KR101102841B1 - Hybridoma cell lines, monoclonal antibodies produced from the hybridoma cell lines, foot-and-mouth disease virusfmdv detection reagents, fmdv detection kits and detection method for fmdv neutralizing antibodies - Google Patents

Abstract

The present invention provides specific immunoreactivity and virus neutralization ability against hybridoma cell line producing foot and tail disease virus O type VP1 site A, which produce monoclonal antibodies that are common to type O, A and Asia type 1 of foot and mouth virus. Hybridoma cell line producing monoclonal antibody, hybridoma cell line producing monoclonal antibody showing virus neutralizing ability and specific immunoreactivity against foot-and-mouth virus type A VP1 site A, produced by each of the hybridoma cell lines The present invention relates to a monoclonal antibody, a foot-and-mouth diagnostic reagent comprising the monoclonal antibody, a foot-and-mouth diagnostic kit comprising the foot-and-mouth diagnostic reagent, and a foot-and-mouth virus neutralizing antibody detection method or foot-and-mouth diagnostic method.

According to the present invention, it is not necessary to capture foot-and-mouth virus diagnostic antigens using the respective antibodies against foot-and-mouth virus serotypes, and the foot-and-mouth disease that can be cultured only in a laboratory equipped with a conventional shielding facility by using a recombinant foot-and-mouth virus structural protein as a diagnostic antigen. Compared to the method using the virus, it is very convenient, and using monoclonal antibodies specific to foot-and-mouth virus serotypes, it is possible to detect neutralizing antibodies without the hassle of using secondary antibodies for each livestock. And the diagnostic method of the present invention is superior to the detection sensitivity and specificity compared to the prior art.

Foot-and-mouth disease viruses, hybridomas, monoclonal antibodies, neutralizing antibodies, diagnostic reagents, diagnostic kits, diagnostic methods, recombinant structural proteins

Description

HYBRIDOMA CELL LINES, MONOCLONAL ANTIBODIES PRODUCED FROM THE HYBRIDOMA CELL LINES, FOOT-AND-MOUTH DISEASE VIRUS (FMDV) DETECTION REAGENTS, FMDV DETECTION KITS AND DETECTION METHOD FOR FMDV NEUTRALIZING ANTIBODIES}

The present invention provides specific immunoreactivity and virus neutralization ability against hybridoma cell line producing foot and tail disease virus O type VP1 site A, which produce monoclonal antibodies that are common to type O, A and Asia type 1 of foot and mouth virus. Hybridoma cell line producing monoclonal antibody, hybridoma cell line producing monoclonal antibody showing virus neutralizing ability and specific immunoreactivity against foot-and-mouth virus type A VP1 site A, produced by each of the hybridoma cell lines It relates to a method for detecting foot-and-mouth virus neutralizing antibodies or a foot-and-mouth disease diagnostic method comprising using a monoclonal antibody, a foot-and-mouth diagnostic reagent comprising the monoclonal antibody, a foot-and-mouth diagnostic kit comprising the foot-and-mouth diagnostic reagent, and the monoclonal antibody.

Foot and mouth disease (FMD) is a highly contagious disease of cows, pigs, sheep, and goats with two hoofed ungulates. List A is defined by the OIE. Decided to be a legal epidemic. The Bureau of International Water Affairs evaluates the status of clean countries, and when foot and mouth disease occurs, it causes restrictions on international trade, including trade transactions involving agricultural and livestock products. In Korea, it occurred in March 2000 after 66 years, and recorded more than 200 billion won of economic damage. At present, Korea maintains the status of foot-and-mouth clean-up country, but the outbreak of foot-and-mouth disease is increasing all over the world, especially in neighboring countries of Mongolia and China, the outbreak of foot-and-mouth disease was reported in 2005. Is increasing, and the government is putting all its efforts into quarantine and quarantine for prevention.

Foot-and-mouth disease virus (FMDV) is divided into seven serotypes of virus serotypes, O, A, C, Asia1, SAT1, SAT2 and SAT3 (South African Territories). Are divided into 80 subtypes.

It is found that the appearance of foot-and-mouth disease virus has an icosahedral structure. The foot-and-mouth disease virus belongs to Picornavirnae ( Aphtovirus ) in the picornavir , and the genome expresses one protein and the viral protein is cleaved by the viral protease and is characterized by being mature protein. The viral envelope is composed of VP1, VP2, VP3, VP4 proteins, and nonstructural proteins involved in viral replication and protein maturation include L, 3A, 3B, 3C, 3D, 2A, 2B, 2C, and the like. Capsid protein or structural protein (SP) induces neutralizing antibodies, and non structural protein (NSP) plays the most important role in viral replication and infection, but it is not known to induce neutralizing antibodies have.

The most important neutralizing antigen determinant of the structural protein that causes neutralizing antibodies to foot-and-mouth disease virus is the 141-160 amino sequence named "GH loop" of VP1 derived from P1 protein, first introduced in 1982 by Strohmaier et al. It is named as Site A. Kitson et al. (1990) reported that site A has higher homogeneity of amino acid bases than other neutralizing antigenic determinants, VP2 and VP3, and Pfaff E et al. (1982) can protect viral infections in guinea pigs with recombinant VP1 proteins alone. It has been reported that it can induce sufficient neutralizing antibodies. In addition, the characteristics of monoclonal antibodies known to possess neutralizing ability in FMDV O1 / Kaufbeuren strains were confirmed to react with amino acid residues of 141-160 sites of VP1.

In the meantime, the virus neutralization test (VNT) is used as a standard test among the serological tests on the salvage protein of foot-and-mouth disease, but this requires a shielding facility for culturing the virus and at least 72 hours of test time. Serum has the disadvantage of having low levels of nonspecific positive reactions. Liquid phase blocking (LPB) -ELISA, developed by Hambline in the mid-1980s, has been highly used because it has a high correlation with VNT.However, inactivated viruses must be used and the test method is very complex. There is a disadvantage that is not suitable for mass inspection. In addition, this test technique has been confirmed by Hass et al. (1994) about 4% of the false positive response in the non-vaccine or negative area serum, 18% non-specific response to the specific animal serum.

K.J. Sorensene et al. (1992) developed a neutralizing antibody detection method for African SAT1, 2, and 3 by comparing the results of LPB ELISA through competitive reaction of foot-and-mouth virus 146S and rabbit serum. Mackay et al. (2001) developed a solid phase competition ELISA using foot-and-mouth virus 146S antigen and guinea pig serum to evaluate vaccine antibody levels. According to G. Chenard et al. (2003), neutralization for mass screening using inactivated virus and monoclonal antibody was performed. It was reported that an ELISA kit for antibody testing was developed. However, using inactivated viral antigens has had to be performed only in special facilities in order to produce viral antigens.

In Korea, patent registration (No. 10-0737446) is provided for an antigen for foot-and-mouth virus vaccines that expresses foot-and-mouth virus P1 and 3C simultaneously using a genetic recombination technology (No. 10-0737446). Has been announced (Vaccine, 25 (20), 4112-4121, 2007). However, these techniques have provided only scientific evidence for measuring foot-and-mouth virus neutralizing antibodies, and insufficient information on the epitope of the used antibodies, conditions for capturing the recombinant antigen and maintaining reproducibility. In addition, there was a problem of low specificity and sensitivity.

The present invention has been conceived to solve the above problems, in the diagnosis of foot-and-mouth virus, to produce monoclonal antibodies exhibiting a common immune response to foot-and-mouth virus O type, A type and Asia type 1 to capture the diagnostic antigen. Specific immunoreactivity against hybridoma cell line, foot-and-mouth virus type O VP1 site A and hybridoma cell line producing monoclonal antibody showing viral neutralizing ability, foot-and-mouth virus A type VP1 site A An object of the present invention is to provide a hybridoma cell line producing a monoclonal antibody having a neutralizing ability, and a monoclonal antibody produced by each of the hybridoma cell lines.

In addition, the present invention is to provide a foot-and-mouth virus neutralizing antibody detection method or foot-and-mouth disease diagnostic method comprising using a foot-and-mouth disease diagnostic reagent comprising the monoclonal antibody, foot-and-mouth disease diagnostic kit comprising the foot-and-mouth disease diagnostic reagent and the monoclonal antibody. do.

In order to achieve the above object, the present invention is a hybridoma cell line (KCTC 11337BP) and the hybridoma characterized in that it produces a monoclonal antibody showing immunoreactivity common to foot-and-mouth virus O type, A type and Asia type 1 Provided are monoclonal antibodies produced by the cell line.

In addition, the present invention is produced by the hybridoma cell line (KCTC 11319BP) and the hybridoma cell line characterized in that it produces a monoclonal antibody that exhibits specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus O type VP1 site A To provide monoclonal antibodies.

In addition, the present invention is produced by the hybridoma cell line (KCTC 11318BP) and the hybridoma cell line, characterized in that it produces a monoclonal antibody that exhibits specific immune response and virus neutralizing ability against foot-and-mouth virus A VP1 site A To provide monoclonal antibodies.

Furthermore, the present invention is a foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label. Monoclonal antibodies for the monoclonal antibodies O-type A, type A, and Asia type 1 and monoclonal antibodies that show a common immunoreactivity to the virus type O, type A and Asia type 1 monoclonal antibodies are high It provides a foot-and-mouth diagnostic reagent, characterized in that the monoclonal antibody produced by the bridoma cell line (KCTC 11337BP).

delete

In the present invention, the monoclonal antibody to which the detection label is bound is specifically immunoreactive to the foot-and-mouth virus O type VP1 site A and the virus neutralization specific immunoreactivity and virus neutralization to the monoclonal antibody or foot-and-mouth virus type A VP1 site A. Provided is a foot-and-mouth diagnostic reagent characterized in that the monoclonal antibody showing the ability.

The present invention specifically reacts to any one or more epitopes selected from epitopes represented by amino acid sequences of SEQ ID NOS: 1 to 3, wherein the monoclonal antibodies exhibiting specific immunoreactivity and virus neutralizing ability against the foot-and-mouth virus O type VP1 site A It provides a foot-and-mouth diagnostic reagent, characterized in that the monoclonal antibody.

The present invention provides a foot-and-mouth diagnostic reagent, characterized in that the monoclonal antibody showing specific immunoreactivity and virus neutralizing ability against the foot-and-mouth virus O type VP1 site A is a monoclonal antibody produced by the hybridoma cell line KCTC 11319BP.

The present invention specifically reacts to any one or more epitopes selected from epitopes represented by the amino acid sequence of SEQ ID NOS: 7 to 9, wherein the monoclonal antibody exhibiting specific immunoreactivity and virus neutralizing ability against the foot-and-mouth virus A type VP1 site A It provides a foot-and-mouth diagnostic reagent, characterized in that the monoclonal antibody.

The present invention provides a foot-and-mouth diagnostic reagent, characterized in that the monoclonal antibody showing specific immune response and virus neutralizing ability against the foot-and-mouth virus A type VP1 site A is a monoclonal antibody produced by the hybridoma cell line KCTC 11318BP.

The present invention provides a recombinant foot-and-mouth disease prepared by transforming a host cell with a recombinant baculovirus comprising at least a gene encoding the structural proteins VP1, VP2, VP3 and VP4 constituting the foot-and-mouth virus and a 3C gene which is a non-structural protein. It provides a foot-and-mouth disease diagnostic reagent, characterized in that the virus is a structural protein.

The present invention also provides a foot-and-mouth disease diagnostic kit comprising the foot-and-mouth disease diagnostic reagent.

In addition, the present invention comprises the steps of immobilizing a monoclonal antibody for antigen capture on a solid phase support; Washing and removing antibodies not attached to the solid phase support; Reacting and immobilizing a foot-and-mouth virus diagnostic antigen to the solid phase support; Washing and removing antigens not attached to the solid phase support; And reacting the monoclonal antibody bound to the detection label with the foot-and-mouth virus diagnostic antigen immobilized on a solid phase support, wherein the monoclonal antibody for capturing the antigen is immune common to foot-and-mouth virus O, A and Asia 1 A monoclonal antibody that is reactive and is used for the monoclonal antibody to which the detection label is bound is a monoclonal antibody or foot-and-mouth virus type A VP1 site A that exhibits specific immunoreactivity and virus neutralization ability against foot-and-mouth virus O type VP1 site A. It is a monoclonal antibody that exhibits specific immunoreactivity and virus neutralization ability against, and the foot-and-mouth virus diagnostic antigen is a recombinant baculovirus comprising at least a gene encoding the structural proteins VP1, VP2, VP3 and VP4 and a 3C gene which is a nonstructural protein. Recombinant foot-and-mouth disease prepared by transforming host cell The recombinant foot-and-mouth virus structural protein is a virus-containing protein, and the foot-and-mouth virus whose viral titer is measured as a control group, and the recombinant foot-and-mouth virus structural protein is used as an experimental group, and the antigenic activity of the recombinant foot-and-mouth virus structural protein is quantitatively added. It provides a foot-and-mouth virus neutralizing antibody detection method characterized in that.

delete

delete

delete

The recombinant foot-and-mouth virus structural protein, the monoclonal antibody for capturing the antigen on the solid phase support, and then reacted with the foot-and-mouth virus antigens measured viral titers (T) as a control to form an antigen-antibody complex In response to the monoclonal antibody conjugated with the detection label to the antigen-antibody complex, the detection result (P) of the control group was measured, and the recombinant foot-and-mouth virus structure instead of the foot-and-mouth virus in which the virus titer (T) was measured in the experimental group. Diluting the protein by a predetermined dilution factor, immobilizing the monoclonal antibody for capturing the antigen on the solid phase support, and then reacting with the diluted recombinant foot-and-mouth virus structural protein to form an antigen-antibody complex, wherein the antigen-antibody complex is Test of the experimental group by reacting the monoclonal antibody bound to the detection label After measuring the resultant value (S), the antigen activity (Tx) was calculated from the formula, and the effective antigen amount of the recombinant foot and mouth virus structural protein was determined from the average value of the antigen activity according to the dilution factor of the recombinant foot and mouth virus structural protein. It provides a foot-and-mouth virus neutralizing antibody detection method characterized in that the addition.

According to the present invention, it is not necessary to capture the antigen by using each antibody against the inactivated foot-and-mouth virus serotype, thereby eliminating the trouble of the conventional neutralizing antibody test.

In addition, monoclonal antibodies specific to foot-and-mouth virus serotypes can be used to detect neutralizing antibodies without the hassle of using secondary antibodies for each livestock.

According to the present invention, neutralizing antibodies can be measured very effectively in animals infected with foot-and-mouth virus vaccines and animals infected outdoors, and neutralizing antibodies can be tested quickly and simply regardless of species.

The present invention uses the recombinant foot-and-mouth virus structural protein as a diagnostic antigen, which is more convenient than the method of using the foot-and-mouth virus as a diagnostic antigen which can be cultured only in a laboratory equipped with a conventional shielding facility.

The present invention is applied to a variety of techniques such as blocking enzyme immunoassay (Blocking ELISA), competitive enzyme immunoassay (Competitive ELISA) technology that can test neutralizing antibodies against foot-and-mouth disease virus with excellent detection sensitivity and specificity compared to the prior art Do.

Therefore, the present invention is effective for the investigation of a wide range of infected animals such as livestock surveillance in areas and countries where foot-and-mouth disease occurs or is being vaccinated, and for the follow-up management of vaccine animals. Prediction also contributes to the prevention of foot-and-mouth disease and the spread of proliferation and provides a very useful technology for the diagnostic use of veterinary drugs in industry.

In addition, the diagnostic reagents and diagnostic kits of the present invention are safer than the conventional foot-and-mouth virus neutralizing antibody test using infectious foot-and-mouth virus antigens, and thus export to Europe, Latin America, Asia, Russia and other global markets in which domestic and foot-and-mouth disease occurs. Since it can be promoted, it is effective to improve the technology of the animal disease diagnosis reagents and diagnostic kits in Korea, and to help acquire foreign currency.

In order to achieve the above object, it is specific for the monoclonal antibody and the hybridoma cell line (KCTC 11337BP) and foot-and-mouth virus O type VP1 site A which produce immunoreactivity common to foot-and-mouth virus O type, A type and Asia type 1, and the same Monoclonal antibody with immunological and viral neutralizing ability and hybridoma cell line (KCTC 11319BP) producing the same, monoclonal antibody with specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus A type VP1 site A and high A bridoma cell line (KCTC 11318BP) is provided.

Meanwhile, the present invention includes a foot-and-mouth diagnostic reagent and a foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label. Provides a foot-and-mouth disease diagnostic kit.

In addition, the present invention comprises the steps of immobilizing a monoclonal antibody for antigen capture on a solid phase support; Washing and removing antibodies not attached to the solid phase support; Reacting and immobilizing a foot-and-mouth virus diagnostic antigen to the solid phase support; Washing and removing antigens not attached to the solid phase support; And reacting the monoclonal antibody bound to the detection label with the foot-and-mouth virus diagnostic antigen immobilized on a solid phase support, wherein the monoclonal antibody for capturing the antigen is immune common to foot-and-mouth virus O, A and Asia 1 It provides a foot-and-mouth virus neutralizing antibody detection method characterized in that the monoclonal antibody showing a reactivity.

Hereinafter, the present invention will be described in detail. At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art. Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

First, the present invention provides a hybridoma cell line, characterized in that for producing foot-and-mouth disease or foot-and-mouth virus neutralizing antibody production, monoclonal antibodies exhibiting immunoreactivity common to foot-and-mouth virus O, A and Asia 1 are produced. Selected. The hybridoma cell line is produced by immunizing the foot-and-mouth trivalent vaccine to mice, and fusion of lymphocytes isolated from the tumor cells with SP2 / O-Ag14 (KCTC H38012, Korea Research Institute of Bioscience and Biotechnology). The fusion cell line was deposited on May 21, 2008 under the “Budapest Treaty on Microorganisms” to the Korea Cell Line Bank and received accession number KCTC 11337BP.

The monoclonal antibodies produced by the hybridoma cell line KCTC 11337BP show immunoreactivity common to foot-and-mouth virus O, A and Asia 1, and the immunoglobulin subtype is IgG1. Monoclonal antibodies produced by the hybridoma cell line KCTC 11337BP and exhibiting immunoreactivity common to foot-and-mouth virus O, type A and Asia type 1 are common to the rescue protein P1 of foot-and-mouth virus O, type A and Asia type 1 It can be used to capture diagnostic antigen in foot-and-mouth diagnostic reagents.

On the other hand, for application to the diagnosis of foot-and-mouth virus O type, a hybridoma cell line characterized by producing a monoclonal antibody showing a specific immune response and virus neutralizing ability against the foot-and-mouth virus O type VP1 site A was prepared and selected. . The hybridoma cell line was immunized with the mouse foot-and-mouth virus type VP1 site A peptide and fused with lymphocytes isolated from the tumor cell line SP2 / O-Ag14 (KCTC H38012, Korea Research Institute of Bioscience and Biotechnology) Is produced by. The fusion cell line was deposited on April 23, 2008 to the Bank of Korea Cell Line under the "Budapest Treaty on Microorganisms" and received accession number KCTC 11319BP.

The monoclonal antibody produced by the hybridoma cell line KCTC 11319BP specifically responds to site A of the structural protein VP1 of foot-and-mouth virus O type, and the immunoglobulin subtype is IgG2a. The monoclonal antibody produced by the hybridoma cell line KCTC 11319BP and specifically reacting against site A of the rescue protein VP1 of foot-and-mouth virus O type neutralizes foot-and-mouth virus O-type in foot-and-mouth diagnostic reagents, thus neutralizing the foot-and-mouth virus O type. It can be used to detect antibodies.

On the other hand, for application to the diagnosis of foot-and-mouth virus A type, a hybridoma cell line was produced and selected for producing monoclonal antibodies showing specific immune response and virus neutralizing ability against foot-and-mouth virus A type VP1 site A. . The hybridoma cell line was immunized with the mouse foot-and-mouth virus type VP1 site A peptide and fused with lymphocytes isolated from the tumor cell line SP2 / O-Ag14 (KCTC H38012, Korea Research Institute of Bioscience and Biotechnology) Is produced by. The fusion cell line was deposited on April 23, 2008 by the Bank of Korea Cell Line under the "Budapest Treaty on Microorganisms" and received accession number KCTC 11318BP.

The monoclonal antibody produced by the hybridoma cell line KCTC 11318BP specifically responds to site A of the structural protein VP1 of foot-and-mouth virus A type, and the immunoglobulin subtype is IgG2b. The monoclonal antibody produced by the hybridoma cell line KCTC 11318BP and specifically reacting against site A of the rescue protein VP1 of foot-and-mouth virus A type neutralizes foot-and-mouth virus A by neutralizing foot-and-mouth disease reagents. It can be used to detect antibodies.

The present invention provides a foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label.

At this time, the monoclonal antibody for capturing the diagnostic antigen is preferably a monoclonal antibody showing the immunoreactivity common to foot-and-mouth virus O type, A type and Asia type 1. It shows the immunoreactivity common to foot-and-mouth virus O, A and Asia type 1, eliminating the need for capturing antigens with each antibody against foot-and-mouth virus serotypes, eliminating the hassle of conventional neutralizing antibody tests. Because it can. In addition, the monoclonal antibody exhibiting immunoreactivity common to foot-and-mouth virus O type, A type and Asia type 1 is preferably a monoclonal antibody produced by the hybridoma cell line KCTC 11337BP.

On the other hand, inactivated foot-and-mouth virus and recombinant foot-and-mouth virus can be used as the diagnostic antigen. However, it is preferable to use recombinant foot-and-mouth virus structural protein rather than inactivated foot-and-mouth virus as a diagnostic antigen. In order to use inactivated foot-and-mouth virus, foot-and-mouth virus can only be cultured in a laboratory equipped with a shielding facility. The recombinant foot-and-mouth virus structural protein is a recombinant cell prepared by transforming a host cell with a recombinant baculovirus comprising at least a gene encoding the structural proteins VP1, VP2, VP3, and VP4 constituting the foot-and-mouth virus and a 3C gene which is a non-structural protein. Preferred foot and mouth virus. This is because it is safe using recombinant baculovirus which enables the production of subunits composed of structural proteins that are not infectious by genetic engineering. The recombinant foot-and-mouth virus structural protein of the present invention can be prepared according to the method disclosed in Patent No. 10-0737446.

In addition, the monoclonal antibody to which the detection label is bound has a specific immunoreactivity against the foot-and-mouth virus O type VP1 site A and a specific immunoreactivity and virus-neutralizing ability against the monoclonal antibody or foot-and-mouth virus type A VP1 site A. Monoclonal antibodies that can be used can be used. For the detection of neutralizing antibody against foot-and-mouth virus O type or the diagnosis of foot-and-mouth disease, monoclonal antibody showing specific immunoreactivity and virus-neutralizing ability against foot-and-mouth virus O type VP1 site A was used and neutralized to foot-and-mouth virus A type For the detection of antibodies or the diagnosis of A-type foot-and-mouth disease, monoclonal antibodies showing specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus A type VP1 site A are used.

The monoclonal antibody that exhibits specific immunoreactivity and virus neutralization ability against the foot-and-mouth virus O type VP1 site A is a monoclonal antibody that specifically responds to any one or more epitopes selected from epitopes represented by amino acid sequences of SEQ ID NOs: 1 to 3. Can be. In this case, the monoclonal antibody showing the specific immune response and virus neutralizing ability against the foot-and-mouth virus O type VP1 site A is produced by the hybridoma cell line KCTC 11319BP, and specific immune response to the foot-and-mouth virus O type VP1 site A It is preferable that it is a monoclonal antibody which shows virus neutralizing ability.

In addition, the monoclonal antibody exhibiting specific immunoreactivity and virus neutralization ability against the foot-and-mouth virus A type VP1 site A specifically reacts with any one or more epitopes selected from epitopes represented by amino acid sequences shown in SEQ ID NOs: 7 to 9. It may be a monoclonal antibody. In this case, the monoclonal antibody showing the specific immune response and virus neutralizing ability against the foot-and-mouth virus A type VP1 site A is produced by the hybridoma cell line KCTC 11318BP, and specific immune response to the foot-and-mouth virus A type VP1 site A. It is preferable that it is a monoclonal antibody which shows virus neutralizing ability.

The detection label and the reagent for measuring the activity of the detection label may be any one of color development, fluorescence, and luminescence properties by reaction, and to measure the various detection labels used in the general immunoassay and the activity measurement according to each detection label. Reagents can be used.

The foot-and-mouth disease diagnostic reagent of the present invention is a reagent for use in general immunoassay in addition to the diagnostic antigen, monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label. It may be included as. More specifically, antibody immobilized solution, antigen immobilized solution, blocking solution, test sample diluent, wash solution to remove serum reaction and non-specific reaction of antibody, positive and negative control reagent to validate the test, the detection label And a stopper for stopping the reaction for measuring the activity of the. In addition, various solid phase supports used in general immunoassay methods, such as plates, microplate wells, plastic test tubes, glass beads, plastic beads, and the like may also be included.

The foot-and-mouth disease diagnostic kit of the present invention may be a kit including the foot-and-mouth disease diagnostic reagent of the present invention.

For the foot-and-mouth disease diagnosis or foot-and-mouth virus neutralizing antibody detection method of the present invention, it is preferable to use Enzyme immunoassay (EIA) or radioimmunoassay (RIA). More preferably, enzyme immunoassay is used. However, the present invention is not limited to the above-mentioned detection method, and the antigen is obtained by reacting a test sample collected from a complex of a monoclonal antibody for detecting foot-and-mouth virus neutralizing antibody, a monoclonal antibody for capturing an antigen, a diagnostic foot-and-mouth virus and an antigen of the present invention. Any method can be used as long as it can detect an antibody response.

The test sample means a biological sample collected from a subject. It is preferable to use intercellular body fluids or serum, and samples obtained by treating the antigens of foot-and-mouth virus or autoantibodies against the foot-and-mouth virus are collected and used.

More specific foot-and-mouth disease diagnostic method of the present invention comprises the steps of immobilizing a monoclonal antibody for antigen capture on a solid phase support; Washing and removing antibodies not attached to the solid phase support; Reacting the foot and mouth virus diagnostic antigen to the solid phase support; Washing and removing antigens not attached to the solid phase support; And reacting the monoclonal antibody bound to the detection label with the foot-and-mouth virus diagnostic antigen immobilized on a solid-phase support, wherein the monoclonal antibody for capturing the diagnostic antigen is produced by the hybridoma cell line KCTC 11337BP, and the foot-and-mouth virus O Monoclonal antibodies with immunoreactivity common to type A, type A and Asia type 1 are used.

The foot-and-mouth disease diagnostic method of the present invention using a competitive enzyme-linked immunoassay includes the first step of immobilizing a monoclonal antibody for capturing a diagnostic antigen on a plate; A second step of washing and removing antibodies not attached to the plate of the first step; A third step of quantifying and adding the recombinant foot-and-mouth virus structural protein antigen to the plate of the second step and then reacting and immobilizing; A fourth step of washing and removing foot and mouth antigens not attached to the third step plate; A fifth step of reacting by adding a test sample to the plate of the fourth step; A sixth step of washing and removing the test sample which is not specifically bound to the foot-and-mouth disease antigen fixed to the fifth step plate; A seventh step of adding a monoclonal antibody for detecting foot-and-mouth virus neutralizing antibody bound to the enzyme having the detection label bound to the plate of the sixth step; An eighth step of washing and removing monoclonal antibodies for detecting foot-and-mouth virus neutralizing antibodies that do not bind to foot-and-mouth antigens immobilized on the plate; And evaluating the level of neutralizing antibody against foot-and-mouth virus in the test sample by adding a substrate reacting with the enzyme to the plate of the eighth step.

In addition, the recombinant foot-and-mouth virus structural protein can be added quantitatively by calculating the antigenic activity of the recombinant foot-and-mouth virus structural protein using the foot-and-mouth virus whose virus titer was measured as a control group, and using the recombinant foot-and-mouth virus structural protein as an experimental group. . Since the recombinant antigen cannot measure the amount of the target protein expressed, and it is difficult to quantitatively measure the activity of the antigen, it is preferable to quantify and add the recombinant foot and mouth virus structural protein antigen for reproducible immobilization. Therefore, in the present invention, the foot-and-mouth virus in which the virus titer was measured was used as a control group, and then the antigenic activity of the recombinant foot-and-mouth virus structural protein was measured, and then the amount of the effective antigen was quantified.

More preferably, the recombinant foot-and-mouth virus structural protein, the monoclonal antibody for capturing the antigen on the solid phase support, and then reacted with the foot-and-mouth virus antigens measured virus titer (T) as a control to the antigen-antibody complex Formed and reacting the antigen-antibody complex with a monoclonal antibody conjugated with the detection label to measure the detection result (P) of the control group, and the recombinant foot-and-mouth virus instead of the foot-and-mouth virus in which the virus titer (T) was measured as an experimental group. Diluting the viral structural protein by a predetermined dilution factor to fix the monoclonal antibody for capturing the antigen on the solid phase support, and then react with the diluted recombinant foot-and-mouth virus structural protein to form an antigen-antibody complex, wherein the antigen-antibody complex The monoclonal antibody to which the detection label is bound reacts with After measuring the detection result value (S) of the test group, the antigen activity (Tx) was calculated from Equation 1 below, and the recombinant foot-and-mouth virus structural protein was obtained from an average value of the antigenic activity according to the dilution factor of the recombinant foot-and-mouth virus structural protein. The effective antigen amount of may be determined and added.

[Equation 1]

Hereinafter, the present invention will be described in more detail with reference to specific examples. The present invention is not limited by the following examples, and it will be apparent to those skilled in the art that various changes or modifications can be made within the spirit and scope of the present invention.

[ Example  One] Monoclonal antibodies  To produce Hybridoma  write

Hybridoma cell lines producing monoclonal antibodies were obtained by the method of Kohell G and Milstein C. (Nature 256, 495-497, 1975). .

SP2 / O-Ag14 (KCTC H38012, Korea Research Institute of Bioscience and Biotechnology), a myeloma cell line, was supplied with 5% carbon dioxide gas using DMEM (GibcoBRL, USA) medium supplemented with 10% fetal bovine serum Incubated at 37 ℃ in an incubator.

VP1 site A peptide of foot-and-mouth virus O type (SEQ ID NO: 3, PVTNRGDLQVLTQKAARTLP, Petrone, Korea), VP1 site A peptide of foot-and-mouth virus A type (SEQ ID NO: 7, PGAGRRGDLGPLAARTAAQLPA, Petrone, Korea) and Foot and mouth disease trivalent vaccines were inoculated into the muscles of 6-week-old BALB / c mice (Orient, Korea). After immunization, spleens of mice were aseptically collected and crushed to recover only lymphocytes. Isolated lymphocytes and tumor cells were induced to fuse lymphocytes with tumor cells using PEG 1500 (Sigma, USA). After two weeks of fusion, the supernatants were examined and the cell lines with positive responses were diluted and cloned in 96-well tissue culture plates. The selection process is briefly described as follows.

The inactivated foot-and-mouth virus antigen was diluted in an appropriate amount in a carbonate bicarbonate buffer (Sigma USA), adsorbed onto an ELISA plate (Greiner, Germany), and fixed. The reaction was carried out for a time. Thereafter, the cells were washed four times with phosphate buffer, and the anti mouse HRP conjugate (KPL, Germany) was reacted for 1 hour. Washed 4 times with phosphate buffer, TMB (3 ', 3', 5 ', 5'-Tetramethyl benzidine, Moss Inc, USA) was added to the colorant for 10 minutes and the reaction stop solution (0.5MH ₂ SO ₄ , Sigma , USA) was added to stop the color reaction, and the absorbance at 450 nm was measured by using a reader (ELISA Reader, EMAX, Molecular device, USA), hybrid cell line and foot-and-mouth virus that respond to foot-and-mouth virus O and A, respectively. Cell lines that responded to Type O, Type A, and Asia Type 1 were selected and cloned (see Table 1).

As shown in Table 1, hybridoma cell lines capable of specifically responding to inactivated foot-and-mouth virus O type antigen and producing the monoclonal antibody 76.5E, an immunoglobulin subtype IgG2a, were selected to the Korea Cell Line Bank on April 23, 2008. Deposited under the "Budapest Treaty on Microorganisms" and received accession number KCTC 11319BP.

In addition, a hybridoma cell line capable of specifically responding to inactivated foot-and-mouth virus virus type A antigen and producing monoclonal antibody 6G43, an immunoglobulin subtype IgG2b, was selected on April 23, 2008 to the Korea Cell Line Bank. Deposited under the "Budapest Treaty" and received accession number KCTC 11318BP.

In addition, a hybridoma cell line capable of responding to inactivated foot and mouth virus type O, type A, and Asia type 1 and producing monoclonal antibody 70.17, which is an immunoglobulin subtype IgG1, was selected by the Korea Cell Line Bank on May 21, 2008. Was deposited under the "Budapest Treaty on Microorganisms" and received accession number KCTC 11337BP.

Immunogen Monoclonal
Antibodies Inactivated foot and mouth virus antigens
(OD 450 nm)
Subtype O A Asia 1 Control FMDV O VP1 Site A peptide (VP1-3) 76.5E 2.338 0.081 0.070 0.048 IgG2a FMDV A VP1 Site A peptide (VP1-4) 6G43 0.081 3.779 0.084 0.068 IgG2b FMDV Vaccine 70.17 0.811 1.072 0.915 0.091 IgG1

FMDV O VP1 Site A peptide: Foot and mouth virus VP1 O type A peptide, FMDV O / SKR / 2002 (Amino Acid-PVTNRGDLQVLTQKAARTLP, SEQ ID NO: 3)

FMDV A VP1 Site A peptide: foot-and-mouth virus VP1 type A peptide, FMDV A22 / India / 1/7/77 (Amino Acid-PGAGRRGDLGPLAARTAAQLPA, SEQ ID NO: 7)

[ Example  2]: Monoclonal antibody  Specific antigen determination site analysis

In order to confirm the antigenic determinant site of the monoclonal antibody of the present invention, a peptide produced by foot-and-mouth virus type (O, A, Asia1) VP1 antigen determinant and VP2, VP3, VP4 was prepared with a synthetic peptide combined with ovalbumin (Petron , Korea) as a result of reactivity, it was found that the monoclonal antibody 76.5E of the present invention responds to the specific antigenic determinant of the site A of VP1 of foot-and-mouth virus O type as shown in Table 2.

In addition, it was found that monoclonal antibody 6G43 responded to specific antigenic determinants of site A of VP1 of foot-and-mouth virus A type.

In addition, the monoclonal antibody 70.17 was found to have a characteristic that does not respond to the major immune antigen determination site of foot-and-mouth virus structural protein.

Monoclonal antibody
Foot-and-mouth disease virus synthetic peptide ELISA
OVA
O type A type Asia Type 1 VP1-1 VP1-2 VP1-3 VP2 VP3 VP4 VP1-4 VP1-5 VP1-6 VP1-7 VP1-8 VP1-9 76.5E 2.960 1.094 2.869 0.044 0.045 0.048 0.045 0.047 0.048 0.052 0.067 0.047 0.051 6G43 0.054 0.054 0.062 0.054 0.055 0.061 2.374 1.084 0.067 0.056 0.077 0.063 0.068 70.17 0.047 0.049 0.050 0.046 0.054 0.054 0.047 0.046 0.051 0.049 0.064 0.049 0.080

1) VP1-1: FMDV O1 / Kaufbeuren (SEQ ID NO: 1: AVPNLRGDLQVLAQKVATLP)

2) VP1-2: FMDV O1 / Manisa / Turkey / 69 (SEQ ID NO 2: VANVRGDLQVLAQKAARALP)

3) VP1-3: FMDV O / SKR / 2002 (SEQ ID NO: 3: PVTNRGDLQVLTQKAARTLP)

4) VP2: FMDV O / SKR / 2002 (SEQ ID NO: 4: DKKTEETTLLEDRIL)

5) VP3: FMDV O / SKR / 2002 (SEQ ID NO: 5: PPGMPPKTPEAAAH)

6) VP4: FMDV O / SKR / 2002 (SEQ ID NO. 6: GSTDTTSTHTTNTQN)

7) VP1-4: FMDV A22 / India / 1/7/77 (SEQ ID NO: 7: PGAGRRGDLGPLAARTAAQLPA)

8) VP1-5: FMDV A24 / Cruz / Brazil / 55 (SEQ ID NO: 8: GGSGRRGDMGSLAARVVKQLPA)

9) VP1-6: FMDV Asia1 / Vaccine (SEQ ID NO. 9: TGPTRRGDLAVLAQRVSNRLP)

10) VP1-7: FMDV Asia1 / Taiwan (SEQ ID NO: 10: ETTTRRGDLAAIAQRVNRQLPTS)

11) VP1-8: FMDV Asia1 / Mongolia / 2005 (SEQ ID NO: 11: EESSRRGDLAALARRVNNRLP)

12) VP1-9: FMDV Asia1 / Shamir (SEQ ID NO: 12: ETTSRRGDMAALAQRLSARLP)

13) OVA: Ovalbumin

[ Example  3]: Monoclonal antibody  virus Neutralization  analysis

In order to confirm virus neutralization of monoclonal antibodies against foot-and-mouth virus, the virus neutralization test (VNT) was performed according to the method of Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2004) of the Office of International Water Services (OIE). Virus neutralization titers of monoclonal antibodies were measured.

Four viruses diluted with a concentration of 100 TCID ₅₀ after diluting the monoclonal antibody prepared by the present invention from 16-fold to binary steps (FMDV O / SKR / 2002, FMDV O / SKR / 2000, FMDV A22 / Shamir, FMDV Asia1) / Mongolisa / 2005) in the same amount, and reacted for 1 hour at 37 ℃ inoculated in IBRS cells and incubated for 2-3 days. Virus neutralizing titers yielded the inverse of the concentration of the maximal dilution factor where no cell degeneration was observed.

In addition, in order to confirm the cross-reactivity, the virus neutralization ability was evaluated in the same manner as the swine Vesicular Disease Virus (SVDV), Vesicular Stomatitis Virus (VSV).

As a result, as shown in Table 3, the monoclonal antibody 76.5E of the present invention was confirmed to have neutralizing ability only against foot-and-mouth virus O type, and was not cross-reactive with foot-and-mouth virus A, Asia1, SVDV, and VSV.

In addition, it was confirmed that the monoclonal antibody 6G43 of the present invention had neutralizing ability only against foot-and-mouth virus A, and had no cross-reactivity with foot-and-mouth virus O, Asia1, SVDV, and VSV.

In addition, the monoclonal antibody 70.17 of the present invention was confirmed to have no neutralizing ability against foot-and-mouth virus O type, A type, Asia1 type, SVDV, VSV.

Monoclonal antibody
Virus neutralization titer Foot-and-mouth disease virus serotypes O / SKR / 2000 O / SKR / 2002 A22 / Shamir Asia1 / Mongolisa / 2005 SVDV VSV 76.5E 256 724 <16 <16 <16 <16 6G43 <16 <16 64 <16 <16 <16 70.17 <16 <16 <16 <16 <16 <16

[ Example  4]: for capturing diagnostic antigens Monoclonal antibody  Selection

In order to select monoclonal antibodies capable of capturing foot-and-mouth virus diagnostic antigens, three inactivated viruses (type O, type A, and Asia type 1) were added to a monoclonal antibody-immobilized plate, followed by inoculation of foot-and-mouth trivalent vaccine. As a result of confirming reactivity by adding serum, it was found that 70.17 monoclonal antibody was the most responsive and captured a certain amount of antigen regardless of virus serotype (Table 4).

Therefore, 70.17 was selected as a monoclonal antibody capable of capturing the foot and mouth virus diagnostic antigen.

Monoclonal antibody
Inactivated foot and mouth virus antibody (O.D.450nm) O / SKR / 2000 O1 manisa A Iraq Asia1 / Shamir 76.5E 0.719 0.775 0.299 0.305 6G43 0.314 0.289 1.024 0.987 70.17 1.140 1.569 1.167 1.159

[ Example  5]: recombinant foot and mouth virus Structural protein  production

In the same manner as the recombinant baculovirus production method of p-FastBacDual P13C of Patent No. 10-0737446, the P1 gene of foot-and-mouth virus A / Iraq was cloned to prepare p-FastBacDual A P13C recombinant baculovirus.

In order to produce each recombinant foot-and-mouth virus structural protein (r-FMDV O P13C, r-FMDV A P13C), inoculated with recombinant baculovirus at 0.1moi in SF-9 cells and cultured for 4-6 days When more than 90% appeared cells were frozen and thawed three times, the supernatant was harvested by centrifugation for 20 minutes at 3,000rpm. Store at -20 ° C until use.

[ Example  6]: genetic recombination Structural protein  Capture and immobilization

(1) Establishment of recombinant structural protein assay

In the case of transgenic antigens, the fusion protein for purification is not included in order to induce the tertiary structure of the expressed protein to be synthesized similar to the viral structure, so that it is impossible to measure the amount of the target protein expressed and the activity of the antigen. Because it is difficult to measure quantitatively, we attempted to establish a quantitative assay that can measure protein activity in order to establish reproducible immobilization techniques.

To this end, monoclonal antibody 70.17 was immobilized on a plate at a concentration of 2 ug / ml and a double antibody sandwich ELISA (DAS ELISA) using monoclonal antibody 76.5E-horseradish peroxidase (HRP) conjugate was used. It was set by quantitative method.

That is, the foot-and-mouth disease virus antigen whose virus titer was measured was set as a standard sample, the recombinant antigen was diluted and reacted, and the monoclonal antibody 76.5E-HRP conjugate was reacted to read the absorbance.

At this time, the absorbance value (P) at the virus titer (T) 10 ^5.0 TCID ₅₀ / ml was determined as 1.0 Unit based on the amount of the effective antigen, and the absorbance value (S) of the recombinant antigen was measured to determine the absorbance value ratio (S). / P) was calculated. The calculated absorbance ratio (S / P) was multiplied by the dilution factor (Dilution factor) of the recombinant antigen was converted into units (see Equation 1).

The mean antigen amount of the recombinant structural protein was quantified by calculating the average from the Unit value of the recombinant antigen reflecting the dilution degree. The recombinant structural protein was diluted to an effective antigen amount of 1.0 Unit and used as a diagnostic antigen (Table 5).

Dilution factor
Genetic foot-and-mouth virus SP antibody (Batch 0501-01) O.D. S / P Unit 20 0.988 (S) 1.01 20.3 40 0.561 (S) 0.57 23.0 60 0.390 (S) 0.40 24.0 80 0.286 (S) 0.29 23.5 Virus (10 ^5.0 TCID ₅₀ / ml)-control 0.975 (P) 1.00 1.0

That is, as shown in Table 5, as a result of reacting the foot-and-mouth virus with a titer (T) of 10 ^5.0 TCID ₅₀ / ml as a control, the absorbance (P) was found to be 0.975, which was determined as an effective antigen amount of 1.0Unit.

After diluting the recombinant recombinant protein 20 times, 40 times, 60 times, and 80 times, the absorbance was measured. As a result, S values were 0.988, 0.561, 0.390, and 0.286, respectively, and the S / P values were 1.01. , 0.57, 0.40, 0.29, and the effective antigen amount was calculated by multiplying the dilution factor.

As a result, the effective antigen amount of the recombinant structural protein was found to be 20.3 Unit, 23.0 Unit, 24.0 Unit, 23.5 Unit, and the average effective antigen amount of the recombinant structural protein was determined to be 22.7 Unit.

From this, the recombinant structural protein was diluted so as to have an effective antigen amount of 1.0 Unit, and used as a diagnostic antigen.

2) Capture and Immobilization of Recombinant Structural Proteins

1 is a schematic diagram showing the steps of immobilizing a recombinant foot-and-mouth virus structural protein antigen to a solid-phase support (antibody-fixing plate) using a monoclonal antibody for capturing diagnostic antigen, capture and immobilize the foot-and-mouth virus structural protein in this order Was carried out.

That is, the monoclonal antibody 70.17, which can capture the foot-and-mouth virus structural protein as a diagnostic antigen, is diluted to a concentration of 1 to 4 µg / ml, added to the antibody fixation solution of Table 6, homogenized by stirring at room temperature for at least 3 hours, and then ELISA plate. Alternatively, an appropriate amount was dispensed into the fixing device, and the monoclonal antibody was immobilized on the plate by reacting at 4-10 ° C. for 18 hours or more.

After the reaction, the non-immobilized antibody was washed with the washing solution of Table 6, and the blocking solution of Table 6 was added to block the non-adhered site, followed by 2 hours of reaction at room temperature, followed by washing with the washing solution.

Thereafter, the recombinant structural protein was diluted and added to the antigen-fixed solution shown in Table 6 so as to be 1 unit, and then immobilized by reacting for 2 hours at room temperature. After drying, sealed with silica gel and stored at 4 ~ 8 ℃.

Types and Compositions of Buffers for Fixation of Recombinant Foot-and-mouth Virus Structural Protein Antigens Buffer Reagent Composition Antibody Fixation Solution 1.0 M Bicarbonate (Bicarbobate);
0.5M Carbonate; pH 9.0 Washing liquid NaCl 0.137 M; KH ₂ PO ₄ 1.4 mM; Na ₂ HPO ₄ 7H ₂ O 8.1 mM; KCl 2.7 mM; Detergent 0.05%, pH 7.4 Blocking solution Tris-HCl 17.8 mM; Tris-Base 2.2 mM; NaCl 0.15M, Bovine Serum Albumin 5%; Gelatin 1%; Detergent 0.05%; pH 8.0 Antigen Fixation Solution NaCl 0.137 M; KH ₂ PO ₄ 1.4 mM; Na ₂ HPO ₄ 7H ₂ O 8.1 mM; KCl 2.7 mM; Bovine Serum Albumin 1%; pH 7.4

[ Example  7]: composition of foot and mouth diagnostic reagents

First, the monoclonal antibodies 76.5E and 6G43 selected in Example 3 were conjugated with the monoclonal antibody according to the use manual of the peroxidase conjugation kit (Roche, Germany), and then diluted to a concentration of 50 to 500 ng for detection. Made with reagents.

In addition, the sample dilution buffer (Tris-base 20mM: Bovine Serum Albumin 1%; Triton X-100, 0.1%; EDTA 0.1%; Tween 20 0.1%, pH 7.5) is a foot-and-mouth virus genetic recombination in which neutralizing antibodies are immobilized in the sample. The conditions that can be most strongly reacted to the antigen were searched and formed.

In addition, a positive control to be used for the test validity assay provided sera of 90 or more neutralizing antibody titers against foot-and-mouth virus, and a negative control provided sera with a neutralizing antibody titer of zero.

As a color developing reagent, TMB purchased from MOSS of USA was used, and 0.5MH ₂ SO ₄ was used as a reaction stop solution.

Figure 2 is a photograph showing the configuration of a diagnostic reagent for testing the foot-and-mouth virus neutralizing antibody of the present invention and a diagnostic kit comprising the same.

[ Example  8]: setting of criteria

As a result criterion, the absorbance was measured at 450 nm using an absorbance reader, and the absorbance (S) and negative control absorbance (N) ratio of the test serum were calculated by Equation 2 below. A positive value was determined when the S / N value was 0.6 or less, and a negative value when greater than 0.6.

[Equation 2]

SN ratio Value (SN) = [average absorbance of the test sample / average absorbance of the negative control]

[ Example  9]: Test for foot-and-mouth virus neutralizing antibody test

The test for foot-and-mouth virus O type using the diagnostic reagent showed a sensitivity of 98.1% for the positive group and a specificity of 98.7% for the negative serum. In addition, similar results were shown regardless of the livestock, it was found that the ability to measure foot-and-mouth virus neutralizing antibodies of the present invention is very excellent (Table 7).

group
Breeder
FMDV O AB ELISA responsiveness Specificity Positive serum Negative serum system (%) (%) positivity
(N = 154)



small 106 One 107 99.1 pig 19 One 20 95.0 Goat 21 One 22 95.5 amount 3 0 3 100.0 Etc 2 0 2 100.0 Total / average 151 3 154 98.1 voice
(N = 384)



small One 127 128 99.2 pig 0 149 149 100.0 Goat 0 65 65 100.0 amount 4 27 31 87.1 deer 0 11 11 100.0 Total / average 5 379 384 98.7

As a result of testing the diagnostic reagent of the present invention on the foot-and-mouth virus O type with a commercially available product (CEDI), it was confirmed that the present invention is 0.7% higher in detection sensitivity and 1.6% higher in specificity as shown in Table 8. It was found to be excellent.

group
Breeder
CEDI FMDV O AB ELISA responsiveness Specificity Positive serum Negative serum system (%) (%) positivity
(N = 76)



small 29 0 29 100.0 pig 18 2 20 90.0 Goat 22 0 22 100.0 amount 3 0 3 100.0 Etc 2 0 2 100.0 Total / average 74 2 76 97.4 voice
(N = 34)


small 0 13 13 100.0 pig 0 4 4 100.0 Goat 0 11 11 100.0 amount One 5 6 83.3 Total / average One 33 34 97.1

And, as a result of checking the foot-and-mouth disease vaccines area serum as shown in Table 9, 16.3%, than the standard test (VNT) for the antibody detection ratio of foot-and-mouth disease diagnostic reagent according to the present invention the conventional foot-and-mouth disease virus type O, commercially available (CEDI ^® FMDV 6.4% better than Type O ELISA.

group
Chinese Antibody Titer FMDV O AB ELISA CEDI FMDV Type O ELISA positivity voice system positivity voice system positivity voice system Positive 113 0 113 112 One 113 34 One 35 Cut-off 0 40 40 26 14 40 28 11 39 Total 113 40 153 138 15 153 62 12 74 % Positive 73.9 90.2 83.8

Positive: Serum 45 times higher than neutralizing antibody

Cut-off: Less than 45 times neutralizing antibody titer 8 times serum

Meanwhile, according to the diagnostic method published by Um et al. (Vaccine, 2007, 25 (20), 4112-4121), as shown in Table 10, one of four samples of pig serum vaccinated with foot-and-mouth vaccine was negatively determined. The positive S / N distribution ranged from 0.25 to 0.57. However, according to the present invention, all four samples were determined to be positive, and all of the S / Ns were also 0.30 or less, indicating that the sensitivity and the specificity were excellent.

Serum
SNT against FMDV O Method by moth FMDV O AB ELISA (invention) OD SN result OD SN result SM01 512 0.291 0.25 positivity 0.086 0.10 positivity SM02 512 0.371 0.32 positivity 0.086 0.10 positivity ST01 128 0.672 0.57 positivity 0.213 0.24 positivity ST02 128 2.072 1.76 voice 0.273 0.30 positivity PC 0.111 0.09 0.1035 0.11 NC 1.176 1.00 0.9045 1.00

SNT: Serum Neutralizing Titer

SM01, SM02: Foot-and-mouth disease vaccine (O1 Manisa) inoculated pig serum

ST01, ST02: Porcine serum inoculated with foot and mouth disease trivalent vaccine (O1 Mannisa, Asia1 Shamir, A Iraq)

PC: positive control

NC: negative contrast

In addition, as shown in Table 11, the S / N value was increased in inverse proportion to the neutralizing antibody titer in the antiserum against foot-and-mouth virus A type, and thus, it was found that the neutralizing antibody titer for type A could be measured.

FMDV A AB ELISA SNT against FMDV A Serotype 1024 512 256 128 64 32 16 8 0 OD 0.116 0.155 0.254 0.472 0.859 1.141 1.381 1.535 1.617 S / N 0.07 0.10 0.16 0.29 0.53 0.71 0.85 0.95 1.00

SNT: Serum Neutralizing Titer

Therefore, the test method according to the present invention can be applied not only to the antibody test for foot-and-mouth virus O type and A type, but also to C type, Asia type 1, etc. Can be.

On the other hand, a range of modifications, variations and substitutions are possible with respect to the foregoing disclosure, and in some cases only some of the features of the invention may be used. Accordingly, the appended claims should be construed broadly and in accordance with the spirit and scope of the invention.

1 is a schematic diagram showing a step of immobilizing a recombinant foot-and-mouth virus structural protein antigen to a solid phase support (antibody fixation plate) using a monoclonal antibody for diagnostic antigen capture.

Figure 2 is a photograph showing the configuration of the foot-and-mouth disease diagnostic kit of the present invention.

<110> JENOBIOTECH Inc.          National Veterinary Research and Quarantine Service <120> Hybridoma cell lines, monoclonal antibodies produced from the          hybridoma cell lines, Foot-and-mouth disease virus (FMDV)          detection reagents, FMDV detection kits and detection method for          FMDV neutralizing antibodies <130> PA08-017 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> PRT <213> FMDV O1 / Kaufbeuren <400> 1 Ala Val Pro Asn Leu Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Val   1 5 10 15 Ala Thr Leu Pro              20 <210> 2 <211> 20 <212> PRT <213> FMDV O1 / Manisa / Turkey / 69 <400> 2 Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala   1 5 10 15 Arg Ala Leu Pro              20 <210> 3 <211> 20 <212> PRT <213> FMDV O / SKR / 2002 <400> 3 Pro Val Thr Asn Arg Gly Asp Leu Gln Val Leu Thr Gln Lys Ala Ala   1 5 10 15 Arg Thr Leu Pro              20 <210> 4 <211> 15 <212> PRT <213> FMDV O / SKR / 2002 <400> 4 Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu   1 5 10 15 <210> 5 <211> 14 <212> PRT <213> FMDV O / SKR / 2002 <400> 5 Pro Pro Gly Met Pro Pro Lys Thr Pro Glu Ala Ala Ala His   1 5 10 <210> 6 <211> 15 <212> PRT <213> FMDV O / SKR / 2002 <400> 6 Gly Ser Thr Asp Thr Thr Ser Ser His His Thr Thr Asn Thr Gln Asn   1 5 10 15 <210> 7 <211> 22 <212> PRT <213> FMDV A22 / India / 1/7/77 <400> 7 Pro Gly Ala Gly Arg Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg Thr   1 5 10 15 Ala Ala Gln Leu Pro Ala              20 <210> 8 <211> 22 <212> PRT <213> FMDV A24 / Cruz / Brazil / 55 <400> 8 Gly Gly Ser Gly Arg Arg Gly Asp Met Gly Ser Leu Ala Ala Arg Val   1 5 10 15 Val Lys Gln Leu Pro Ala              20 <210> 9 <211> 21 <212> PRT <213> FMDV Asia1 / Vaccine <400> 9 Thr Gly Pro Thr Arg Arg Gly Asp Leu Ala Val Leu Ala Gln Arg Val   1 5 10 15 Ser Asn Arg Leu Pro              20 <210> 10 <211> 23 <212> PRT <213> FMDV Asia1 / Taiwan <400> 10 Glu Thr Thr Thr Arg Arg Gly Asp Leu Ala Ala Ile Ala Gln Arg Val   1 5 10 15 Asn Arg Gln Leu Pro Thr Ser              20 <210> 11 <211> 21 <212> PRT <213> FMDV Asia1 / Mongolia / 2005 <400> 11 Glu Glu Ser Ser Arg Arg Gly Asp Leu Ala Ala Leu Ala Arg Arg Val   1 5 10 15 Asn Asn Arg Leu Pro              20 <210> 12 <211> 21 <212> PRT <213> FMDV Asia1 / Shamir <400> 12 Glu Thr Thr Ser Arg Arg Gly Asp Met Ala Ala Leu Ala Gln Arg Leu   1 5 10 15 Ser Ala Arg Leu Pro              20  

Claims (20)

A hybridoma cell line deposited with Accession No. KCTC 11337BP characterized by producing monoclonal antibodies exhibiting immunoreactivity common to foot-and-mouth virus O-types, A-types and Asia 1-type. Monoclonal antibody produced by the hybridoma cell line of claim 1. A hybridoma cell line deposited with accession no. KCTC 11319BP, characterized by producing monoclonal antibodies exhibiting specific immunoreactivity and virus neutralizing capacity against foot-and-mouth virus O type VP1 site A. Monoclonal antibody produced by the hybridoma cell line of claim 3. A hybridoma cell line deposited with Accession No. KCTC 11318BP, characterized by producing monoclonal antibodies exhibiting specific immunoreactivity and virus neutralizing capacity against foot-and-mouth virus A type VP1 site A. Monoclonal antibody produced by the hybridoma cell line of claim 5. delete A foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label, The monoclonal antibody for capturing the diagnostic antigen is a monoclonal antibody exhibiting a common immune response to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibody for foot-and-mouth virus O-type, A-type and Asia type 1 is a monoclonal antibody produced by a hybridoma cell line deposited with accession number KCTC 11337BP. The method of claim 8, The monoclonal antibody to which the detection label is bound exhibits specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus O type VP1 site A, and specific immunoreactivity and virus neutralization capacity against monoclonal antibody or foot-and-mouth virus type A VP1 site A. Foot and mouth diagnostic reagents, characterized in that the monoclonal antibody. A foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label, The monoclonal antibody for capturing the diagnostic antigen is a monoclonal antibody exhibiting a common immune response to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibody to which the detection label is bound exhibits specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus O type VP1 site A, and specific immunoreactivity and virus neutralization capacity against monoclonal antibody or foot-and-mouth virus type A VP1 site A. Monoclonal antibody, The monoclonal antibody that exhibits specific immunoreactivity and virus neutralization ability against the foot-and-mouth virus O type VP1 site A is a monoclonal antibody that specifically responds to any one or more epitopes selected from epitopes represented by amino acid sequences of SEQ ID NOs: 1 to 3. Foot-and-mouth disease diagnostic reagent. A foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label, The monoclonal antibody for capturing the diagnostic antigen is a monoclonal antibody exhibiting a common immune response to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibody to which the detection label is bound exhibits specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus O type VP1 site A, and specific immunoreactivity and virus neutralization capacity against monoclonal antibody or foot-and-mouth virus type A VP1 site A. Monoclonal antibody, The monoclonal antibody for foot-and-mouth virus O type VP1 site A is a monoclonal antibody produced by a hybridoma cell line deposited with Accession No. KCTC 11319BP. A foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label, The monoclonal antibody for capturing the diagnostic antigen is a monoclonal antibody exhibiting a common immune response to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibody to which the detection label is bound exhibits specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus O type VP1 site A, and specific immunoreactivity and virus neutralization capacity against monoclonal antibody or foot-and-mouth virus type A VP1 site A. Monoclonal antibody, The monoclonal antibody that exhibits specific immunoreactivity and virus neutralization ability against the foot-and-mouth virus A type VP1 site A is a monoclonal antibody that specifically reacts with at least one epitope selected from epitopes represented by amino acid sequences of SEQ ID NOs: 7 to 9. Foot-and-mouth disease diagnostic reagent. A foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label, The monoclonal antibody for capturing the diagnostic antigen is a monoclonal antibody exhibiting a common immune response to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibody to which the detection label is bound exhibits specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus O type VP1 site A, and specific immunoreactivity and virus neutralization capacity against monoclonal antibody or foot-and-mouth virus type A VP1 site A. Monoclonal antibody, A monoclonal antibody for foot-and-mouth virus A type VP1 site A is a monoclonal antibody produced by a hybridoma cell line deposited with the accession number KCTC 11318BP, the monoclonal antibody showing a specific immunoreactivity and virus neutralizing ability. A foot-and-mouth diagnostic reagent containing a diagnostic antigen, a monoclonal antibody for capturing the diagnostic antigen, a detection label, a monoclonal antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label, The monoclonal antibody for capturing the diagnostic antigen is a monoclonal antibody exhibiting a common immune response to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibodies exhibiting immunoreactivity common to the foot and mouth virus type O, type A and Asia type 1 are monoclonal antibodies produced by a hybridoma cell line deposited with accession number KCTC 11337BP, The monoclonal antibody to which the detection label is bound exhibits specific immunoreactivity and virus neutralizing ability against foot-and-mouth virus O type VP1 site A, and specific immunoreactivity and virus neutralization capacity against monoclonal antibody or foot-and-mouth virus type A VP1 site A. Monoclonal antibody, The diagnostic antigen is a recombinant foot-and-mouth virus structural protein prepared by transforming a host cell with a recombinant baculovirus comprising at least a gene encoding the structural proteins VP1, VP2, VP3 and VP4 constituting the foot-and-mouth virus and a 3C gene which is a non-structural protein. Foot-and-mouth disease diagnostic reagent. A foot-and-mouth diagnostic kit comprising the foot-and-mouth diagnostic reagent of any one of claims 8-14. delete delete delete Immobilizing the monoclonal antibody for antigen capture on the solid phase support; Washing and removing antibodies not attached to the solid phase support; Reacting and immobilizing a foot-and-mouth virus diagnostic antigen to the solid phase support; Washing and removing antigens not attached to the solid phase support; And Reacting the detection label-bound monoclonal antibody with the foot-and-mouth virus diagnostic antigen immobilized on a solid phase support, The monoclonal antibody for capturing the antigen is a monoclonal antibody showing the immunoreactivity common to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibody used in the monoclonal antibody to which the detection label is bound has a specific immunoreactivity against foot-and-mouth virus O type VP1 site A and a specific immunoreactivity against monoclonal antibody or foot-and-mouth virus A type VP1 site A that exhibits virus neutralization ability. It is a monoclonal antibody that shows virus neutralizing ability, The foot-and-mouth virus diagnostic antigen is a recombinant foot-and-mouth virus structural protein prepared by transforming a host cell with a recombinant baculovirus comprising at least a gene encoding the structural proteins VP1, VP2, VP3 and VP4 and a non-structural protein 3C gene, The recombinant foot-and-mouth virus structural protein, A foot-and-mouth virus neutralizing antibody detection method, characterized in that the foot-and-mouth virus of which virus titer was measured as a control, and the recombinant foot-and-mouth virus structural protein as an experimental group, was calculated and added quantitatively to calculate the antigenic activity of the recombinant foot-and-mouth virus structural protein. Immobilizing the monoclonal antibody for antigen capture on the solid phase support; Washing and removing antibodies not attached to the solid phase support; Reacting and immobilizing a foot-and-mouth virus diagnostic antigen to the solid phase support; Washing and removing antigens not attached to the solid phase support; And Reacting the detection label-bound monoclonal antibody with the foot-and-mouth virus diagnostic antigen immobilized on a solid phase support, The monoclonal antibody for capturing the antigen is a monoclonal antibody showing the immunoreactivity common to foot-and-mouth virus O type, A type and Asia type 1, The monoclonal antibody used in the monoclonal antibody to which the detection label is bound has a specific immunoreactivity against foot-and-mouth virus O type VP1 site A and a specific immunoreactivity against monoclonal antibody or foot-and-mouth virus A type VP1 site A that exhibits virus neutralization ability. It is a monoclonal antibody that shows virus neutralizing ability, The foot-and-mouth virus diagnostic antigen is a recombinant foot-and-mouth virus structural protein prepared by transforming a host cell with a recombinant baculovirus comprising at least a gene encoding the structural proteins VP1, VP2, VP3 and VP4 and a non-structural protein 3C gene, The recombinant foot-and-mouth virus structural protein, The monoclonal antibody for capturing the antigen was immobilized on the solid support, and then reacted with the foot-and-mouth virus antigen whose virus titer (T) was measured as a control to form an antigen-antibody complex, and the detection label was added to the antigen-antibody complex. To measure the detection result (P) of the control by reacting the combined monoclonal antibody, In the experimental group, the recombinant foot-and-mouth virus structural protein instead of the foot-and-mouth virus in which the virus titer was measured was diluted by a predetermined dilution factor to fix the monoclonal antibody for capturing the antigen on the solid-phase support, and then the diluted recombinant foot-and-mouth virus structure React with the protein to form an antigen-antibody complex, and react the antigen-antibody complex with the monoclonal antibody bound to the detection label to measure the detection result (S) of the experimental group, The foot-and-mouth virus neutralization is calculated by calculating the antigenic activity (Tx) from the following formula, and determining the effective antigen amount of the recombinant foot-and-mouth virus structural protein from the mean value of the antigenic activity according to the dilution factor of the recombinant foot-and-mouth virus structural protein. Antibody Detection Method.

Images