KR20110115006A - Genetically engineered protein and monoclonal antibody of foot-and-mouth disease virus type sat2 and their application to the diagnostic method - Google Patents

Abstract

The present invention relates to an enzyme-linked immunoassay for preparing foot-and-mouth virus SAT2 recombination protein and monoclonal antibody and detecting foot-and-mouth disease SAT2 antibody using the same.
The purpose of the present invention is to develop a diagnostic antigen that can be safely manufactured in a general laboratory by replacing an inactivated virus with a genetic recombinant protein as a diagnostic antigen, and to produce a monoclonal antibody having a neutralizing titer against foot-and-mouth virus SAT2, a foot-and-mouth virus The present invention provides a new diagnostic method for rapidly detecting SAT2 antibody.
More specifically, P1 and 3C protease genes corresponding to the foot-and-mouth virus SAT2 structural protein were cloned into one expression vector, and then expressed in insect cells to produce virus-like particles. Neutralizing monoclonal antibodies neutralized the virus. Peptides corresponding to epitopes were synthesized and inoculated into mice, and then fused with immune cells and myeloma-derived cells. Using this recombinant protein antigen and monoclonal antibody, a new type of enzyme-linked immunoassay has been developed.
The diagnostic method of the present invention is a competitive enzyme-linked immunoassay method using a recombinant protein diagnostic antigen that can be safely produced in a general laboratory, and can be tested in the same manner regardless of the stocks of cattle, pigs, goats, etc. The ability to quickly determine vaccine antibody titers at the time of determination or vaccination can be used as a new diagnostic alternative to the standardized test for neutralization.

Description

Foot-and-mouth disease virus type 2 recombinant protein antigen, monoclonal antibody and diagnostic method using the same {genetically engineered protein and monoclonal antibody of foot-and-mouth disease virus type SAT2 and their application to the diagnostic method}

The present invention develops an antigen that can be safely produced in a general laboratory by replacing an inactivated virus with a genetic recombinant protein as a diagnostic antigen, and selects a monoclonal antibody that specifically binds to the foot-and-mouth virus SAT2, foot-and-mouth virus SAT2 It relates to a new diagnostic method for the rapid detection of type antibodies.

Foot-and-mouth disease is a highly contagious animal disease that infects cows, pigs, sheep and goats. Once that has a devastating effect on international trade, it causes considerable economic damage. The causative agent is a foot-and-mouth virus containing 8,500 base pairs of single-stranded RNA, and four proteins of VP1, VP2, VP3, and VP4 come together to form a viral envelope. Foot-and-mouth disease is divided into seven serotypes: O, A, Asia 1, C, SAT (South African territories) 1, SAT2 and SAT3.

In a foot-and-mouth disease test, an antigen test is essential, but in the absence of an antigen test sample, serological tests are used to confirm the infection. In particular, in order to rapidly detect an antibody formed in a host animal after infection or to accurately measure a vaccine antibody titer after vaccination, a test method for measuring a structural protein antibody rather than a conventional non-structural protein antibody test method is required.

As a diagnostic method for detecting structural protein antibodies, the International Water Bureau recommends neutralization test as a standard diagnostic method, but it takes about 3 days and it is difficult to test a large amount of samples. Enzyme-linked immunoassays have been developed to overcome. Hamblin et al. (1986) developed liquid-phase blocking enzyme-linked immunosorbent assay (LPB ELISA) using inactivated foot-and-mouth virus and rabbit and guinea pig polyclonal antibodies. Since the polyclonal antibody is used as a diagnostic antibody, there is a disadvantage in that a difference in antibody titer occurs every production. In particular, there is a risk that foot and mouth disease can occur outdoors due to the outflow of the virus in the process of handling live viruses. According to Valarcher et al. (2008), there were cases of foot-and-mouth disease caused by in-lab foot-and-mouth virus outbreaks in Germany in 1987 and 1988 and in Russia in 1993. This is an example of a leak.

In order to overcome the drawbacks mentioned in the prior art, Eom et al. (2004) prepared a recombinant protein antigen using P1, which is a precursor protein of foot-and-mouth virus O type, and 3C, which is a proteinase, and applied it to the foot-and-mouth type O antibody diagnostic method. . In addition, Koo et al. (2009) approached the same strategy for foot-and-mouth disease Asian type 1 and developed a recombinant protein diagnostic antigen, and reported the development of a diagnostic technique to detect Asian type 1 specific antibodies by combining Asian type 1 specific monoclonal antibodies.

However, there are no examples of antibody diagnosis using genetically engineered diagnostic antigens for SAT2 type in Africa among serotypes of 7 foot and mouth disease.

Therefore, the present invention attempted the artificial expression of diagnostic antigen in baculovirus-infected insect cells using the foot-and-mouth virus SAT2-type protein precursor P1 and 3C protease gene. In addition, a monoclonal antibody specific for foot-and-mouth virus SAT2 type was prepared as a competitive antibody to provide a new diagnostic method for rapidly detecting foot-and-mouth disease SAT2 type antibody in addition to the above diagnostic antigen.

More specifically, after the cloning of the rescue protein precursor P1 gene and the 3C protease gene of the foot-and-mouth virus SAT2 type into one expression vector, a recombinant baculovirus DNA was produced and expressed in insect cells to diagnose a protein. A monoclonal antibody used as an antigen and a competition reaction was prepared by synthesizing a peptide corresponding to the neutralizing epitope of the virus and inoculating mice with cell fusion of immune cells and myeloma-derived cells. A new type of enzyme-linked immunoassay was developed using a recombinant protein diagnostic antigen and a monoclonal antibody.

The diagnostic method of the present invention is a competitive enzyme-linked immunoassay method that can be tested in the same manner regardless of cattle, pigs, goats, or livestock, and determines whether the foot-and-mouth virus SAT2 is infected or rapidly measures vaccine antibody titers during vaccination. As a result, it can be used as a new diagnostic method that can replace the standard neutralization test method and the existing LPB ELISA.

Technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description of the present invention. .

In order to achieve the above object, the present invention provides a foot-and-mouth virus SAT2 virus-like particle expressed by infecting the baculovirus produced by the genetic recombination method to insect cells.

It provides monoclonal antibody 2E42 which specifically binds to foot-and-mouth virus SAT2 type and has a virus neutralizing titer.

Fusion of the foot-and-mouth virus SAT2-type VP1 specific peptide site to the mouse, and produced by cell fusion between the isolated immune B cells and mouse myeloma-derived cells to produce monoclonal antibody 2E42 that specifically binds to the foot-and-mouth virus SAT2 type Cell line (Accession No. KCLRF-BP-00227) is provided.

In addition, the enzyme-linked immunoassay method using the recombinant protein diagnostic antigen and monoclonal antibody 2E42 provides a diagnostic method for detecting foot-and-mouth virus SAT2-specific antibody by competition between test serum and monoclonal antibody 2E42.

The diagnostic method is specifically

(a) reacting SAT2-type monoclonal antibody 2B30 or VP1 antiserum with a 96-well microplate;

(b) washing and removing the antibody not attached to the plate;

(c) reacting the foot and mouth virus SAT2 type recombinant protein as a diagnostic antigen;

(d) washing and removing protein antigens not attached to the plate;

(e) reacting the test serum sample;

(f) washing and removing the test serum sample not attached to the plate;

(g) competitively reacting the SAT2-specific monoclonal antibody to which the horseradish peroxidase enzyme is bound;

(h) washing and removing monoclonal antibodies not attached to the plate; And

(i) measuring the level of foot-and-mouth virus SAT2 antibody in the test serum by measuring the absorbance of the color reaction by the enzymatic reaction; It can be configured as.

In the present invention, a new type of enzyme-linked immunoassay has been developed using diagnostic antigens prepared using the P1 gene and the 3C protease gene corresponding to the rescue protein of foot-and-mouth virus Asian type 1 and monoclonal antibodies for competition. As a result of evaluating the effectiveness, the results were comparable with the existing diagnostic methods. Thus, in the field of foot-and-mouth disease SAT2 type, it was the first case in the world that the use of recombinant protein antigen and monoclonal antibody showed the same result as the diagnosis method using inactivated virus diagnostic antigen and antiserum.

The diagnostic method of the present invention is a competitive enzyme-linked immunoassay method that can be tested in the same manner regardless of cattle, pigs, goats, or livestock. Because it can be measured, it can be used as a new diagnostic method that can replace the standard neutralization test or the LPB ELISA that uses the inactivated virus.

Unlike the existing diagnostic methods, the diagnostic method of the present invention does not handle live viruses, and thus, in advance, the possibility of a virus leaking out due to an accident caused by the handling of the virus may be prevented in advance.

As a result, it is possible to easily produce foot-and-mouth virus SAT2-type antigens in general laboratories by using genetically engineered protein diagnostic antigens. Therefore, it is possible to supply diagnostics quickly in emergency situations and ultimately to protect domestic livestock industry by blocking the influx of foot-and-mouth disease. Can be.

In Figure 1 (a) is a picture of the SAT2 gene P1 and 3C inserted into the cloning vector and cut with restriction enzyme (B) is inserted into the transfer vector after the treatment with the same restriction enzyme. (C) is finally a recombinant baculovirus gene produced and confirmed by PCR. Stratagene cloning vector (BamHI & SpeI) amino acid alteration site in relation to (A): A491V, Y164H; In connection with (b) pFastBacDual vector (BamHI &SpeI); In relation to (c), it corresponds to Recombinant bacmid (M13F (-40) / M13R).
2 is a diagram illustrating the difference in reactivity with SAT2 VP1 serum from non-infected cells (a) and insect cells (b) inoculated with recombinant baculovirus by fluorescent antibody method. Normal Sf9 cells with respect to (a); (B) Corresponds to the recombinant baculovirus inoculated Sf9 cells.
3 is a diagram showing whether the protein expression by SAT2 foot-and-mouth disease virus VP1, VP2, VP3 antiserum after the expression of recombinant P1 and 3C in insect cells. Recombinant P13C protein in connection with 1; 2 corresponds to inactivated virus (LPB ELISA diagnostic antigen).
Figure 4 shows the results of Western blotting analysis of samples taken from the upper layer of the centrifuge tube after ultrafast centrifugation with a cesium chloride concentration gradient (5-40%) on the left side, and fractions 7-11 on the right side. It is a picture of virus-like particles observed by electron microscopy of the precipitate formed after the ultrafast centrifugation. The red arrow is the intact 25 nm size virus like particle and the black arrow is the 10 nm size pentamer virus like particle. The left side corresponds to Western blotting analysis for samples taken from the upper layer of the centrifuge tube after ultrafast centrifugation with a cesium chloride concentration gradient (5-40%). On the right is a virus-like particle observed by electron microscopy of samples 7, 8 and 9 collected after ultra-centrifugation (red arrows are intact 25 nm-sized virus-like particles and black arrows are 10 nm-sized penta). Mercury virus analog particles).
5 is a diagram illustrating the difference in reactivity between positive and negative serum between the recombinant protein diagnostic antigen and inactivated virus of the present invention by applying the existing diagnostic method LPB ELISA. Comparison of the reactivity between the recombinant protein antigen and the inactivated virus diagnostic antigen by applying the control serum included in the conventional diagnostic LPB ELISA kit.
FIG. 6 is a diagram illustrating the difference in reactivity with SAT2 monoclonal antibody 2E42 in normal IBRS-2 cells (a) and IBRS-2 cells (b) inoculated with SAT2 foot-and-mouth virus by fluorescent antibody method. (A) corresponds to normal Sf9 cells. (B) corresponds to SAT2 (ZIM 5/81) inoculated IBRS-2 cells.
Figure 7 is the enzyme-linked immunoassay established by combining the recombinant protein antigen and monoclonal antibody 2E42 of the present invention to the standard serum (strong positive, weak positive, cut-off, negative serum) obtained from the British Institute for Foot and Mouth Standards Figure showing the difference in responsiveness.
8 is a diagram showing the reaction inhibition (%) distribution of the results measured by the enzyme-linked immunoassay method of the present invention for each 88 heads of cattle, pigs, and goats being raised in Korea.
9 is a result of comparing the initial antibody detection rate between the enzyme-linked immunoassay of the present invention and the conventional diagnostic method LPB ELISA for the serum collected by date after inactivating SAT2 foot-and-mouth disease virus inoculated in goats.

The present invention relates to a foot-and-mouth virus SAT2 type recombinant protein antigen, a monoclonal antibody, and a method for diagnosing a worm.

More specifically, the present invention relates to an enzyme-linked immunoassay for the detection of foot-and-mouth disease SAT2 antibody using foot-and-mouth virus SAT2 type recombinant protein and SAT2 type monoclonal antibody.

Preferably, the recombination protein may be a protein produced in insect cells using a baculovirus containing the foot and mouth virus SAT2 type P1 gene and 3C gene.

In addition, the monoclonal antibody may be 2E42 that competes with the antibodies in the serum of the foot-and-mouth virus SAT2-type infectious agent or vaccine.

The present invention also relates to a fusion cell line producing the monoclonal antibody (Accession No. KCLRF-BP-00227).

Also,

(a) reacting the foot-and-mouth virus SAT2-type monoclonal antibody or VP1 antiserum to a 96-well microplate;

(b) washing and removing the antibody not attached to the plate;

(c) reacting the foot and mouth virus SAT2 type recombinant protein as a diagnostic antigen;

(d) washing and removing protein antigens not attached to the plate;

(e) reacting the test serum sample;

(f) washing and removing the test serum sample not attached to the plate;

(g) competitively reacting the SAT2-type neutralizing monoclonal antibody (2E42) to which the horseradish peroxidase enzyme is bound;

(h) washing and removing monoclonal antibodies not attached to the plate; And

(i) a method for detecting foot-and-mouth virus SAT2-type antibody, comprising measuring the absorbance of the color reaction by the enzymatic reaction and measuring the level of the foot-and-mouth virus SAT2-type antibody in the test serum.

Hereinafter, the drawings will be described in detail.

In Figure 1 (a) is a picture of the SAT2 gene P1 and 3C inserted into the cloning vector and cut with restriction enzyme (B) is inserted into the transfer vector after the treatment with the same restriction enzyme. (C) shows the final production of genetically modified baculovirus gene and confirmed by PCR.

Figure 2 is a diagram illustrating the difference in reactivity with SAT2 VP1 serum from non-infected cells (A) and insect cells (B) inoculated with recombinant baculovirus by fluorescent antibody method.

Figure 3 shows the expression of the protein expression of the recombinant P1 and 3C in insect cells after expression of SAT2 foot-and-mouth virus VP1, VP2, VP3 antiserum.

Figure 4 shows the results of Western blotting analysis of the samples taken from the upper layer of the centrifuge tube after ultrafast centrifugation with a cesium chloride concentration gradient (5-40%) on the left side and fractional samples 7-11 on the right side. It is a picture of virus-like particles observed by electron microscopy of the precipitate formed after the ultrafast centrifugation. The red arrow is the intact 25 nm size virus like particle and the black arrow is the 10 nm size pentamer virus like particle.

5 is a diagram illustrating the difference in reactivity between positive and negative serum between the recombinant protein diagnostic antigen and inactivated virus of the present invention by applying the existing diagnostic method LPB ELISA.

Fig. 6 is a diagram illustrating the difference in reactivity with SAT2 monoclonal antibody 2E42 in normal IBRS-2 cells (a) and IBRS-2 cells (b) inoculated with SAT2 foot-and-mouth virus by fluorescent antibody method.

Figure 7 is the enzyme-linked immunoassay established by combining the recombinant protein antigen and monoclonal antibody 2E42 of the present invention to the standard serum (strong positive, weak positive, cut-off, negative serum) obtained from the British Institute for Foot and Mouth Standards Figure showing the difference in responsiveness. Since the lower reaction rate of the cutoff serum was 40%, the negative determination criterion of the present invention was determined to be 40%.

8 is a diagram showing the reaction inhibition (%) distribution of the results measured by the enzyme-linked immunoassay method of the present invention for each 88 heads of cattle, pigs, and goats being raised in Korea. Both pigs and goats showed less than 40% inactivation, and only two bovine serum showed less than 40% inactivation.

9 is a result of comparing the initial antibody detection rate between the enzyme-linked immunoassay method of the present invention and the conventional diagnostic method LPB ELISA in the serum collected by date after inactivating SAT2 foot-and-mouth disease virus inoculated in goats Indicated.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Example 1 Gene Recombination Foot-and-mouth Virus SAT2 Protein Expression

Viral RNA was extracted from IBRS-2 cells infected with foot and mouth virus SAT2 (ZIM5 / 81; GenBank EF134951) using the RNeasy extraction kit (Qiagen). Complementary DNA was prepared using a random hexamer and AccuPower RT Premix (Bionia), and the gene was amplified by applying nPfu DNA polymerase (Enzynomics) as a cDNA template. The forward primer of the P1 gene is 5 'GCA GGA TCC ATG GGA GCG GGA CAG TCA TCA GCA 3' and the reverse primer is 5 'GCC ACT AGT TTA TTG TTT CTC GAC GCC AAT 3'. 5 'GAT TCT CGA GAT GAG CGG TGC CCC CCC GAC CGA C 3' and the reverse primer is 5 'AAC AGC ATG CTA CTC ATG GTG TGG TTC AGG GTC 3'. A gene amplifier was used to amplify the gene for the above primers and the conditions were as follows. That is, after denaturing at 95 for 2 minutes, the cycle was repeated 35 times using 9530 seconds, 55 30 seconds, and 72 minutes as the basic cycle. At the end of the period, 72 to 10 minutes of extension amplification. The result was confirmed as in FIG. 1 (a), and the amplified gene was inserted into pFastBacDual vector (Invitrogen), which is a transition vector. The thus prepared transfer vector was identified as shown in FIG. 1 (b), and the vector was transformed into Escherichia coli containing baculovirus gene bacmid DNA to confirm the recombinant recombinant baculovirus nucleic acid as shown in FIG. After extracting the nucleic acid and injected into insect cells (Sf9) to produce a recombinant baculovirus.

After inoculation of recombinant baculovirus to the insect cells cultured in the cell culture flask, when cell deformation was observed, the cells were crushed by freezing and thawing three times, and the supernatant was collected by centrifugation at 10,000 xg for 30 minutes and used as a diagnostic antigen. .

In order to confirm the expression of the protein was carried out a fluorescent antibody method. When cell degeneration was observed after the inoculation of foot-and-mouth virus SAT2 on the slide, the cells were washed three times with phosphate buffer solution and dried, and then fixed with the same amount of acetone and methanol for 10 minutes. After washing with phosphate buffer solution it was reacted with SAT2 VP1 peptide serum at 37 for 1 hour. After washing with phosphate buffer solution, the FITC-bound anti-mouse antibody was reacted with a secondary antibody for 1 hour and observed for fluorescence. As a result, as shown in Fig. 2 (b), the positive response was confirmed in the fluorescent antibody method using the SAT2 VP1 peptide antiserum.

Since it was confirmed that the recombinant protein was successfully expressed, Western blotting was applied as another identification method. That is, the protein samples were electrophoresed in NuPAGE Novex Bis-Tris gels (Invitrogen) using an Xcell SureLock mini-cell device (Invitrogen) according to the user manual. The isolated proteins were transferred to the nitrocellulose membrane, and blocked with the sample-diluted solution [Tris-buffered saline containing 0.05% Tween 20 (TBST) & 5% skim milk] for 1 hour at room temperature, followed by the anti-airborne virus SAT2 VP1, VP2, VP3 antiserum. After diluting 1: 100 with the sample dilution solution, the reaction was carried out for 1 hour. After washing three times with a washing solution (Tris-buffered saline containing 0.05% Tween 20), the goat anti-rabbit antibody conjugated with alkaline phosphatase was added by diluting 1: 1,000 with a sample dilution solution. After reacting for 1 hour at room temperature, the solution was washed 5 times with a washing solution, and color development was performed with BCIP / NBT solution. As shown in FIG. 3, since only the VP1 band was observed without the P1 band, which is a structural protein precursor, it was confirmed that P1 was cleaved by 3C protease. In addition, bands were also identified in the VP2 and VP3 antiserum, indicating that the SAT2 structural protein was expressed and cleaved into unit proteins such as VP1, VP2, VP3. After ultrafast centrifugation (100,000 × g, 6 hours) of the expression protein with a cesium chloride concentration gradient (5-40%), Western blotting was performed on samples taken 0.5 ml from the top of the centrifuge tube. Likewise, 10 nm-sized pentamers (five individual proteins) virus-like particles were observed along with intact virus-like particles consisting of 60 individual 25 nm-sized proteins.

Example 2 Antigen Evaluation of Recombinant Protein

To assess the suitability of the recombinant protein as a diagnostic antigen, the antigenicity between the inactivated virus antigen and the recombinant protein of the present invention using control serum (strong positive, weak positive, negative serum) included in the existing LPB ELISA As a result of the evaluation, as shown in FIG. 5, the recombinant protein showed a similar reactivity to the inactivated foot-and-mouth virus SAT2-type antigen against three control serum.

Example 3 Preparation of Fusion Cell Line Producing Foot-and-mouth Virus SAT2 Monoclonal Antibody

Peptides (CTS TAI RGD RAV LAA KYA NAK HEL P) based on amino acid sequence of foot-and-mouth disease virus SAT2-type ZIM 5/81 (GenBank No. EF134951) amino acid sequence of VP1 139 to 162 Made in After peptide synthesis, the cells were bound to KLH and intraperitoneally inoculated into mice four times. After the fifth intravenous injection, spleens were collected and used for cell fusion.

Cell fusion was carried out as follows using a general method using polyethylene glycol. The collected lymphocytes were washed with serum free medium, mixed with a mouse myeloma-derived cell line (SP2 / 0-Ag14 mouse myeloma cell line) in a 5: 1 ratio, and then fused with polyethylene glycol 1500. . After completion of the fusion, the cells were appropriately diluted in proliferation medium (D-MEM, HAT (Hypoxanthine Aminopterin Thymidine, 10% Fetal Bovine Serum), and then 100 aliquots were prepared in 96-well microplates containing mouse celiac cells. Cultured in a% carbon dioxide incubator.

Selection of fusion cell lines producing monoclonal antibodies against foot-and-mouth virus SAT2 was performed as follows. As a result of the cell fusion, the culture supernatants of the wells in which the fusion cells proliferated were collected and subjected to indirect enzyme-linked immunoassay using a peptide antigen. 0.8 cells per well were added and incubated until monoclonal was formed. Wells were selected in which only one clone was propagating while producing antibodies against foot-and-mouth virus A1. The ascites produced by inoculating the selected fusion cell line into the abdominal cavity of the valgus mice were harvested, and only IgG antibodies were purified using ImmunoPure IgG Purification Kit (Pierce, USA) and used in the experiment. Finally, the selected monoclonal antibody 2E42 is IgG1, and as shown in FIG. 6, the reactivity with the viral antigen was confirmed.

Example 4 Establishment and Evaluation of Enzyme-linked Immunoassay Using Genetic Recombinant Protein Antigen and SAT2 Monoclonal Antibody

The enzyme-linked immunoassay for detecting foot-and-mouth virus SAT2-type antibody using a recombinant protein antigen and monoclonal antibody 2E42 is as follows.

As an antibody capable of capturing SAT2 antigen in 96-well microplate, another monoclonal antibody 2B30 or VP1 antiserum was diluted 2200 times in carbonate buffer (pH 9.6) and 50 per well in 96-well microplate (Maxisorp). Aliquoted and left for 4 to 16 hours. The next day, the coated microplates were washed three times with a washing solution (10 mM phosphate buffered buffer containing 0.05% Tween 20) to wash off unattached antibodies, and then to remove foot and mouth virus SAT2 gene. Recombinant protein antigen was diluted 1:16 with a blocking solution (a solution containing 0.05% Tween 20 and 5% skim milk in 10 mM phosphate buffer solution), 50 divided per well and then reacted at 37 for 1 hour. After washing three times with the washing solution to remove the unattached protein antigen, the test serum was diluted 1: 5 with the blocking solution, 50 divided per well and then reacted for 37 to 1 hour. After washing again three times with the washing solution to remove the antibody in the serum that did not bind to the antigen, dilution of 2E42, a SAT2 monoclonal antibody conjugated with horseradish peroxidase enzyme to a 2 / ml concentration with a blocking solution and 50 divided per well And then reacted at 37 for 1 hour. After washing five times with the washing solution, and reacted for about 15 minutes by adding O-Phenylenediamine solution and 0.015% hydrogen peroxide solution as a coloring agent. When the absorbance of the serum-excluded control group was estimated to be around 1.2, 50 / well of 1.25N sulfuric acid solution was added to stop the color reaction, and then the absorbance was measured at 492 nm. The final results were analyzed by converting the absorbance inhibition of the test well to the absorbance of the wells to which only monoclonal antibody was added without serum. The result of the reaction was determined to be positive for foot-and-mouth virus SAT2-type antibody when the wells that inhibited the reaction of 50% or more than the absorbance of the monoclonal antibody control well, that is, the percentage of inhibition of inhibition (PI) of 50% or more.

% Reactivity (%) = 100 × [(Control Well Absorbance-Test Serum Absorbance) / Control Well Absorbance]

In this case, the control well absorbance is the absorbance of the well to which only the monoclonal antibody 2E42 was added without serum.

[Test Example 1] Validation of diagnostic method of the present invention applying international standard serum and establishment of anegative judgment criteria

As a result of applying the diagnostic method of the present invention to SAT2 type serum (strong positive, weak positive, cutoff, negative serum) obtained from Ferbright, UK, serum can be accurately classified based on 40% inhibition of reaction.

Test Example 2 Evaluation of Specificity Using Negative Serum

The result of measurement by enzyme-linked immunoassay of the present invention for 88 heads of cattle, pigs, and goats domestically raised in Korea is shown in the distribution diagram (%) as shown in FIG. 8. Both pigs and goats showed less than 40% inactivation, and only two bovine serum showed less than 40% inactivation. Of the total 264 heads, only two of them were positive, indicating 99.2% specificity.

[Test Example 3] Evaluation of the effectiveness of the diagnostic method for the inactivated virus test vaccination goat serum

In order to evaluate the initial antibody detection rate of the diagnostic method of the present invention, serum was collected by date (0, 4, 7, 10, 14, 21, 28) after inoculation of SAT2 inactivated virus to goats. As a result of comparing the initial antibody detection rate between the enzyme-linked immunoassay and the conventional diagnostic LPB ELISA, two out of four heads were positive at 7 days after inoculation and all four were positive at 10 days. This was the same as the existing diagnostic method, LPB ELISA.

On the basis of the embodiments with reference to the accompanying drawings it was described in detail. However, this is not intended to limit the scope of the present invention to this intended to illustrate the present invention, there are various equivalents that can replace them. In addition, the terms or words used in the specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly define the concept of terms in order to best explain their invention in the best way. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that it can.

Of the seven serotypes of foot-and-mouth disease, the SAT2 type in Africa has not been introduced with antibody diagnostic techniques using genetically engineered diagnostic antigens.

Therefore, the present invention attempted the artificial expression of diagnostic antigen in baculovirus-infected insect cells using the foot-and-mouth virus SAT2-type protein precursor P1 and 3C protease gene. In addition, since monoclonal antibodies specific for foot-and-mouth virus SAT2 type were prepared as a competitive reaction antibody, a new diagnostic method for rapidly detecting foot-and-mouth disease SAT2 type antibody was provided in addition to the above-described diagnostic antigen, thereby providing excellent industrial availability.

In addition, the diagnostic method of the present invention is a competitive enzyme-linked immunoassay method, which can be tested in the same manner regardless of cattle, pigs, goats, or livestock, so that it is easy to determine whether or not a foot-and-mouth virus SAT2 infection is carried out or to quickly vaccine vaccine titer at vaccination. It can be used as a new diagnostic method that can replace the standard neutralization test or the LPB ELISA that uses the inactivated virus.

Therefore, the diagnostic method of the present invention does not handle live viruses, unlike conventional diagnostic methods, it is possible to block in advance the concern that the virus may leak out due to an accident caused by the virus handling. As a result, it is possible to easily produce foot-and-mouth virus SAT2-type antigens in general laboratories by using genetically engineered protein diagnostic antigens. Therefore, it is possible to supply diagnostics quickly in emergency situations and ultimately to protect domestic livestock industry by blocking the influx of foot-and-mouth disease. It can be said that the industrial availability is very excellent.

Korea Cell Line Research Foundation KCLRFBP00227 20100107

<110> Korea (National Veterinary Research and Quarantine Service) <120> Genetically engineered protein and monoclonal antibody of foot          and mouth disease virus type SAT2 and their application to the          diagnostic method for Artiodactyla <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer of P1 gene <400> 1 gcaggatcca tgggagcggg acagtcatca gca 33 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of P1 gene <400> 2 gccactagtt tattgtttct cgacgccaat 30 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> forward primer of 3C gene <400> 3 gattctcgag atgagcggtg cccccccgac cgac 34 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of 3C gene <400> 4 aacagcatgc tactcatggt gtggttcagg gtc 33

Claims (5)

Enzyme-linked immunoassay for the detection of foot-and-mouth disease SAT2 antibody using foot-and-mouth virus SAT2 type recombinant protein and SAT2 type monoclonal antibody.
The method of claim 1, wherein the recombinant protein is an enzyme-linked immunoassay for the detection of foot-and-mouth disease SAT2 antibody, characterized in that the protein produced in insect cells using a baculovirus containing the foot-and-mouth virus SAT2 type P1 gene and 3C gene.
[Claim 2] The enzyme-linked immunoassay for detecting foot-and-mouth disease SAT2 antibody according to claim 1, wherein the monoclonal antibody is 2E42 which competes with the antibody in the serum of the foot-and-mouth virus SAT2-type infectious agent or vaccine object.
A fusion cell line producing the monoclonal antibody of claim 1 (Accession No. KCLRF-BP-00227).
Foot-and-mouth disease virus SAT2 antibody detection method comprising the following steps.
(a) reacting the foot-and-mouth virus SAT2-type monoclonal antibody or VP1 antiserum to a 96-well microplate;
(b) washing and removing the antibody not attached to the plate;
(c) reacting the foot and mouth virus SAT2 type recombinant protein as a diagnostic antigen;
(d) washing and removing protein antigens not attached to the plate;
(e) reacting the test serum sample;
(f) washing and removing the test serum sample not attached to the plate;
(g) competitively reacting the SAT2-type neutralizing monoclonal antibody (2E42) to which the horseradish peroxidase enzyme is bound;
(h) washing and removing monoclonal antibodies not attached to the plate; And
(i) measuring the level of foot-and-mouth virus SAT2 antibody in the test serum by measuring the absorbance of the color reaction by the enzymatic reaction.

Images