KR101814758B1 - Diagnosis Method for Classical Swine Fever(CSF) using E2 proteins of CSF-Virus and its Specific Monoclonal Antibodies, and Diagnostic Kit using the method - Google Patents

Abstract

The present invention relates to a method for diagnosing porcine cholera virus infection using a porcine cholera virus E2 protein and a monoclonal antibody specific thereto, and a diagnostic kit for implementing the method. The immunoassay using the lateral flow assay of the present invention is a rapid kit, which enables rapid diagnosis and low cost compared to the conventional enzyme immunoassay. Therefore, the diagnostic method and the diagnostic kit of the present invention can replace the conventional method for diagnosing porcine cholera infection.

Description

(Diagnostic Method for Classical Swine Fever (CSF) using E2 Protein and Specific Monoclonal Antibody of Porcine Cholera Virus and Diagnostic Kit for Implementing the Method Monoclonal Antibodies, and Diagnostic Kit using the method}

The present invention relates to a method for diagnosing porcine cholera virus infection using a porcine cholera virus E2 protein and a monoclonal antibody specific thereto, and a diagnostic kit for implementing the method.

Classical Swine Fever (CSF) is an infectious disease caused by swine cholera virus, and pigs are the only natural host. In the case of pig-shaped cholera, it is a disease of high mortality and morbidity with symptoms such as high fever, skin rash, appetite deficiency, constipation, diarrhea, leukocytosis, Livestock is an epidemic.

The porcine cholera virus is an RNA virus belonging to Flaviviridae (Pestivirus), and the size of the virus is known to be 12.3 to 12.5 Kb. Genomic RNA consists of 5'-enoncoding resion (NCR), single open reading frame (ORF) and 3'-NCR. Single ORFs are cleaved with the structural proteins of the virus, capsid C, Erns, E1 and E2, and the nonstructural proteins Npro, p7, NS2-3, NS4A, NS4B, NS5A and NS5B, respectively. E2 protein is the main structural protein that constitutes the envelope of porcine cholera virus. It is known that E2 protein plays an important role in defense mechanism of porcine cholera immunologically by inducing virus neutralizing antibody reaction. In Europe, studies on marker vaccines using E2 have been conducted, and their use under certain conditions has been recommended.

The existing blocking ELISA requires several steps and is time-consuming and requires expensive equipment. Therefore, it is difficult to perform a simple and rapid diagnosis, and the cost of the rapid kit There are expensive disadvantages. On the other hand, the immunoassay using a lateral flow assay is a method of measuring the presence or absence of an antigen or an antibody with a fluorescent laser, which enables rapid diagnosis and is less costly than enzyme immunoassay. In addition, it has better sensitivity and specificity than the existing gold rapid kit, which uses a lot of indirect methods, and it has the merit that it can be convenient and more accurate than the gold system which is confirmed by the naked eye because the result can be expressed numerically.

Therefore, the present invention aims to develop a method of diagnosing porcine cholera virus infection by immunoassay using lateral flow assay and a diagnostic kit using the same.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have endeavored to develop a diagnostic method for swine cholera infection which can easily and rapidly diagnose whether or not a porcine cholera infection has occurred. As a result, a method for diagnosing porcine cholera virus infection using the E2 protein of pig cholera virus and a monoclonal antibody specific thereto has been developed and a diagnostic kit using the same has been completed.

It is therefore an object of the present invention to provide a method for diagnosing porcine cholera virus infection.

It is still another object of the present invention to provide a diagnostic kit using the diagnostic method.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a diagnostic kit capable of judging whether or not a porcine cholera virus (CSFV) is infected.

The present inventors have made efforts to develop a quick, simple and accurate diagnostic kit as compared with the conventional enzyme immunoassay (EISA). As a result, it was confirmed that swine cholera infection can be diagnosed quickly and accurately by immunoassay using a lateral flow assay.

Immunoassay using a lateral flow assay is a method of measuring the presence or absence of an antigen and an antibody using a fluorescent laser. The procedure is simple compared to the enzyme immunoassay, but its accuracy is not different from the enzyme immunoassay The development of a diagnostic kit utilizing this in the field is being carried out.

 As used herein, the term " antigen of porcine cholera virus " means all or part of the structure and non-structural proteins of porcine cholera virus. The genome of the porcine cholera virus is composed of a single RNA positive strand, which is initially translated into a single polypeptide and then sequentially cleaved by the viral protease encoded by the virus in the infected cell To generate the structural proteins capsid C, Erns, E1 and E2 and the non-structural proteins Npro, p7, NS2-3, NS4A, NS4B, NS5A and NS5B.

E2 protein is the main structural protein that constitutes the envelope of porcine cholera virus. It is known that E2 protein plays an important role in defense mechanism of porcine cholera immunologically by inducing virus neutralizing antibody reaction. In Europe, studies on marker vaccines using E2 have been conducted, and their use under certain conditions has been recommended. In view of these advantages, the present invention uses the E2 structural protein of porcine cholera virus as an antigen.

According to one embodiment of the present invention, the porcine cholera virus antigen used in the present invention is the E2 structural protein of porcine cholera virus represented by SEQ ID No. 1.

As used herein, the term " detector antigen " is a conjugate of a signal for generating a signal to the porcine cholera virus antigen, and detects whether an antibody against a porcine cholera virus antigen is present in a sample to be examined . That is, if an antibody to the antigen exists in the test sample, it will react with the detector antigen to form a complex, and if it does not exist, the detector antigen will remain in its original form since it does not cause any reaction. Therefore, if the complex can be distinguished from the antigen present in its original form, the presence of the antibody against the porcine cholera virus antigen in the test sample can be confirmed.

In order to confirm whether the antigen-antibody reaction normally occurs in the diagnostic kit, the control antigen is used simultaneously with the detector antigen as a control. Antigens used as control antigens should not form any complexes with the serum of the individual to be tested, so proteins isolated from other species should be used. For example, if the subject to be examined is cow or pig, the control antigen is preferably a protein isolated from rabbit. According to some embodiments of the present invention, rabbit IgG (rabbit IgG) was used as a control antigen in a diagnostic kit.

Signals that generate signals include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties (For example, gold), antibodies (for example, gold), magnetic particles, radioactive isotopes (P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase) , Streptavidin, and biotin. The binding of the label can be carried out through various methods conventionally practiced in the art. The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency and nanocrystals. According to some embodiments of the present invention, a fluorescent label is used as a label for generating a signal, and specifically, fluorescent particles such as Alexa Fluor 647 Carboxylic acid succinimidyl ester (Invitrogen) were used.

As used herein, the term " binder that specifically binds to the antigen of porcine cholera virus " is a substance that specifically binds to the antigen of the specific porcine cholera virus used in the diagnostic kit of the present invention, It should be able to compete with antibodies in the sera of virus infected individuals. The phrase " competitively reacted " means that the antibody in the serum of the individual infected with porcine cholera virus and the binding agent recognize the same epitope of the specific porcine cholera virus antigen, for example, the antibody in the serum of a porcine cholera virus- When reacting with an antigen to form a complex, it means that this complex no longer reacts with the binder. In other words, when the subject to be diagnosed is infected with porcine cholera virus, the antibody is generated in the serum of the infected individual and forms a complex with the detectable antigen, so that the antibody does not bind to the binding agent. Since the same antibody is not present, the detector antigen binds to the binding agent.

The binding agent is preferably an aptamer or a monoclonal antibody. An aptamer is an oligonucleic acid or peptide molecule that binds to a specific target molecule, and is a peptide aptamer consisting of DNA, RNA or XNA aptamers consisting of short oligonucleotides and short peptide domains. Unlike polyclonal antibodies, monoclonal antibodies bind to the same epitope because they are made from the same immune cells. Therefore, if a detectable antigen-specific monoclonal antibody is used as a binding agent, when the detector antigen forms a complex with an antibody in the serum of a porcine cholera-infected individual, it can no longer bind to a monoclonal antibody as a binding agent. On the other hand, in the case of an animal not infected with foot-and-mouth disease, since the antibody against the antigen of porcine cholera virus is not present in the serum, the detectable antigen binds to the binding agent which is a monoclonal antibody.

According to some embodiments of the present invention, E2 protein of porcine cholera virus was used as foot-and-mouth virus antigen and monoclonal antibody specific to E2 protein was used as a binding agent.

As used herein, the term " reaction part " means a part where the reaction solution in which the detector antigen and the sample to be tested are reacted and the reaction occurs by contacting the binding agent. That is, when the subject to be examined is infected with foot-and-mouth disease, the antibody against the porcine cholera virus present in the serum in the individual forms a complex with the detectable antigen and does not bind to the binding agent in the reaction part. However, if not infected, a detectable antigen that has not formed any complex binds to the binding agent in the reaction site.

The reaction part contains a binder as a control in addition to the above binder. The binding agent as the control group is used to confirm whether the antigen-antibody reaction is normally occurring in the reaction part, and an aptamer or an antibody that specifically binds to the control antigen can be used. It is more preferable to use a polyclonal antibody rather than a monoclonal antibody since it is important that the antigen-antibody reaction certainly occurs under any condition, unlike in the case of the detection of the detector antigen-antibody. According to some embodiments of the present invention, rabbit immunoglobulin (rabbit IgG) was used as a control antigen and anti-rabbit IgG was used as a control binding agent.

As used herein, the term " strip " means that the sample pad, the opening membrane, and the absorption pad are superimposed so that the reaction solution, in which the detection antigen and the sample to be examined are reacted in advance, flows in a certain direction from the sample pad to the opening membrane, To react with the binder of the reaction part. The development film is characterized by being at least one material selected from the group consisting of nylon, polyester, cellulose, polysulfone, polyvinylidene difluoride, cellulose acetate, polyurethane, glass fiber and nitrocellulose. It is not. According to some embodiments of the present invention, a nitrocellulose membrane was used as the preform.

The method of producing the strip is as follows. First, the binding agent specifically binding to the antigen of porcine cholera virus and the binding agent as the control group are fixed so as to be positioned at regular intervals in the middle part of the opening membrane corresponding to the reaction part. Attach the sample pad and the absorbing pad to both ends of the reaction part. Assembling the completed strip into a suitable housing completes the diagnostic kit.

According to another aspect of the present invention, the present invention provides a method for diagnosing a porcine cholera virus infection comprising the steps of:

(a) contacting a sample to be detected with a detector antigen comprising an antigen of a porcine cholera virus, wherein the detector antigen is labeled with a signal generating tag;

(b) bringing the reaction solution of (a) into contact with a reaction part immobilized with a binding agent that specifically binds to an antigen of the porcine cholera virus, wherein the reaction part includes, in addition to the part where the binding agent is immobilized, A binding agent as a control group for confirmation is included;

(c) analyzing each signal of a portion of the reaction unit in which the binding agent that specifically binds to the antigen of the porcine cholera virus is immobilized and a portion of the binding agent as a control, to which the binding agent is immobilized, using a signal analyzer;

(d) judging whether or not a pig cholera infection is caused by using the measured signal value.

Since the diagnostic method of the present invention includes a process of using the kit for diagnosing porcine cholera virus infection described above, the description of the diagnostic kit of the present invention is omitted in order to avoid the excessive complexity of the present invention.

As used herein, the term " sample " refers to a blood, plasma, or serum sample obtained from an individual who is to be diagnosed as having a porcine cholera virus infection.

The term " signal analyzer " in the present specification means a device capable of analyzing a signal bound to a detector antigen in the aforementioned kit for diagnosing porcine cholera virus infection, and the analysis device differs depending on the type of the label used. According to some embodiments of the present invention, a fluorescent label is used as a label for generating a signal, and thus a fluorescent reader is used as a signal analyzer.

Hereinafter, a method for judging whether or not a porcine cholera virus is infected by using a measured signal value is as follows. First, you should set a cut-off value that is the basis for distinguishing between the infected and non-infected individuals of the pig cholera virus. For this purpose, a number of samples which have already been confirmed to have been infected with swine cholera virus through other diagnostic methods are analyzed through the diagnostic method of the present invention. As a result of the analysis, the signal value that distinguishes the infected object from the non-infected object is cut off. In this process, the signal value used as the control group is used to correct the signal value of the sample.

According to some embodiments of the present invention, the signal value in the reaction part in which the binding agent that specifically binds to the antigen of swine cholera virus is divided by the signal value in the reaction part where the binding agent used as the control group is located, And cut-off was determined.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for diagnosing porcine cholera virus infection using the E2 protein of porcine cholera virus and a monoclonal antibody specific thereto.

(b) The diagnostic method of the present invention is a rapid kit using immunoassay using lateral flow assay.

(c) The present invention provides a diagnostic kit using the diagnostic method.

Brief Description of the Drawings Fig. 1 is a diagram showing an outline of a working principle of a porcine cholera virus infection diagnosis kit.
2 is a view showing a strip structure of a kit for diagnosing foot-and-mouth disease virus infection.
FIG. 3 is a chart summarizing the results of analyzing 395 samples to determine the cut-off for judging whether swine cholera virus infection of a swine specimen is detected in a diagnostic kit.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Production of porcine cholera virus E2 recombinant protein

To make the recombinant protein (rE2) of E2 protein of porcine cholera virus (CSFV), the E2 gene of CSFV ALD strain was amplified using PCR. The primers for the PCR reaction are as follows:

Forward (5'-GGA TCC CGG CTA GCC TGC AAG GAA GAT-3`)

Reverse (5'-GGA TCC TTC TGC GAA GTA ATC TGA-3`)

The amplified cDNA was ligated to a pAcgp67B baculovirus vector to prepare an expression vector pAcgp67B-E2. The prepared expression vector was transformed into E. coli DH5-a cells and then cultured in an agar plate to isolate the plasmid from the colonies formed. Plasmid-introduced cells were mass-propagated to obtain plasmids, and Sf 21 insect cells were co-transfected with baculovirus linear DNA. Transformation was confirmed by observing the presence of cytopathic effect. Plaque purification was performed to isolate the recombinant baculovirus in the transformed culture supernatant to select recombinant baculovirus

Immunocytochemical staining (ICS) and indirect immunofluorescent antibody technique (SEM) were performed using an anti-E2 monoclonal antibody to confirm the expression of E2 protein in Sf 21 cells infected with recombinant baculovirus , IFA) (Figs. 2 and 3).

Since the CSFV rE2 protein is secreted out of the cell as a soluble protein, 0.5-1 moi of the recombinant baculovirus selected in Sf21 cells is inoculated and cultured for 5-6 days. Then, the supernatant is treated with ammonium sulfate (ammonium sulfate) After precipitation, the protein was purified using His-tagging column (manufacturer GE healthcare). As a result of purification, a protein of 46 kDa was obtained.

Preparation of monoclonal antibodies

The recombinant protein (rE2) prepared according to the above method was used to immunize the mouse to produce a monoclonal antibody. The concentration of CSFV LOM (vaccine strain) (CSFV used as an antigen for vaccination) was set at 0.5 mg / ml, and CSFV LOM (vaccine strain) and 200 μl of adjuvant (Freund's Adjuvant , FA) were mixed and injected into Balb / C mouse abdominal cavity three times at intervals of two weeks. One week after the third immunization, 100 μl of FMDV r3AB was injected into the abdominal cavity and used for cell fusion 4 days later.

SP2 / 0 Ag14 (ATCC, USA) was used as a cell line for cell fusion. The cells were cultured in D-MEM / HAT & HT (manufactured by GIBCO) medium and PEG method using spleen cells was used as a fusion method. Two clones were screened for CSFV LOM (0.5 ug / ㎖) and non - responsive to normal cell lysate. Only single IgG isotyping reagent (Isotyping reagent, Sigma) was used to screen only IgG class except IgM class.

Detector antigen production

200 μl of 1 mg / ml CSFV rE2 as a raw material of the detector antigen and 1 mg / ml of rabbit IgG (Arista) as a raw material of the control antigen were prepared. As fluorescent particles, 1 mg of Alexa Fluor 647 succinimidyl ester (Invitrogen) was dissolved in 100 μl of DMSO (Sigma). 20 μl of 1 M carbonate buffer (pH 9.0) was added to each of the detector antigen and the control antigen solution, and 4 μl of the fluorescent particle solution was added thereto, followed by reaction at room temperature for 1 hour. Purification was carried out using a mini-column packed with 3 ml of sephadex G-25 resin (Amersham biosciences) to separate the complex. 100 μl of each eluted fraction was collected, and the protein was quantified to collect only fractions having a protein concentration of 100 μg / ml or more. Appropriate amount was dispensed and stored at 20 ℃.

Manufacture of diagnostic kits

The strip was made to overlap the sample pad (Ahlstrom), the membrane (Millipore) and the absorption pad (Ahlstrom) so that the sample reacted with the capture antibody while flowing in a certain direction.

The manufacturing method of the diagnostic kit according to the manufacturing order is as follows.

1) Antibody preparation

0.5% sucrose (Sigma) and 0.1% NaN ₃ (Sigma) were added to 1 mg / ml anti-rabbit IgG (Arista) and 2 mg / ml CSFV LOM mAb, respectively.

2) Antibody dispensing and fixation

The membrane was attached to a backing card (PJ Corp.), anti-rabbit IgG was applied to the control line, and 7F22 was applied to the test line at a bed speed of 10 cm / sec and a dispensing volume of 0.9 μl / cm 2. Followed by drying in a dehumidifying cabinet for more than 24 hours to fix the antibody to the membrane.

3) Pad attaching and cutting

When the antibody was immobilized, the sample pad and the absorbent pad were attached to the predetermined positions, respectively, and then cut at intervals of 4 mm using a rotary slitter.

4) Assembly and packing

The finished strip was assembled into a housing and sealed in an aluminum pouch with silica gel.

Detection reagent production

To aid the stability of the detector and control antigens, 2% nonfat milk (Difco), 0.4% Tween-20 (Sigma) and 0.01% NaN ₃ were added to the PBS buffer followed by anti-rabbit IgG control antigen ng / ml, and the rE2 detector antigen was added at 125 ng / ml.

How to use the diagnostic kit

How to use the diagnostic kit is as follows.

1) Add 40 μl of the sample to be tested to 40 μl of the detection reagent, mix well and react at room temperature for 2 minutes.

2) Remove the diagnostic kit and place it on a flat surface. Add 70 μl of reaction solution 1) to the sample injection port.

3) After loading for 12 minutes at room temperature, measure the fluorescence value using a fluorescent reader.

4) Test line Fluctuation value / Control line The ratio (T / C) of fluorescence value is obtained.

(5) If the T / C value obtained in (4) exceeds the cut-off value, it is judged to be positive if the value is below the cut-off value.

Setting the cut-off value

A total of 395 swine serum specimens were tested with VDPro CSFV E2 Ab ELISA (Median Dinostick), and 186 positive samples read as E2 antibody positive and 209 negative samples read as negative E2 antibody were compared with the developed CSFV E2 Ab The TG-ROC curves were used to determine the optimal sensitivity and specificity for the T-C ratio cut-off. Respectively.

positivity voice Specificity responsiveness 0-0.1 31 - 100 16.49 0.1-0.2 30 - 100 32.45 0.2-0.3 30 - 100 48.4 0.3-0.4 25 - 100 61.7 0.4-0.5 13 - 100 68.62 0.5-0.6 16 - 100 77.13 0.6-0.7 10 One 99.52 82.45 0.7-0.8 5 - 99.52 85.11 0.8-0.9 8 2 98.55 89.36 0.9-1.0 8 2 97.58 93.62 1.0-1.1 2 5 95.17 94.68 1.1-1.2 5 15 87.92 97.34 1.2-1.3 4 37 70.05 99.47 1.3-1.4 - 56 43 99.47 1.4-1.5 - 49 19.32 99.47 1.5-1.6 - 16 11.59 99.47 1.6-1.7 One 14 4.83 100 1.7-1.8 - 6 1.93 100 1.8-1.9 - 2 0.97 100 1.9-2.0 - One 0.48 100 2.0-2.1 - One 0 100 Sum 188 207 - -

Repeatability test between strips

The pigs' negative samples were tested repeatedly 4 times according to the POCT method, and the CV% values were determined. The POCT method was performed by mixing the sample and the conjugate buffer in a ratio of 1: 1, reacting for 5 minutes, loading on a strip, developing for 12 minutes, and measuring fluorescence using a reader.

- T / C Test Control group #One 1.42 198 140 #2 1.31 188 143 # 3 1.23 178 145 #4 1.33 187 140 Aver. 1.32 188 142 STDEV 0.08 8 2 CV% 5.89 4.46 1.67

Strip reproducibility test showed CV% value within 6%.

Comparative analysis with existing diagnostic methods

In order to compare with the existing test methods, the blocking ELISA and the diagnostic kit of the present invention were compared in terms of detection limit, sensitivity and specificity.

1. Comparison of detection limits

The CSFV E2 monoclonal antibody was tested by POCT using a calibrator prepared by binary dilution from 10 μg / ml. The POCT method was performed by mixing the sample and the conjugate buffer in a ratio of 1: 1, reacting for 5 minutes, loading on a strip, developing for 12 minutes, and measuring fluorescence using a reader.

The cut-off value set above was compared with the ELISA results, and the detection limit concentration was confirmed using the concentration of the spiking monoclonal antibody. The comparison results are summarized in Table 3.

- Test method Fluorescent POCT Blocking ELISA T / C ratio P / N % PC P / N Antibody concentration
(μg / ml) 10 0.38 P 85.25 P 5 0.64 P 82.79 P 2.5 0.85 P 80.33 P 1.25 1.07 P 71.31 P 0.63 1.18 N 53.28 P 0.31 1.39 N 29.51 N 0.16 1.36 N 15.57 N 0 1.38 N -1.64 N

Fluorescent POCT showed a detection limit twice as low as that of blocking enzyme immunoassay.

2. Comparison of Sensitivity and Specificity

In order to compare the antibody detection efficiency with the conventional assay, 57 sera determined to be positive and 175 sera judged to be negative when tested according to the blocking enzyme immunoassay (blocking ELISA) (Table 4). As a result, the sensitivity was 100% and the specificity was 99.4%.

Fluorescent POCT (1.1 cut-off) synthesis positivity voice ELISA positivity 178 8 186 voice 11 198 209 synthesis 189 206 395 responsiveness
(Positive / (positive + false negative) 95.7% Specificity
Voice / (voice + false positive) 94.7%

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> MEDIAN Diagnostics Inc. <120> Diagnosis Method for Classical Swine Fever using E2 proteins of          CSF-Virus and its Specific Monoclonal Antibodies, and Diagnostic          Kit using the method <130> PN140131 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 406 <212> PRT <213> CSFV LOM E2 Amino Acid <400> 1 Ala Ser Thr Thr Ala Phe Leu Ile Cys Leu Ile Lys Val Leu Arg Gly   1 5 10 15 Gln Ile Val Gln Gly Val Ile Trp Leu Leu Leu Val Thr Gly Ala Gln              20 25 30 Gly Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Leu Ser Ser Thr          35 40 45 Asn Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Glu      50 55 60 Glu Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ile  65 70 75 80 Cys Val Ala Gly Ser Phe Lys Ile Thr Ala Leu Asn Val Val Ser Arg                  85 90 95 Arg Tyr Leu Ala Ser Leu His Lys Glu Ala Leu Pro Thr Ser Val Thr             100 105 110 Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly         115 120 125 Asp Asp Phe Gly Phe Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val     130 135 140 Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu 145 150 155 160 Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser                 165 170 175 Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys             180 185 190 Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu         195 200 205 Asp Leu Phe Tyr Cys Asn Trp Gly Gly Asn Trp Thr Cys Val Lys Gly     210 215 220 Glu Pro Val Val Tyr Ala Gly Gly Leu Val Lys Gln Cys Arg Trp Cys 225 230 235 240 Gly Phe Asp Phe Asn Glu Pro Asp Gly Leu Pro His Tyr Pro Ile Gly                 245 250 255 Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr             260 265 270 Asp Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu         275 280 285 Cys Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg     290 295 300 Leu Gly Pro Met Pro Cys Arg Pro Lys Glu Thr Val Ser Ser Glu Gly 305 310 315 320 Pro Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Lys                 325 330 335 Asn Lys Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu             340 345 350 Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Val Thr Asp Arg His         355 360 365 Ser Asp Tyr Phe Ala Glu Phe Val Val Leu Val Val Val Ala Leu Leu     370 375 380 Gly Gly Arg Tyr Val Leu Trp Leu Ile Val Thr Tyr Ile Val Leu Thr 385 390 395 400 Glu Gln Pro Ala Ala Gly                 405 <210> 2 <211> 1218 <212> DNA <213> CSFV LOM E2 DNA <400> 2 gcatcaacca cggcattcct catctgcttg ataaaagtat taaggggaca gatcgtgcaa 60 ggtgtgatat ggctgctact agtaactggg gcacaaggcc ggctagcctg caaggaagat 120 tacaggtacg cactatcatc aaccaatgag atagggctac tcggggccgg aggtctcacc 180 accacctggg aagaatacaa ccacgatttg caactgaatg acgggaccgt taaggccatt 240 tgcgtggcag gttcctttaa aatcacagca cttaatgtgg tcagtaggag gtatttggca 300 tcattgcata aggaggcttt acccacttct gtgacattcg agctcctgtt cgacgggacc 360 aacccatcaa ctgaggaaat gggagatgac ttcgggttcg ggctgtgccc gtttgatacg 420 agtcctgttg tcaagggaaa gtacaataca accttgttga acggtagtgc tttctatctt 480 gtctgtccaa tagggtggac gggtgttata gagtgcacag cagtgagccc aacaactctg 540 agaacagaag tggtaaagac cttcaggagg gacaagccct ttccgcacag aatggattgt 600 gtgaccacaa cagtggaaaa tgaagattta ttctactgta attggggggg caactggaca 660 tgtgtgaaag gtgaaccagt ggtctacgcg ggggggctag taaaacaatg cagatggtgt 720 ggctttgact tcaatgagcc tgacggactc ccacactacc ccataggtaa gtgcattttg 780 gcaaatgaga caggttacag aatagtggat tcaacagact gtaacagaga tggtgttgta 840 atcagcacag aggggagtca tgagtgcttg atcggtaaca caaccgtcaa ggtgcatgca 900 tcagatgaaa gactgggccc catgccatgc agacctaaag agaccgtctc tagtgaagga 960 cctgtaagga aaacttcctg tacattcaac tacgcaaaaa ctttgaagaa caagtactat 1020 gagcccaggg acagctattt ccagcaatat atgcttaagg gcgagtatca gtactggttt 1080 gacctggacg tgactgaccg ccactcagat tacttcgcag aatttgttgt cttggtggtg 1140 gtagcactgt taggaggaag atatgtcctg tggttaatag tgacctacat agttttaaca 1200 gaacaacccg ccgctggt 1218 <210> 3 <211> 27 <212> DNA <213> rE2 Forward primer <400> 3 ggatcccggc tagcctgcaa ggaagat 27 <210> 4 <211> 24 <212> DNA <213> rE2 Reverse primer <400> 4 ggatccttct gcgaagtaat ctga 24

Claims (11)

A kit for diagnosing a porcine cholera virus (CSFV) infection comprising:
(a) a detectable antigen coupled to a fluorescent label that generates a signal comprising the E2 protein of porcine cholera virus as an antigen; And
(b) a reaction unit immobilized with a monoclonal antibody comprising SEQ ID NO: 1, which is specific to the E2 protein of the porcine cholera virus and compete with an antibody in the serum of a porcine cholera virus-infected individual.
delete delete delete 2. The method of claim 1, wherein the strip comprises a sample pad, a structure in which a sample is contacted with one end of the sample pad and a capillary tube moves the sample, and an absorbent pad is in contact with one end of the tube, Wherein the pad is located between the sample pad and the absorbent pad.
A method for diagnosing a porcine cholera virus (CSFV) infection comprising the steps of:
(a) contacting the sample to be detected with a fluorescently labeled detector antigen, which comprises a signal-generating E2 protein of porcine cholera virus, as an antigen;
(b) reacting the reaction solution of (a) with a monoclonal antibody immobilized on a monoclonal antibody of SEQ ID NO: 1, which is specific to the E2 protein of the porcine cholera virus and competitively reacts with an antibody in the serum of a porcine cholera virus- , &Lt; / RTI &gt;
The reaction unit includes a portion where the binding agent is immobilized as a control group for confirming whether or not the monoclonal antibody is normally reacted, in addition to the portion to which the monoclonal antibody is immobilized;
(c) analyzing each signal of a portion in which the monoclonal antibody that specifically binds to the antigen of the porcine cholera virus is immobilized in the reaction unit and a portion in which the binding agent is immobilized as a control, using a signal analyzer; And
(d) using the measured signal value to determine whether or not the infection is a porcine cholera virus.
delete delete delete The diagnostic method according to claim 6, wherein the binding agent as a control group for confirming the normal reaction is anti-rabbit IgG.
7. The method of claim 6, wherein the signal analyzer is a fluorescent reader.

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