KR20120022287A - Elisa system and the kit on the n3 protein of low pathogenic avian influenza virus - Google Patents

Abstract

PURPOSE: A composition for detecting N3 protein of low pathogenic avian influenza virus is provided to accurately distinguish H9N2 positive group and H9N3 vaccine group. CONSTITUTION: A composition for detecting neuraminidase N3 protein of low pathogenic avian influenza virus contains a complementary monoclonal antibody of sequence number 2. An ELISA kit for detecting neuraminidase N3 protein of low pathogenic avian influenza virus contains the composition. An ELISA method for detecting the neuraminidase N3 protein comprises: a step of binding neuraminidase N3 protein antigen of sequence number 1 to a plate; and a step of binding the complementary monoclonal antibody to a blood sample and neuraminidase N3 protein.

Description

ELISA system and the kit on the N3 protein of Low Pathogenic Avian Influenza virus}

The present invention is directed to detecting N3 protein of a low pathogenic avian influenza virus, which comprises a competition monoclonal antibody against neuraminidase N3 of the Low Pathogenic Avian Influenza (LPAI) virus, represented by SEQ ID NO: 2. A composition for use, an enzyme immunoassay kit, and an enzyme immunoassay method. Accordingly, the H9N2 positive group and the H9N3 vaccination group, including field infection, were correctly distinguished.

Influenza is caused by an infection of the Influenza virus. Influenza viruses are RNA viruses and belong to the orthomyxovirus. Virus types are classified into A, B, and C types according to the antigenicity of nucleoprotein (NP) and matrix protein, which accounts for 35 to 45% of viral particles (Palese P, Young JF. Variation of influenza A, B and C viruses.Science, 215: 1468-1474, 1982., Schild GC, Oxford JS, Newman RW.Evidence for antigenic variation in influenza A nucleoprotein.Virology, 93: 569-557, 1981.) Among these, influenza A virus is subtyped by surface antigens hemagglutinin (HA) and neuraminidase (NA). In HA, there are 16 subtypes (H1-H16) that the virus attaches to somatic cells, and in NA there are 9 subtypes (N1-N9) (Rohm C, Zhou N, Suss J, et al. Characterization of a novel influenza hamegglutinin, H15: criteria for determination of influenza A subtypes.Virology, 217: 508-516, 1996.). HA subtypes that occur mainly in humans are H1, H2, H3, and NA types are N1, N2. As well as humans of all ages, pigs and birds can be infected.

Low Pathogenic Avian Influenza (LPAI) virus in Korea is H9 type, and first occurred in five farms in three regions, including Hwaseong, Gyeonggi-do in March 1996, and has spread to laying hen farms throughout the country. And various deaths have caused enormous losses to the poultry industry. Control of low-pathogenic avian influenza has been very urgent, but inoculation of live vaccines has the potential to cause viral mutations, low efficacy of deadly poison vaccines, inability to block horizontal spread, and incompatibility between outdoor and vaccine strains. Due to the shortcomings of vaccination in the domestic poultry industry, vaccination was not actively promoted, but the economic loss caused by low-pathogenic avian influenza was mitigated since March 2007.

However, domestic isolates and vaccinates are H9N2, the same serotype, and the vaccination group and the field infection group are not distinguishable serologically, making it more difficult to predict and control low-pathogenic avian influenza than before vaccination. Therefore, DIVA (Differentiation Infected from Vaccinated Animals) using recombinant virus as a vaccine strain that maintains protection against H9N2 by maintaining H9 serotype that is directly linked to defense and can be used as an indicator of differentiation by different type N The need for successful disease control by the method has been raised.

DIVA methods for controlling low pathogenic avian influenza include H7N1 and H7N3 inactivated vaccines, which are currently applied in Italy, and iIFAT (indirect fluorescent antibody method) for detecting N1 and N3. However, iIFAT is inadequate for large-scale inspection due to the disadvantages such as time-consuming inspection and lack of objectivity in analyzing results. Therefore, it is necessary to develop a serum test suitable for large-scale tests such as ELISA.

In general, the enzyme-linked immunosorbent assay (ELISA) system is a method of binding an enzyme to an antibody to confirm an antigen-antibody reaction. The method is simple, inexpensive, and can be analyzed in large quantities. It has become one of the widely used antigen-antibody assays. In particular, it is a method of increasing its use because it has a very sensitive reaction such as RIA (Radio ImmunoAssay) but does not use radioactivity as in RIA.

The inventors have developed a serological diagnostic kit that can detect N3 protein to accurately distinguish between H9N2 positive group and H9N3 vaccination group, including field infection.

The present invention is a composition for detecting N3 protein of low pathogenic avian influenza virus, enzyme immunoassay, comprising a competitive monoclonal antibody against neuraminidase N3 type of low pathogenic avian influenza (LPAI) virus Kits and enzyme immunoassay methods.

The present invention relates to a composition for detecting neuraminidase N3 protein of a low pathogenic avian influenza virus comprising a competitive monoclonal antibody represented by SEQ ID NO: 2.

Another aspect of the present invention is an enzyme immunoassay kit for detecting a neuraminidase N3 protein of a low pathogenic avian influenza virus, comprising the composition for detection.

Another aspect of the invention provides a method for binding a neuraminidase N3 protein antigen represented by SEQ ID NO: 1 to a plate; And

A neuralinase N3 of the low pathogenic avian influenza virus, characterized in that it comprises the step of binding a competitive blood sample and a competitive monoclonal antibody against the neuraminidase N3 protein represented by SEQ ID NO: 2 to the bound antigen. It is an enzyme immunoassay for protein detection.

In the enzyme immunoassay, the antigen concentration is preferably 25ng / well to 300ng / well.

In addition, in the enzyme immunoassay method, it is preferable that the blood sample is undiluted serum or serum diluted to a concentration of 1: 800.

Hereinafter, the present invention will be described in more detail.

The ELISA system can be classified into four types: direct ELISA, indirect ELISA (i-ELISA), competitive ELISA (c-EILSA), and sandwich ELISA. The direct method refers to a method of binding an antigen to a plate and binding an enzyme directly to an antibody that reacts with the antigen. The indirect method binds an antigen to a plate and then binds the antibody that reacts with the antigen. Refers to a method in which an enzyme is bound to an antibody and attached to an enzyme-linked antibody that reacts with the antibody. Competition method refers to the binding of the antigen to the plate and the antibody that reacts with the antigen, the antibody in combination with the pre-cultured antigen is bound, and the enzyme is bound to the bound antibody and the enzyme-linked antibody reacts with the antibody. . In addition, the sandwich method refers to the test by first binding the antibody to the plate and then the antigen that reacts with the antibody, and then attaches an enzyme-binding antibody that reacts with the antigen by binding an enzyme to the bound antigen.

The present invention provides a diagnostic kit for distinguishing the outdoor infection group and the vaccination group. That is, a diagnostic kit using N3 protein as an antigen was prepared to specifically detect only antibodies against neuraminidase of H9N3.

In addition, the present invention prepared a c- ELISA kit for detecting antibodies against the neuraminidase N3 type of Low Pathogenic Avian Influenza (LPAI) virus.

To prepare the ELISA, the concentration of neuraminidase N3 protein to be used in the ELISA was determined by checkerboard titration, and the appropriate dilution factor of test serum or monoclonal antibody was obtained and normalized to ELISA. In addition, a positive determination reference value was obtained through ELISA for negative sera, and when a value higher than the reference value was obtained, it was determined that the antibody contained an antibody to the analysis target protein.

In the present invention, instead of the H9N2 vaccine, a recombinant H9N3 inactivating vaccine was inoculated to distinguish the H9N2 field infection group from the vaccination group. That is, the vaccinated group inoculated with the H9N3 inactivating vaccine and the H9N2 field infection group were divided by using the same H (H9) serotype but different N serotypes. At this time, a composition and kit for detecting an antibody against N3 were used, which responded exclusively to different N serotypes, respectively.

Through the N3 c- ELISA according to the present invention, it was possible to effectively distinguish between the H9N2 vaccinated group or the infected group and the H9N3 vaccinated group, it was also possible to detect the group inoculated with the H5N3 test vaccine.

When applying the recombinant H9N3 inactivated vaccine together with the N3 c -ELISA kit developed in this study, it is possible to control the disease using the DIVA method and eradicate H9N2 type low pathogenic avian influenza in Korea. Will be able to advance

1 shows the results of screening competition antibodies from MAb clones using N2 / N3 i- ELISA.
2 shows the results of screening for competing antibodies using N3 c- ELISA.
3 shows the results of optimization experiments for N3 antigen dilution by tile titration.
4 shows the results of optimization experiments on competitive MAb dilutions by tile titration.
Figure 5 shows the N3 c- ELISA results for the change in PI value of the diluted serum, the horizontal baseline shows a positive determination reference value.
Fig. 6 shows the distribution of PI values of SPF chickens with or without inoculation with H9N3 or H9N2 as a result of N3 c- ELISA, and the horizontal baseline shows a positive determination reference value.
7 is N3 c -ELISA result, represents a PI value of a normal distribution Laying farm not inoculated or inoculated with the vaccine, the horizontal reference line shows a positive determination reference value.

The present invention is described in more detail based on the following examples. The following examples are intended to illustrate the invention, not to limit the invention. All documents disclosed in the present invention are incorporated by reference.

Example

Example 1 Preparation of H9N3 Recombinant Virus

Avian Influenza virus (AIV) H5N1 subtype antiserum (National Veterinary Research and Quarantine Service (NVRQS), Anyang) and ADL0408 (H5N3) virus were mixed and reacted at 37 ° C. 0.1 ml of the reaction mixture and 0.1 ml of A / Chicken / Korea / VI072378 / 2007 (H9N2) virus were mixed and cultured in 10-day-old specific pathogen free (SPF) eggs.

HA test, HI test and quantitative real-time PCR (RT-PCR) were performed to select the urea solution showing the closest result to H9N3 and passaged to SPF eggs.

Subsequent passages over 30 times were performed using genetic tests and antisera, followed by H9N3 virus production and proliferation in chickens was also confirmed by sequencing of the virus released after challenge. The prepared H9N3 virus was deposited on May 13, 2010 to the Korea Institute of Bioscience and Biotechnology (KCTC 11697BP).

Example 2 Preparation of Zadok Vaccine

Recombinant virus was purified using limiting dilution for use as a vaccine strain. That is, after diluting the recombinant virus culture with 10 decimals and culturing them in SPF eggs, the urinary fluid of maximum dilution factor showing hemagglutination was collected. The collected urinary cavity fluid was diluted 10 times and cultured in SPF eggs. This serial passage of the virus was used as a seed venom for vaccine production. After inactivating the virus culture with formalin, the virus titer was appropriately adjusted, and then mixed with an adjuvant to prepare a dead poison oil vaccine. It was.

Example 3. Extraction of N3 Protein Using Gel Chromatography

H9N3 virus prepared by the method of Example 1 was inoculated into 600 specific pathogen free (SPF) oocytes 9-11 days old to purify the NA protein to be used as an antigen in the ELISA method. After inoculation, the incubator was incubated for 40 hours to collect allantoic fluid. As a result, 7,000 ml of the virus culture was obtained. The culture was passed through a column filled with Superose 12 prep grade gel (GE Healthcare Inc., UK) through concentration and pronase treatment. PBS (pH. 7.4) was used as a solvent of the gel chromatography, and the eluent was collected by dropping 20 drops in one tube to collect a total of 100 tubes. In order to extract the NA-containing tube from the tube containing the separation solution, the proteins were separated by size by SDS-PAGE and confirmed by coomasie staining. The sequence number of N3 is shown in SEQ ID NO: 1.

Example 4 Development of an N3 Competition ELISA Kit

Example 4-1. Preparation of monoclonal antibodies for use with N3 competition-ELISA and selection of competing antibodies

N3 protein purified by the gel chromatography method of Example 3 was immunized with mice, and nine monoclonal antibodies (MAbs) were obtained from spleen cells. Four clones (3D6, 6F12, 3C8, 1C9) were selected as the cell culture medium of these clones by N2 / N3 i- ELISA and low titer to N2, but high titer to N3. 1). These antibodies were combined with biotin and then used as competing antibodies in the experiment of N3 c -ELISA.

N3 competition-ELISA ( c- ELISA) test using positive serum from H9N3 vaccination chicken as a test serum resulted in a percent inhibition of 3C8, 6F12, 1C9, and 3D6, respectively. %, 1.6%, 1.0%, 5.3% was confirmed (Fig. 2). The 3C8 showing the highest PI value was selected as the competing MAb. The sequence of 3C8 is shown in SEQ ID NO: 2.

Example 4-2. N3 c -ELISA standardization test

A checkerboard titration was performed to determine the optimal concentration of N3 protein to be used as antigen in N3 c -ELISA. As a result, no saturation point was found even at 50ng / well. 3) Since the result of c- ELISA was very unstable when the antigen was lowered to a concentration of 25ng / well or less, 50ng / well of the antigen was used.

In addition, as a result of performing a tile titration method to determine the appropriate dilution fold of the competitive MAb to be used for c- ELISA, a concentration of 1: 1000 was determined to be appropriate (FIG. 4). To determine the optimal concentration of test serum, c- ELISA was performed after diluting the SPF family sera inoculated with recombinant H9N3 inactivated vaccine and the non-vaccinated SPF family sera up to 128-fold, starting with the stock solution. . As a result, the PI values of undiluted serum were 72% and 52%, but the 2-fold dilution of serum decreased the PI value to 50% and 19%, respectively. Inhibition ability was almost lost. In view of the above results, it was judged that test serum was appropriate to use undiluted stock solution (FIG. 5).

N3 c -ELISA test was performed as follows. The purified N3 protein was diluted with PBS containing 0.05% sodium azide, 50ul was dispensed into each well of the ELISA plate, and then reacted at 4 ° C for 16 hours to coat the antigen. The coated ELISA plate was used after washing five times with sterile distilled water. 50 μl of test serum was dispensed into each well of an ELISA plate coated with N3 protein, and then 50 μl of competitive MAb diluted to an appropriate concentration was reacted at 37 ° C. for 1 hour. After washing five times with sterile distilled water, 100ul of peroxidase-labeled streptavidin was dispensed into each well and reacted at 37 ° C for 1 hour. The ELISA plate was washed five times again with sterile distilled water, and then 100 μl of TMB Microwell Peroxidase Substrate (KPL, USA) was dispensed into each well for 15 minutes at room temperature. Then 1M H ₃ PO ₄ was added to stop the reaction and the absorbance was measured at 450nm.

Example 4-3. Calculation of PI and Positive Criteria of N3 Competitive ELISA

N3 c- ELISA was performed on the sera of the group that had not been vaccinated and did not detect antibodies in the HI test as the serum was negative. Percent inhibition (PI) values were calculated by substituting the measured absorbance into the following formula.

 * PI = 100-OD (optical density) value of test sample / OD value of negative control x 100

The mean PI value and the deviation of the negative serum were obtained, and the positive reference value was calculated by the following equation.

 * Criterion of determination = mean percent inhibition of negative serum + 2 x standard deviation

As a result of 50 negative serum tests, the positive reference value of PI value was 6.13%.

Example 4-4. Efficacy test of N3 competition ELISA

In order to investigate whether the N3 c- ELISA established in this study can detect the group vaccinated with the H9N3 vaccine, the c- ELISA was performed using the SPF family and the outdoor farm serum according to the conditions and methods established in this study. Table 1, Table 2).

Table 1 below shows the positive rate of N3 c-ELISA for SPF chickens inoculated with H9N3 or N9N2. In Table 1 below, H5N1 control serum was obtained from the National Veterinary Quarantine Service (NVRQS).

group Serum water Vaccination Age Sampling age N3 c -ELISA Unvaccinated 10 Week 7 1/10 H9N3 vaccine 10 4 Weeks Week 7 7/10 H9N2 vaccine 4 4 Weeks Week 7 0/4 H5N1 control serum 2 0/2

Table 2 below shows the positive rate of N3 c-ELISA for the layer laid with H9N3 or N9N2. At this time, all chicken breeds are Hyline brown.

Farm Flock size Vaccination age (week) Sampling age (week) group N3 c -ELISA A 28,000 17 21 Unvaccinated 1/10 - 21 Inoculation (H9N3) 6/10 B 80,000 18 30 Unvaccinated 0/10 - 30 Inoculation (H9N3) 9/10 C 20,000 10 14 Unvaccinated 1/10 - 14 Inoculation (H9N3) 9/10 D 20,000 - 54 Before infection 0/10 - 60 Infection (H9N2) 0/10 E 20,000 16 14 Before vaccination 0/10 - 32 Inoculation (H9N2) 0/10 F 20 5 16 Unvaccinated 1/9 - 16 Inoculation (H5N3) 10/10 G 20,000 - 10 Negative Control 0/10

The SPF family was inoculated with the recombinant H9N3 inactivated vaccine of Example 2, and blood was collected and subjected to N3 c- ELISA. As a result, 7 out of 10 water samples showed absorbance higher than the positive reference value. N3 c- ELISA was performed after inoculation of the Vaccine Research Institute, Poulshot Pullul H9N2), and all four numbers showed low PI values. In this way, the SPF group was able to effectively distinguish between the H9N2 vaccination group and the H9N3 vaccination group by the N3 c- ELISA method (Fig. 6, Table 1).

In addition, 24 out of 30 sera of three farms inoculated with H9N3 vaccine showed positive PI values. In addition, the average PI values of the H9N2 infected group and the H9N2 vaccinated group were -2.89 and -2.71, respectively, which were lower than the average PI value of the non-vaccinated group, -0.13. In the above experimental results, the N3 c- ELISA kit accurately distinguished the H9N2 positive group and the H9N3 vaccination group including the outdoor infection (Fig. 7, Table 2).

The blood samples from the H5N3 inactivated vaccine group were tested by N3 c- ELISA method, and all 10 cells showed positive PI values, whereas the PI value of H5N1 positive sera was -0.31. All of the negative values were lower than the PI value of -0.13. Based on the above experimental results, it was judged that the N3 c- ELISA kit could be effectively used for the detection of the group inoculated with the H5N3 test vaccine (FIG. 8, Table 2).

Although the above has been described with reference to a preferred embodiment of the present invention, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention described in the claims below. It will be appreciated.

Korea Research Institute of Bioscience and Biotechnology KCTC11697BP 20100513

<110> Chungbuk National University Industry-Academic Cooperation Foundation          Choong Ang Vaccine Lab. <120> ELISA system and the kit on the N3 protein of Low Pathogenic          Avian Influenza virus <130> IPDC36934 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 450 <212> PRT <213> Avian influenza virus <400> 1 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Val Val Asn Thr Thr   1 5 10 15 Leu Ser Ile Ile Ala Leu Leu Ile Gly Val Gly Asn Leu Ile Phe Asn              20 25 30 Thr Val Ile His Asp Lys Ile Gly Asp His Gln Thr Val Val Tyr Pro          35 40 45 Thr Thr Ile Ala Pro Val Val Pro Asn Cys Ser Asp Thr Ile Arg Ala      50 55 60 Glu Thr His Phe Lys Ser Ser Leu Pro Leu Cys Pro Phe Arg Gly Phe  65 70 75 80 Phe Pro Phe His Lys Asp Asn Ala Ile Arg Leu Gly Glu Asn Lys Asp                  85 90 95 Val Ile Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Asn Asp Asp Cys             100 105 110 Trp Ser Phe Ala Leu Ala Gln Gly Ala Leu Leu Gly Thr Lys His Ser         115 120 125 Asn Gly Thr Ile Lys Asp Arg Thr Pro Tyr Arg Ser Leu Ile Arg Phe     130 135 140 Pro Ile Gly Thr Ala Pro Val Leu Gly Asn Tyr Lys Glu Ile Cys Val 145 150 155 160 Ala Trp Ser Ser Ser Ser Cys Phe Asp Gly Lys Glu Trp Met His Val                 165 170 175 Cys Met Thr Gly Asn Asp Asn Asp Ala Ser Gly Gln Ile Met Tyr Ala             180 185 190 Gly Lys Met Thr Asp Ser Ile Lys Ser Trp Arg Lys Asp Ile Leu Arg         195 200 205 Thr Gln Glu Ser Glu Cys Gln Cys Ile Asp Gly Thr Cys Val Val Ala     210 215 220 Val Thr Asp Gly Pro Ala Ala Asn Ser Ala Asp His Arg Ile Tyr Trp 225 230 235 240 Ile Arg Glu Gly Lys Val Ile Lys Tyr Glu Asn Ile Pro Lys Thr Lys                 245 250 255 Ile Gln His Leu Glu Glu Cys Ser Cys Tyr Val Asp Ile Asp Val Tyr             260 265 270 Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Trp Met Arg         275 280 285 Ile Asn Asn Glu Thr Ile Leu Glu Thr Gly Tyr Val Cys Ser Lys Phe     290 295 300 His Ser Asp Thr Pro Arg Pro Ala Asp Pro Ser Thr Val Ser Cys Asp 305 310 315 320 Ser Pro Ser Asn Val Asn Gly Gly Pro Gly Val Lys Gly Phe Gly Phe                 325 330 335 Lys Thr Gly Asn Asp Val Trp Leu Gly Arg Thr Val Ser Thr Ser Gly             340 345 350 Arg Ser Gly Phe Glu Ile Ile Lys Val Thr Glu Gly Trp Ile Asn Ser         355 360 365 Pro Asn His Ala Lys Ser Val Thr Gln Thr Leu Val Ser Asn Asn Asp     370 375 380 Trp Ser Gly Tyr Ser Gly Ser Phe Ile Val Glu Asn Asn Gly Cys Phe 385 390 395 400 Gln Pro Cys Phe Tyr Ile Glu Leu Ile Arg Gly Arg Pro Asn Lys Asn                 405 410 415 Asp Asp Val Ser Trp Thr Ser Asn Ser Ile Val Thr Phe Cys Gly Leu             420 425 430 Asp Asn Glu Pro Gly Ser Gly Asn Trp Pro Asp Gly Ser Asn Ile Gly         435 440 445 Phen met     450 <210> 2 <211> 309 <212> PRT <213> mouse <400> 2 Gly Cys Cys Ala Cys Cys Cys Thr Gly Thr Cys Thr Gly Thr Gly Ala   1 5 10 15 Cys Thr Cys Cys Ala Gly Gly Ala Gly Ala Thr Cys Gly Thr Gly Thr              20 25 30 Cys Thr Cys Thr Cys Thr Thr Thr Cys Cys Thr Gly Cys Ala Gly Gly          35 40 45 Gly Cys Cys Ala Gly Cys Cys Gly Gly Ala Gly Thr Ala Thr Thr Ala      50 55 60 Gly Cys Gly Ala Cys Thr Ala Cys Thr Thr Ala Cys Ala Cys Thr Gly  65 70 75 80 Gly Thr Ala Thr Cys Ala Ala Cys Ala Ala Ala Ala Ala Thr Cys Ala                  85 90 95 Cys Ala Thr Gly Ala Gly Thr Cys Thr Cys Cys Ala Ala Gly Gly Cys             100 105 110 Thr Thr Cys Thr Cys Ala Thr Cys Ala Ala Ala Thr Ala Thr Gly Cys         115 120 125 Thr Thr Cys Cys Cys Ala Ala Thr Cys Cys Ala Thr Cys Thr Cys Thr     130 135 140 Gly Gly Gly Ala Thr Cys Cys Cys Cys Thr Cys Cys Ala Gly Gly Thr 145 150 155 160 Thr Cys Ala Gly Thr Gly Gly Cys Ala Gly Thr Gly Gly Ala Thr Cys                 165 170 175 Ala Gly Gly Gly Thr Cys Ala Gly Ala Thr Thr Thr Cys Ala Cys Thr             180 185 190 Cys Thr Cys Ala Cys Thr Ala Thr Cys Ala Ala Cys Ala Gly Thr Gly         195 200 205 Thr Gly Gly Ala Ala Cys Cys Thr Gly Ala Ala Gly Ala Thr Gly Thr     210 215 220 Thr Gly Gly Ala Ala Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr 225 230 235 240 Cys Ala Ala Ala Ala Thr Gly Gly Thr Cys Ala Cys Ala Gly Cys Thr                 245 250 255 Thr Thr Cys Cys Thr Cys Thr Cys Ala Cys Gly Thr Thr Cys Gly Gly             260 265 270 Thr Gly Cys Thr Gly Gly Gly Ala Cys Cys Ala Ala Ala Cys Thr Gly         275 280 285 Gly Ala Ala Cys Thr Gly Ala Ala Ala Cys Gly Gly Gly Cys Thr Gly     290 295 300 Ala Thr Gly Cys Thr 305

Claims (6)

A composition for detecting neuraminidase N3 protein of a low pathogenic avian influenza virus comprising a competitive monoclonal antibody represented by SEQ ID NO: 2.
An enzyme immunoassay kit for detecting a neuraminidase N3 protein of a low pathogenic avian influenza virus, comprising the composition for detection of claim 1.
Binding the neuraminidase N3 protein antigen represented by SEQ ID NO: 1 to the plate; And
A neuralinase N3 of the low pathogenic avian influenza virus, characterized in that it comprises the step of binding a competitive blood sample and a competitive monoclonal antibody against the neuraminidase N3 protein represented by SEQ ID NO: 2 to the bound antigen. Enzyme immunoassay method for protein detection.
The method of claim 3,
An enzyme immunoassay for detecting neuraminidase N3 protein of low pathogenic avian influenza virus, characterized in that the concentration of the antigen is 25ng / well to 300ng / well.
The method of claim 3,
An enzyme immunoassay for detecting neuraminidase N3 protein of low-pathogenic avian influenza virus, characterized in that the blood sample is a serum of a stock solution.
The method of claim 3,
An enzyme immunoassay for detecting the neuraminidase N3 protein of a low pathogenic avian influenza virus, wherein the blood sample is serum diluted to a concentration of 1: 800.

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