaggga tattccttgg tgggaataga ccctttcaaa 960 ctacttcaaa atagccaaat atacagccta atcagaccta acgagaatcc agcacacaag 1020 agtcagctgg tgtggatggc atgccattct gctgcatttg aagatttaag attgttaagc 1080 ttcatcagag ggacaaaagt atctcctcgg gggaaactgt caactagagg agtacaaatt 1140 gcttcaaatg agaacatgga taatatggga tcgagcactc ttgaactgag aagcgggtac 1200 tgggccataa ggaccaggag tggaggaaac actaatcaac agagggcctc cgcaggccaa 1260 accagtgtgc aacctacgtt ttctgtacaa agaaacctcc catttgaaaa gtcaaccatc 1320 atggcagcat tcactggaaa tacggaggga agaacttcag acatgagggc agaaatcata 1380 agaatgatgg aaggtgcaaa accagaagaa gtgtcattcc gggggagggg agttttcgag 1440 ctctcagacg agaaggcaac gaacccgatc gtgccctctt ttgatatgag taatgaaggg 1500 tcttatttct tcggagacaa tgcagaagag tacgacaatt aaggaaaaat acccttgttt 1560 ctact 1565  

Claims (16)

(a) an influenza antigen adsorption plate in which a recombinant nucleoprotein antigen is bound to a microplate in which a monoclonal antibody to a nucleoprotein of avian influenza virus is adsorbed; And (b) A diagnostic kit for avian influenza virus antibodies comprising a monoclonal antibody against an avian influenza virus nucleoprotein and an enzyme-labeled antibody conjugated with an enzyme. The kit according to claim 1, further comprising a negative control serum, a positive control serum, a wash solution, a substrate solution for measuring enzyme activity, and an enzyme reaction stop solution. The kit according to claim 1, wherein the avian influenza virus is a domestic influenza avian influenza (A / CK / Korea / ES / 03 (H5N1)). The kit of claim 1, wherein the monoclonal antibody is specific for H1 to H15 type viruses. The method of claim 1, wherein the recombinant nucleoprotein is prepared by introducing a nuclear protein gene cloned from avian influenza virus (A / CK / Korea / MS96 / 96 (H9N2)) into baculovirus and then inoculated it into insect cells And characterized in that the kit. The kit according to claim 5, wherein the nucleoprotein gene has a nucleotide sequence represented by SEQ ID NO: 3. The method of claim 1, wherein the enzyme is β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycose oxidase, hexokinase, malate dehydro Kit, characterized in that selected from the group consisting of genease, glucose-6-phosphate dihydrogenase and invertase. 8. The kit of claim 7, wherein said enzyme is a peroxidase. The method of claim 1, wherein the antibody for detecting an avian influenza virus is detected in any one selected from the group consisting of duck, turkey, pig, chicken, goose, guinea pole, quail, gray partridge, red partridge, pheasant, swan and horse. Kit characterized in that. The test sample and the enzyme-labeled antibody of claim 1 are added simultaneously to the avian influenza antigen adsorption plate of claim 1 to allow the antibody and the enzyme-labeled antibody to react competitively, and then to determine the presence of the antibody in the test sample by adding a colorant. A diagnostic method for avian influenza virus antibody, characterized in that. The method of claim 10, further comprising: (a) dispensing the test sample, negative control serum and positive control serum into the influenza antigen adsorption plate of claim 1, and simultaneously treating and reacting the enzyme-labeled antibody of claim 1; (b) washing the plate in which the reaction is completed in step (a) and treating the substrate solution; And (c) treating the reaction stop solution to the plate of step (b) and measuring the absorbance. 12. The method according to claim 11, wherein the negative control serum in step (a) is tested using 3 wells and the average absorbance value is 1.8000 or more. 12. The method of claim 11, wherein the positive control serum of step (a) is tested using two wells and has an average absorbance value of 0.2000 or less. 12. The method of claim 11, further comprising calculating a PI value from the absorbance of the test sample measured in step (c) using the following equation. PI value = [1- (sample absorbance / negative control mean absorbance)] X 100 15. The method according to claim 14, characterized in that it is determined as positive when the calculated PI value is greater than or equal to the criterion value and negative when less than. The method of claim 15, wherein the criterion is 50 for chicken, duck, quail, goose, guinea pole, gray partridge, red partridge, pheasant, swan, horse and pig, and 85 for turkey. Way.

Images