KR20120028665A - Antibody for detecting neuraminidase of swine influenza and use thereof - Google Patents

Abstract

PURPOSE: An epitope of swine influenza virus neuraminidase is provided to detect type 1 or 2 of neuraminidase antigen and to easily diagnose infection type. CONSTITUTION: An epitope polypeptide molecule of swine influenza virus neuraminidase contains a polypeptide having amino acid sequence selected from sequence numbers 1-12. A kit for detecting swine influenza virus contains the antibody which specifically recognizes the epitope polypeptide molecule. A method for detecting swine influenza virus comprises: a step of preparing a sample; a step of applying one or more antibodies which specifically recognizes the epitope polypeptide molecule; and a step of testing antigen-antibody reaction.

Description

Antibody for Detecting Neuraminidase of Swine Influenza and Use Thereof}

The present invention provides an epitope of a swine influenza virus neuraminidase, an antibody prepared using the same, an antibody prepared using the same, a detection kit for a swine influenza virus neuraminidase type comprising the antibody, a pig using the antibody It relates to a method for detecting influenza virus neuraminidase type, and a vaccine composition comprising the epitope.

Swine Influenza was first identified as influenza type A H1N1 virus in 1930, and H1N1 virus samples obtained by the US Centers for Disease Control and Prevention (CDC) extracted from the H1N1 influenza virus. The virus has been found to be a mixed species not found in pigs, birds and humans. In the process of combining avian influenza and human influenza with the existing swine influenza virus, a genome modification occurs and a virus spread between pigs is spreading among humans.

Currently, the World Health Organization (WHO) also uses the alert stage as the current pandemic alert stage, with efficient and sustained human and pandemic stages, the highest level in stage 5, where there is meaningful evidence of human-to-human infection. There is still a great deal of international influenza in the country, including a review to upgrade to six levels of human infection. Influenza virus causes diseases in animals such as humans, horses, birds and pigs, and these species have been reported to infect each other.

In particular, the pig tracheal epithelium is a receptor for avian influenza virus (receptor, α-2,3-N-acetylneuraminic acid-galactose linkages) and human influenza virus (receptor, α-2,6-N-acetylneuraminic acid-galactose linkages) are expressed, and this double sensitivity is associated with pigs as a mixing vessel for the emergence of new influenza viruses with pandemic potential in humans.

 The spread of influenza virus species has been studied a lot, and research with great interest is focused on whether swine influenza virus, which has a short life cycle, acts as a reservoir due to less antigenic changes and less mutagenicity than human strains. Recent studies have shown that pandemic was caused by swine-like influenza viruses, and humans have been infected with swine influenza virus.

On the contrary, in some cases, the pigs were infected with human influenza virus, and antibodies to human H1N1 subtype variants were detected in pig serum, and these reports indicate that the spread of influenza virus species has been studied.

The pandemic of influenza A strains may be attributed to the spontaneous reassortment of influenza viruses of human and non-human strains in a common host. Pigs are susceptible to human influenza virus and may serve as potential reservoirs of the H1N1 and H3N2 subtypes. Scholtissek et al suggested that pigs are infected with different subtypes of viruses, and that they are important for the emergence of reassortment virus and the development of new pandemics by exchanging genes with each other during the growth process. These facts suggest that swine influenza virus plays an important role in the development of human influenza virus.

The surveillance system and diagnosis system of avian influenza virus have already been established according to many years of experience. However, the surveillance system and diagnosis system of swine influenza virus have not been developed. In addition, the existing commercialized avian influenza virus diagnostic kit is uncertain because of the low sensitivity (10 ^4.5 ~ 10 ^5.5 EID / mL) and the possibility of nonspecific reaction of pig specimens.

Therefore, it is urgent to establish a continuous on-site monitoring system for swine influenza virus, and to establish a continuous surveillance system for swine influenza virus that can be generated from pigs by analyzing the change pattern and the extent of infection of the swine influenza virus by region and time. It is required to do

In response to this need, one example of the present invention provides an antigenic specific epitope of the swine influenza virus neuraminidase protein comprising a specific portion of the swine influenza virus neuraminidase sequence.

Another example provides an antibody prepared by using the antigen-type specific epitope of the swine influenza virus neuraminidase protein as an antigen.

Another example provides a swine influenza virus neuraminidase antigen type detection kit comprising the antibody.

Another example provides a method of detecting a swine influenza neuraminidase antigen type by reacting the antibody with a sample comprising swine influenza and detecting a positive response to the antibody.

Another example provides a composition for preventing and / or treating swine influenza infection comprising the antibody.

Another example provides a vaccine composition for swine influenza comprising the epitope, a DNA molecule encoding the same, an expression vector comprising the same, and / or a transformed cell transformed with the expression vector.

We searched for 12 amino acid sequences that are part of the swine influenza virus neuraminidase sequence and have specificity according to neuraminidase antigen type (see Table 1 and Figure 1), and their epitope regions ( The present invention was completed by confirming the use as an epitope). In the present invention, the amino acid sequence searched as the epitope is characterized by specific neuraminidase antigen-type type, in particular with respect to neuraminidase antigen type 1 or type 2.

Thus, one embodiment of the present invention provides a polypeptide molecule of the epitope of swine influenza virus neuraminidase comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12 of Table 1.

Antigenic Sites of Swine Flu Virus Neuraminidase Protein SEQ ID NO. Amino acid sequence Length (aa) Type One FFLTQGALLNDK 12 NA1 2 IITDTIKSWRNNILR 15 NA1 3 NLEYQIGYICSG 12 NA1 4 GSFVQHPELTG 11 NA1 5 TQGALLNDKHSNGT 14 NA1 6 TDTIKSWRNNILRTQES 17 NA1 7 RPCFWVELIRGRPKE 15 NA1 8 ITGFAPFSKDNS 12 NA2 9 SSYVCSGLVGDTPR 14 NA2 10 SNSIVVFCGTSGTYGTGSWPD 21 NA2 11 NRCFYVELIRGR 12 NA2 12 GTSGTYGTGSWPDG 14 NA2

In one embodiment, the epitope polypeptide molecule is an epitope of a swine influenza virus neuraminidase antigen type 1 protein comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7. It may be a polypeptide molecule. In another embodiment, the epitope polypeptide is an epitope of a swine influenza virus neuraminidase antigen type 2 protein comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 8 to 12 It may be a polypeptide molecule.

As used herein, an 'antigenic site polypeptide molecule' is a swine influenza virus neuraminidase antigen type 1 or 2 of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. It is a physical representation of the use of a protein as an epitope.

Another example of the present invention is a swine influenza virus neuramy that specifically recognizes (or specifically binds) an epitope comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. Provide antibodies against idases. In one example, the antibody specifically comprises at least one epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, porcine influenza virus neuraminidase. It may be an antibody specific for antigen type 1 protein. In another example, the antibody is a swine influenza virus neuraminid that specifically recognizes one or more epitope polypeptide molecules comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12. It may be an antibody specific for an azeotype 2 protein.

The swine influenza virus neuraminidase antigen type 1 protein-specific antibody is swine influenza virus neuraminidase antigen type 1 comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7. It may be prepared by a conventional method using a polypeptide molecule containing an epitope of a protein as an antigen. In addition, the antibody specific for the swine influenza virus neuraminidase antigen type 2 protein comprises swine influenza virus neuraminidase antigen type comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12. It may be prepared by a conventional method using a polypeptide molecule containing an epitope of the type 2 protein as an antigen.

 For example, the epitope of the swine influenza virus neuraminidase antigen type 1 protein comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7 or SEQ ID NOs: 8 to 12 Prepared by injecting a peptide molecule comprising the epitope of the swine influenza virus neuraminidase antigen type 2 protein comprising a polypeptide consisting of an amino acid sequence selected from the group into an animal commonly used, such as a rabbit or mouse as an antigen May be an antibody.

The antibody may be an antibody that is a polyclonal antibody or a monoclonal antibody. In one embodiment, the antibody, i.e., the antibody specific for the swine influenza virus neuraminidase antigen type 1 protein or the antibody specific for the swine influenza virus neuraminidase antigen type 2 protein, comprises said swine influenza virus neuraminidase. It may be a monoclonal antibody produced by a conventional method by hybridoma cells obtained by a conventional method from a mouse injected with a polypeptide containing an epitope of the idase antigen type 1 or type 2 protein. In another embodiment, the antibody comprises at least two epitope domains of swine influenza virus neuraminidase antigen type 1 protein comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, or SEQ ID NO: 2 or more of the swine influenza virus neuraminidase antigen type 2 protein comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of a mixture of two or more, such as rabbit or mouse It may be a polyclonal antibody prepared by a conventional method by administering to the animal used as.

The antibody thus obtained specifically recognizes specific epitopes specific for each type of neuraminidase antigen of the swine influenza virus, preferably swine influenza virus, and thus detects swine influenza virus, preferably swine. Detection of neuraminidase antigenic type (type 1 or type 2) of influenza viruses is possible.

Therefore, another example of the present invention provides a kit for detecting swine influenza virus comprising at least one of the above antibodies. In one embodiment, the kit is swine influenza comprising at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7. Kit for detecting viral neuraminidase antigen type 1 (or for detecting swine influenza virus of neuraminidase antigen type 1). In another embodiment, the kit comprises swine influenza comprising at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12. Kit for detecting viral neuraminidase antigen type 2 (or for detecting swine influenza virus of neuraminidase antigen type 2). In another embodiment, the kit comprises at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7; And at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 12, porcine influenza virus neuraminidase anti It may be a circular type 1 and type 2 kit for simultaneous detection.

The kit may further include a labeling means for confirming the reaction between the antibody included and the antigen (antigen determination site) in the sample to be applied thereto. The reaction confirmation label means is not particularly limited, for example, immunochromatography, flow cytometry, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) , Enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA) can be any means commonly used to perform a method selected from the group consisting of.

The method for detecting the labeling means may use a method known to those skilled in the art according to the type of labeling factor, and specifically, the naked eye, a detection device, a detection reagent, or the like may be used. The detection reagent may be provided by a predetermined labeling reagent, an auxiliary specific binding member, and / or a signaling system, which enables the external identification of the presence of an analyte to be tested by the naked eye or other apparatus. It may comprise a component. Labeled detection reagents are well known to those of ordinary skill in the art. Examples of such labels include catalysts, enzymes (e.g., phosphatase, peroxidase, etc., and more specific examples include basic phosphatase and red pepper peroxidase used in conjunction with enzyme substrates), enzyme substrates (e.g. , Nitrobluetetrazolium, 3,5 ', 5,5'-tetranitrobenzidine, 4-methoxy1naphthol, 4chloro1naphthol, 5-bromo-4-chloro-3-indolylphosphate, chemiluminescent enzyme Substrates include dioxetane and derivatives and analogs thereof, fluorescent compounds (e.g., fluorescein, phycobiliprotein, rhodamine and the like and derivatives and analogues), chemiluminescent compounds , Metal sol, dye sol, particulate latex, color indicator, color matter contained in liposomes, non-metal sol such as carbon sol and selenium have. For example, as a labeling means, enzymes include horse radishperoxidase (HRP), fluorescent materials include FITC (Fluoresceinthiocarbamyl ethylenediamine), and light emitting materials include luminol, isoluminol and lucigenin, and radioisotope. Elements include, but are not limited to, 125I, 3H, 14C, and 131I. Labeling by the labeling means can be confirmed using a substrate for measuring the enzyme or a tool for measuring fluorescence, luminescence or radiation. In one embodiment, the antibody may be prepared as an ELISA kit or a strip kit.

Based on the detection of swine influenza virus of the above-mentioned antibody, preferably the detection of swine influenza virus by type of neuraminidase antigen type (type 1 or type 2), another example of the present invention uses the antibody Provided is a method for detecting swine influenza virus. More specifically, the detection method

Preparing a biological sample;

Applying to the sample at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12; And

Testing antigen-antibody responses

It may include.

In one embodiment, the detection method is applied to one or more antibodies that specifically recognize epitope polypeptide molecules comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 7 to a sample. Swine influenza virus neuraminidase antigen type 1 detection (or neuraminidase antigen type 1 swine influenza virus detection) method comprising the steps of: In another embodiment, the detection method applies to the sample at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12. Swine influenza virus neuraminidase antigen type 2 detection (or neuraminidase antigen type 2 swine influenza virus detection) method comprising the steps of: In another embodiment, the detection method comprises one or more antibodies that specifically recognize epitope polypeptide molecules comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7 in a sample; Swine influenza virus neuramy, comprising applying at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12 Simultaneous detection of idase antigen type 1 and type 2 (or simultaneous detection of neuraminidase antigen type 1 and type 2 swine influenza viruses).

The sample refers to any biological or environmental sample requiring the presence of swine influenza virus and / or swine influenza virus neuraminidase antigen type. For example, the sample may be, but is not limited to, biological samples such as saliva, body fluids such as blood, cells, tissues, and the like isolated from animals, preferably birds or mammals.

If a positive reaction is detected in the antigen-antibody reaction, it may be determined that the swine influenza virus neuraminidase is present. Positive reactions of the antigen-antibody reactions can be detected through conventional enzymatic reactions, fluorescence, luminescence and / or radiation detection. More specifically, immunochromatography, flow cytometry, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA) ), Fluorescence immunoassay (FIA), luminescence immunoassay (LIA) and the like. Labeling factors used herein are as described in the kit section above.

In another example, the present invention provides an antibody that specifically recognizes one or more epitope polypeptide molecules comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12 as an active ingredient. It provides a composition for preventing and / or treating swine influenza virus infection comprising. In one embodiment, the composition comprises a neuramy comprising at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7. It may be a composition for preventing and / or treating swine influenza virus infection of idase antigen type 1. In another embodiment, the composition comprises a neuramy comprising at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12 It may be a composition for preventing and / or treating swine influenza virus infection of the idase antigen type 2. In another embodiment, the composition comprises one or more antibodies and SEQ ID NO that specifically recognize epitope polypeptide molecules comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7 : Porcine influenza virus of the neuraminidase antigen type 1 comprising at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of 8 to 12, and It may be a composition for the prevention and / or treatment of neuraminidase antigen type 2 swine influenza virus infection.

In addition, the epitope having a amino acid sequence of SEQ ID NO: 1 to 12 of the present invention is a site specific for swine influenza virus, preferably swine influenza virus neuraminidase antigen type 1 or type 2, and swine influenza Since it effectively produces antibodies specific for the viral neuraminidase antigen type, the epitope is swine influenza virus, preferably swine influenza virus of neuraminidase antigen type 1 or neuraminidase antigen type 2 It is also useful as a vaccine against swine flu virus.

Thus, another example of the present invention is an epitope polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, a DNA molecule encoding the polypeptide molecule, an expression comprising the DNA molecule It provides a vaccine composition for swine influenza virus comprising as an active ingredient a vector, and at least one selected from the group consisting of transformed cells transformed with the expression vector. In one embodiment, the vaccine composition comprises an epitope polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, a DNA molecule encoding the polypeptide molecule, an expression comprising the DNA molecule It may be a vaccine composition for the swine flu virus of neuraminidase antigen type 1 comprising at least one selected from the group consisting of a vector and transformed cells transformed with the expression vector as an active ingredient. In another embodiment, the vaccine composition comprises an epitope polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12, a DNA molecule encoding the polypeptide molecule, an expression comprising the DNA molecule It may be a vaccine composition for the swine influenza virus of the neuraminidase antigen type 2 comprising the vector, and at least one selected from the group consisting of transformed cells transformed with the expression vector as an active ingredient.

In another embodiment, the vaccine composition comprises an epitope polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, a DNA molecule encoding the polypeptide molecule, the DNA molecule At least one selected from the group consisting of expression vectors and transformed cells transformed with the expression vectors; And SEQ ID NO: 8 to 12 epitope polypeptide molecule comprising an amino acid sequence selected from the group consisting of, a DNA molecule encoding the polypeptide molecule, an expression vector comprising the DNA molecule, and the expression vector It may be a vaccine composition against the neuraminidase antigen type 1 swine influenza virus and neuraminidase antigen type 2 swine influenza virus comprising at least one selected from the group consisting of transformed transformed cells as an active ingredient. .

The composition for preventing and / or treating swine influenza virus infection, and the vaccine composition may be administered by conventional routes, such as, but not limited to, intravascular administration, subcutaneous administration, peritoneal administration, and the like. Can be administered. In addition, the compositions may further include additives such as excipients commonly used. Routes of administration, dosages, additives and the like of such compositions are those that can be readily determined by one of ordinary skill in the art.

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

In the present invention, a monoclonal and polyclonal antibody capable of artificially preparing a peptide antigen using the nucleotide sequence of the swine influenza virus, which is popular in Korea and abroad, and specifically reacting to the swine influenza virus using rabbits and mice. Produced. The amino acid sequence of each antigenic type of the acquired swine influenza virus was compared and analyzed using a bioinformatics tool. In particular, the amino acid sequence of the neuraminidase protein, which is closely related to the antigenic type of swine influenza virus, was subjected to multiple alignments using a bioinformatics tool program, and the mutations and conservative parts of the neuraminidase were identified. In addition, about 10 amino acid sites were selected for each type by analyzing the characteristics of antigenicity and hydrophilicity, and 5 to 7 peptides were selected for each type having strong hydrophilicity and high antigenicity.

The peptide antigen was also used to prepare polyclonal and monoclonal antibodies in rabbits and mice, respectively. In order to evaluate the reactivity of the monoclonal and polyclonal antibodies produced according to the present invention with the swine influenza virus neuraminidase protein, 13 kinds of viruses (1 to 8) synthesized antigen-specific peptides and influenza virus neuraminidase types 1 to 8 ( Table 2) The immunological reaction with the prepared antibody and the like was evaluated using ELISA. Both the peptide monoclonal and polyclonal antibodies produced in the present invention were confirmed by ELISA assay specifically reacting with swine influenza virus neuraminidase antigen type 1 and type 2, respectively.

The detection method using antibodies can be said to be a detection method that overcomes the limitations of such molecular biological assays and requires fast, simple and relatively low quality control. Sensitive and specific monoclonal and polyclonal antibodies are essential and essential for the development of new swine influenza virus diagnostics using antibodies. Therefore, developing antibodies that react with various genotypes of swine influenza virus is the key to immunological analysis. The development of specific monoclonal and polyclonal antibodies that react with the swine influenza virus can be used as an important research material that can be used for not only diagnostic methods but also for transcription and replication of viruses.

Swine influenza was first identified in influenza type A H1N1 virus in 1930, and H1N1 virus samples of the latest pandemic influenza virus from the US Centers for Disease Control and Prevention (CDC) have been analyzed. It has been found to be a mixed species not found in pigs, birds, and humans. In the process of combining avian influenza and human influenza with the existing swine influenza virus, a genome modification occurs and a virus spread between pigs is spreading among humans. With the recent pandemic of the influenza virus, the World Health Organization (WHO) has raised the alert level to six levels of efficient and persistent human-to-human infection, the highest pandemic level.

As such, the swine influenza virus is increasingly important as an important cause of pandemic influenza of domestic and foreign influenza, but it is very difficult to diagnose and research due to the lack of sensitive and specific antibodies and the lack of standardized diagnostic methods. .

The present invention provides a method for producing an antibody that selectively recognizes swine influenza virus neuraminidase antigen-type specific epitopes, and a producer of the antibody.

In one example, the antibody producer can be prepared by a conventional method using a cell expressing the epitope according to the present invention as an antigen.

For example, the method for producing an antibody producer may include (a) immunizing the antigen-determining polypeptide molecule or a cell expressing the antigen-determining site with an animal such as a mouse or a rabbit, and (b) immunizing the antigen-determining site; Obtaining splenocytes producing specific antibodies, and (c) fusing the splenocytes with myeloma cells to select hybridoma cells producing the specific antibodies.

The antibody producer may be cultured in vitro or injected in vivo to separate the antibody. For example, the antibody producer can be inserted into the abdominal cavity of a mouse, and the produced antibody can be separated and purified from ascites. Isolation and purification of the antibody can be carried out using affinity chromatography, such as ion exchange chromatography (DEAE or DE52), anti-immunoglobulin column or protein A column, in the culture supernatant or ascites.

In the present invention, 12-21 peptide antigens showing conservative and antigenicity of swine influenza virus neuraminidase antigen type 1 and type 2 proteins were prepared and monoclonal and polyclonal antibodies were prepared using mice and rabbits. It was. The prepared monoclonal and polyclonal antibodies can be used as a tool to quickly and accurately identify the swine influenza virus neuraminidase antigen-type. Such antibodies not only provide basic tools for studying biological properties for swine influenza virus neuraminidase antigen-type analysis but also have important functions that can be applied to diagnosis.

In the present invention, the amino acid sequence of the swine influenza virus epidemic strain isolated in Korea and the swine influenza virus amino acid sequence of the foreign swine influenza virus strain registered in the Genebank is conserved while using the bioinformatics tool. Amino acid sites capable of discriminating between antigens of type 1 and type 2 were successfully obtained, and the presence of antigenic properties and their presence on the surface were studied. In the present invention, the neuraminidase amino acid sequence region that is conservative and can distinguish only antigenic type 1 and type 2 is the antigenic type of other types other than type 1 and 2 except for the site showing strong hydrophobicity by analyzing hydrophilicity and hydrophobicity. The consensus of amino acid sequences that could induce cross-reactions with each other was analyzed to exclude sites with limitations in the production of antibodies for differentiation.

Therefore, 12 candidate peptide sequences were obtained, prepared, and injected to prepare and evaluate monoclonal and polyclonal antibodies using mice and rabbits. To see the immunological reactivity of the prepared monoclonal and polyclonal antibodies, the reactivity with the prepared peptide was first tested. As a result, it was confirmed that all kinds of monoclonal and polyclonal antibodies reacted specifically with the injected peptide. And in order to analyze the reactivity and cross-reaction between the antibody produced in the next step and the swine influenza virus neuraminidase, the enzyme directly immunized with the swine influenza virus of swine influenza virus neuraminidase antigen type 1 to type 8 of Table 2 As a result of the assay (ELISA, Enzyme Linked Immuno-Sorbent Assay), at least eight monoclonal antibodies capable of distinguishing neuraminidase antigen type 1 and type 2 were prepared.

In addition, two polyclonal antibodies in response to neuraminidase antigen type 1 and two polyclonal antibodies in response to neuraminidase antigen type 2 produced in the present invention showed strong reactivity. It was possible.

From these results, it can be seen that both monoclonal and polyclonal antibodies of the present invention react very sensitively and specifically to swine influenza virus neuraminidase antigen type 1 and type 2. Therefore, the monoclonal and polyclonal antibodies developed in the present invention are important diagnostic materials that can be used for the screening and monitoring of swine influenza infection in the future, and distinguish between the rapid and rapid swine influenza virus neuraminidase antigen types 1 and 2 in the future. It is expected to be used in the development of diagnostic kits.

The present invention provides antigen-type 1 and type 2 epitopes of swine influenza virus neuraminidase protein, antibodies prepared using the same, and differential detection technology of swine influenza virus neuraminidase using the antibody. As the antibody is very sensitive and specifically reacts, it can be used as an important diagnostic baseline that can be used for screening and monitoring influenza virus infection in swine, and in the future, swine influenza virus neuraminidase It may be used in the development of a kit for differential detection and diagnosis of antigens type 1 and type 2.

1A and 1B are graphs obtained as a result of Predicted transmembrane segments for sequence of influenza virus neuraminidase antigen type 1 (1a) and type 2 (1b).
2A and 2B are graphs showing the hydrophilic (2a) / hydrophobic (2b) parameters of influenza virus neuraminidase antigen type 1 and type 2 proteins.
FIG. 3A shows the antigenic peptide of influenza virus neuraminidase antigen type 1, and FIG. 3B shows the antigenic peptide of influenza virus neuraminidase antigen type 2. FIG.
4A-4C show epitopes of neuraminidase antigen type 1 and type 2 finally selected for the preparation of monoclonal / polyclonal antibodies (indicated in red and underlined).
5 is a schematic diagram illustrating a method of preparing monoclonal / polyclonal antibodies in the Examples.
6a to 6d show the reactivity test results of the influenza virus neuraminidase antigen type 1 polyclonal antibody prepared by mixing SEQ ID NOs: 1 to 4, and FIGS. 6E to 6G are prepared by mixing SEQ ID NOs: 5 to 7 It is a graph showing the reactivity test results of one influenza virus neuraminidase antigen type 1 polyclonal antibody.
7A to 7C show the reactivity test results of the influenza virus neuraminidase antigen type 2 polyclonal antibody prepared by mixing SEQ ID NOs: 8 to 10, and FIGS. 7D to 7F show the mixture of SEQ ID NOs: 8, 11, and 12 It is a graph showing the reactivity test results of influenza virus neuraminidase antigen type 2 polyclonal antibody produced by the above.
FIG. 8A shows the reactivity test results of the monoclonal antibody of influenza virus neuraminidase antigen type 1, and FIG. 8B shows the reactivity test results of the monoclonal antibody of influenza virus neuraminidase antigen type 2.
9 is an exploded perspective view of the swine influenza virus neuraminidase antigen type 1 and type 2 diagnostic kit according to the present invention.
Figure 10 shows the structure of the strip constituting the diagnostic kit of the present invention.
Figure 11 is a photograph of the swine influenza virus neuraminidase antigen type 1 and type 2 differential diagnosis kit according to the present invention.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

Peptide  Epitope determination

For differentiation of swine influenza virus neuraminidase antigen type 1 and type 2 homology with multialign using the bioinformatics program (DNAMAN version 6 (Lynnon Corporation) or Vector NTI Suite 7.1 (informax, Inc.) The area was examined, and the procedure for selecting the area with high antigenicity at the site was performed.

First, in order to develop antibodies by type for differentiation of influenza virus neuraminidase (NA) antigens, the base and amino acid sequences of domestic and overseas swine and human infected influenza viruses are obtained from overseas GenBank (US) and genome databases. The serotype multialign was performed to select more than 10 conservative amino acid sequences as homologous regions by domain of each neuraminidase protein site. At the same time, the same influenza virus was divided into hypervariable regions and highly conserved regions by analyzing the year and region of virus mutations.

Based on these results, antigenic epitope selection was simulated using a DNA bioversion program (DNAMAN version 6 (Lynnon Corporation) or Vector NTI Suite 7.1 (informax, Inc.)) in a highly conserved region. Secondary structures and antigenic sites are ranked through and five or more epitopes (epitopes) were determined and used for antibody production. The ranking method was performed by selecting a site capable of differentiating between antigen types among the conserved sites, and then ranking the features after listing the degree of similarity, function as an epitope, hydrophilicity, or hydrophobic characterization. By using the functions included in the DNAMAN program, the results of the predicted transmembrane segments for sequence, the hydrophilicity and hydrophobic parameters, and the antigenic peptide were derived. FIG. 1A and FIG. 1B (above Predicted transmembrane segments for sequence), FIG. 2A and FIG. 2B. (Above hydrophilicity and hydrophobicity parameters) and 3A and 3B (antigenic peptide).

GenBank listed type-specific protein sequences compared to select homologous and distinguishable sites of neuraminidase antigen-specific influenza virus amino acid sequences are shown in Table 3 below.

No. NA Type Accession Number One NA1 EU296600 2 NA2 EU296608 3 NA3 AB546182 4 NA4 HM849002 5 NA6 CY073454 6 NA7 HM849014 7 NA8 HM589207

The final selected neuraminidase antigen type 1 and 2 epitopes are shown in FIGS. 4A-4C (indicated in red and underlined).

Twelve kinds of epitopes shown in FIGS. 4A to 4C are shown below.

SEQ ID NO. Amino Acid Sequence Length (aa) Type

1 FFLTQGALLNDK 12 NA1

2 IITDTIKSWRNNILR 15 NA1

3 NLEYQIGYICSG 12 NA1

4 GSFVQHPELTG 11 NA1

5 TQGALLNDKHSNGT 14 NA1

6 TDTIKSWRNNILRTQES 17 NA1

7 RPCFWVELIRGRPKE 15 NA1

8 ITGFAPFSKDNS 12 NA2

9 SSYVCSGLVGDTPR 14 NA2

10 SNSIVVFCGTSGTYGTGSWPD 21 NA2

11 NRCFYVELIRGR 12 NA2

12 GTSGTYGTGSWPDG 14 NA2

< Example  2>

Swine Flu Virus Peptide  only/ Polyclonal  Construction of Antibodies

The neuraminidase antigen type 1 and type 2 specific peptides shown in Table 1 above were conjugated to pre-activated KLH (keyhole limpet hemocyanin, Pierce) and pre-activated BSA (bovine serum albumine, Pierce) for immunological reactivity. To appear. The conjugated peptide thus prepared was added to mice and rabbits to prepare monoclonal antibodies and polyclonal antibodies, respectively. Monoclonal and polyclonal antibodies were successfully generated. The monoclonal and polyclonal antibodies generated from each peptide are described in Table 4 below. In the case of polyclonal antibodies, two neuraminidase antigen types 1 and 2 were prepared using rabbits, and monoclonal antibodies were 4 types of 4 types 1 and 4 types of 4 types 2 monoclonal antibody cells. Was investigated.

<2-1> from mouse Monoclonal  Construction of Antibodies

In order to prepare an antibody specific for antigen type 1, the above-mentioned peptide antigens of SEQ ID NOS: 1 to 4 were mixed and introduced into a mouse to prepare monoclonal antibodies, and to prepare antibodies specific for antigen type 2, Monoclonal antibodies were prepared by mixing the peptide antigens of SEQ ID NOs: 8 to 10 mentioned above into mice.

More specifically, as the first immunization, 250μℓ of the peptide synthesized to have the sequence shown in Table 1 above was mixed with the same amount (250μℓ) of complete Freund's adjuvant (Sigma) to make an emulsifier, and then, female BALB / c The mice were injected into intraperitoneal injection (ip). Six mice were injected with 500 μg of the synthesized peptide per animal. Two weeks later as a second immunization, the same technique was injected. However, incomplete Freund's adjuvant (Sigma) was used, and the second and third immunizations (two weeks after the second immunization) were administered in the same manner with the same amount of antigen as the first immunization.

Finally, two weeks later, the antigen (the same amount as in the third administration) was mixed in PBS and injected into the vein of the tail (travenous injection, iv). Three days after the last immunization, cell fusion was performed to prepare cells that produce monoclonal antibodies (see Examples <2-3> and <2-4>). The specific method of manufacturing a monoclonal antibody is typically shown in FIG.

<2-2> In the rabbit Polyclonal  Preparation of antibodies

The polyclonal antibody was prepared by mixing three types of peptide antigens, each of seven types 1 and 7 of SEQ ID NOs: 1 to 7 and two types of two types of SEQ ID NOs: 8 to 12, respectively. The synthetic peptides secured above were administered to rabbits (New Zealand White, Female, 2-2.2 kg).

At the first dose, prepare the antigen in 1mg / 400μℓ PBS, mix the same amount of complete Freund's adjuvant (Sigma), and make an emulsion. If a large amount is administered at one site, purulent-pus may be formed, and if severe, granulomas are induced). After 2 weeks, the second dose was the same as the first dose, but incomplete Freund's adjuvant (Sigma) was used. The third dose was the same as the second dose, and the last four doses were administered directly to the vein about 100 μL in the vein.

After the last administration, blood was collected through the vein and the antibody titer was tested by the following method and reached a high level (ELISA test showed that the serum was diluted 1000 times or more to find the titer that can be measured). When blood was collected and allowed to stand at 37 ° C. for 30 to 60 minutes, blood cells coagulated to form a blood clot, thereby removing plasma. The prepared plasma was centrifuged at 4 ° C., 10000 g for 10 minutes, and the supernatant was taken to separate and purify antibodies as follows.

Antibodies were purified from the obtained supernatants using immunoaffinity chromatography. That is, the supernatant was loaded onto an agarose beads column (Pierce), which was conjugated with Protein A, which was equilibrated with a 5-fold volume of phosphate (PBS). After washing the column with bed volumn phosphate (PBS), the polyclonal antibody was eluted with 0.1M Glycine, pH3.0 buffer solution. Eluted polyclonal antibody was added to the 1/10 volume of 1M Tris, pH8.0 again for neutralization. Finally, the pure polyclonal antibody was isolated and then dialyzed in phosphate buffer (PBS) to isolate and purify the antibody.

<2-3> Serum antibody test during antigen administration (antibody titering )

Four days after the third immunization, blood was collected from the tail vein and serum was obtained, and serial dilutions were performed in phosphate buffer (PBS) at concentrations of 1/100000, 1/5000, 1/1000 and 1/100. ELISA, Enzyme Linked Immuno-Sorbent Assay) was used to determine the formation of antibodies. If the measured optical density (OD) value is good compared to the absorbance of the negative control mouse (serum of the non-antigen-injected mouse), and the absorbance is 2 or more at 1/100 dilution, the immunization was reported to be successful. Fusion was prepared. Immunization was again attempted after 2 weeks if the antibody titer was low.

<2-4> Splenocytes and  Fusion of myeloma cells ( Cell fusion )

After 3-4 days of final immunization, the spleens of mice were aseptically removed, and the spleens were excised with a 26-gauge needle and suspended in cold DMEM (Dulbecco's Modified ed Eagle's medium, Gibco) without serum. . The suspension was collected in a 50 ml tube and allowed to stand for 1-2 minutes. The supernatants were collected and centrifuged three times for 10 minutes at 1,000 rpm with DMEM. Precipitated lymphocytes were mixed with mouse myeloma cells (myeloma, Sp2 / 0-Ag14, Korea Cell Line Bank) at a ratio of 7: 1 to 10: 1 based on the number of cells, and centrifuged once with DMEM preheated to 37 ° C. After washing, the supernatant was completely removed using a pasteur pipet.

1 ml of 50% (w / v) polyethylene glycol 1,500 (PEG 1500; Boehringer Mannheim), previously warmed to 37 ° C., was added to the mixed cells over 1 minute. After adding PEG, the test tube containing the mixed cells was shaken for 1 to 2 minutes to induce cell fusion. After 5 ml of DMEM was added thereto over 5 minutes, 10 ml of DMEM was slowly added again, and allowed to stand for 5 minutes in a 37 ° C constant temperature water bath. The mixed solution was centrifuged at 1,000 rpm for 10 minutes and the supernatant was removed. Precipitated cells were suspended in DMEM supplemented with HAT supplement (0.1 mM hypoxanthine, 0.4 µM aminopteri, 16 µM thymidine, Sigma) and 20% fetal bovine serum (Gibco) at a concentration of 10 ⁶ cells / ml. This suspension was dispensed in a 96-well microplate (Nunc) 100 μl per well. The prepared wells were incubated in a 37 ° C. constant temperature incubator, and medium was changed every 3 to 4 days. Two weeks after fusion, HT supplements (100μ hypoxanthine, 16μM thymidine, Sigma) and 10% fetal bovine serum were added to DMEM. After 3 weeks, only 10% fetal bovine serum was added to DMEM.

Hybridoma screening was selected based on the specificity and reactivity results in the following <2-5> (absorbance criteria).

Monoclonal antibodies were purified using immunoaffinity chromatogarphy. That is, the supernatant was loaded onto an agarose beads column (Pierce), which was conjugated with Protein A, which was equilibrated with a 5-fold volume of phosphate (PBS). After washing the column with bed volumn phosphate (PBS), the monoclonal antibody was eluted with 0.1M Glycine, pH3.0 buffer solution. Eluted monoclonal antibody was added to the 1/10 volume of 1M Tris, pH8.0 again for neutralization. Finally, the pure monoclonal antibody was isolated and then dialyzed in phosphate buffer (PBS) to isolate and purify the antibody.

<2-5> stage / Polyclonal  Antibody ELISA Specificity test

For specificity analysis of mono / polyclonal antibodies, the synthesized peptides were diluted to 1 ㎍ / ml in coating buffer (KOMA BIOTECH INC, K0331021), added 200 ㎕ per well, and coated by overnight incubation at 4 ° C. Wash the coated plate five times with washing solution (PBST, KOMA BIOTECH INC, K0331041, K0331051), add Blocking solution (1% BSA / PBS, KOMA BIOTECH INC, K0331032) 250 well per well, and then at room temperature Blocking was performed by reacting for a time.

The blocked plate was washed again five times using the washing solution, and 200 μl of the poly / monoclonal antibody culture solution (medium: DMEM) was dispensed by 200 μl per well, and 200 μl of the diluted solution (cell culture medium DMEM) was added to the well for the negative control group. After the reaction at room temperature for 60 minutes.

After the reaction of the sample was completed, the plate was washed five times with a washing solution, and a reagent (1: 5,000 (v / v), Antibody Incorporated, USA) in which HRP (Horse reddish peroxidase) was fused to anti-mouse IgG was well used. After 200 μl of sugar was added, the mixture was reacted at room temperature for 1 hour, and washed with a washing solution (PBST, 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na ₂ HPO ₄ , 1.76 mM K ₂ HPO ₄ , 0.5% Tween). Induce the color reaction with the TMB Substrate kit (KOMA BIOTECH INC, K0331060), stop the reaction with stop solution (2 M, H ₂ SO ₄ ), and absorb the light at 450 nm with ELISA reader (TECAN). Measured and analyzed.

An absorbance value that is three times or more the absorbance of the negative control group was determined to be negative or positive based on the minimum value of the positive reaction. Of the mono / polyclonal antibodies generated in each peptide, only those with an optical density (OD) greater than 1 were selected.

< Example  3>

only/ Polyclonal  Antibody ELISA Screening and Characterization

<3-1> ELISA Antibody Screening Through

The peptide antibodies selected in Example <2-5> are summarized in Table 4 below. Four monoclonal peptide antibodies against swine influenza virus neuraminidase antigen type 1 (NA1M # 1-NA1M # 4) and four monoclonal peptide antibodies against type 2 (NA2M # 1-NA2M #) as described in Table 4. 4) A total of 12 antibodies, including 2 polyclonal peptide antibodies against antigen type 1 (NA1P # 1, NA1P # 2) and 2 polyclonal peptide antibodies against type 2 (NA2P # 1, NA2P # 2) Subjects were subjected to antigen-antibody reactions.

The single / multi-antigen for the monoclonal antibodies selected - antibody reaction, 10 ⁵ TCID ₅₀ of the Influenza A / Korea / 01/2009 H1N1 ( local isolates, initially the new influenza A (H1N1) virus isolated from Korean people, disease control NCBI Taxonomy , distributed by National Institutes of Health, Influenza Virus Team ID: 644289) and Influenza A / Swine / Korea / Can04 / 2005 H3N2 ( domestic isolates, the National Veterinary Research and Quarantine Service, NCBI Taxonomy ID : 538544, currently used to produce three inactivated vaccines for swine influenza, diluted 1/10 in coating buffer (KOMA BIOTECH INC, K0331021), added 200 μl per well, and coated by overnight incubation at 4 ° C. Except that, it was carried out in the same manner as in Example <2-5>.

The results thus obtained are shown in Table 4 below.

Antibody clones H1N1 H3N2 Epitope peptides 450nm O.D 450nm O.D Monoclonal
Peptide Antibodies NA1M # 1 1.312 0.110 N1-1 (SEQ ID NO: 1) NA1M # 2 1.426 0.103 N1-2 (SEQ ID NO: 2) NA1M # 3 1.82 0.109 N1-4 (SEQ ID NO: 4) NA1M # 4 1.78 0.101 N1-4 (SEQ ID NO: 4) NA2M # 1 0.102 1.87 N2-1 (SEQ ID NO: 8) NA2M # 2 0.103 1.91 N2-1 (SEQ ID NO: 8) NA2M # 3 0.102 1.781 N2-2 (SEQ ID NO: 9) NA2M # 4 0.102 1.902 N2-2 (SEQ ID NO: 9) Polyclonal
Peptide Antibodies NA1P # 1 1.42 0.99 N1-1 ~ 4 (SEQ ID NOs: 1, 2, 3, 4) NA1P # 2 1.37 0.98 N1-5 ~ 7 (SEQ ID NOs: 5, 6, 7) NA2P # 1 0.102 1.53 N2-1 ~ 3 (SEQ ID NOs: 8, 9, 10) NA2P # 2 0.103 1.61 N2-1,4 ~ 5 (SEQ ID NOs: 8, 11, 12)

As can be seen in Table 4, differential detection between neuraminidase antigen type 1 and type 2 was possible using the swine influenza virus neuraminidase antigen type 1 and type 2 specific antibodies according to the present invention, respectively. . However, no antibodies to SEQ ID NO: 3 and SEQ ID NO: 10 were made. The excellent monoclonal antibody confirmed the reactivity was used to confirm whether the neuraminidase antigen type 1 to type 8 can be discriminated. Selected monoclones for antigen type 1 include NA1M # 1, N1-2 (SEQ ID NO: 2), which responds to N1-1 (SEQ ID NO: 1), and NA1M # 2, N1-4 (SEQ ID). NO: 4) NA1M # 3 and NA1M # 4 in response, and selected monoclonals for antigen type 2 were NA2M # 1 and NA2M # 2, N2-2 in response to N2-1 (SEQ ID NO: 8) NA2M # 3 and NA2M # 4 responsive to (SEQ ID NO: 9). Antibody type 1 and type 2 polyclonal antibodies (NA1P # 1, NA1P # 2, NA2P # 1, NA2P # 2) were all highly reactive and selected.

<3-2> ELISA Antibody Characterization Through

For characterization of selected mono / polyclonal antibodies, synthesized peptides were diluted to 1 ㎍ / ml in coating buffer (KOMA BIOTECH INC, K0331021), added 200 ㎕ per well and coated overnight at 4 ℃. I was. Wash the coated plate five times with washing solution (PBST, KOMA BIOTECH INC, K0331041, K0331051), add Blocking solution (1% BSA / PBS, KOMA BIOTECH INC, K0331032) 250 well per well, and then at room temperature Blocking was performed by reacting for a time.

The blocked plate was washed again five times with the washing solution, and the poly / monoclonal antibody was adjusted to 1 mg / ml and 1/100, 1 / 1,000, 1 / 5,000, 1 / 10,000, with phosphorylation buffer (PBS). Dilute to 1 / 50,000 and 1 / 100,000 and dispense 200μl per well, and the negative control group dilute mouse IgG (1mg / ml, ARISTA BIOLOGICALS INC.) To 1 / 1,000 with phosphorylated buffer (PBS) and dispense 200μℓ to the well. After the reaction at room temperature for 60 minutes.

After the reaction of the sample was completed, the plate was washed five times with a washing solution and a reagent (1: 5,000 (v / v), Antibody Incorporated, in which HRP (Horse reddish peroxidase) was fused to anti-mouse IgG or anti-rabbit IgG) , USA) was added 200 μl per well and then reacted at room temperature for 1 hour, followed by washing (PBST, 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na ₂ HPO ₄ , 1.76 mM K ₂ HPO ₄ , 0.5% Tween). Washed. Induce the color reaction with the TMB Substrate kit (KOMA BIOTECH INC, K0331060), stop the reaction with stop solution (2 M, H ₂ SO ₄ ), and absorb the light at 450 nm with ELISA reader (TECAN). Measured and analyzed.

The absorbance results thus obtained are shown in Table 4, FIGS. 6A to 6G, 7A to 7F, 8A, and 8B.

< Example  4>

Swine Flu Virus Neuraminidase Antigenic type  Diagnostic Strips and Diagnosis with Type 1 and Type 2 Specific Antibodies Of kit  Produce

<4-1> Swine Flu Virus Neuraminidase Antigenic type  Membranes coated with type 1 and type 2 specific antibodies ( membrane Preparation of

The selected monoclonal for antigen type 1 in <Example 3> is NA1M # 1 in response to N1-1 (SEQ ID NO: 1), NA1M # 2 in response to N1-2 (SEQ ID NO: 2) , NA1M # 3 and NA1M # 4 in response to N1-4 (SEQ ID NO: 4), and selected monoclonals for antigenic type 2 were NA2M # 1 in response to N2-1 (SEQ ID NO: 8) and NA2M # 3 and NA2M # 4 in response to NA2M # 2, N2-2 (SEQ ID NO: 9).

The respective polyclonal antibodies of antigen type 1 and type 2 are NA1P # 1, NA1P # 2, NA2P # 1, NA2P # 2.

The mono / polyclonal antibody thus selected was used as a test line at a concentration of 0.5 to 2.5 mg / ml, and a control line was used as goat anti-mouse IgG (Arista Biologicals Inc.).

The mono / polyclonal antibody of the test line and the control solution of the control line were coated on a nitro cellulose membrane (Millipore) using a dispensing instrument (KINAMETICS, USA) and dried overnight in a low humidity laboratory or in a pan for at least 5 hours. I was. Plates of the prepared membranes were stored in a sealed container or a low humidity laboratory with a desiccant (Silica gel, TPG).

<4-2> Swine Flu Virus Neuraminidase Antigenic type  Type 1 and type 2 specific antibody-gold ( Gold ) Manufacture of conjugate

The mono / polyclonal antibody selected in <Example 3> was mixed well while stirring to have a final concentration of 4 to 20 µg / ml, and was added dropwise to a gold solution (colloidal Gold, 1 OD). The solution was stirred again for 15 minutes. Thereafter, 10% BSA solution was added to the gold particle suspension. After another 15 minutes of stirring, the bound gold solution was centrifuged at 6,000 g for 30 minutes, and the supernatant was discarded to remove unbound recombinant protein. The pellet was resuspended after addition of 5 mM Sodium Tetraborate (pH 7.2) with BSA. The suspension was centrifuged again and the final pellet was adjusted to an absorbance of 10 +/− 1 at spectrophotometer 530 nm in 5 mM Sodium Tetraborate (pH 7.2) with 1% BSA. Influenza virus neuraminidase antigen type 1 and type 2 specific specific antibody-Gold conjugates were prepared.

<4-3> Preparation of gold conjugated treatment pad

The gold conjugated treatment pad was prepared by adding trehalose (SIGMA) to a final concentration of 5% (v / v) to the gold conjugate prepared in Example 4-2.

Specifically, for the diagnostic strip using swine influenza virus neuraminidase antigen type 1 and type 2 specific antibodies, swine influenza virus prepared in Example 4-2 on 0.5 x 20 cm glass fiber. Neuraminidase antigen type 1 and type 2 specific antibody-Gold conjugates were prepared to a final concentration of 1.0 to 2.5 OD (Optical Density).

<4-4> absorption pad and Sample pad  Produce

The absorbent pad and the sample pad were dried so that the reaction solution could be absorbed well, and were prepared by the process of cutting the pad, treating the reaction solution (generally dipping), drying, and storing according to methods known in the art.

<4-5> device  Assembly

Each of the membranes and pads prepared above were combined to overlap the sample pad, the gold pad (gold conjugate pad), the nitrocellulose membrane, and finally the absorbent pad, respectively, from the right side, and were sized to the immunoassay device (device). Cut to size (see FIG. 10). The strips thus cut were finally put in the lower plate of the simultaneous diagnosis immunoassay device (combo device, application number 10-2008-0075645), and the top plate was covered to prepare a simultaneous diagnosis kit (see FIG. 9).

<4-6> Product Composition

The immunoassay device and sample development (PBS, 50mM KCl, 300mM NaCl, 0.5% Tween20, 0.1% NaN3) described above consisted of the final product (see Figure 11).

< Test Example  1>

Swine Flu Virus Neuraminidase Antigenic type  Efficacy test of neuraminidase differential diagnostic strip using type 1 and type 2 specific antibodies

The differential diagnosis kit prepared according to Example 4 was used to diagnose the differentiation of the 12 antigen-type influenza viruses of Table 2 above.

Each of the differentiation diagnostic kits prepared in <Example 4> and the genotype sequence results of the antigen-type viruses were examined, and the results are shown in Table 5.

Evaluation of Swine Influenza Virus Neuraminidase Antigen Type 1 and Type 2 Differential Diagnostic Kits of the Invention on Antigen-Specific Influenza Virus Specimens Antigenic type Strain HA Titer Response result NA1 NA2 H1N1 A / hvPR8 / 34 (H1N1) 8 + - H2N2 A / Singapore / 1/57 (H2N2) 3 - + H3N8 A / duck / Ukraine / 1/63 (H3N8) 4 ~ 5 - - H4N6 A / duck / Czechoslovakia / 1956 (H4N6) 7 - - H5N3 A / duck / Hong Kong / 820/80 (H5N3) 5 - - H6N5 A / shearwater / Australia / 1/1972 (H6N5) 6 ~ 7 - - H7N1 A / duck / Hong Kong / 301/78 (H7N1) 6 + - H8N4 A / turkey / Ontario / 6118/1968 (H8N4) 4 ~ 5 - - H9N2 A / turkey / Wisconsin / 66 (H9N2) 5 - + H10N7 A / chicken / Germany / N '/ 49 (H10N7) 8 - - H11N6 A / duck / England / 1/1956 (H11N6) 7-8 - - H12N5 A / duck / Alberta / 60/1976 (H12N5) 6 - - H13N6 A / gull / Maryland / 704/77 (H13N6) 6 - -

As described in Table 5, the diagnostic kit using the swine influenza virus neuraminidase antigen type 1 and type 2 specific antibodies of the present invention, showed a result consistent with the gene base sequence results.

Therefore, it can be seen that the swine influenza virus neuraminidase antigen type 1 and type 2 specific antibodies of the present invention are very useful for developing a diagnostic kit for discriminating influenza virus neuraminidase antigen type 1 and type 2.

In FIG. 9, each code | symbol is as follows.
1: immunoassay device 2: assay strip
3: lower case 4: upper case
3A, 3B: first and second strip supports
4A, 4B: first and second strip counterparts
21: sample pad 22: membrane
221: inspection line 222: control line
23: absorption pad 24: short side
25: long side 31: short side fixing part
32: long side fixing part 33: lower blocking part
41: Sample inlet 42: Result display window
43: character indicator

<110> National Veterinary Research and Quarantin Service (NVRQS) <120> Antibody for Detecting Neuraminidase of Swine Influenza and Use          Therof <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 1 Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys   1 5 10 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 2 Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg   1 5 10 15 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 3 Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly   1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 4 Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly   1 5 10 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 5 Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr   1 5 10 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 6 Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu   1 5 10 15 Ser     <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 7 Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu   1 5 10 15 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 8 Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser   1 5 10 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 9 Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg   1 5 10 <210> 10 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 10 Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr   1 5 10 15 Gly Ser Trp Pro Asp              20 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 11 Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg   1 5 10 <210> 12 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 12 Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly   1 5 10

Claims (23)

An epitope polypeptide molecule of a swine influenza virus neuraminidase, comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. The method of claim 1,
SEQ ID NO: comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of 1 to 7, which is the epitope of the swine influenza virus neuraminidase antigen type 1 protein,
Polypeptide molecules. The method of claim 1,
SEQ ID NO: comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of 8 to 12, which is the epitope of a swine influenza virus neuraminidase antigen type 2 protein,
Polypeptide molecules. An antibody that specifically recognizes the epitope polypeptide molecule of claim 1. The method of claim 4, wherein
The antibody specifically recognizes one or more epitope polypeptide molecules comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, and porcine influenza virus neuraminidase antigen type 1 The antibody specific for type protein. The method of claim 4, wherein
Said antibody is the swine influenza virus neuraminidase antigen type 2 which specifically recognizes at least one epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12. An antibody specific for a protein. The method according to any one of claims 4 to 6,
The antibody is a monoclonal antibody or a polyclonal antibody. A kit for detecting swine influenza virus comprising at least one antibody of claim 4. The method of claim 8,
SEQ ID NO: 1 or 7 comprising at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of swine influenza virus neuraminidase antigen type Detecting type 1,
Pig Influenza Virus Detection Kit. The method of claim 8,
SEQ ID NO: comprising at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12, porcine influenza virus neuraminidase antigen type Detecting type 2,
Pig Influenza Virus Detection Kit. The method of claim 8,
At least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7; And
At least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12
Including,
Simultaneous detection of swine influenza virus neuraminidase antigen type 1 and type 2,
Pig Influenza Virus Detection Kit. Preparing a sample;
Applying to the sample at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12; And
Testing antigen-antibody responses
Comprising, swine flu virus detection method. The method of claim 12,
Applying to the sample at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7,
Detecting swine influenza virus neuraminidase antigen type 1,
Swine Flu Virus Detection Method. The method of claim 12,
Applying to the sample at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12,
Detecting swine influenza virus neuraminidase antigen type 2,
Swine Flu Virus Detection Method. The method of claim 12,
The sample consists of one or more antibodies and SEQ ID NOs: 8 to 12 that specifically recognize an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7. Applying at least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group,
Simultaneous detection of swine influenza virus neuraminidase antigen type 1 and type 2,
Swine Flu Virus Detection Method. At least one selected from the group consisting of a polypeptide molecule of claim 1, a DNA molecule encoding the polypeptide molecule, an expression vector comprising the DNA molecule, and a transformed cell transformed with the expression vector. Vaccine composition for swine influenza virus comprising as a component. The method of claim 16,
The epitope polypeptide molecule comprises one or more polypeptides consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 1-7,
It has an effect on the swine influenza virus of the neuraminidase antigen type 1,
Vaccine composition. The method of claim 16,
The epitope polypeptide molecule comprises one or more polypeptides consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 8 to 12,
It has an effect on the swine influenza virus of the neuraminidase antigen type 2,
Vaccine composition. The method of claim 16,
The epitope polypeptide molecule is at least one polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7 and at least one polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 12 It contains a peptide,
It has an effect on the swine influenza virus of the neuraminidase antigen type 1 and the swine influenza virus of the neuraminidase antigen type 2,
Vaccine composition. Composition for preventing or treating swine influenza virus infection comprising the antibody of claim 4 as an active ingredient. The method of claim 20,
At least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7;
It has an effect on the swine influenza virus of the neuraminidase antigen type 1,
Composition for preventing or treating swine influenza virus infection. The method of claim 20,
At least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12,
It has an effect on the neuraminidase antigen type 2 swine influenza virus,
Composition for preventing or treating swine influenza virus infection. The method of claim 20,
One or more antibodies that specifically recognize an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7 and SEQ ID NOs: 8-12 At least one antibody that specifically recognizes an epitope polypeptide molecule comprising a polypeptide consisting of an amino acid sequence,
It has an effect on the swine influenza virus of the neuraminidase antigen type 1 and the swine influenza virus of the neuraminidase antigen type 2,
Composition for preventing or treating swine influenza virus infection.

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