KR101922234B1 - 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

The present invention relates to a kit for detecting an antibody against swine fever virus, a method for determining the presence or absence of a swine fever virus antibody and / or antibody titer using the same, and a method for determining whether a vaccine is administered, In addition to being able to measure the presence or absence of antibodies to fever viruses, it is possible to quantitatively and quickly measure the presence of porcine fetal maternal antibody in porcine pigs using a portable quantitative assay device using a magnetic field, It is very useful because it is possible. In particular, it is required to provide efficient information on the determination of vaccination against piglets in farms. Since the present invention can quantitatively detect maternal antibodies, it is possible to appropriately respond to such demands, It can inhibit the invention of fever virus and also contribute to the economic gain of pig farmers.

Description

TECHNICAL FIELD The present invention relates to a kit for detecting an antibody against swine fever virus, and a method for measuring the presence and / or antibody titer of an antibody against swine fever virus using the same, OR ANTIBODY TITER AGAINST CLASSICAL SWINE FEVER VIRUS USING THE SAME}

The present invention relates to a kit for detecting an antibody against swine fever virus, a swine fever virus antibody using the same, and / or a method for measuring antibody titer and a method for determining vaccine administration.

The Classical Swine Fever Virus (CSFV) is an RNA virus belonging to the genus Flaviviridae and Pestivirus . It is about 40-50nm in size. The swine fever virus gene is a positive sense single strand RNA. It is about 12.3 kb in size. It synthesizes about 3900 amino acids in the code and constructs virus structural proteins. The proteins of the swine fever virus are 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 swine fever virus produce antibodies against ERNS, E2, and NS3 structural proteins of viruses. Especially, among these proteins, E2 protein, which constitutes the envelope of swine fever virus, is a major factor involved in the generation of antibodies that strongly neutralize virus (Edwards et al., 1991. Vet. Microbiol. 29: 101-108, Weiland et al., 1992. J. Virol. 66: 3677-3682).

 If swine fever virus infection is acute, it indicates clinical symptoms such as high fever, skin eruption, appetite deficiency, constipation, diarrhea and back numbness, and suppresses immune function such as leukocyte reduction and eventually causes death. However, It is possible to defend (Van Oirschot. 1999. In Diseaseof swine. P.159-172). In pigs infected with chronic CSF, neutralizing antibodies are detected for a few days after 4 weeks of infection and then disappear (Depner et al. , 1966. Eur. J. Vet Path. 2: 61-62, Mengeling et al., 1969. Am. J. Vet. Res. 30: 409-417). Although piglets infected with low pathogenic CSFV through the placenta show constant viremia, they are immune tolerant and rarely produce specific antibodies (Van Oirschot, 1999. In Diseaseof swine. P.159- 172).

The maternal transit antibody is delivered to piglets through colostrum, is usually absorbed most 24-48 hours, and usually decreases gradually over a half-life of 12-19 days (Kim et al., 1982. Res. (1), (2), (3), (4), (6) and (7) of the present study, (Launais et al., 1978. Vet. Microbio. 3: 31-43). Inoculation of vaccine against piglets with a maternal transit antibody level of more than 64-fold from sows' slaughter is associated with immune interference and is limited by active immunization (Lai et al., 1981. J. Chinese Soc. 6: 77-81), and vaccination is recommended at a time when the maternal antibody is reduced to a certain level, ie, less than 32-fold. In the case of vaccine intramuscular vaccination, which is now widely practiced, if the maternal blood transfusion antibody level is more than 64 times, the vaccine does not give enough immune response by a single vaccination due to considerable interference of maternal antibody, Inoculation should be carried out. Since individuals who are not immunized by the first vaccination are more likely to be infected with the pathogenic virus until the second vaccination, the risk of developing CSF is increased for farms that are raising such pigs. Therefore, quantitative measurement of maternal antibody before vaccination is important to increase the effectiveness of the vaccine and lower the incidence of CSF.

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

However, in the case of the neutralizing antibody test which can measure the quantitative antibody titer, specialized human and experimental equipments are needed and the detection time is long and it is not suitable for outdoor use. Specifically, it is difficult to treat a large amount of serum samples. Pig blood is collected, and only the serum components are collected by centrifugation and then used for immobilization at 56 ° C for 30 minutes or longer. The examination time is 3-5 days, There was a problem requiring manpower and it was not suitable for use in pig farms.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a kit for detecting the presence or absence of antibodies to swine fever virus and antibodies in a field, And a method for determining whether or not vaccination is possible without being influenced by the infection of swine fever virus and the immune interference of the maternal antibody in piglets do.

In order to solve the above problems, the present inventors have established a method for determining the presence or absence of a porcine febrile virus virus and / or the activity of a maternal antibody in a piglet, As a result of research efforts, the use of a conjugate conjugated with a porcine fever virus antigen and a magnetic nanoparticle was used to detect the presence or absence of antibodies against swine fever virus, and furthermore, quantitatively the neutralizing antibody titer in piglets was 16 times, 32 times, 64 times , And 128 times, respectively. Thus, a rapid diagnostic kit capable of discriminating whether or not a pig fever virus vaccine can be administered has been developed, and the present invention has been completed. Accordingly, a conventional neutralizing antibody test (NPLA , Neutralizing peroxidase-linked assay (AGA) This study has suggested a method to measure the maternal antibody level in the field more quickly and easily.

An embodiment of the present invention provides a sample pad for dispensing a sample;

Conjugation pads on which swine fever virus E2 antigen-magnetic nanoparticle conjugates are adsorbed;

A membrane showing the detection result; And

Absorbing pad for absorbing the sample developing solution

And a kit for detecting an antibody against swine fever virus.

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

Another embodiment of the present invention is a method for preparing a sample, comprising: preparing a sample;

Reacting the specimen with a porcine fever virus E2 antigen-nanomagnetic bead conjugate; And

Measuring the magnetic value of the sample bound to the porcine febrile virus E2 antigen-nanomagnetic bead conjugate using a magnetic sensor

Lt; RTI ID = 0.0 > pandemic < / RTI > virus.

Another embodiment of the present invention is a method for preparing a sample, comprising: preparing a sample;

Reacting the specimen with a porcine fever virus E2 antigen-nanomagnetic bead conjugate; And

Measuring the magnetic value of the sample bound to the porcine febrile virus E2 antigen-nanomagnetic bead conjugate using a magnetic sensor

Lt; RTI ID = 0.0 > a < / RTI > swine fever virus vaccine.

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

According to one embodiment of the present invention, a sample pad for dispensing a sample; Conjugation pads on which swine fever virus E2 antigen-magnetic nanoparticle conjugates are adsorbed; A membrane showing the detection result; And an absorbent pad for absorbing the sample developing solution.

Pigs infected with swine fever virus produce antibodies against ERNS, E2, and NS3 structural proteins of viruses. Especially, among these proteins, E2 protein, which constitutes the envelope of swine fever virus, is a major factor involved in the generation of antibodies that strongly neutralize virus (Edwards et al., 1991. Vet. Microbiol. 29: 101-108, Weiland et al., 1992. J. Virol. 66: 3677-3682).

Therefore, E2 protein, which is known to produce the highest level of neutralizing antibody in swine fever virus infection, can be used as a target antigen for diagnosis of swine fever virus infection, detection of antibody presence therefrom, measurement of antibody titer, and the like have. In addition, the reference antigen can be used without any particular limitation as long as it does not cross-react with the swine fever virus antigen, preferably, a feline leukemia virus (FeLV) antigen Can be used.

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

The magnetic nanoparticles are used to form a conjugate with the target antigen E2 and the antibody against the control antigen FeLV, respectively, and each conjugate formed adsorbs on the conjugation pad. Then, the antibody bound to the target can be qualitatively and quantitatively detected by measuring the magnetic value after the magnetic nanoparticle formed with the conjugate, preferably the nanomagnetic bead, reacts with the specimen.

Accordingly, one embodiment of the present invention provides a method of conjugating to a magnetic nanoparticle using an E2 protein as an antigen to detect a porcine fever virus antibody in a stallion and a porcine fetal bovine transfer antibody in a piglet.

The conjugation between the E2 antigen and the magnetic nanoparticles is preferably performed by activating the magnetic nanoparticles; And reacting the activated magnetic nanoparticles with a porcine fever virus E2 antigen.

The step of activating the magnetic nanoparticles is preferably a step of precipitating the magnetic nanoparticles diluted with buffer with a magnet, removing the supernatant and suspending the pellet twice to 5 times, preferably 3 times ; And suspending the pellet obtained through the repeating process using an active buffer, followed by reacting at 30 to 40 ° C for 5 to 15 minutes.

The buffer used for diluting the magnetic nanoparticles and suspending the pellets in the activation step is not particularly limited, but MES (0.1 M 2- (N-morpholino) ethanesulfonic acid) buffer may be preferably used. The active buffer may be an active buffer (0.1 M MES, 0.5 M NaCl, pH 6.0) supplemented with 1-ethyl-3- (3-dimethylaminopropyl carbodiimide hydrochloride) and sulfo-NHS (N-hydroxylsulfosuccinimide) .

The step of reacting the activated magnetic nanoparticles with the E2 antigen is preferably carried out by shaking the activated magnetic nanoparticles and the E2 antigen in a PB buffer at 30 to 40 DEG C for 1 to 3 hours; Adding 0.1 to 0.5% (w / v) of BSA active buffer and shaking culture at 30 to 40 DEG C for 20 to 40 minutes.

Conjugate formation with the reference antigen can also be carried out in the same way.

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

As described above, the kit for detecting an antibody against swine fever virus according to an embodiment of the present invention uses magnetic nanoparticles as a conjugation, thereby making it possible to provide a progressive (non-invasive) method for completely replacing the conventional colloidal gold conjugation ≪ / RTI >

That is, by using magnetic nanoparticles, preferably nanomagnetic beads, which are not gold particles, it is possible to quantitatively measure antibody titers by measuring magnetic values using a portable quantitative analyzer (magnetic sensor) using a magnetic field, In addition to the determination of the presence or absence of the antibody, the antibody titer can be measured and the vaccine can be identified through the same. In addition, the present method is more effective than the conventional method of measuring gold particle conjugation using optical It has the ability to improve sensitivity and specificity due to contamination. This can be widely applied to all lateral flow immunoassays.

The specimen is preferably a body fluid secreted out of the body and includes blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomit or feces and more preferably, But not limited to, blood.

The kit for detecting an antibody against swine fever virus may preferably detect an E2 antibody against swine fever virus infection using an antigen-antibody reaction between a specimen and the E2 antigen, and preferably, May be used to detect porcine fever virus.

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

The swine fever virus E2 antigen is integrated into the test line 3 to the nitrocellulose membrane 4, which is attached in strip form to a backing plate 7. Further, on the other side of the nitrocellulose membrane, antigens which are not reactive with the pig fever virus antibody are integrated with a control line (5). Preferably a feline leukemia virus (FeLV) antigen that has been confirmed to have no cross-reactivity to the virus in the pig.

A conjugation pad (2) adsorbing an E2 antigen-magnetic nanoparticle conjugate capable of reacting with a porcine fever virus E2 antibody present in a specimen sample is provided on one end of the nitrocellulose membrane on which the test line and the control line are integrated, And an absorption pad 6 is formed on the other end of the nitrocellulose membrane so as to partially absorb the specimen sample and to expand the nitrocellulose membrane through the nitrocellulose membrane. A specimen pad 1 capable of dropping a specimen sample is placed on the upper portion of one end of the conjugation pad 2 (opposite to the end in contact with the upper end of the membrane) so as to partially overlap the upper end of the conjugation pad. The thus constructed analytical strip can be embedded in a case divided into a lower part and an upper part to finally complete the immunoassay (refer to FIGS. 2 and 5)

As described above, a method of detecting the presence or absence of an antibody using the prepared immunoassay device can be carried out as follows, for example, when a nano-magnetic bead is used as magnetic nanoparticles (see FIGS. 3 to 5).

First, a sample of a pig's eye sword prepared in advance is dropped on the sample pad 1. Preferably, 40 [mu] l of the sample of the gut is added, but not limited thereto. After the spotting, a swine blood sample was obtained from the conjugate pad (2) where the E2 antigen-nano magnetic bead conjugate was adsorbed and the porcine fever virus antigen (3) was adhered via the nitrocellulose membrane (4) The nano magnetic bead conjugate is also absorbed through the nitrocellulose membrane 4, and the anti-feline leukemia virus antibody (α-FeLV antibody) adsorbed to the conjugation pad 2 in the same manner is absorbed through the nitrocellulose membrane 4 And moves in the direction of the pad 6. When the swine fever virus E2 antibody is present in the blood sample, the E2 antigen is adhered to the nitrocellulose membrane (4) after the primary antigen-antibody reaction with the E2 antigen-nanomagnetic bead conjugate on the conjugation pad (2) The test line (3) causes a secondary antigen-antibody reaction, in which nano-magnetic bead particles (E2 antigen-nanomagnetic bead conjugate) sensitized to the antigen show a specific yellow color. Thereby, it is possible to judge positive (antibody oil) or negative (no antibody) according to the presence or absence of the antibody in the blood of the pig.

The reaction with the antigen-antibody reaction and the E2 antigen-nanomagnetic bead conjugate is carried out in a buffer. Examples of the buffer include PBS, Tris, or borate buffer, and preferably PBS buffer (PBS, 0.5 % BSA, 300 mM NaCl, 0.1% NaN ₃ , pH 7.2).

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

Preferably, the method is a method for measuring the presence or absence of an antibody. When the test line of the kit is yellow, it is judged that the antibody is present (positive), and when it is not yellow, the antibody is absent It can be judging.

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

Preferably, the antibody titer measuring method further comprises, after the magnetic value measuring step, judging that the antibody titer is 1:32 or less when the magnetic value is 509 mV or less and the antibody titer is 1:64 or more when the magnetic value is 827 mV or more And a step of judging that the user is a user.

The magnetic value used as a reference for determining the antibody titer may be based on a value measured in a piglet (piglet) which is about 3 weeks old. However, when it is difficult to accurately determine whether the antibody titer belongs to 1:32 or 1:64 when the magnetic value measured at this time is an intermediate value exceeding 509 mV and less than 827 mV, , It is judged that the antibody titer is 1:32 or less when the measured magnetic value becomes 509 mV or less by repeating the measurement at intervals of one week from the point of about 3 weeks of the first measurement day.

Refers to the highest dilution of an antibody that reacts with an antigen of the antibody to form a precipitate aggregate, which may mean, but not limited to, the activity of a maternal antibody specifically expressed in piglets.

A maternal antibody is an immune antibody that transits from a maternal to a fetus. It is transmitted to piglets through colostrum, is usually absorbed within 24-48 hours, decreases gradually through a half-life of 12-19 days Kim et al., 1984. Res. Rept. OR: 26: 53-55), 30-35 days of age (Kim et al., 1982. Res. Rep. (Launais et al., 1978. Vet. Microbiol. 3: 31-43), because the immune interference caused by maternal antibody is weakened. Inoculation of vaccine against piglets with a maternal transit antibody level of more than 64-fold from sows' slaughter is associated with immune interference and is limited by active immunization (Lai et al., 1981. J. Chinese Soc. 6: 77-81), and vaccination is recommended at a time when the maternal antibody is reduced to a certain level, ie, less than 32-fold. In the case of vaccine intramuscular vaccination, which is now widely practiced, if the maternal blood transfusion antibody level is more than 64 times, the vaccine does not give enough immune response by a single vaccination due to considerable interference of maternal antibody, Inoculation should be carried out. Since individuals who are not immunized by the first vaccination are more likely to be infected with the pathogenic virus until the second vaccination, the risk of developing CSF is increased for farms that are raising such pigs. Therefore, quantitative measurement of maternal antibody before vaccination is important to increase the effectiveness of the vaccine and lower the incidence of CSF.

Another embodiment of the present invention is a method for quantitatively measuring porcine fever virus antibody in pigs and porcine fetal bovine transfer antibody in porcine pigs using the magnetic quantitative analysis device using a magnetic field using the E2 antigen-magnetic nanoparticle conjugate Provide a detection kit that can be used.

The magnetic nanoparticles may preferably be nanomagnetic beads, and may be applied in the same manner as described in the kit for detecting antibodies against swine fever virus according to one embodiment of the present invention.

The specimen may be blood, serum, plasma, lymph fluid, tissue fluid, urine, tear, saliva, milk, vomit, feces or the like, preferably separated from piglets, It does not.

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

As described above, the titer of the maternal antibody is very important in determining vaccine administration (or inoculation), and therefore, the magnetic value of a sample combined with the porcine fever virus E2 antigen-magnetic nanoparticle conjugate can be measured , The vaccine administration can be determined.

Preferably, the method may further include determining that vaccination is possible when the magnetic value is 509 mV or less, and determining that the vaccine administration is impossible when the magnetic value is 827 mV or more.

The magnetic value used as a standard for judging whether or not the vaccine is administered may preferably be measured using a specimen separated from piglets of about three weeks old. However, when the measured magnetic value is more than 509 mV and corresponds to an intermediate region of less than 827 mV, it is preferably measured at intervals of one week from the time point of about 3 weeks after birth, which is the first measurement day as in the antibody titer measurement method described above , And when the measured magnetic value is 509 mV or less, it can be judged that the vaccine can be administered from a time point to an object.

The specimen may be blood, serum, plasma, lymph fluid, tissue fluid, urine, tear, saliva, milk, vomit, feces or the like, preferably separated from piglets, It does not.

As described above, the use of the detection kit according to the present invention can measure the presence or absence of an antibody against swine fever virus in blood, and more particularly, to a portable quantitative assay device using porcine fever maternal antibody It is very useful because it can be measured quickly and quantitatively by using the sensor. In particular, it is required to provide efficient information on the determination of vaccination against piglets in farms. Since the present invention can quantitatively detect maternal antibodies, it is possible to appropriately respond to such demands, It can inhibit the invention of fever virus and also contribute to the economic gain of pig farmers.

FIG. 1 schematically shows the quantitative and qualitative detection of antibodies against swine fever virus according to an embodiment of the present invention.
2 is a schematic exploded perspective view of a kit for detecting swine fever virus antibody according to an embodiment of the present invention.
FIG. 3 schematically shows a structure of a strip constituting a kit for detecting porcine fowl virus antibody according to an embodiment of the present invention.
FIG. 4 schematically shows the results obtained when the kit for detecting swine fever virus antibody according to an embodiment of the present invention is reacted with a specimen, when positive and negative.
FIG. 5 shows the results of testing the neutralized antibody samples according to the fourth embodiment of the present invention by using the kits of the second embodiment of the present invention.
6 is a graph showing the results of Example 4 of the present invention by numerically quantifying magnetic values using a magnetic sensor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

Example 1 : On swine fever virus  Production of target antigens and reference antigens for

A. As a target antigen Swine fever virus E2  Production of antigens

The swine fever virus recombinant E2 antigen was prepared according to Example 2 of Korean Patent No. 10-0543691 and was supplied from JBT (Jeno Biotech). For simplicity, a forward primer (5'-GGATCCCGGCTAGCCTGCAAGGAAGAT-3 ') (SEQ ID NO: 1) and a reverse primer (5'-GGATCCTTCTGCGAAGTAATCTGA-3') were prepared in order to clone the ALD- (Reverse Transcription Polymerase Chain Reaction (RT-PCR)) was performed with 30 cycles of 92 ° C for 40 seconds, 50 ° C for 40 seconds, and 72 ° C for 60 seconds, Was transfected into baculovirus transfer vector pAcGP67B (Pharmingen) and co-transfected with baculovirus DNA (Pharmingen) into insect cell Sf21 (Invitrogen) to transform E2 protein Baculovirus BacE2TNALD was constructed. BacE2TNALD was inoculated again into insect cells and cultured for 6 days, and then the supernatant was harvested. The harvested supernatant was dialyzed against phosphate buffer (PBS, pH 7.2) after treatment with a binary ethylenimine (BEI), and used for the preparation of the kit according to the present invention.

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

As a control antigen, a feline leukemia virus (FeLV) antigen confirmed to have no cross-reactivity to swine fever virus was used. The p27 antigen protein of FeLV was prepared as follows according to Example 2 of Korean Patent No. 10-1069391 previously filed and registered by the present inventors. A template oligonucleotide for the p27 antigen gene was synthesized (Bionics, KOREA) based on the NCBI GeneBank data (ACCESSION: M18247) nucleotide sequence registered with the NCBI gene bank. The template oligonucleotides were introduced into pGEX-4T-1 (GE Health care) using restriction endonucleases EcoRI (New England Biolabs) and SalI (New England Biolabs) to express the FeLV p27 antigen protein Respectively. The binding products were transformed in BL21 or Rosetta (Stratagene) recipient cells and inoculated into LB medium supplemented with 100 ug / ml of ampicillin and 0.5 mM IPTG (isopropylthiogalactoside) in order to induce expression, and then cultured in a shaking incubator shaking orbital incubator) overnight. The pellet was suspended, suspended in cold buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA) and 1 mM PMSF, and pulverized by ultrasonication. Then, the pellet was centrifuged (10,000 rpm, And the protein was purified by glutathione affinity chromatography. The water-soluble cell lysate was separated by filtration on a glutathione sepharose 4B (Pharmacia) solution equilibrated with PBS. The soluble protein was purified by using a 0.45um syringe filter (Sartorius) It was loaded into the column. After washing the column with 3 volumes bed volume of PBS, the fusion protein was eluted with a buffer solution of 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0. The purified fusion protein was used for the kit preparation according to the present invention after 0.2-μm 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  Antigenic Nitrocellulose Membrane ( membrane ) Produce

The swine fever virus recombinant E2 antigen prepared in Example 1. A above was dialyzed with 2 mM sodium tetraborate at 4 ° C for 12 hours and dialyzed against a buffer solution containing 0.1% trehalose (0.1 g trehaloe / 100 ml dH ₂ O), were mixed in 10mM PB (phosphate buffer)) was used as a test line, the above embodiment 1. the cats prepared in B leukemia virus (FeLV) antigens for 2mM sodium dialysis for 12 hours at 4 ℃ in tetraborate The mixture was mixed with buffer (0.1% trehalose, 10 mM PB) at a concentration of 1 mg / ml and used as a reference line. The control solution of the recombinant antigen and the control line of the test line was injected into a nitrocellulose membrane (MDI, India) using a dispensing apparatus (KINAMETICS, USA) as shown in Table 1. Thereafter, the membrane was dried overnight in a low humidity laboratory or dried in a fan for at least 5 hours to prepare a test line to which a porcine fever virus E2 antigen was adhered and a control line to which a cat leukemia virus (FeLV) antigen was adhered. The prepared membrane plates were stored in a sealed container or a low humidity laboratory with a desiccant.

Swine fever virus antigen injection condition division Inspection line Korean ship antigen Swine fever virus (E2)
0.7 mg / ml Feline leukemia virus (p27)
1 mg / ml density( ul / cm ) 0.9 0.9 Bed speed ( cm / s) 7 7

B. Swine fever virus E2 Antigen and feline leukemia virus p27  Nanomagnetism adsorbed on each antibody Bead Conjugate  Produce.

To conjugate the pig fever virus E2 antigen to the nanomagnetic beads, the nanomagnetic beads were activated as follows.

After dilution with 0.1% (v / v) 10 ml MES (0.1 M 2- (N-morpholino) ethanesulfonic acid, Sigma) buffer was added to 333 μl of nanomagnetic beads (3% Carboxyl- Adembeads 0212, Ademtech, France) , The supernatant was removed, and the process of suspending the pellet in the MES buffer was repeated three times. The final collected pellet was resuspended in 1 ml of active buffer (0.1 ml) containing 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, 0.5 M NaCl, pH 6.0) and reacted for 10 minutes at 37 ° C. Finally, the nanomagnetic beads activated at 1% (v / v) concentration were washed twice with 10 mM PB buffer After washing, 10 ml of PB buffer was added to adjust the concentration to 0.1% (v / v), followed by addition of swine fever virus E2 antigen (50 μl (1 mg / ml)) and shaking culture at 37 ° C. for 2 hours (0.1 M MES, 0.5 M NaCl, pH 6.0) to which 0.5% (0.5 g BSA / 100 ml dH2O) BSA had been added was added to a concentration of 0.1% (v / v) After three washes with storage buffer (0.1% (w / v) BSA, 0.1% (w / v) NaN ₃ ), the storage buffer was added to a concentration of 1% (v / v) , And stored in a refrigerator to produce a kit according to the present invention. The feline leukemia virus antibody (50 ug, FeLV p27 Antibody, MFLV-20M-8D11, ICL) was also conjugated with nanomagnetic beads in the same manner as described above.

C. Swine fever virus E2  Antigen-adsorbed nanomagnetic Bead Conjugation  Manufacture of pads

The thus prepared 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 were each 0.13% (v v / v) and 0.05% (v / v), and 5% (w / v) trehalose (Sigma) was added thereto. The resulting solution was titrated with distilled water to a total volume of 900 μl, Pad (0.5 x 20 cm glass fiber). It was then dried in a low humidity laboratory overnight or in a fan for at least 5 hours. The prepared conjugate pad was stored in a container sealed with a desiccant or in a low humidity laboratory.

D. Absorbent pads and Sample pad  Produce

The absorbent pad and the sample pad were dried by a method known in the art to remove the reaction solution, and the pad was cut, treated with a reaction solution (generally dipping), dried, and stored.

E. device  Assembly

Each of the membranes and pads prepared as described above was combined with the sample pad, the conjugation pad (nanomagnetic bead conjugate treating pad), the nitrocellulose membrane and finally the absorbing pad in the overlapping manner from the right side and immersed in an immunoassay device And cut into strips of the size appropriate for the size. The thus cut strip was finally placed in the lower plate of the immunoassay device (single device) and covered with the upper plate to prepare a diagnostic kit (see FIG. 2).

F. Product Configuration

(PBS, 0.5% (w / v) BSA, 0.1% (w / v) NaN ₃ , pH 7.2) as the above-described immunoassay apparatus and the test solution were constructed as final products (see FIG. 5) .

Example  3: Nano Magnetic Bead Kit  Used At the prosperity Swine fever virus  Detection of antibodies

In order to compare the antibody detection efficiency with the conventional test method, 30 sera judged to be positive for swine fever virus and 30 sera judged to be negative according to the existing ELISA (Jeno Biotech, Korea) method were used as specimens, Were tested with the kit manufactured according to the method of < RTI ID = 0.0 >

After the sample was reacted with the prepared kit, 10 minutes after the test, the result was first confirmed through the naked eye of the experimenter. The magnetic value was measured six times per one sample using a magnetic sensor (Care-U, LG innotek) The maximum and minimum values were excluded and the mean value of the remaining values was obtained and quantified quantitatively. As a result, the sensitivity (relative sensitivity) was 100%, and the specificity (relative specificity) was 100%, and the agreement rate with the conventional method was very high (see Table 2 and Table 3).

Comparison of Antibody Detection Efficiency in Swine Fever Virus ELISA Positive (+) voice(-) The present invention kit Positive (+) 30 0 voice(-) 0 30 system 30 30 Relative sensitivity (%)
Relative specificity (%) 100
100

Comparison test result of diagnosis of swine fever virus antibody No . Specimen name ELISA SensPERT  (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 - -

CPC = Positive Control Average Absorbance - Negative Control Average Absorbance) - Sample to positive ratio = {(Kidney sample average absorbance - Negative control mean absorbance) / CPC (corrected positive control)

Example  4: Nano Magnetic Bead Kit  Used Swine fever virus  Neutralizing antibody of standard sample By activity Magnetic value  Quantification.

Neutralizing antibody samples for the diagnosis of swine fever virus antigens were obtained from the Agriculture, Forestry and Fisheries Inspection Headquarters and were serially diluted to a factor of 256, which was a serial dilution of a 1024-fold neutralizing antibody-positive sample of swine fever virus to the pathogen-free pig serum (26250084, Invitrogen) 128 times, 64 times, 32 times, and 16 times, respectively. The thus-prepared neutralizing antibody samples were tested using the kit prepared according to the method of Example 2. The test of each sample and the kit of Example 2 was performed for a total of ten times.

After the sample was reacted with the prepared kit, 10 minutes after the elapse of 10 minutes, the result was first visually confirmed by the experimenter (see FIG. 5). The magnetic value was quantitatively measured using a magnetic sensor (Care-U, LG innotek) And the results are shown in Table 4 and FIG. 6 below. Magnetic value quantification of each standard sample was repeated 10 times with 1 set of 256 from batches of each lot and batch. Six measurements were made per sample to exclude the maximum and minimum values, and the remaining values The average value was obtained.

Quantitative analysis of neutralizing antibody with porcine fever virus titres using magnetic sensor 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 FIG. 6, it was confirmed that the magnetic value was increased according to the antibody value of the swine fever virus titer. Specifically, the magnetic value was 483.958 ± 24.9989 mV at 1:32 (32 times) of the porcine fever virus neutralizing antibody titer, and 754.875 ± 71.7058 mV at 1:64 (64 times).

If the vaccine can be vaccinated at a time when the fetal dengue virus maternal transit antibody is reduced to a certain level, that is, less than 32 times, if the maternal antibody is at least 64 times higher, vaccination with the maternal antibody will be sufficient Since the immune response can not be given, it is possible to establish a criterion for dividing vaccine-able piglets and pigs that can not be vaccinated through the measured magnetic potency values according to the present experiment (see FIG. 6).

That is, as shown in FIG. 6, when the magnetic value is less than about 509 mV, it is judged that the vaccine is possible because the titer of the swine fever virus neutralizing antibody is 1:32 or less. When the magnetic value is about 827 mV or more, : It can be judged that vaccination is not possible because it is 64 or more. In addition, according to the criteria set forth in this example, when the magnetic value is less than about 186 mV, the negative value is less than about 186 mV and less than 394 mV, the potency is 1:16, the less than about 827 mV and less than 1294 mV is the potency 1: 128, the less than about 1294 mV and less than 2556 mV And 1: 256, respectively.

However, when the measured magnetic value is a value corresponding to an intermediate region of more than 509 mV and less than 827 mV, which is the judgment criterion, the titer of the antibody titer is about 1:32 , It is difficult to accurately judge whether it belongs to 1:64 or not. The measurement is repeated at intervals of one week from the third measurement, which is the first measurement day, at the time when the measured magnetic value becomes 509 mV or less, : 32 or less.

As described above, using the kit of the present invention, an effective immune response can be given by inoculating the vaccine after confirming the time when the titer of the maternal antibody decreases to a magnetic value of 509 mV or less.

Example  5: Swine fever virus  Nano Magnetic Bead Of kit  Clinical results.

Magnetic values were quantified by using a nano magnetic bead kit for outdoor groups in which neutralizing antibodies were known by the neutralizing antibody method, and outdoor groups having various neutralizing antibody values were subjected to immunization according to the criteria set forth in Example 4 And the other two groups.

First, a virus-neutralizing test (Neutralizing peroxidase) was performed to measure the inhibition of viral proliferation in cultured PK-15 cells (National Veterinary Research and Quarantine Service) cultured by stair-dilution of test sera from an outdoor military sample (National Veterinary Research and Quarantine Service) VL titer) against swine fever virus was identified through the -linked assay (NPLA) (C. Terpstra et al., Veterinary microbiology 16 (2): 123-128 1988)). As a result, the negative samples and neutralizing antibody titer were 1:16 (10 species), 1:32 (10 species), 1:64 (10 species), 1:128 (10 species) and 1: 256 Clinical samples were obtained. Magnetic values were measured using a magnetic sensor (Care-U, LG innotek) for clinical samples secured with various titers. The results are shown in Tables 5 and 6, and the titers and measured magnetic values of the samples were compared And finally confirmed whether clinical specimens having different neutralizing antibody titers were also classified according to the magnetic value-by-titer value measured in Example 4. [

Quantitative Analysis of Neutralizing Antibody of Clinical Trial by Swine Fever Virus Titer Using Magnetic Sensor sample VN Titer
(According to the present invention mV Reference value ) Measure Magnetic value
 ( mV ) Magnetic value  Results based on criteria 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 of neutralizing antibody titer derived from swine fever virus neutralization test method and titer derived from 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 specimen count (TP) / (Positive specimen count (TP) + False negative specimen count (FN)) X100

** Number of actual voice samples (TN) / (Number of actual voice samples (TN) + Number of false positive samples (FP)) X100

As shown in Tables 5 and 6, the antibody titer derived from the magnetic value standards prepared in Example 4 and the neutralizing antibody titer of the clinical sample were compared with respect to the negative samples and the titer samples, Three of the samples showed magnetic titers with 1:16 neutralizing antibody titers. At 1:16, one of the ten samples showed negative magnetic titers. At 1:32, two of the ten samples A magnetic value with a neutralizing antibody titer of 1:64 appeared. At 1:64, 3 of the 10 samples showed magnetic titers with 1: 128 neutralizing antibody titers. In both 1: 128 and 1: 256 samples, the magnetic values were 1: 128 and 1: 256 neutralizing antibodies And the quantitative titer of the neutralizing antibody by the magnetic value showed 88% relative sensitivity and 73% relative specificity compared with the antibody titer according to the neutralization test (Table 5) ).

However, regarding the differentiation of vaccine subjects, the magnetic values of clinical specimens belonging to the vaccinated subjects 1:16 and 1:32 were mostly consistent with the neutralizing antibody titers obtained by the neutralizing antibody test, 32 clinical samples were found to have a magnetic value with a 1:64 neutralizing antibody titer. In addition, the magnetic values of clinical samples belonging to 1:64 and 1: 256 non-vaccinated subjects showed similar tendency to those of neutralizing antibodies derived from neutralizing antibody test. However, 3 of 1:64 and 1: One clinical sample belonging to 256 was found to have a magnetic value with a 1: 128 neutralizing antibody titer.

As a result, it can be confirmed that about 96% (58/60) of accuracy is obtained with respect to the distinction between the vaccination target group and the non-vaccination group by the magnetic value, and the kit according to the present invention and the magnetic value It was found that the antibody titer of swine fever virus, which is efficient and accurate, can be measured and the vaccine can be discriminated according to the criteria.

In FIG. 2, the symbols are as follows.
1. Immunoassay 2. Analytical strip
3. Lower Case 4. Upper Case
5. Sample pad 6. Conjugation pad
7. Inspection line
9. Membrane 10. Absorbent pad
11. Sample inlet 12. Results display window
In FIG. 3, the symbols are 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 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 dispensing the sample;
Swine fever virus E2 antigen - conjugation pad with nanomagnetic beads adsorbed;
A membrane showing the detection result; And
Absorbing pad for absorbing the sample developing solution
A kit for detecting antibodies against swine fever virus.
The method according to claim 1,
Wherein the specimen is blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomit, or feces.
The method according to claim 1,
Wherein the membrane comprises a test line to which a porcine fever virus E2 antigen has been fixed and a control line to which a feline leukemia virus (FeLV) antigen has been fixed.
A method for measuring the presence or absence of an antibody against swine fever virus or the activity of an antibody using the kit of claim 1.
5. The method of claim 4,
To measure the presence or absence of an antibody,
Wherein when the test line of the kit shows yellow, it is determined that the antibody is present, and when it is not yellow, the antibody is judged to be absent.
Preparing a sample;
Reacting the specimen with a porcine fever virus E2 antigen-nanomagnetic bead conjugate; And
Measuring the magnetic value of the sample bound to the porcine febrile virus E2 antigen-nanomagnetic bead conjugate using a magnetic sensor
Lt; RTI ID = 0.0 &gt; pandemic &lt; / RTI &gt; virus.
7. The method according to claim 6, wherein after the magnetic value measuring step
When the magnetic value is 509 mV or less, it is judged that the antibody titer is 1:32 or less,
And determining that the antibody titer is 1:64 or more when the magnetic value is 827 mV or more.
8. The method according to claim 6 or 7,
Wherein the specimen is blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomit, or feces.
Preparing a sample;
Reacting the specimen with a porcine fever virus E2 antigen-nanomagnetic bead conjugate; And
Measuring the magnetic value of the sample bound to the porcine febrile virus E2 antigen-nanomagnetic bead conjugate using a magnetic sensor
&Lt; / RTI &gt; wherein said method comprises the steps of:
10. The method according to claim 9, wherein after the magnetic value measuring step
When the magnetic value is 509 mV or less, it is judged that the vaccine can be administered.
And determining that vaccination is not possible if the magnetic value is greater than or equal to 827 mV.
11. The method according to claim 9 or 10,
Wherein the specimen is blood, serum, plasma, lymph, tissue fluid, urine, tears, saliva, milk, vomit, or feces separated from piglets to be vaccinated.

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