KR20130055044A - Kit for detection of antibody against classical swine fever virus, and detecting method of the antibody and/or antibody titer against classical swine fever virus using the same - Google Patents

Abstract

PURPOSE: A kit for detecting an antibody for swine fever virus is provided to identify the presence of the antibody, and to quantitatively and quickly measure a maternal antibody for swine fever virus in a piglet using a portable quantitative analyzer. CONSTITUTION: A kit for detecting an antibody for swine fever virus comprises: a sample pad; a conjugate pad in which swine fever virus E2 antigen-nano magnetic bead is conjugated; a membrane which shows detection results; and an adsorption pad. The presence or titer of the antibody for swine fever virus is measured using the kit. The yellow line of the kit indicates the presence of the antibody. A method for measuring the titer of the antibody comprises: a step of preparing a sample; and a step of reacting the sample with the swine fever virus E2 antigen-nano magnetic bead conjugate; and a step of measuring the magnetism value of swine fever virus E2 antigen-nano magnetic bead using a magnetic sensor.

Description

KIT FOR DETECTION OF ANTIBODY AGAINST CLASSICAL SWINE FEVER VIRUS, AND DETECTING METHOD OF THE ANTIBODY AND / OR ANTIBODY TITER AGAINST CLASSICAL SWINE FEVER VIRUS USING THE SAME}

The present invention relates to a kit for detecting antibodies against swine fever virus, a method for measuring the presence and / or antibody titer of swine fever virus antibodies using the same, and a method for determining whether or not to administer a vaccine.

Swine fever virus (CSFV) is an RNA virus belonging to the genus Flaviviridae and Pestivirus . Its size is about 40 ~ 50nm, and the swine fever virus gene is positive sense single strand RNA, which is about 12.3 kb in size. The code synthesizes about 3900 amino acids and constitutes a viral structural protein. The swine fever virus protein consists of Npro, Capsid, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. (Kummerer et al., 2000. Vet. Microbiol. 77: 117-128, Moenning et al., 2003. Vet. Journal 165: 11-20)

Pigs infected with the swine fever virus produce antibodies against the ERNS, E2, and NS3 structural proteins of the virus, and in particular, the E2 protein, which constitutes the envelope of the swine fever virus, is a major player involved in producing antibodies that strongly neutralize the virus. Known as protective antigen (Edwards et al., 1991. Vet. Microbiol. 29: 101-108, Weiland et al., 1992. J. Virol. 66: 3677-3682).

 In case of swine fever virus infection, acute type shows clinical symptoms such as high fever, skin redness, appetite deficiency, constipation, diarrhea and posterior palsy, and the immune function such as leukocyte reduction is suppressed and eventually dies. Defense is possible (Van Oirschot. 1999. In Disease of swine. P. 159-172). Blood neutralizing antibodies in infected pigs are detected within 2 weeks (usually 6-11 days), and in pigs infected with chronic CSF, neutralizing antibodies are observed for several days after 4 weeks of infection, after which they disappear (Depner et al. , 1966. Eur. J. Vet. Path. 2: 61-62, Mengeling et al., 1969. Am. J. Vet. Res. 30: 409-417). Piglets delivered after infection with low-pathogenic CSFV through the placenta continue to develop viremia, but become immune tolerant and rarely produce specific antibodies (Van Oirschot. 1999. In Disease of swine. P. 159-) 172).

Maternal transit antibodies are delivered to piglets via colostrum, usually absorbed most frequently within 24-48 hours, gradually decreasing after a half-life of 12-19 days (Kim et al., 1982. Res. Rep. Korea.Ins) Ani.Sci. 2: 62-70, Kim et al., 1984. Res.Rept. ORD. 26: 53-55), after 30-35 days of age, immune interference by maternal transducing antibodies is weakened. Vaccination time has been suggested (Launais et al., 1978. Vet. Microbio. 3: 31-43). Vaccination of piglets with maternal transgenic antibodies derived from delivery sows more than 64-fold has immune interference and is constrained by active immune formation (Lai et al., 1981. J. Chinese Soc. Vet. Sci. 6: 77-81), vaccination is recommended at a time when maternal transducing antibodies are reduced to a certain level, ie less than 32-fold. In the case of vaccine vaccination, which is widely practiced at present, when the level of maternal transfecting antibody is 64 times or more, a single vaccination alone does not give sufficient immune response due to significant interference of maternal transducing antibody, Inoculation should be performed. Individuals not immunized with the first vaccination are more likely to be infected with the pathogenic virus until the second vaccination, so farms with more pigs are at increased risk of developing CSF. Therefore, in order to increase the effectiveness of the vaccine and to lower the incidence of CSF, it is important to quantitatively measure maternal transfer antibodies before vaccination.

Conventional methods for detecting antibodies to swine fever include virus neutralization test (VN test), enzyme linked immunosorbent assay (ELISA) and latex (latex) aggregation reaction, gold conjugate Lateral flow immunochromatography using a colloidal gold conjugate was used. However, the only method of quantitatively measuring the titer of maternal transfer antibody in piglets is the neutralizing antibody test (NPLA, Neutralizing peroxidase linked assay), and in the case of ELISA or latex agglutination reaction, only the presence of antibody is qualitatively. Can be detected.

However, the neutralizing antibody test that can measure the quantitative antibody titer requires specialized manpower and experimental equipment, and the detection time is long, which is not suitable for outdoor use. Specifically, it is difficult to process a large amount of serum samples, pig blood is collected and only serum components are collected by Winsim Separation, and then used for at least 30 minutes at 56 ° C. There was a problem requiring manpower, so it was not suitable for use in pig farms.

Accordingly, the present invention has been made to solve the problems of the prior art, an antibody detection kit that can quickly and easily measure the presence and antibody titers of porcine fever virus antibody and the presence and / or antibody of the antibody using the same To provide a method for measuring the titer, and to provide a method for determining whether the vaccine can be efficiently vaccinated without affecting the immune interference phenomenon of maternal transfer antibodies in pigs and pigs infected with swine fever virus do.

In order to solve the above problems, the present inventors have established a method for measuring the titer of the swine fever virus antibody in pigs and / or maternal transfer antibody in piglets and accordingly determining whether swine fever virus is infected and whether the vaccine can be administered. As a result of the research efforts, the conjugated conjugate of the swine fever virus antigen and the magnetic nanoparticles is used to detect the presence or absence of antibodies to swine fever virus, as well as to quantitatively neutralize antibody titers 16, 32 and 64 times in piglets. And, it can measure the value corresponding to 128 times, through which to develop a rapid diagnostic kit that can determine whether the swine fever virus vaccine can be administered to complete the present invention, according to the conventional neutralizing antibody test (NPLA To replace the Neutralizing peroxidase linked assay. This suggests that faster and simpler method of measuring maternal transfer antibody values in the field.

Accordingly, one embodiment of the present invention, the sample pad for dropping the sample;

Conjugation pads with porcine fever virus E2 antigen-magnetic nanoparticle conjugates adsorbed;

A membrane showing a detection result; And

Absorbent pad that absorbs sample development

It provides a kit for detecting antibodies to swine fever virus comprising a.

Another embodiment of the present invention provides a method for measuring the presence and / or titer of an antibody against swine fever virus using the kit.

Another embodiment of the present invention, preparing a sample;

Reacting the sample with a swine fever virus E2 antigen-nano magnetic bead conjugate; And

Measuring the magnetic value of the sample bound to the swine fever virus E2 antigen-nano magnetic bead conjugate using a magnetic sensor

It provides a method for measuring the antibody titer to porcine fever virus comprising a.

Another embodiment of the present invention, preparing a sample;

Reacting the sample with a swine fever virus E2 antigen-nano magnetic bead conjugate; And

Measuring the magnetic value of the sample bound to the swine fever virus E2 antigen-nano magnetic bead conjugate using a magnetic sensor

It provides a method for determining whether or not porcine fever virus vaccine administration comprising a.

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

According to one embodiment of the invention, the sample pad for dropping the sample; Conjugation pads with porcine fever virus E2 antigen-magnetic nanoparticle conjugates adsorbed; A membrane showing a detection result; And there is provided a kit for detecting antibodies to swine fever virus, including an absorption pad absorbing the sample development solution.

Pigs infected with the swine fever virus produce antibodies against the ERNS, E2, and NS3 structural proteins of the virus, and in particular, the E2 protein, which constitutes the envelope of the swine fever virus, is a major player involved in producing antibodies that strongly neutralize the virus. Known as protective antigen (Edwards et al., 1991. Vet. Microbiol. 29: 101-108, Weiland et al., 1992. J. Virol. 66: 3677-3682).

Therefore, for diagnosing swine fever virus infection, deriving the presence of antibodies thereto, and measuring the titer of the antibody, E2 protein, which is known to produce the highest level of neutralizing antibody upon swine fever virus infection, may be preferably used as a target antigen. have. In addition, the control antigen for this may be used without particular limitation as long as there is no cross-reaction with the swine fever virus antigen, preferably feline leukemia virus (FeLV) antigen that has been confirmed that there is no cross-reaction to the swine fever virus This can be used.

The membrane may be used as a material of a conventional diagnostic strip as a part showing the detection result, for example, one selected from various synthetic polymers such as nitrocellulose, cellulose, cellulose acetate, polyethylene, and the like. Preferably, a nitrocellulose membrane may be used, and may also preferably include a test line to which porcine fever virus E2 antigen is fixed and a control line to which feline leukemia virus (FeLV) antigen is fixed.

Magnetic nanoparticles are used to form conjugates, respectively, with the target antigen E2 and the antibody against the control antigen FeLV, each conjugate formed to adsorb to a conjugate pad. After the magnetic nanoparticles, preferably nano-magnetic beads, which form the conjugate react with the sample, the antibody bound to the target can be qualitatively and quantitatively detected by measuring the magnetic value to be expressed.

Thus, one embodiment of the present invention provides a method for conjugating with magnetic nanoparticles using E2 protein as an antigen to detect swine fever virus antibodies in pigs and swine fever maternal transition antibodies in piglets.

Conjugation of the E2 antigen with the magnetic nanoparticles preferably comprises activating the magnetic nanoparticles; And reacting the activated magnetic nanoparticles with swine fever virus E2 antigen.

The activating of the magnetic nanoparticles preferably includes repeating the process of removing the supernatant and suspending the pellet 2 to 5 times, preferably three times, after precipitating the magnetic nanoparticles diluted with the buffer with a magnet. ; And the pellet obtained through the repeating process may be made by including a step of reacting for 5 to 15 minutes at 30 to 40 ℃ after floating using an active buffer.

The buffer used for dilution and pellet suspension of the magnetic nanoparticles in the activation step is not particularly limited, but preferably MES (0.1M 2- (N-morpholino) ethanesulfonic acid) buffer may be used. In addition, the active buffer may be an active buffer (0.1M MES, 0.5M NaCl, pH6.0) to which EDC (1-ethyl-3- (3-dimethylaminopropyl carbodiimide hydrochloride) and Sulfo-NHS (N-hydroxylsulfosuccinimide) is added. .

The step of reacting the activated magnetic nanoparticles with the E2 antigen preferably comprises the steps of shaking culture the activated magnetic nanoparticles and E2 antigen at 30 to 40 ℃ in PB buffer for 1 to 3 hours; The BSA activation buffer may be added by adding 0.1 to 0.5% (w / v) to shake the culture at 30 to 40 ° C. for 20 to 40 minutes.

Conjugation with the control antigen can also be performed in the same manner.

The magnetic nanoparticles may preferably have a diameter of 200 nm (CV max 20%), preferably nano magnetic beads may be used.

As such, the antibody detection kit for swine fever virus according to an embodiment of the present invention is a progressive that can completely replace the conventional gold particle (colloidal gold) conjugation, by using the magnetic nanoparticles as a conjugation Provide a method.

That is, by using magnetic nanoparticles, preferably nano magnetic beads, not gold particles, quantitative antibody titers can be measured by measuring magnetic values using a portable quantitative analyzer (magnetic sensor) using a magnetic field. In addition to determining the presence or absence of antibodies, furthermore, it is possible to measure antibody titers and to determine whether vaccination is possible through this method. Also, the present method is more effective than optical measurement for conventional gold particle conjugation. Advances in sensitivity and specificity due to contamination This can be widely applied to all lateral flow immunoassays.

The sample is preferably a body fluid secreted into the body, and includes blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomiting or feces, and more preferably obtained from adult pigs. It may be, but is not limited to, one blood.

The kit for detecting antibodies to swine fever virus may preferably be to detect an E2 antibody against swine fever virus infection by using an antigen-antibody reaction between a specimen and the E2 antigen, and preferably, an immunochromatography method It may be to detect swine fever virus using.

The principle of the detection kit according to an embodiment of the present invention will be described with reference to FIG. 3.

The swine fever virus E2 antigen is accumulated on a nitrocellulose membrane (4) attached to a lamination card (Backing plate, 7) in the form of a strip with a test line (3). On the other side of the nitrocellulose membrane, the control line 5 also accumulates antigens that are not reactive with the swine fever virus antibody. Preferably, feline leukemia virus (FeLV) antigens are identified that have been found to have no cross reaction to the virus in pigs.

Conjugation pads (2) adsorbed with E2 antigen-magnetic nanoparticle conjugates capable of reacting with swine fever virus E2 antibodies present in a sample sample on one end of the nitrocellulose membrane in which the test and control lines are integrated And an absorbent pad (6) which absorbs a sample sample on top of the other end of the nitrocellulose membrane to facilitate deployment through the nitrocellulose membrane. An upper portion of one end of the conjugation pad 2 (opposite to the end in contact with the upper end of the membrane) is positioned so that the sample pad 1 capable of dropping the sample sample overlaps with the upper part of the conjugation pad. The assay strip thus prepared may be embedded in a case divided into a lower part and an upper part to finally complete an immunoassay device (see FIGS. 2 and 5).

As described above, a method for detecting the presence of an antibody using the manufactured immunoassay device may be performed as follows, for example, when nano magnetic beads are used as magnetic nanoparticles (see FIGS. 3 to 5).

First, a test sample of a pig prepared in advance is dropped on the specimen pad 1. Preferably, 40 ul of test sample may be loaded, but is not limited thereto. After instillation, the pig blood sample was placed in a conjugate pad (2) adsorbed with an E2 antigen-nano magnetic bead conjugate to the place where the swine fever virus antigen (3) was fixed through the nitrocellulose membrane (4) after the antigen-antibody reaction. In the same way, anti-feline leukemia virus antibody (α-FeLV antibody) -nano magnetic bead conjugate adsorbed to the conjugation pad (2) is also absorbed through the nitrocellulose membrane (4). The pad 6 is moved in the direction. If swine fever virus E2 antibody is present in the blood sample, the E2 antigen is fixed in the process of moving the nitrocellulose membrane 4 after the primary antigen-antibody reaction with the E2 antigen-nano magnetic bead conjugate on the conjugation pad 2. In the test line 3, a secondary antigen-antibody reaction is caused, which is characterized by the yellow magnetic beads (E2 antigen-nano magnetic bead conjugate) sensitized to the antigen. Thereby, the determination of positive (antibody) or negative (no antibody) depending on the presence or absence of antibodies in pig blood can be made.

The antigen-antibody reaction and the reaction with the E2 antigen-nano magnetic bead conjugate are carried out in a buffer, and for example, PBS, Tris, or borate buffer, etc. may be used, and preferably PBS buffer (PBS, 0.5). % BSA, 300 mM NaCl, 0.1% NaN ₃ , pH 7.2).

Thus, according to another preferred embodiment of the present invention, using the kit for detecting antibodies to swine fever virus, there is provided a method for measuring the presence or absence of the antibody against swine fever virus or the titer of the antibody.

Preferably, the method is a method for measuring the presence or absence of an antibody. If the test line of the kit shows yellow, it is determined that there is an antibody (positive), and if it does not display yellow, the antibody is absent (negative). It may be to judge.

In addition, according to another preferred embodiment of the present invention, preparing a sample; Reacting the sample with a swine fever virus E2 antigen-magnetic nanoparticle conjugate; And it provides a method for measuring the antibody titer against swine fever virus comprising the step of measuring the magnetic value of the sample bound to the swine fever virus E2 antigen-magnetic nanoparticle conjugate using a magnetic sensor.

Also preferably, in the antibody titer measuring method, after the magnetic value measuring step, when the magnetic value is 509 mV or less, the antibody titer is determined to be 1:32 or less, and when the magnetic value is 827 mV or more, the antibody titer is 1:64 or more. The method may further include the step of determining.

The magnetic value that is a standard for determining the antibody titer may be preferably based on a value measured in piglets, which is about three weeks after birth. However, when the magnetic value measured at this time is a value corresponding to an intermediate region exceeding 509 mV and below 827 mV, which is the determination criteria, it is difficult to accurately determine whether the antibody titer belongs to 1:32 or 1:64. The antibody titer can be determined to have an antibody titer of 1:32 or less at the time when the measured magnetic value becomes 509 mV or less by repeating the measurement at a weekly interval from the time of about 3 weeks after birth, which is the first measurement day.

Antibody titer refers to the highest dilution of the antibody that reacts with the antigen to form precipitate aggregates, but is not limited thereto, and may preferably mean the titer of the parental transfer antibody, particularly in piglets.

Maternal antibodies are immune antibodies that transfer from the mother to the fetus and are delivered to the piglets via colostrum, usually absorbed most within 24-48 hours, and gradually diminish through a half-life of 12-19 days ( Kim et al., 1982.Res.Rep.Korea.Ins.Ani.Sci. 2: 62-70, Kim et al., 1984.Res.Rept.ORD.26: 53-55), 30-35 days of age After this time, the immune interference caused by the parental transfer antibody is weakened, and thus it is suggested that the vaccine can be vaccinated (Launais et al., 1978. Vet. Microbio. 3: 31-43). Vaccination of piglets with maternal transgenic antibodies derived from delivery sows more than 64-fold has immune interference and is constrained by active immune formation (Lai et al., 1981. J. Chinese Soc. Vet. Sci. 6: 77-81), vaccination is recommended at a time when maternal transducing antibodies are reduced to a certain level, ie less than 32-fold. In the case of vaccine vaccination, which is widely practiced at present, when the level of maternal transfecting antibody is 64 times or more, a single vaccination alone does not give sufficient immune response due to significant interference of maternal transducing antibody, Inoculation should be performed. Individuals not immunized with the first vaccination are more likely to be infected with the pathogenic virus until the second vaccination, so farms with more pigs are at increased risk of developing CSF. Therefore, in order to increase the effectiveness of the vaccine and to lower the incidence of CSF, it is important to quantitatively measure maternal transfer antibodies before vaccination.

Another embodiment of the present invention is to quantitatively measure porcine fever virus antibodies in pigs, and pig fever maternal transfer antibodies in piglets using a portable quantitative analysis device using magnetic fields using the E2 antigen-magnetic nanoparticle conjugate. It provides a detection kit that can be used.

The magnetic nanoparticles may preferably be nano magnetic beads, which may be applied in the same manner as mentioned in the antibody detection kit for swine fever virus according to the embodiment of the present invention.

The sample may be blood, serum, plasma, lymph, tissue fluid, urine, tear, saliva, milk, vomiting, or feces, as described above, and preferably may be separated from piglets, but is not limited thereto. Not.

According to another preferred embodiment of the present invention, preparing a sample; Reacting the sample with a swine fever virus E2 antigen-magnetic nanoparticle conjugate; And measuring a magnetic value of a sample bound to the swine fever virus E2 antigen-magnetic nanoparticle conjugate using a magnetic sensor.

As mentioned above, the titer of the maternal transfecting antibody is very important in determining whether to administer the vaccine (or inoculation) and therefore the determination of the magnetic value of a sample bound to the swine fever virus E2 antigen-magnetic nanoparticle conjugate. Through this, it is possible to determine whether the vaccine is administered.

Preferably, the method may further include determining that the vaccine can be administered when the magnetic value is 509 mV or less, and determining that the vaccine is impossible when the magnetic value is 827 mV or more.

The magnetic value as a criterion for determining whether the vaccine is administered or not may be measured using a sample separated from the piglets up to about 3 weeks after birth. However, when the measured magnetic value is a value corresponding to an intermediate region of more than 509 mV and less than 827 mV, it is preferably measured at a weekly interval from the point of the first measurement date of about 3 weeks after birth as in the above-described method for measuring antibody titers. When the repeated measured magnetic value is 509 mV or less, it can be determined as the time point or subject to the vaccine administration.

The sample may be blood, serum, plasma, lymph, tissue fluid, urine, tear, saliva, milk, vomiting, or feces, as described above, and preferably may be separated from piglets, but is not limited thereto. It doesn't work.

As described above, using the detection kit according to the present invention, it is possible to measure the presence or absence of an antibody against swine fever virus in blood, and in particular, a portable quantitative analysis device using porcine fever maternal transfer antibody in a piglet using a magnetic field. It is very useful because it can be measured quickly and quantitatively by using. In particular, it is required to provide efficient information for determining whether or not to be vaccinated for piglets in the farm. The present invention can quantify maternal transfer antibodies so that this requirement is adequately met, thereby enabling efficient vaccination. Suppressing the invention of fever virus can also contribute to the economic benefit of pig breeders.

Figure 1 schematically shows a quantitative and qualitative detection process of the antibody against swine fever virus according to an embodiment of the present invention.
Figure 2 schematically shows an exploded perspective view of a kit for detecting swine fever virus antibody according to an embodiment of the present invention.
Figure 3 schematically shows the structure of the strip constituting the kit for detecting swine fever virus antibody according to an embodiment of the present invention.
Figure 4 schematically shows the results of the positive and negative, respectively, when the swine fever virus antibody detection kit according to an embodiment of the present invention reacted with the sample.
FIG. 5 shows the results of testing neutralizing antibody samples by titer according to Example 4 of the present invention using the kit of Example 2 of the present invention.
6 is a graph showing quantitatively quantifying a magnetic value using a magnetic sensor of the result according to Example 4 of the present invention.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments to help understand the present invention. However, the following examples are merely to illustrate the present invention is not limited to the scope of the present invention.

Example 1 : Swine fever virus  Target and Control Antigen Preparation

A. As a Target Antigen Swine Fever Virus E2  Production of antigens

Swine fever virus recombinant E2 antigen was prepared according to Example 2 of Korean Patent No. 10-0543691, and was provided from Genobiotech (JBT, Jeno Biotech). Briefly describing the preparation method, in order to clone the E2 gene of the ALD strain, a forward primer (5'-GGATCCCGGCTAGCCTGCAAGGAAGAT-3 ') (SEQ ID NO: 1) and a reverse primer (5'-GGATCCTTCTGCGAAGTAATCTGA-3') Using (SEQ ID NO: 2), a reverse transcription polymerase chain reaction (RT-PCR) was carried out for 30 cycles of 40 seconds at 92 ° C, 40 seconds at 50 ° C, 60 seconds at 72 ° C, and 1021bp. DNA was isolated and cloned into pAcGP67B (Pharmingen), a baculovirus transfer vector, which was cotransfected into insect cell Sf21 (Invitrogen) with baculovirus DNA (Pharmingen) to express E2 protein. Baculovirus BacE2TNALD was produced. BacE2TNALD was again inoculated into insect cells and cultured for 6 days, followed by harvesting supernatant. The harvested supernatant was treated with a virus inactivator (Binary ethylenimine: BEI), and then dialyzed in phosphate buffer (PBS, pH 7.2) to use the kit according to the present invention.

B. Feline leukemia virus as a control antigen ( FeLV ) p27  Production of antigens

As a control antigen, feline leukemia virus (FeLV) antigen, which was found to have no cross reaction to porcine fever virus, was used. The p27 antigen protein of FeLV was prepared as follows according to Example 2 of Korean Patent No. 10-1069391, to which the inventors have previously filed. Template oligonucleotides for the p27 antigen gene were synthesized (Bionics, KOREA) based on the base sequence registered in the NCBI Gene Bank (NCBI GeneBank data, ACCESSION: M18247). A template oligonucleotide was introduced into pGEX-4T-1 (GE Health care) using restriction nucleic acid endonucleases EcoRI (New England Biolabs) and SalI (New England Biolabs) to express the FeLV p27 antigen protein. It was. The binding product was transformed in BL21 or Rosetta (Stratagene) recipient cells, and then inoculated in LB medium to which 100 μg / ml of ampicillin and 0.5 mM IPTG (isopropylthiogalactoside) were added to induce expression, followed by shaking at 20 ° C. ( Incubated overnight in a shaking orbital incubator. Pellets were collected and suspended by adding Cold buffer (50 mM Tris, pH7.5, 150 mM NaCl, 1 mM EDTA) and 1 mM PMSF, ground by ultrasonication, and then centrifuged (10,000 rpm, 30 minutes). Water soluble lysates were prepared, filtered with a 0.45 um Syringe filter, Sartorius, and then purified by glutathione affinity chromatography, and the soluble cell lysates were glutathione Sepharose 4B (Pharmacia) equilibrated with PBS. It was loaded into the column. After washing the column with bed volume PBS, the fusion protein was eluted with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 buffer solution. Purified fusion protein was used for kit preparation according to the invention after 0.2um filtering.

Example 2 : Swine Fever Virus  Assay strips for antibody detection and Of kit  Produce

A. Swine Fever Virus  Antigen and feline leukemia virus ( FeLV ) p27  The antigen is fixed Nitrocellulose Membrane ( membrane ) Produce

Example 1. The swine fever virus recombinant E2 antigen prepared in A was dialyzed at 2 ° C. with 2 mM sodium tetraborate at 4 ° C. for 12 hours, and adjusted to a concentration of 0.7 mg / ml in a buffer solution (0.1% trehalose (0.1 g trehaloe / 100). ml dH ₂ O) and 10 mM PB (phosphate buffer) were mixed and used as test lines. Example 1 Cat leukemia virus (FeLV) antigen prepared in B was dialyzed in 2 mM sodium tetraborate at 4 ° C. for 12 hours. The mixture was adjusted to 1 mg / ml concentration and used as a control line in a buffer solution (0.1% trehalose, 10 mM PB). The recombinant antigen of the test line and the control solution of the control line were sprayed onto the nitro cellulose membrane (MDI, India) using a dispensing apparatus (KINAMETICS, USA) as shown in Table 1. Then, the membrane was dried in a low humidity laboratory overnight or in a pan for at least 5 hours to prepare a membrane including a test line in which porcine fever virus E2 antigen was fixed and a control line in which feline leukemia virus (FeLV) antigen was fixed. Plates of the prepared membranes were stored in containers sealed with desiccant or in low humidity laboratories.

Swine Fever Virus Antigen Injection Conditions division Inspection line Contrast antigen Swine Fever Virus (E2)
0.7 mg / ml Feline Leukemia Virus (p27)
1 mg / ml density( ul Of cm ) 0.9 0.9 Bed speed ( cm / s) 7 7

B. Swine Fever Virus E2 Antigens and Feline Leukemia Virus p27  Nano Magnetics Adsorbed to Antibodies Bead Of conjugate  Produce.

In order to conjugate the swine fever virus E2 antigen to the nano magnetic beads, the nano magnetic beads were activated as follows.

10 ml MES (0.1M 2- (N-morpholino) ethanesulfonic acid, Sigma) buffer was added to 333 ul (3% Carboxyl-Adembeads 0212, Ademtech, France) nano magnetic beads and diluted to 0.1% (v / v) After precipitation by using the supernatant was removed, the process of floating the pellet in MES buffer was repeated three times. The pellet collected at the end of the process was mixed with 0.32 mg / ml EDC (1-ethyl-3- (3-dimethylaminopropyl carbodiimide hydrochloride, Sigma) and 0.725 mg / ml Sulfo-NHS (N-hydroxylsulfosuccinimide, Thermo). M MES, 0.5M NaCl, pH6.0), and reacted for 10 minutes at 37 ° C. Finally, activated nano magnetic beads at 1% (v / v) concentration twice with 10 mM PB buffer. After washing, 10 ml of PB buffer was added to adjust the concentration to 0.1% (v / v), and swine fever virus E2 antigen was added (50 ul (1 mg / ml)), followed by shaking culture at 37 ° C. for 2 hours. After shaking, add 0.5% (0.5 g BSA / 100 ml dH2O) BSA-activated active buffer (0.1M MES, 0.5M NaCl, pH6.0) to a concentration of 0.1% (v / v) at 37 ° C. Shake incubation for 30 minutes, washed three times with storage buffer (0.1% (w / v) BSA, 0.1% (w / v) NaN ₃ ), and then added to the storage buffer to a concentration of 1% (v / v) , Refrigerated to produce a kit according to the invention For control, feline leukemia virus antibody (50 ug, FeLV p27 Antibody, MFLV-20M-8D11, ICL) was also conjugated with nano magnetic beads in the same manner as described above.

C. Swine Fever Virus E2  Anti-Adsorbed Nano Magnetics Bead Conjugation  Manufacture of pads

1% (v / v) swine fever virus E2 antigen-nano magnetic bead conjugate solution and 1% (v / v) feline leukemia virus p27 antibody-nano magnetic bead conjugate solution prepared as described above were respectively 0.13% (v / v). / v) and diluted to 0.05% (v / v), 5% (w / v) Trehalose (Sigma) is added, titrated with distilled water to a total volume of 900 ul and conjugated with a pipette The pads (0.5 x 20 cm glass fiber) were evenly adsorbed. It was then dried overnight in a low humidity laboratory or in a pan for at least 5 hours. The prepared conjugate pad was stored in a sealed container or a low humidity laboratory with desiccant.

D. Absorption Pads Sample pad  Produce

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

E. device  Assembly

Each of the membranes and pads prepared above were combined with a sample pad, a conjugation pad (nano magnetic bead conjugated treatment pad), a nitrocellulose membrane, and finally an absorption pad, respectively, from the right side to the immunoassay device (device). Cut into strip sizes that fit the size. The strip thus cut was finally put in the lower plate of the immunoassay device (single device), and the top plate was covered to prepare a diagnostic kit (see FIG. 2).

F. Product Composition

The immunoassay device and the sample development solution PBS buffer (PBS, 0.5% (w / v) BSA, 0.1% (w / v) NaN ₃ , pH 7.2) described above were configured as the final product (see FIG. 5). .

Example  3: nano magnetic Bead Kit  Used In sex Swine Fever Virus  Detection of antibodies

In order to compare the antibody detection efficiency with the conventional assay, Example 2 using 30 sera determined as pig swine fever virus positive and 30 sera as negative according to the existing ELISA (Jeno Biotech, Korea) method as a sample Inspected with a kit prepared according to the method.

After 10 minutes after the specimen was reacted with the prepared kit, the results were first confirmed by the naked eye of the experimenter. The maximum and minimum values were excluded, and the mean values of the remaining values were calculated and quantified. As a result, the sensitivity (relative sensitivity) was 100%, the specificity (relative specificity) was confirmed to be 100%, the agreement rate with the existing test method was very high (see Table 2 and Table 3).

Comparison of swine fever virus antibody detection efficiency ELISA Positive (+) voice(-) Invention Kit Positive (+) 30 0 voice(-) 0 30 system 30 30 Relative Sensitivity (%)
Relative specificity (%) 100
100

Swine fever virus antibody diagnostic comparison test result No . Sample name ELISA SensPERT  (The present invention) Kit ) % SP ^* result mV ± SD result One D1 107.1 positivity 2500 ± 0 positivity 2 D2 99.6 positivity 1271.1 ± 18.3 positivity 3 D4 82.1 positivity 2500 ± 0 positivity 4 D5 67.1 positivity 1376.2 ± 63.5 positivity 5 D6 96.4 positivity 2500 ± 0 positivity 6 D7 46.1 positivity 2500 ± 0 positivity 7 D8 65.4 positivity 2362 ± 51.7 positivity 8 D9 85.0 positivity 2500 ± 0 positivity 9 D10 73.6 positivity 2500 ± 0 positivity 10 D11 60.7 positivity 1589.2 ± 34.2 positivity 11 D12 96.1 positivity 1521.9 ± 2.2 positivity 12 D13 79.6 positivity 1891.7 ± 61.1 positivity 13 D14 110.7 positivity 2500 ± 0 positivity 14 D15 81.8 positivity 2277.7 ± 4.6 positivity 15 D16 121.8 positivity 2500 ± 0 positivity 16 D17 63.2 positivity 2500 ± 0 positivity 17 D18 83.6 positivity 1569.2 ± 33.5 positivity 18 D19 83.2 positivity 1368.3 ± 24.3 positivity 19 D20 84.6 positivity 2500 ± 0 positivity 20 D21 85.0 positivity 2339.1 ± 30.4 positivity 21 D22 92.9 positivity 768.3 ± 24 positivity 22 D23 123.6 positivity 2500 ± 0 positivity 23 D24 86.4 positivity 2500 ± 0 positivity 24 D25 78.2 positivity 1784.7 ± 44.3 positivity 25 D26 96.4 positivity 1774.2 ± 132.5 positivity 26 D27 80.0 positivity 819.3 ± 22.7 positivity 27 D28 56.1 positivity 2301.2 ± 31 positivity 28 D29 101.1 positivity 820.6 ± 30.9 positivity 29 D30 76.4 positivity 2308.2 ± 12.8 positivity 30 D31 107.1 positivity 2390 ± 12.8 positivity 31 J1 -1.0 voice 66.8 ± 3.2 voice 32 J2 4.2 voice 62.3 ± 7.6 voice 33 J3 -2.1 voice 58.8 ± 3.4 voice 34 J4 -1.0 voice 57 ± 7.6 voice 35 J5 0.3 voice 68.2 ± 2.6 voice 36 J6 -1.4 voice 56.6 ± 6.98 voice 37 J7 -2.4 voice 63.7 ± 5.2 voice 38 J8 1.0 voice 57.2 ± 3.1 voice 39 J9 0.3 voice 84.6 ± 1.7 voice 40 J10 -1.4 voice 56.6 ± 4.4 voice 41 J11 -2.4 voice 54.3 ± 1.9 voice 42 J12 1.0 voice 52.7 ± 2.1 voice 43 J13 0.3 voice 51.6 ± 6.7 voice 44 J14 -1.4 voice 50.1 ± 5.6 voice 45 J15 -1.7 voice 68.2 ± 5.0 voice 46 J16 -1.4 voice 58.1 ± 3.3 voice 47 J17 -0.3 voice 62.5 ± 5.0 voice 48 J18 -1.0 voice 53.1 ± 1.9 voice 49 J19 2.1 voice 62 ± 6.8 voice 50 J20 0.0 voice 104.4 ± 6.8 voice 51 J21 -0.7 voice 63.6 ± 14.2 voice 52 J22 -2.4 voice 113.1 ± 10.6 voice 53 J23 -2.8 voice 53.7 ± 7.9 voice 54 J24 0.3 voice 49.4 ± 3.2 voice 55 J25 1.4 voice 56.6 ± 5.9 voice 56 J26 2.4 voice 48.6 ± 6.8 voice 57 J27 -2.1 voice 59.4 ± 4.2 voice 58 J28 5.6 voice 52.4 ± 3.7 voice 59 J29 -2.1 voice 42.1 ± 1.4 voice 60 J30 -1.0 voice 86.5 ± 7.5 voice PC 100.0 positivity - - NC 0.0 voice - -

* Sample to positive ratio = {(test sample average absorbance-negative control mean absorbance) / CPC (corrected positive control) X 100, CPC = positive control mean absorbance-negative control mean absorbance}

Example  4: nano magnetic Bead Kit  Used Swine Fever Virus  Neutralization antibody of standard sample By potency Magnetic value  Quantification.

Neutralizing antibody samples for antibody diagnosis by swine fever virus titer were provided by the Ministry of Agriculture, Forestry and Fisheries Quarantine and Inspection Division, and serially diluting swine fever virus 1024-fold neutralizing antibody positive sample to pathogen-free pig serum (26250084, Invitrogen). Samples corresponding to 128, 64, 32, and 16 times were prepared. Thus prepared titer neutralizing antibody samples were examined using a kit prepared according to the method of Example 2. Inspection of each sample with the kit of Example 2 was performed a total of 10 times.

Ten minutes after the specimens were reacted with the prepared kit, the results were first confirmed by the naked eye of the experimenter (see FIG. 5). Secondly, the magnetic values were quantitatively determined using a magnetic sensor (Care-U, LG innotek). It was quantified by Table 4 and shown in FIG. The quantification of the magnetic value of each standard sample was repeated 10 times, using a set of 1: 256 from the buffer for each lot and batch, and 6 measurements were taken per sample to exclude the maximum and minimum values. The average value was calculated | required.

Quantitative Analysis of Neutralizing Antibodies by Porcine Fever Virus Titer Using Magnetic Sensors No VN titer Magnetic measurement value ± SD  ( mV ) range( mV ) One - 16 ± 3.3
<186 2 Buffer 143.592 ± 42.1646 3 16 292.133 ± 101.699 <394 4 32 483.958 ± 24.9989 <509 5 64 754.875 ± 71.7058 <827 6 128 1120.64 ± 173.353 <1294 7 256 2303.77 ± 252.37 <2556

As shown in Table 4 and Figure 6, it was confirmed that the magnetic value increases according to the antibody value of each swine fever virus titer. Specifically, when the swine fever virus neutralizing antibody titer was 1:32 (32-fold), the magnetic value was 483.958 ± 24.9989 mV, and when 1:64 (64-fold), the magnetic value was 754.875 ± 71.7058 mV.

When vaccination with a vaccine is possible at a certain level, i.e., when it is reduced to 32 times or less, or when the level of maternal transfer antibody is 64 times or more, sufficient vaccination may be necessary due to interference of maternal transfer antibody. Because of the impossibility of imparting an immune response, it was possible to prepare a criterion for dividing vaccinable piglets and vaccinated piglets through the titer-specific magnetic values measured according to the present experiment (see FIG. 6).

That is, as shown in Figure 6, when the magnetic value is less than about 509 mV porcine fever virus neutralizing antibody titer of 1:32 or less judging that vaccination is possible, if the magnetic value is about 827 mV or more titer 1 : It is more than 64 and can judge that vaccination is impossible. In addition, according to the criteria provided in the present embodiment, when the magnetic value is less than about 186 mV, the negative, about 186 mV or more and less than 394 mV, the potency 1:16, about 827 mV or more, and less than 1294 mV, the titer 1: 128, about 1294 mV or more and less than 2556 mV It can be determined that the titer is 1: 256, respectively.

However, when the measured magnetic value is a value corresponding to the middle region of more than 509 mV and less than 827 mV, the titer of antibody titer is 1:32 in the piglets at about 3 weeks of age, when the magnetic value is measured. It is difficult to accurately determine whether or not belong to 1:64, the antibody titer is 1 when the measured magnetic value is 509 mV or less by repeating the measurement at a weekly interval from the first measurement of about 3 weeks after birth. It judges that it is: 32 or less.

As described above, an effective immune response can be given by inoculating a vaccine after confirming the time when the titer of the parental transfer antibody falls below the magnetic value of 509 mV using the kit of the present invention.

Example  5: Swine Fever Virus  Nano magnetic Bead Of kit  Clinical results.

Magnetic field quantification was carried out using a nano magnetic bead kit for the outdoor groups known to the neutralizing antibody value by the neutralizing antibody method, and the outdoor groups having various neutralizing antibody values were vaccinated according to the criteria prepared in Example 4 above. It was checked whether it was divided into group and non-target group.

First, a virus neutralization test (Neutralizing peroxidase) to examine the antibody titer by measuring the degree of virus inhibition in PK-15 cells (National Veterinary Quarantine Service) cultured by diluting the test serum of an outdoor group sample (National Veterinary Quarantine Service). -Neutralization titer against swine fever virus (Virus Neutralization Titer, VN titer) was confirmed by -linked assay (NPLA) (see C. Terpstra et al., Veterinary microbiology 16 (2): 123-128 1988). As a result, voice samples and neutralizing antibody titers were 1:16 (10 species), 1:32 (10 species), 1:64 (10 species), 1: 128 (10 species), and 1: 256 (10 species). Clinical samples were obtained. The magnetic values (Care-U, LG innotek) were measured for the clinical samples obtained by various titers, and the results are shown in Tables 5 and 6 below, and the titers and measured magnetic values of the samples were compared. Finally, it was confirmed whether clinical samples having different neutralizing antibody titers were also distinguished according to the magnetic value criteria for each titer measured in Example 4 above.

Quantitative Analysis of Clinical Sample Neutralizing Antibodies by Porcine Fever Virus Titer Using Magnetic Sensors sample VN Titer
(According to the invention mV Reference value ) Measure Magnetic value
 ( mV ) Magnetic value  Result according to standard One voice
(<186) 117.5 voice 2 99.6 voice 3 119.4 voice 4 128.4 voice 5 115 voice 6 110.9 voice 7 204.1 1:16 8 180.7 voice 9 207 1:16 10 205.3 1:16 11 1:16
(<394) 262.6 1:16 12 233.7 1:16 13 238.4 1:16 14 258.1 1:16 15 258.1 1:16 16 245.7 1:16 17 251.5 1:16 18 258.7 1:16 19 176.6 voice 20 210.5 1:16 21 1:32
(<509) 403.6 1:32 22 411.8 1:32 23 438.2 1:32 24 429.2 1:32 25 464.7 1:32 26 483.8 1:32 27 496.8 1:32 28 467.2 1:32 29 517.2 1:64 30 527.8 1:64 31 1:64
(<827) 816.2 1:64 32 860.3 1: 128 33 851.4 1: 128 34 769.1 1:64 35 791.2 1:64 36 785.4 1:64 37 795.5 1:64 38 721.3 1:64 39 759 1:64 40 828.4 1: 128 41 1: 128
(<1294) 1164.8 1: 128 42 1205 1: 128 43 1240.1 1: 128 44 978 1: 128 45 841.3 1: 128 46 942.6 1: 128 47 894.7 1: 128 48 891.7 1: 128 49 917.9 1: 128 50 1027.4 1: 128 51 1: 256
(<2556) 1866.4 1: 256 52 2367.7 1: 256 53 2138 1: 256 54 2135.4 1: 256 55 2391.7 1: 256 56 2362 1: 256 57 2351.3 1: 256 58 2500 1: 256 59 2496.1 1: 256 60 2500 1: 256

Comparison of specificity and sensitivity between the titer derived from the neutralizing antibody titer derived from the swine fever virus neutralization assay and the magnetic value
division VN Titer positivity voice Sum
Magnetic Kit positivity 44 (TP) 3 (FP) 47 voice 6 (FN) 7 (TN) 13 Sum 50 10 60 Relative sensitivity ^* 88% Relative specificity ^** 70%

* Actual Positive Sample Count (TP) / (Actual Positive Sample Count (TP) + False Negative Sample Count (FN)) X 100

** Actual Negative Samples (TN) / (Actual Negative Samples (TN) + False Positives (FP)) X100

As shown in Table 5 and Table 6, as a result of comparing the antibody titer derived according to the magnetic value criteria prepared in Example 4 and the neutralizing antibody titer of the clinical sample with respect to the negative sample and the titer-specific sample, 10 in the negative sample Three of the samples showed magnetic values with 1:16 neutralizing antibody titers, 1:16 had negative magnetic values, and 1:32 had two magnetic values. Magnetic values with 1:64 neutralizing antibody titers were shown. In addition, the magnetic values of 1: 128 neutralizing antibody titers were found in three of ten samples at 1:64, and the magnetic values of 1: 128 and 1: 256 neutralizing antibodies were found in all samples of 1: 128 and 1: 256. It was confirmed that the titer is consistent with the titer, and the quantitative titer classification of the neutralizing antibody by the magnetic value showed 88% relative sensitivity and 73% relative specificity compared to the antibody titer classification by the neutralization test method (Table 5). ).

However, regarding the differentiation of vaccines, the magnetic values of clinical samples belonging to the vaccinated groups 1:16 and 1:32 were mostly consistent with the neutralizing antibody titers derived by the neutralizing antibody test. Two clinical samples belonging to 32 were identified as having magnetic values with 1:64 neutralizing antibody titers. In addition, the magnetic values of clinical samples belonging to the 1:64 and 1: 256 non-vaccinated groups showed a tendency to coincide with the neutralizing antibody titers derived by the neutralizing antibody test. One clinical sample belonging to 256 was identified as having a magnetic value with a 1: 128 neutralizing antibody titer.

In conclusion, it can be confirmed that the accuracy of about 96% (58/60) is shown with respect to the classification of the target group and the non-vaccinated group by the magnetic value, the kit according to the present invention and the magnetic value derived using the same According to the criteria, it was possible to measure the antibody titer of the swine fever virus efficiently and accurately and to discriminate against the vaccine.

In Fig. 2, each symbol is as follows.
1. Immunoassay Device 2. Assay Strip
3. lower case 4. upper case
5. Sample pad 6. Conjugation pad
7. Inspection Line 8. Contrast Line
9. Membrane 10. Absorption pad
11. Sample Inlet 12. Result Display Window
In FIG. 3, each code | symbol is as follows.
1. Sample pad 2. Conjugation pad
3. Inspection Line 4. Nitrocellulose Membrane
5. Contrast line 6. Absorption pad
7. Lamination card (Backing plate)

<110> VETALL CO., LTD. <120> KIT FOR DETECTION OF ANTIBODY AGAINST CLASSICAL SWINE FEVER          VIRUS, AND DETECTING METHOD OF THE ANTIBODY AND / OR ANTIBODY TITER          AGAINST CLASSICAL SWINE FEVER VIRUS USING THE SAME <130> DPP20115609KR <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for E2 gene clonning <400> 1 ggatcccggc tagcctgcaa ggaagat 27 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for E2 gene clonning <400> 2 ggatccttct gcgaagtaat ctga 24

Claims (11)

A sample pad for dropping a sample;
Conjugation pads with porcine fever virus E2 antigen-nano magnetic beads adsorbed;
A membrane showing a detection result; And
Absorbent pad that absorbs sample development
Kit for detecting antibodies to swine fever virus comprising a.
The method of claim 1,
The sample is blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomiting, or feces.
The method of claim 1,
Wherein the membrane comprises a test line to which porcine fever virus E2 antigen is fixed and a control line to which feline leukemia virus (FeLV) antigen is fixed.
Method for measuring the presence or absence of the antibody to the swine fever virus using the kit of claim 1 or the titer of the antibody.
5. The method of claim 4,
As measuring the presence or absence of an antibody,
When the test line of the kit is yellow, it is determined that there is an antibody, and if it is not yellow, it is determined that there is no antibody.
Preparing a sample;
Reacting the sample with a swine fever virus E2 antigen-nano magnetic bead conjugate; And
Measuring the magnetic value of the sample bound to the swine fever virus E2 antigen-nano magnetic bead conjugate using a magnetic sensor
Method for measuring the antibody titer against swine fever virus comprising a.
The method of claim 6, wherein after the magnetic value measuring step
When the magnetic value is 509 mV or less, it is determined that the antibody titer is 1:32 or less.
If the magnetic value is at least 827 mV, the method further comprises determining that the antibody titer is at least 1:64.
8. The method according to claim 6 or 7,
The sample is blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomiting, or feces.
Preparing a sample;
Reacting the sample with a swine fever virus E2 antigen-nano magnetic bead conjugate; And
Measuring the magnetic value of the sample bound to the swine fever virus E2 antigen-nano magnetic bead conjugate using a magnetic sensor
Method for determining whether or not porcine fever virus vaccine administration comprising a.
10. The method according to claim 9, wherein after the magnetic value measuring step
If the magnetic value is 509mV or less, it is determined that the vaccine can be administered,
If the magnetic value is 827 mV or greater, further comprising determining that vaccine administration is impossible.
11. The method according to claim 9 or 10,
The sample is blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomiting, or feces isolated from piglets subject to vaccine administration.

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