KR100543691B1 - Detection kit for the classical swine fever antibody using immunochromatography method - Google Patents

Detection kit for the classical swine fever antibody using immunochromatography method Download PDF

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KR100543691B1
KR100543691B1 KR1020020079085A KR20020079085A KR100543691B1 KR 100543691 B1 KR100543691 B1 KR 100543691B1 KR 1020020079085 A KR1020020079085 A KR 1020020079085A KR 20020079085 A KR20020079085 A KR 20020079085A KR 100543691 B1 KR100543691 B1 KR 100543691B1
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cholera virus
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송재영
최은진
박종현
안동준
차상호
권준헌
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Abstract

본 발명은 항원-항체 반응을 이용하여 신속하게 돼지콜레라바이러스에 대한 항체의 존재유무를 검출할 수 있도록 고안된 검출 키트 및 이를 이용한 돼지콜레라바이러스 항체의 검출 방법에 관한 것이다.The present invention relates to a detection kit designed to rapidly detect the presence or absence of an antibody against porcine cholera virus using an antigen-antibody reaction and a method for detecting porcine cholera virus antibody using the same.

돼지콜레라, 면역크로마토그라피, 골드 콘쥬게이트, 유전자 재조합Porcine cholera, immunochromatography, gold conjugate, genetic recombination

Description

면역 크로마토그래피법을 이용한 돼지콜레라바이러스 항체의 검출 키트 {Detection kit for the classical swine fever antibody using immunochromatography method} Detection kit for the classical swine fever antibody using immunochromatography method             

도 1은 돼지콜레라바이러스 항원의 정제 모식도를 나타낸 것이고,1 shows a schematic diagram of purification of porcine cholera virus antigen,

도 2는 항체검출 모식도이며,2 is a schematic diagram of antibody detection.

도 3은 검출 키트 모식도이고,3 is a schematic diagram of a detection kit,

도 4a는 돼지 혈액내에 돼지콜레라바이러스에 대한 항체가 존재할 경우 양성 또는 음성반응이 나타난 모식도이고,Figure 4a is a schematic diagram showing a positive or negative reaction when there is an antibody against porcine cholera virus in pig blood,

도 4b는 돼지 혈액내에 돼지콜레라바이러스에 대한 항체가 존재할 경우 양성 또는 음성반응이 나타난 결과 사진이다.Figure 4b is a result photo showing a positive or negative reaction when the antibody to porcine cholera virus in the pig blood.

본 발명은 항원-항체 반응을 이용하여 신속하게 돼지콜레라바이러스에 대한 항체의 존재유무를 검출할 수 있도록 고안된 검출 키트 및 이를 이용한 돼지콜레라 바이러스 항체의 검출 방법에 관한 것이다. The present invention relates to a detection kit designed to rapidly detect the presence or absence of an antibody against porcine cholera virus using an antigen-antibody reaction and a method for detecting porcine cholera virus antibody using the same.

종래 돼지콜레라에 대한 항체를 검출하는 방법으로는 바이러스 중화시험(virus neutralization : VN test), 효소면역항체를 이용한 엘리자(enzyme linked immunosorbent assay : ELISA) 및 라텍스(latex) 응집반응 등을 사용하였다. 그러나 이들 방법은 전문화된 인력 및 실험 장비를 필요로 하며 검출시간도 길어 야외에서 사용하기에는 부적합하였다. 구체적으로, 중화시험법은 항체량을 간접적으로 측정할 수 있지만, 돼지혈액을 채취하여 혈청성분만을 원심분리로 모은 다음 56℃에서 30분 이상 비동화하여 사용해야 하고 검사시간도 3 내지 5일이 걸릴 뿐 아니라 전문화된 인력을 필요로 하는 문제점이 있다. 또한, 엘리자 및 라텍스 응집반응을 이용한 항체검출 방법은 대량의 혈청 샘플을 처리할 수 있는 장점이 있지만, 4 내지 5시간 이상이 소요되며 전문적인 인력과 결과 판독용 장비를 필요로 하므로 양돈장에서 사용하기에는 부적합하였다.Conventional methods for detecting antibodies to porcine cholera were virus neutralization (VN test), enzyme linked immunosorbent assay (ELISA) and latex (latex) aggregation reactions using enzyme-immunized antibodies. However, these methods require specialized manpower and experimental equipment and have long detection times, making them unsuitable for outdoor use. Specifically, the neutralization test method can indirectly measure the amount of antibody, but the pig blood is collected and collected by centrifugation of only serum components, and then used for at least 30 minutes at 56 ° C. In addition, there is a problem that requires specialized personnel. In addition, the antibody detection method using Eliza and latex aggregation reaction has the advantage of processing a large amount of serum samples, but it takes more than 4 to 5 hours and requires professional personnel and equipment for reading results, so it is not suitable for use in pig farms. It was inappropriate.

이에 본 발명자들은 이와 같이 불편한 기존의 항체검출법을 보다 간편하고 효과적인 방법에 의해 대체하고자 노력하였으며, 그 결과 항원-항체 반응을 이용한 검출 키트를 새로 고안하여 이를 사용하면 소기의 목적이 달성될 수 있음을 발견하고 본 발명을 완성하게 되었다. Therefore, the present inventors have tried to replace the existing antibody detection method which is inconvenient by a simpler and more effective method, and as a result, a newly designed detection kit using an antigen-antibody reaction can be used to achieve the desired purpose. Discovered and completed the present invention.

따라서 본 발명은 돼지콜레라바이러스 항체를 검출하기 위한 검출 키트를 제공함을 목적으로 한다. Accordingly, an object of the present invention is to provide a detection kit for detecting porcine cholera virus antibody.                         

본 발명은 또한, 이러한 검출 키트를 사용하여 돼지콜레라바이러스 항체를 검출하는 방법을 제공한다. The present invention also provides a method for detecting porcine choleravirus antibodies using such a detection kit.

본 발명에 따른 검출 키트의 원리를 도 3을 참조하여 간단히 설명하면 다음과 같다. 먼저, 스트립(strip) 형태의 니트로셀룰로스막(5)에 미리 정제된 돼지콜레라바이러스의 단백질 항원(1)을 고착시킨다. 이때, 바람직하게는 돼지콜레라바이러스의 구성단백질 중 돼지에서 항체를 가장 잘 생성하게 하는 단백질로 알려져 있어 항체검출용으로 가장 적절한 항원으로 판단되는 돼지콜레라바이러스의 E2 단백질을 사용한다. 또한, 니트로셀룰로스막의 다른 한편에는 항돼지IgG 항체(2)를 고착시킨다. 이와 같이 작제된 니트로셀룰로스막을 미리 준비된 가검물인 돼지 혈액 샘플과 접촉시킨다. 접촉은 돼지 혈액 샘플을 샘플 로딩 홀(4)을 통하여 샘플 패드(9)에 점적함으로써 이루어진다. 점적 후, 돼지 혈액 샘플은 니트로셀룰로스막(5)을 통하여 돼지콜레라바이러스 항원(1)이 고착된 곳으로 이동하게 되며, 동일한 방식으로 콘쥬게이트 패드(8)에 흡착된 항돼지IgG(anti-swine IgG) 골드(gold) 콘쥬게이트 역시 흡수 패드(3) 방향으로 이동하게 된다. 혈액 샘플내에 돼지콜레라바이러스 항체가 존재하면 이동하는 과정에 항원이 고착된 부위(1)에서 항원-항체 반응이 이루어지고, 항원-항체 결합물은 또한 항체에 감작된 골드 입자(항돼지IgG 골드 콘쥬게이트)와 반응하여 특유의 적자색을 나타내게 된다. 이에 따라, 돼지 혈액내의 항체 존재 유무에 따른 양성(항체 유) 또는 음성(항체 무)의 판정을 할 수 있다. The principle of the detection kit according to the present invention is briefly described with reference to FIG. 3 as follows. First, the purified protein antigen 1 of porcine cholera virus is fixed to the strip nitrocellulose membrane 5. At this time, it is preferable to use the E2 protein of porcine cholera virus, which is known to be the protein which makes the best antibody in pigs among the constituent proteins of porcine cholera virus, and is determined to be the most suitable antigen for antibody detection. In addition, on the other side of the nitrocellulose membrane, the anti-pig IgG antibody 2 is fixed. The nitrocellulose membrane thus prepared is contacted with a pig blood sample which is a prepared specimen. Contact is made by dropping a pig blood sample into the sample pad 9 through the sample loading hole 4. After instillation, the pig blood sample is transferred to the place where the pig choleravirus antigen 1 is fixed through the nitrocellulose membrane 5, and in the same manner, anti-swine IgG (anti-swine) adsorbed to the conjugate pad 8 IgG) gold conjugates also move in the direction of the absorbent pad (3). The presence of porcine choleravirus antibodies in the blood sample results in an antigen-antibody reaction at the site of antigen immobilization (1) during migration, and the antigen-antibody combination is also a gold particle (anti-pig IgG gold conjugate) sensitized to the antibody. Gate) to produce a characteristic reddish purple color. Thereby, the determination of positive (antibody) or negative (no antibody) depending on the presence or absence of antibodies in pig blood can be made.

항원-항체 반응 및 항돼지IgG 골드 콘쥬게이트와의 반응은 완충액중에서 이루어지게 되며, 바람직하게는 트리스 완충액(TBST: Tris 50mM, NaCl 150mM, Tween 20 0.1%, pH 7.2)을 이용한다. Antigen-antibody reactions and reactions with anti-pig IgG gold conjugates are carried out in buffer, preferably using Tris buffer (TBST: Tris 50 mM, NaCl 150 mM, Tween 20 0.1%, pH 7.2).

이하, 본 발명을 하기 실시예를 통하여 보다 구체적으로 설명한다. 그러나, 이들 실시예는 본 발명에 대한 이해를 돕기위한 것일 뿐, 어떤 의미로든 본 발명의 범위가 이들 실시예로 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention in any sense is not limited to these examples.

실시예 1Example 1

돼지혈액의 채취 및 처리Collection and Processing of Porcine Blood

돼지로부터 혈액을 직접 채혈하여 적혈구 제거용 필터를 통과시킨 혈청, 또는 돼지 귀정맥, 꼬리정맥 또는 도축시에 채취한 혈액을 셀룰로오스 흡수지에 흡수시킨 후 이를 500㎕의 트리스 완충액(Tris buffered saline: TBS, Tris 50mM, NaCl 150mM, pH 7.2, tween 20 0.1%)이 담겨진 투명한 튜브에 10분간 침지시켜 혈청성분만을 용출시킨 것을 검사혈액으로 사용하였다. Blood collected directly from pigs was passed through a filter for removing red blood cells, or blood collected at the time of hog vein, tail vein, or slaughter was absorbed into cellulose absorbent paper, and 500 μl of Tris buffered saline (TBS, Tris 50mM, NaCl 150mM, pH 7.2, tween 20 0.1%) was immersed in a transparent tube for 10 minutes to elute only serum components were used as test blood.

실시예 2Example 2

돼지콜레라바이러스 항원의 제조Preparation of Porcine Cholera Virus Antigen

돼지콜레라바이러스 ALD주의 E2 유전자를 클로닝하기 위하여 다음과 같은 실험을 수행하였다. 역전사중합효소 체인 반응(Reverse transcription polymerase chain reaction; RT-PCR)에 의해 증폭시키기 위하여 포워드 프라이머(forward primer)로서 5'-GGA TCC CGG CTA GCC TGC AAG GAA GAT-3'를 사용하고 안티센스 프라이머(antisense primer)로서 5'-GGA TCC TTC TGC GAA GTA ATC TGA-3'를 사용하였다. PCR 은 92℃에서 40초, 50℃에서 40초, 72℃에서 60초 동안 반응시키는 것을 1 사이클로하여 Thermocycler (PerkinElmer)에서 30 사이클 반응시켰으며, 아가로스 겔 전기영동에 의해 증폭된 유전자를 확인하였다. 증폭된 유전자를 전기영동하여 1021 bp 길이의 DNA 단편을 분리한 다음 pGemT (Promega) 및 Baculovirus 트랜스퍼 벡터인 pAcGP67B (Pharmingen)에 클로닝하여 pAcGP67BE2TNALD를 제작하였다. pAcGP67BE2TNALD와 바큘로바이러스 DNA(Pharmingen 사에서 구입)를 곤충세포 Sf21(Invitrogen 사에 구입)에 공형질감염 (cotransfection)시켜 E2 단백질을 발현하는 유전자 재조합 바큘로바이러스 BacE2TNALD를 제작하였다. BacE2TNALD를 곤충세포에 접종하여 6일간 배양한 후 상층액을 수확하였다. 6000 rpm에서 20분간 원심분리한 후 상층액을 수확하여 바이러스 불활화제(Binary ethylenimmine : BEI)로 처리하고, 인산완충액(10mM 포스페이트, pH 7.2)에 투석하고, 분주하고, 동결건조한 다음, 4℃에 보관하면서 본 발명에 따른 키트 제작에 사용하였다.In order to clone the E2 gene of porcine cholera virus ALD strain, the following experiment was performed. 5'-GGA TCC CGG CTA GCC TGC AAG GAA GAT-3 'as a forward primer for amplification by Reverse transcription polymerase chain reaction (RT-PCR) and antisense primer 5'-GGA TCC TTC TGC GAA GTA ATC TGA-3 'was used as the primer). PCR was carried out for 30 cycles in Thermocycler (PerkinElmer) by reacting for 40 seconds at 92 ° C, 40 seconds at 50 ° C, and 60 seconds at 72 ° C, and confirmed amplified genes by agarose gel electrophoresis. . 1021 bp long DNA fragments were isolated by electrophoresis of the amplified gene, and then cloned into pGemT (Promega) and Baculovirus transfer vector pAcGP67B (Pharmingen) to prepare pAcGP67BE2TNALD. pAcGP67BE2TNALD and baculovirus DNA (purchased from Pharmingen) were cotransfected into insect cell Sf21 (purchased from Invitrogen) to produce a recombinant baculovirus BacE2TNALD expressing the E2 protein. BacE2TNALD was inoculated into insect cells and cultured for 6 days, followed by harvesting supernatant. After centrifugation at 6000 rpm for 20 minutes, the supernatant was harvested, treated with a virus inactivator (Binary ethylenimmine: BEI), dialyzed in phosphate buffer (10 mM phosphate, pH 7.2), aliquoted, lyophilized, and then dried at 4 ° C It was used to make the kit according to the invention while storing.

실시예 3Example 3

돼지콜레라바이러스 항원이 고착된 니트로셀룰로우스막 스트립 제조Preparation of Nitrocellulose Membrane Strips with Porcine Cholera Virus Antigens

실시예 2에서 준비된 돼지콜레라바이러스 유전자 재조합 E2 단백질이 인산완충액에 녹아 있는 용액을 분사기(BioDot, USA)에 넣고 표 1에서와 같은 조건으로 포아사이즈(pore size) 3 내지 5㎛의 니트로셀룰로스막에 분사하였다. 돼지콜레라 바이러스 항원이 분사되는 위치는 니트로셀룰로스막에 혈액완충액이 침지되는 위치에서 14mm 정도 떨어진 위치이며, 대조 항원으로 사용하는 항돼지IgG 항체(구입처: Cappel 56970)의 인산완충용액을 돼지콜레라바이러스 항원이 분사된 위치에서 10mm 떨어진 위치에 표 1에서와 같은 조건으로 분사하였다. 항원이 분사된 니트로셀룰로스 막을 45℃에서 60분간 건조시킨 즉시 접착제가 도포된 폴리프로필렌 플레이트 막(backing plate)에 부착시켰다. 항돼지IgG 항체가 고정된 위치에서 7mm 상단 위치에 흡수지(밀리포어사, absorbent pad)를 위치시키고, 니트로셀룰로스막 하단에 도 3에서와 같이 콘쥬게이트 패드를 이어서 샘플패드(밀리포어사 absorbent pad)를 부착시켰다. 이를 도 3의 규격과 같이 4x60mm로 절단하여 돼지콜레라바이러스 항체검사용 키트를 완성하였다. The solution of porcine choleravirus recombinant E2 protein prepared in Example 2 was dissolved in phosphate buffer solution into the injector (BioDot, USA) and placed in a nitrocellulose membrane having a pore size of 3 to 5 μm under the conditions as shown in Table 1. Sprayed. The position where the swine cholera virus antigen is injected is about 14mm away from the position where the blood buffer solution is immersed in the nitrocellulose membrane. The injection was carried out under the same conditions as in Table 1 at a position 10 mm away from this injection position. The antigen sprayed nitrocellulose membrane was dried at 45 ° C. for 60 minutes and immediately attached to an adhesive coated polypropylene plate membrane. Place the absorbent paper (Millipore, absorbent pad) at the top position of 7mm from the anti-pig IgG antibody fixed position, and then the conjugate pad as shown in Figure 3 at the bottom of the nitrocellulose membrane, the sample pad (Millipore absorber pad) Was attached. This was cut into 4x60mm as in the standard of Figure 3 to complete the kit for porcine cholera virus antibody test.

[표 1] TABLE 1

돼지콜레라바이러스 항원 분사 조건 Porcine choleravirus antigen injection conditions

구분division 항돼지IgG 항체Anti-Pig IgG Antibodies 돼지콜레라바이러스 항원Porcine choleravirus antigen 파라미터parameter 농도(㎕/cm)Concentration (μl / cm) drop size(nm)drop size (nm) 농도(㎕/cm)Concentration (μl / cm) drop size(nm)drop size (nm) 0.750.75 31.2531.25 1One 31.531.5

실시예 4Example 4

항돼지IgG가 흡착된 콜로이드 골드 콘쥬게이트의 제작Preparation of Colloidal Gold Conjugates Adsorbed with Anti-Pig IgG

<항돼지IgG(anti-swine IgG : Cappel, 56970)가 흡착된 골드 콘쥬게이트의 제작><Preparation of Gold Conjugate Adsorbed Anti-swine IgG (Cappel, 56970)>

항돼지IgG(anti-swine IgG : Cappel, 56970)를 2mM Borax buffer(Sigma, pH 9.0)에 2㎍/㎕ 농도가 되도록 투석하고 최종적으로 0.1㎍/㎕ 농도가 되도록 희석하였다. 한편, 직경 40nm의 콜로이드 골드(BBI, CG40)는 530nm에서의 흡광도가 0.957이 되도록 희석하고 pH 9.0이 되도록 100mM K2CO3로 조절하였다. 준비된 골드 용액에 항돼지IgG 용액을 신속히 첨가하고 (혼합비 ; 골드 용액 : 항돼지IgG 용액= 1:1 , v:v) 혼합액을 5분간 교반하였다. 1/10 volume의 10% 소혈청알부민(BSA, pH 9.0)을 첨가하고 10분간 교반하였다. 항체가 흡착된 골드 입자는 5000rpm에서 50분간 원심분리로 침전시켜 수확하고 이를 다시 1% BSA가 함유된 트리스 완충액(50mM Tris, 150mM NaCl, pH 8,2)에 항돼지IgG가 첨가된 골드용액 volume의 1/10로 부유시켰다. Anti-pig IgG (anti-swine IgG: Cappel, 56970) was dialyzed to 2 μg / μl concentration in 2 mM Borax buffer (Sigma, pH 9.0) and finally diluted to 0.1 μg / μl concentration. On the other hand, colloidal gold (BBI, CG40) having a diameter of 40nm was diluted so that the absorbance at 530nm to 0.957 and adjusted to 100mM K 2 CO 3 to pH 9.0. The anti-pig IgG solution was added quickly to the prepared gold solution (mixing ratio; Gold solution: anti-pig IgG solution = 1: 1, v: v) and the mixed solution was stirred for 5 minutes. 1/10 volume of 10% bovine serum albumin (BSA, pH 9.0) was added and stirred for 10 minutes. Gold particles to which the antibody was adsorbed were harvested by centrifugation at 5000 rpm for 50 minutes, and again, gold solution volume containing anti-pig IgG was added to Tris buffer containing 1% BSA (50 mM Tris, 150 mM NaCl, pH 8,2). 1/10 of the was suspended.

<항돼지IgG 골드 콘쥬게이트가 흡착된 콘쥬게이트 패드의 제작><Production of conjugate pads to which anti-pig IgG gold conjugates are adsorbed>

상기와 같이 제작된 항돼지IgG 골드 콘쥬게이트 용액 300㎕를 피펫을 이용하여 콘쥬게이트 패드(100x7mm)에 고르게 분사하여 흡착시켰다. 45℃에서 60분간 콘쥬게이트 패드를 건조시킨 후 실시예 3에서 제작한 항체 검사용 스트립에 도 3에 나타낸 바와 같이 유전자재조합 E2 단백질이 고착된 니트로셀룰로스막과 겹치게 부착시키고 여기에 샘플흡수용 패드를 부착시켰다. 완성된 스트립은 도 3에 나타낸 바와 같이 플라스틱 하우징에 넣어 사용하였다.300 μl of the anti-pig IgG gold conjugate solution prepared as described above was adsorbed by evenly spraying onto a conjugate pad (100 × 7 mm) using a pipette. After drying the conjugate pad at 45 ° C. for 60 minutes, the antibody test strip prepared in Example 3 was overlapped with the nitrocellulose membrane to which the recombinant E2 protein was fixed as shown in FIG. 3, and the sample absorption pad was attached thereto. Attached. The finished strip was used in a plastic housing as shown in FIG.

실시예 5Example 5

실시예 3 및 4에서 제작한 돼지콜레라 항원이 고착된 니트로셀룰로스 스트 립을 사용하여 다음과 같이 항체 검출을 시도하였다. Antibody detection was attempted using the nitrocellulose strips to which the porcine cholera antigens prepared in Examples 3 and 4 were fixed.

플라스틱하우징의 샘플로딩 부위에 실시예 1에서 준비한 혈액 샘플을 50㎕ 떨어뜨리고 트리스 완충액(Tris 50mM, NaCl 150mM, 0.1% Tween 20, pH 7.2 : TBST)을 200㎕ 추가한 다음 10분간 반응시켰다. 그 결과, 도 4에 나타낸 바와 같이, 니트로셀룰로스 스트립의 항원이 고착된 위치에 선상으로 적자색이 발현되는 동시에 항돼지IgG가 고착된 부위에 선상으로 적자색이 발현되는 경우 양성으로 판정하며 항원이 고착된 위치에는 선상으로 색깔이 발현되지 않고 항돼지IgG가 고착된 위치에만 선상으로 적자색이 발현되는 경우 음성으로 판정하였다. 50 μl of the blood sample prepared in Example 1 was dropped onto the sample loading site of the plastic housing, 200 μl of Tris buffer (Tris 50 mM, NaCl 150 mM, 0.1% Tween 20, pH 7.2: TBST) was added, and the reaction was performed for 10 minutes. As a result, as shown in FIG. 4, when the reddish violet color is expressed linearly at the position where the antigen of the nitrocellulose strip is fixed, and the reddish violet is expressed linearly at the site where the anti-pig IgG is fixed, it is determined as positive and the antigen is fixed. Negative red color was detected in the line where only the position where anti-pig IgG was fixed.

실시예 6Example 6

키트를 이용한 돼지콜레라 항체의 검출Detection of Porcine Cholera Antibodies Using Kits

기존 검사법과의 항체검출 효율을 비교하기 위하여, 기존 ELISA 법에 의할 때 양성으로 판정된 490개의 혈청과 음성으로 판정된 139개의 혈청을 실시예 3 및 4의 방법에 따라 제작된 키트를 이용하여 검사하였다. 그 결과, 민감성은 97.6%였으며 특이성은 95%로 확인되어 기존 검사법과의 일치율이 매우 높게 나타났다 (표 2 참조).In order to compare the antibody detection efficiency with the conventional assay, 490 sera determined as positive and 139 sera determined as negative by the conventional ELISA method were prepared using the kits prepared according to the methods of Examples 3 and 4. Inspected. As a result, the sensitivity was 97.6% and the specificity was 95%, which showed a high agreement with the existing test method (see Table 2).

[표 2]TABLE 2

항체검출 효율성 비교Antibody Detection Efficiency Comparison

ELISAELISA 양성(+)Positive (+) 음성(-)voice(-) 면역크로마토그라피법 (골드 콘쥬게이트) Immunochromatography (Gold Conjugate) 양성(+)Positive (+) 478478 77 음성(-)voice(-) 1212 132132 계system 490490 139139 민감성 : 118/123 → 97.6% 특이성 : 49/53 → 95.0%         Sensitivity: 118/123 → 97.6% Specificity: 49/53 → 95.0%

한편, 돼지콜레라바이러스와 같은 바이러스속에 속하는 BVDV(Bovine Viral Diarrheal Virus) 및 BDV(Border Disease Virus)에 대한 항체(입수처 : 국립수의과학검역원)와의 교차반응성을 조사한 결과 BVDV 항체와는 50%의 교차반응성이 확인되었으며 BDV 항체와는 100% 교차반응성을 나타내었다(표 3 참조). On the other hand, cross-reactivity with antibodies to BVDV (Bovine Viral Diarrheal Virus) and BDV (Border Disease Virus) belonging to the genus of the virus such as porcine cholera virus was obtained. Reactivity was confirmed and 100% cross-reactive with BDV antibodies (see Table 3).

[표 3] TABLE 3

교차반응성 비교Cross Reactivity Comparison

출처 source No. No. 면역크로마토그라피법 (골드 콘쥬게이트) Immunochromatography (Gold Conjugate) 교차반응성(%) % Cross reactivity +ve + ve -ve -ve Anti-BVDVAnti-BVDV 기니아피그Guinea pig 77 22 22 5050 돼지pig 33 33 00 Anti-BDVAnti-BDV 기니아피그Guinea pig 66 66 00 100100

+ve : positive : -ve : negative+ ve: positive: -ve: negative

한편, 기존의 ELISA 법에 의해 양성으로 확인된 총 178개의 혈장과 음성혈장 101개에 대해 혈장을 이용한 항체검출 실험을 수행한 결과 민감성 100%, 특이성 99%의 반응성을 보였다(표 4 참조).On the other hand, the antibody detection experiment using plasma was performed on a total of 178 plasma and 101 negative plasmas positively confirmed by the conventional ELISA method, and showed 100% sensitivity and 99% specificity (see Table 4).

[표 4] TABLE 4

혈장을 이용한 항체 검출Antibody Detection Using Plasma

ELISAELISA 양성(+)Positive (+) 음성(-)voice(-) 면역크로마토그라피법 (골드 콘쥬게이트) Immunochromatography (Gold Conjugate) 양성(+)Positive (+) 178178 1One 음성(-)voice(-) 00 100100 계system 178178 101101 민감성 : 178/178 → 100% 특이성 : 100/101 → 99%         Sensitivity: 178/178 → 100% Specificity: 100/101 → 99%

상기 설명한 바와 같이, 본 발명에 따른 검출 키트를 이용하면 돼지 혈액내 돼지콜레라바이러스에 대한 항체의 존재 유무를 신속하게 검사할 수 있을 뿐 아니라 야외에서도 검사가 가능하므로 매우 유용하다. 특히, 돼지콜레라의 경우와 같이 현재 근절사업이 진행 중인 것은 농장에서 신속하게 항체를 검출할 수 있는 방법이 절실하게 요구되는데, 본 발명은 이러한 요구에 적절히 부응하고 있다. As described above, the detection kit according to the present invention is very useful because it is possible not only to quickly test the presence or absence of antibodies to porcine cholera virus in pig blood, but also to test outdoors. In particular, the current eradication project, such as the case of porcine cholera is urgently required a method for detecting the antibody on the farm quickly, the present invention satisfies this need.

Claims (6)

돼지콜레라바이러스 항원 및 항돼지IgG 항체가 고착된 니트로셀룰로스막과 함께 항돼지IgG 항체 콜로이드 골드 콘쥬게이트가 흡착된 콘쥬게이트 패드를 포함함을 특징으로 하는 돼지콜레라바이러스 항체 검출용 키트.A kit for detecting swine cholera virus antibody, comprising: a conjugate pad to which an anti-pig IgG antibody colloid gold conjugate is adsorbed together with a nitrocellulose membrane to which a porcine cholera virus antigen and an anti-pig IgG antibody are fixed. 제1항에 있어서, 돼지콜레라바이러스 항원이 돼지콜레라바이러스의 E2 단백질인 것을 특징으로 하는 돼지콜레라바이러스 항체 검출용 키트.The kit for detecting swine choleravirus antibody according to claim 1, wherein the swine choleravirus antigen is E2 protein of swine cholera virus. 제1항에 있어서, 도 3에 도시한 구조를 갖는 것을 특징으로 하는 돼지콜레라바이러스 항체 검출용 키트.The kit for detecting swine choleravirus antibody according to claim 1, which has a structure shown in FIG. 제1항에 따른 키트를 돼지 혈액 및 완충액과 접촉시킴을 특징으로 하는 돼지콜레라바이러스의 항체 검출 방법.A method for detecting an antibody of swine choleravirus, comprising contacting a kit according to claim 1 with pig blood and a buffer solution. 제4항에 있어서, 완충액이 트리스 완충액인 것을 특징으로 하는 돼지콜레라바이러스 항체 검출 방법. The method of claim 4, wherein the buffer is Tris buffer. 제5항에 있어서, 완충액이 트리스 완충액(TBST: Tris 50mM, NaCl 150mM, Tween 20 0.1%, pH 7.2)인 것을 특징으로 하는 돼지콜레라바이러스 항체 검출 방법. The method of claim 5, wherein the buffer is Tris buffer (TBST: Tris 50 mM, NaCl 150 mM, Tween 20 0.1%, pH 7.2).
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