느타리버섯 세균성갈빈병균 Pseudomonas tolaasii의 효소면역검출법

이향범,전낙범,손동화,유승헌 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-

느타리버섯의 세균성갈반병균인 P. talaasii(PT)를 신속 간편하게 검출할 수 있는 효소면역측정법(ELISA)을 개발하였다. 특이항체를 생산하기 위하여 PT(5×10 exp (7) cfu)를 Freund's adjuvant와 함께 토끼에 수차례 면역하였으며, 가장 높은 역가를 나타낸 항혈청을 이용하여 비경합 ELISA 및 경합 ELISA를 각각 확립하였다. 표준곡선으로부터 PT의 그 검출한계는 각각 2×10 exp (2) cfu/ml 및 3×10 exp (2) cfu/ml가량으로 나타났다. 교차반응은 비경합 ELISA의 경우 P. agarici, P. reactans 및 비병원성 Pseudomonas속 균주와 1/10^3이하의 매우 미약한 반응을 보였으나 P. salanacearum, Erwinia chrysanthemi, Streptococcus mutans, Xanthomonas citri 및 곰팡이인 Fusarium oxysporum 등 다른 균주와의 반응성이 거의 없었으며, 경합 ELISA의 경우 PT이외에는 같은 속 균주에 대하여도 반응성이 거의 없었다. ELISA를 이용하여 버섯으로부터 분리된 18균주에 대하여 PT의 여부를 조사하였을 때 병원성과 white line 반응성을 동시에 나타내었던 11균주에 대하여만 명확한 양성으로 나타나, 본 연구에서 확립한 ELISA는 간편하고 정확한 PT검출법임이 확인되었다. For simple and rapid detection of Pseudomonas tolaasii (PT), a bacterial brown blotch pathogen of oyster mushroom, enzyme-linked immunosorbent assays (ELISA) were developed. To produce specific antibody, PT (5 × 10 exp (7) cfu) and Freund's adjuvant were subcutaneously immunized into rabbits several times. By using the antiserum showing the highest titer, we established noncompetitive and competitive ELISA's. Standard curves of the ELISA's showed that the detection limits were 2 × 10 exp (2) cfu/ml and 3 × 10 exp (2) cfu/ml, respectively. When investigated by noncompetitive ELISA, cross reactivities of the anti-PT antibodies against P. agarici, P. reactans, and other fluorescent Pseudomonas spp. were very low (<1/10^3), but those against P. solanacearum, Erwinia chrysanthemi, Streptococcus mutans, Xanthomonas citri, and a fungus Fusarium oxysporum were almost none. However, when investigated by competitive ELISA, the reactivities against any other strains except PT were almost none. When the ELISA's were applied to 18 strains derived from mushrooms in order to identify PT, only 11 strains showing both pathogenicity and white line reactivity were obviously positive. These results showed that the ELISA's could be convenient tools to detect PT in accordance with existing methods.

PCR-ELISA법을 이용한 거대세포바이러스 DNA의 조기검출

김민,윤명,임우현,신종희,서순팔,양동욱 대한임상병리학회 1998 대한임상병리학회지 Vol.18 No.3

느타리버섯 세균성갈반병균 Pseudomonas tolaasii의 효소면역검출법

이향범,전낙범,손동화,유승헌 한국산업미생물학회 1998 한국미생물·생명공학회지 Vol.26 No.3

느타리버섯의 세균성갈반병균인 P. tolaasii(PT)를 신속 간편하게 검출할 수 있는 효소면역측정법(ELISA)을 개발하였다. 특이항체를 생산하기 위하여 PT(5×10 exp (7) cfu)를 Freund's adjuvant와 함께 토끼에 수차례 면역하였으며, 가장 높은 역가를 나타낸 항혈청을 이용하여 비경합 ELISA 및 경합 ELISA를 각각 확립하였다. 표준곡선으로부터 PT의 그 검출한계는 각각 2×10 exp (2) cfu/㎖ 및 3×10 exp (2) cfu/㎖가량으로 나타났다. 교차반응은 비경합 ELISA의 경우 P. agarici, P. reactans 및 비병원성 Pseudomonas속 균주와 1/10^3이하의 매우 미약한 반응을 보였으나 P. solanacearum, Erwinia chrysanthemi, Streptococcus mutans, Xanthomonas citri 및 곰팡이인 Fusarium oxysporum 등 다른 균주와의 반응성이 거의 없었으며, 경합 ELISA의 경우 PT이외에는 같은 속 균주에 대하여도 반응성이 거의 없었다. ELISA를 이용하여 버섯으로부터 분리된 18균주에 대하여 PT의 여부를 조사하였을 때 병원성과 white line 반응성을 동시에 나타내었던 11균주에 대하여만 명확한 양성으로 나타나, 본 연구에서 확립한 ELISA는 간편하고 정확한 PT검출법임이 확인되었다. For simple and rapid detection of Pseudomonas tolaasii (PT), a bacterial brown blotch pathogen of oyster mushroom, enzyme-linked immunosorbent assays (ELISA) were developed. To produce specific antibody, PT (5×10 exp (7) cfu) and Freund's adjuvant were subcutaneously immunized into rabbits several times. By using the antiserum showing the highest titer, we established noncompetitive and competitive ELISA's. Standard curves of the ELISA's showed that the detection limits were 2×10 exp (2) cfu/㎖ and 3×10 exp (2) cfu/㎖, respectively. When investigated by noncompetitive ELISA, cross reactivities of the anti-PT antibodies against P. agarici, P. reactans, and other fluorescent Pseudomonas spp. were very low (<1/10^3), but those against P. solanacearum, Erwinia chrysanthemi, Streptococcus mutans, Xanthomonas citri, and a fungus Fusarium oxysporum were almost none. However, when investigates by competitive ELISA, thc reactivities against any other strains except PT were almost none. When the ELISA's were applied to 18 strains derived from mushrooms in order to identify PT, only 11 strain showing both pathogenicity and white line reactivity were obviously positive. These results showed that the ELISA's could be convenient tools to detect PT in accordance with existing methods.

C형 간염바이러스항체 검출을 위한3세대 효소면역측정 시약의 비교

이장혁,서순팔,기승정,송정원,신명근,신종희,양동욱,김세종 대한임상병리학회 1997 대한임상병리학회지 Vol.17 No.4

Reducing background optical density in enzyme-linked immunosorbent assay for detecting nervous necrosis virus (NNV)-specific IgM by immobilizing fish sera

Gye, Hyun Jung,Nishizawa, Toyohiko Elsevier 2018 Aquaculture Vol.485 No.-

<P><B>Abstract</B></P> <P>Enzyme-linked immunosorbent assay (ELISA) for detecting fish antibodies is problematic due to its low reproducibility and high background optical density (OD). In this study, we investigated problematic sources using nervous necrosis virus (NNV, a fish pathogenic virus belonging to the genus <I>Betanodavirus</I>) and sevenband grouper (<I>Hyporthodus septemfasciatus</I>) as a model for detecting specific fish IgM through ELISA. It was revealed that both fish IgM and mammalian immunoglobulins were non-specifically adsorbed to purified NNV particles. This could be the problematic source of the high background OD and low reproducibility in ELISA. ELISA values of naïve fish IgM non-specifically adsorbed to NNV particles immobilized onto ELISA plate wells ranged from 0.09 to 0.15 (high background OD). However, ELISA values of NNV antigens non-specifically adsorbed to naïve fish IgM immobilized onto ELISA plate wells were all <0.03 (low background OD). Thus, we developed a sandwich ELISA by immobilizing fish sera. NNV-specific antibodies could be indirectly detected by detecting NNV antigens captured by fish IgM immobilized onto ELISA plate wells using anti-NNV serum. When anti-NNV and naïve fish sera were subjected to such sandwich ELISA, ELISA values of anti-NNV fish sera ranged from 0.24 to 2.48. Its reproducibility was sufficiently secured based on results obtained from five experiments performed on different days. Conversely, ELISA values of naïve fish sera were all <0.02, suggesting that background OD was completely regulated. The present sandwich ELISA does not require antiserum against fish IgM, meaning that NNV-specific antibodies are detectable from any fish species using only one antiserum against NNV.</P> <P><B>Highlights</B></P> <P> <UL> <LI> ELISA by immobilizing NNV antigens was problematic for detecting specific fish IgM. </LI> <LI> Fish IgM and mammalian immunoglobulins were non-specifically adsorbed to NNV particles. </LI> <LI> Sandwich ELISA by immobilizing fish sera was developed for detecting NNV-specific IgM. </LI> <LI> Reproducibility and background OD were greatly improved in such sandwich ELISA. </LI> </UL> </P>

효소면역측정법을 이용한 Lambda Antigen Tray 키트의 Panel Reactive Antibody 검사 유용성 평가

송선미,박병택,양윤선 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.6

Genedia HCV ELISA 3.0, Rapid 및 Confirm 4.0 시약의 진단적 유용성 평가

임영애,전희선,곽연식,조영식,이동순 대한임상병리학회 1998 대한임상병리학회지 Vol.18 No.2

Babesia gibsoni의 적혈구내 배양법과 진단법 개발에 관한 연구 1. Babesia gibsoni 진단을 위한 간접형광항체법(IFAT)과 효소표지면역검사법(ELISA)

서명득,신용승,Suh, Myung-deuk,Shin, Yong-seung 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.3

Indirect fluorescent antibody test(IFAT) and enzyme-linked imuunosorbent assay (IgG-ELISA) as serological diagnostic tools were conducted to evaluate the usefulness for diagnosis of canine babesiosis infected with Babesia gibsoni in domestic various dog breeds, american pit bullterrier, military shepherd, and mongrel dogs. The results obtained from this study were abstracted as follows. The nonionic detergent Triton X-100 and absorbent bio-bead $SM_2$ were useful reagents for the preparation of pure merozoite antigen of B gibsoni to be used in ELISA. The optimum reaction in ELISA was shown when the protein concentration of ELISA antigen was measured as 625ng/ml and the conjugate concentration was diluted into 1/6000 fold. The average OD value of ELISA in sera determined with negative responses in IFAT was measured as $0.255{\pm}0.051$(490nm) and the cut - off value of OD was determined as 0.399(490nm). The serum antibodies in both of IFAT and ELISA were detected on one week after artificially infected with B gibsoni and these high antibody titers, 512X in IFAT and 1024X in ELISA, were long lasted until 15 weeks after infection. The reproducibility of reaction and stability of the antigen absorbed microtitration polystyrene plate preserved in $4^{\circ}C$ refrigerator and $-20^{\circ}C$ freezer, respectively could be lasted until 135 days after storage. The positive rates in IFAT by dog breeds were shown 8.1%(60/744 heads) in mongrel dogs, 81.3%(78/96 heads) in american pit bullterrier and 15.6%(15/96 heads) in military shepherd, while the positive rate in ELISA shown 17.6%(131/744 heads) in mongrel dogs, 83.3%(80/96 heads) in american pit bullterrier and 36.5%(35/96 heads) in military shepherd, respiectively. In the total of 936 heads surveyed with IFAT and ELISA the positive rates in IFAT and ELISA were 16.4%(153/936 heads) and 26.3%(246/936 heads), respectivily. Agreement of reactions between IFAT and ELISA was shown 82.4% in 936 dog sera. The specificity and sensitivity of ELISA reaction were 83.5% and 76.5%, respectively. From the conclusion obtained in this study it was evaluated that IFAT and ELISA were useful as highly specific, sensitive and stable serelogical tools for the diagnosis of canine babesiosis in Korea.

수입곡물 중의 Aflatoxin B_1 검출을 위한 효소면역측정법의 평가

손동화,박애란,이인원 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.3

곡물 중의 aflatoxin B_1(AFB_1)을 검출하는 방법으로서 효소면역측정법(ELISA)의 활용가능성을 평가하기 위하여, ELISA 분석치를 인위적인 오염치 및 자연오염된 시료의 HPLC 분석치와 비교하였다. 분석대상 시료로는 외국산 면실박(19점), 채종박(11점), 대두박(9점), 옥수수(3점) 등의 수입곡물을 사용하였다. 각 곡물별로 작성한 표준곡선으로부터 실제 곡물시료 중 대체로 1∼100ng/g 농도의 AFB_1이 분석가능함을 알 수 있었다. 이를 기준으로 하여 표준 AFB_1을 인위적으로 오염시킨 시료를 ELISA로 분석하였을 때, 1 ng/g의 저농도에서는 그 회수율이 높았으나(평균 268%) 3 ng/g 이상의 농도에서는 평균 138%(68∼193%)이었으며, 각 분석치의 상대적인 분산도(C.V.)는 평균 7.0%(0∼22%)로 매우 양호하였다. 한편 자연오염된 시료의 ELISA 분석치를 HPLC 분석치와 비교하였을 때, 10 ng/g 이하의 시료에서는 특히 채종박의 경우 두 분석치 간에 차이가 다소 크게 나타났으나, 그 이상의 오염시료에서는 몇몇 예외를 제외하고는 비교적 근사한 수치를 나타내었다. 특히 오염도가 심각한 면실박의 경우 HPLC분석치에 대한 ELISA 분석치의 비율이 평균 153%로 양호하였다. 따라서 본 효소면역측정법은 10 ng/g 이상의 AFB_1이 오염된 곡물시료의 분석에 실용화가 가능한 것으로 나타났다. In order to evaluate an enzyme-linked immunosorbent assay(ELISA) for practical use in detecting aflatoxin B_1(AFB_1) from cereals, we compared AFB_1 concentrations of samples contaminated artificially or naturally that were quantitated by the ELISA with those spiked or quantitated by HPLC. Cotton seed meals(19 items), rape seed meals(11), soybean meals(9), and corns(3) imported from foreign countries were used as sample cereals. The standard curves of each cereal class showed that 1∼100 ng/g of AFB_1 from cereals could be assayed by the ELISA. When artificially contaminated cereals were assayed by ELISA, the average recovery of AFB_1 from samples spiked to 3 ng/g and more was 138%(68∼193%), although that spiked to 1 ng/g was somewhat high(268%). The average C.V. of recovery was 7.O%(O∼22.2%). When naturally contaminated cereals were assayed, the concentrations of AFB_1 below 10 ng/g especially from rape seed meals quantitated by ELISA were much lower than those determined by HPLC. However, the concentrations of 10 ng/g and more from samples, except a few extraordinary samples, quantitated by ELISA were similar to those determined by HPLC, especially in case of cotton seed meals whose average recovery (ELISA/HPLC) was 153%. In conclusion, the ELISA was elucidated such as a practical tool to detect AFB_1 of 10 ng/g and more from cereals.

수입곡물 중의 Alfatoxin $B_1$ 검출을 위한 효소면역측정법의 평가

손동화,박애란,이인원 한국미생물 · 생명공학회 1992 한국미생물·생명공학회지 Vol.20 No.3

곡물 중의 aflatoxin $B_1(AFB_1)$을 검출하는 방법으로서 효소면역측정법(ELISA)의 활용가능성을 평가하기 위하여, ELISA 분석치를 인위적인 오염치 및 자연오염된 시료의 HPLC 분석치와 비교하였다. 분석대상 시료로는 외국산 면실박(19점), 채종박(11점), 대두박(9점), 옥수수(3점) 등위 수입곡물을 사용하였다. 각 곡물별로 작성한 표준곡선으로부터 실제 곡물시료 중 대체로 1-100ng/g 농도의 $AFB_1$분석 가능함을 알 수 있었다. 이를 기준으로하여 표준 $AFB_1$을 인위적으로 오염시킨 시료를 ELISA로 분석 하였을 때, 1 ng/g의 저농도에서는 그 회수율이 높았으나 (평균 265) 3ng/g 이상의 농도에서는 평균 138(68-193)이었으며, 각 분석치의 상대적인 분산도(C.V)는 평균 7.0(0-22)로 매우 양호하였다한편 자연오염된 시료의 ELISA 분석치를 HPLC 분석치와 비교하였을 때, 10ng/g 이하의 시료에서는 특히 채종박의 경우 두 분석치의 간에 차이가 다소 크게 나타났으나, 그 이상의 오염시료에서는 몇몇 예외를 제외하고는 비교적 근사한 수치를 나타내었다. 특히 오염도가 심각한 면실박의 경우 HPLC 분석치에 대한 ELISA 분석치의 비율이 평균 153%로 양호하였다. 따라서 본 효소면역측정법은 10ng/g 이상의 $AFB_1$이 오염된 곡물시료의 분석에 실용화가 가능한 것으로 나타났다. In order to evaluate an enzyme-linked immunosorbent assay(ELISA) for practical use in detecting aflatoxin $B_1(AFB_1)$ from cereals, we compared $AFB_1$ concentrations of samples contaminated artificially or naturally that were quantitated by the ELISA with those spiked or quantitated by HPLC. Cotton seed meals(19 items), rape seed meals(ll), soybean meals(9), and corns(3) imported from foreign countries were used as sample cereals. The standard curves of each cereal class showed that 1-100 ng/g of $AFB_1$ from cereals could be assayed by the ELISA. When artificially contaminated cereals were assayed by ELISA, the average recovery of AFB! from samples spiked to 3 ng/g and more was 138%(68-193%), although that spiked to 1 ng/g was somewhat high(268%). The average C.V. of recovery was 7.0%(0-22.2%). When naturally contaminated cereals were assayed, the concentrations of $AFB_1$ below 10 ng/g especially from rape seed meals quantitated by ELISA were much lower than those determined by HPLC. However, the concentrations of 10 ng/g and more from samples, except a few extraordinary samples. quantitated by ELISA were similar to those determined by HPLC, especially in case of cotton seed meals whose average recovery (ELISA/HPLC) was 153%. In conclusion, the ELISA was elucidated such as a practical tool to detect $AFB_1$ of 10 ng/g and more from cereals.