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1 Journal of Bacteriology and Virology Vol. 38, No. 3 p Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A Yun Jeong Kim, Na-Ri Shin, Jeong-Hee Kim, So-Yeon Yoon, Gi-eun Rhie, Bong Su Kim and Hee-Bok Oh * Center for Infectious Diseases, National Institute of Health, 5-Nokbun-dong, Eunpyung-gu, Seoul , Republic of Korea Received : June 20, 2008 Revised : August 4, 2008 Accepted : August 18, 2008 A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC For the capture ELISA, the optimized amount (2 μg/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 μg/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r 2 ) ranged from ~ for all assays. The inter-variations were typically 0.50~ 6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was ng/ml, which was close to mouse LD 50. In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens. Key Words: BoNT/A, Capture ELISA, Biotinylated monoclonal antibody 서 보툴리눔독소 (Botulinum neurotoxin) 는절대혐기성세균인 Clostridium botulinum에서생성되는독소로 A-G의 7가지혈청형으로구분되며, 인체노출시심각한신경성이완성마비를야기시킴으로써보툴리눔중독증을일으킨다 (3). 보툴리눔독소는음식물에 C. botulinum 포자가오염, 발아하여독소를생성함으로써자연적인보툴리눔 * Corresponding author: Hee-Bok Oh. Center for Infectious Diseases, National Institute of Health, 5-Nokbun-dong, Eunpyung-gu, Seoul , Republic of Korea. Phone: , Fax: hboh@nih.go.kr ** This study was financially supported by an intramural grant from National Institute of Health, Korea. 론 중독증을야기할수있을뿐만아니라전쟁이나생물테러발생시공기중으로보툴리눔독소를인위적으로분사함으로써 inhalational outbreak를야기할수도있다. 보툴리눔독소는자연계에서생성되는독소중가장강력한독성을지니고있을뿐만아니라심각한사회적파급효과를야기할수있는특성으로인해미국 CDC에서생물테러가능물질 category A로분류하였으며, 실제생물테러사용시다수의사람들이동시에독소에노출될수있어이에대한신속하고정확한탐지가중요하다 (8). 보툴리눔독소는 100 kda의 heavy chain과 50 kda의 light chain으로구성되어있으며, 이를둘러싼 accessory protein과함께독소복합체를형성하고있다. 독소작용기작은체내흡수시표적부위인근신경접합부의신경말단에서아세틸콜린의방출을억제함으로써근육마비를 119

2 120 Y J Kim, et al. 일으킨다 (15). 보툴리눔독소의작용은비가역적반응이기때문에보툴리눔중독증에대한진단은보툴리눔독소가완전히결합하기전인임상증상발현초기단계에이루어져야적절하고효과적인임상적대처가가능하므로신속하고민감한진단법의개발이필수적이다. 보툴리눔중독증에대한국제적표준법은 mouse bioassay로서민감도및특이도가높아현재까지전세계적으로사용해오고있으나, 여러동물의희생을요구하고실험결과를얻기까지오랜시간 (3~4일) 이소요된다는단점이있다 (14). 따라서이러한단점을보완하기위하여보툴리눔독소탐색을위한새로운기법연구가지속적으로이루어지고있으며 (2), 이들방법중면역학적기법을바탕으로개발한진단법은생물테러발생시환경혹은인체유래가검물을이용한 massive testing이가능하고스크리닝이용이하다는이점이있다. 면역학적기법을바탕으로하여개발된탐지법중 indirect ELISA법은간단하지만 mouse assay 보다는민감도가낮으며 (14), enzyme-linked coagulation assay (ELCA) 법의경우민감도는높지만, snake venom coagulation factor 및고가의시약들이필요하고실험이매우복잡하다는단점이있다 (6). 최근에는 Food and Drug Administration (FDA) 에서보툴리눔독소 ABEF 형을동시진단할수있는 polyclonal antibody를이용한 capture ELISA법을개발하였으며 (13), Szilagyi 등은 horse polyclonal antibody를이용한 capture ELISA법을개발하여보고한바있다 (16). 따라서본연구에서는국내에서처음으로보툴리눔 A 형독소탐지법을 in vitro 상에서개발하고자하였으며, 민감도및특이도향상을위하여기존의보고에서시도되지않은토끼 polyclonal antibody와 biotin-conjugated monoclonal antibody의조합으로 capture ELISA법을개발하였다. 또한보툴리눔독소를 normal human serum에직접 spiking하여검사하거나 mouse bioassay와의민감도비교를통해본연구에서개발된방법을검증함으로써향후생물테러와관련된다량의샘플을동시스크리닝할수있는지의여부를확인하고자하였다. 재료및방법 1. 보툴리눔 A형독소 (BoNT/A) 정제토끼항체제작및마우스단클론항체제작을위한 BoNT/A는 Malizio (11) 등이제시한방법에의해정제하 여사용하였다. 간단히기술하면, C. botulinum ATCC 을독소생성용배지 (2% casein acid, 1% yeast extract, 0.5% glucose) 에 37 에서 48시간동안혐기배양한후 C. botulinum 배양액에 3 N sulfuric acid를처리하였으며, 침전물에 0.2 M sodium phosphate buffer (ph 6.0) 를첨가하여독소를용출한후원심상층액을 60% ammonium sulfate로재침전하였다. 침전물을다시수거하여 0.05 M sodium citrate buffer (ph 5.5) 로용해시키고, 동일한 buffer 에서투석하여염을제거한후 anion exchange column (Pharmacia, Uppsala, Sweden) 에적용함으로써 BoNT/A를분리정제하였다. 정제독소는 SDS-PAGE 및 Western blot 을실시한후, 마우스에서의 LD 50 를결정함으로써 BoNT/ A의생물활성도를확인하였다. LD 50 는 2-fold dilutions된 5가지서로다른농도의 BoNT/A를 ICR 마우스 (4주령, female) 에그룹당 8마리씩복강내접종한후그생존여부를 Reed and Muench법에의해분석하여결정하였다 (12). 2. 토끼유래 anti-bont/a polyclonal antibody (PAb) 의준비 Capture ELISA를위한 PAb는 BoNT/A toxoid에대하여면역한토끼항혈청으로부터정제하여사용하였다. 먼저, BoNT/A를 formalin을 0.4% 가되도록첨가하여 37 에서 shaking 상태로 5일간방치하여무독화시킨후, BoNT/A toxoid 0.5 mg을 Freund's adjuvant (Sigma, St. Lois, MO, USA) 로흡착시켜토끼대퇴부근육에 2주간격으로 3회면역하였다. 토끼항혈청은최종면역 2주후심장채혈을통해확보하였으며, mouse bioassay법 (1) 을이용하여중화능을확인하였다. 즉, 250 μl의 BoNT/A (10,000LD 50 ) 를동량의 gelatin phosphate buffer (ph 6.2) 혹은동량의토끼항혈청과혼합하여실온에서 1시간동안반응시킨후 2마리 ICR mouse의복강내에각각접종하여마우스생존여부를확인하였다. Capture ELISA를위한 PAb는중화능이확인된토끼항혈청으로부터 protein A affinity column (Amersham Biosciences, Uppsala, Sweden) 을이용하여정제하여준비하였다. 즉, 혈청과 binding buffer (20 mm sodium phosphate, ph 7.0) 를 1:1의비율로혼합하여 column에 loading한후 10 ml의 binding buffer를이용하여세척하였으며 (1 ml/min), column에결합한 IgG는 elution buffer (0.1 M glycine-hcl, ph 2.7) 를사용하여분리한후 SDS-PAGE gel에전기영동함으로써정제도를확인하고

3 Development of Capture ELISA for BoNT/A Detection 121 최종적으로 PBS (ph 7.2) 로투석 농축하여 -20 에보관하였다. 3. Monoclonal antibody (MAb) 의준비및 biotinylation MAb는 C. botulinum ATCC19397로부터정제한 BoNT/A 복합체를항원으로사용하여 ( 주 ) 중겸 ( 안산, 대한민국 ) 에의뢰하여제작하였다. MAb 제조를위한 clone을결정하기위하여 hybridoma cell 중 BoNT/B 및 BoNT/E에대한반응없이 BoNT/A에만결합하는 12개의 clone을 indirect ELISA를이용하여먼저스크리닝한후, 이를 Western blot analysis에적용하여 BoNT/A-binding efficacy 가높은 3개의 clone (16F9, 24D1 및 28B3) 을선별하였다. 선별된 3개의 clone에대한마우스복수액을제조한후이를 capture ELISA에적용하여각각의 antigen capture ability를조사하였다. Capture ELISA를위한 MAb는가장높은 antigen capture ability를가진마우스복수액 (16F9) 으로부터 protein G affinity column (Amercham Biosciences) 을이용하여정제한후 monoclonal antibody isotyping kit (HRP/ABTS) (Pierce, Rockford, IL, USA) 를이용하여 MAb 의 IgG subclass를결정하였다. 민감도개선을위한 biotinylation은 1.5 mg의 MAb를 1 ml의 PBS에용해시킨후 0.02 ml의 10 mm NHS-biotin (Pierce) 과혼합하여 ice에서 2시간방치함으로써수행하였으며, 20 mm ammonium acetate로투석하여결합하지않은 biotin을제거한다음 biotinylated antibody를 0.1 mg 씩 aliquot하여 -20 에보관하였다. 4. Capture ELISA BoNT/A에대한 capture ELISA법은토끼 PAb를 capture antibody로, biotinylated MAb를 detection antibody로사용하여개발하였다. 먼저, 토끼 PAb를 coating buffer (0.1 M sodium carbonate, ph 9.6) 에 2 μg/ml이되도록희석하여 well당 100 μl씩분주하여 4 에하룻밤방치한후, PAbcoated plate를 tween 20이첨가된 PBS (tween 20-PBS) 로 3회세척하였다. 1% BSA를 well당 200 μl씩첨가하여 37 에서 1시간반응시킨다음, tween 20-PBS로다시 3회세척하였다. BoNT/A는 tween 20-PBS 또는 10% normal human serum에 2-fold dilutions하여 100 μl를첨가한후 37 에서 1시간반응시켰다. BoNT/A의탐지를위하여 biotinylated MAb (2 μg/ml) 및 neutravidin-linked alkaline phosphatase (2,000:1, Pierce) 를순서대로 well 당 100 μl씩 분주하여 37 에서 1시간반응시킨다음, 2 mg/ml 2-NPP (Sigma) 를이용하여 30분간발색시킨후 405 nm에서흡광도를측정하였다. BoNT/A에대한 detection limit는 [ 대조군평균값 (3 human sera (10%)) + 2 standard deviation] 를 cut-off로하여결정하였으며, 본실험은 intra-day 및 inter-day에각각 3회이상반복하여재현성및반복성을확인하였다. Capture ELISA법의보툴리눔독소 B형 (BoNT/B) 및 E형 (BoNT/E) 에대한 cross-reactivity는 BoNT/A 대신 Wako사 (Osaka, Japan) 에서구입한 BoNT/B 및 BoNT/E를 2-fold dilutions하여적용함으로써확인하였으며, 실제사람혈청에서의독소탐지여부는 3명의서로다른 10% human normal serum에독소를 31.25, 3.906, ng/ml이되도록첨가한후본방법에적용함으로써확인하였다. 마지막으로 LD 50 와의독소탐지능비교를위하여 Wako 사에서구입한독소, ATCC19397 및 ATCC3502로부터정제한독소를재료및방법 1에서제시한방법과같이 LD 50 를결정한다음본방법에적용하여 detection limit 를비교하였다. 결과 1. Capture ELISA 를위한 standard curve 및 detection limit 결정 BoNT/A 탐지를위한 capture ELISA는토끼 PAb를 capture antibody로, biotin-conjugated MAb를 detection antibody로사용하여개발하였다. 토끼 PAb를정제하기위한토끼항혈청의경우 mouse bioassay를통해 BoNT/A 에대한중화능을보유하고있음을확인하였다. Detection antibody로사용된 MAb는 BoNT/A 복합체중약 38 kda 의 accessory protein에결합하였으며, IgG isotyping test 결과 IgG1 subclass로분류되었다. 또한 PAb 및 MAb에대한 Western blot 결과 BoNT/B 및 BoNT/E에대한교차반응없이 BoNT/A에특이적으로결합함을확인하였다. 본연구에서개발한 capture ELISA는 BoNT/A의농도범위 0~500 ng/ml에대하여적정곡선을나타냈으며 (Fig. 1A) 특히, 0에서 ng/ml 범위에서는직선회귀곡선 ( 상관계수 (r 2 ) = ~0.9999) 을나타냄으로써보툴리눔독소량에대하여비례적으로반응함을확인하였다 (Fig. 1B). 또한 BoNT/A ( mouse i.p. LD 50 /mg) 에대한 detection limit를 [ 대조군평균값 (3 human sera (10%)) + 2

4 122 Y J Kim, et al. standard deviation] 를 cut-off로하여결정한결과 ng/ ml의독소를탐지할수있었다. 2. Inter- 및 intra-assay variation 새로개발한 capture ELISA법의독소탐지에대한실험반복성 (repeatability) 및재현성 (reproducibility) 을확인하기위하여 BoNT/A에대하여 1회실험당 3 replicates (intra-day) 으로 3회 (inter-day) 실험을반복실시하였으며, 그결과는 Table 1과같다. 즉, BoNT/A의농도 0부터 ng/ml에대한 inter- 및 intra-assay variation은흡광도 A (OD 405 ) 절대값의 1~11% 범위로매우안정적인결과를나타내었다. 3. BoNT/B 및 BoNT/E에대한 cross-reactivity BoNT/A에대한 capture ELISA가다른혈청형의독소에대해서 cross-reactivity가있는지조사하였다. Crossreactivity는사람보툴리눔중독증과주로관련된독소혈청형인 BoNT/B 및 BoNT/E에대하여확인하였으며, 0~31.25 ng/ml의각각의독소농도범위에대한흡광도 (OD 405 ) 값은 ~ 범위로서보툴리눔 A형독소 0 ng/ml에서분석된흡광도값인 ~0.1561과차이가없었다 (Fig. 2). 즉, 보툴리눔 A형독소만을특이적으로탐지할수있는것으로확인하였다. 4. BoNT/A-spiking human serum을이용한 capture ELISA Capture ELISA법이환자혈청으로부터의독소탐지능이있는지확인하기위하여 10% normal human serum에인위적으로보툴리눔 A형독소 31.25, 3.91, 0.488, 0 ng/ml B Figure 1. Capture ELISA for detection of BoNT/A. The ELISA was performed with a serial of 2-fold dilutions of BoNT/A ranging from 0 to 500 ng/ml (A) and 0 to ng/ml (B), which were recognized by 2 μg/ml of a biotinylated anti-bont/a MAb. Figure 2. Specificity of the capture ELISA for BoNT/A detection. The ELISA was performed with a serial of 2-fold dilutions of BoNT/X (A, B, and E) ranging from 0 to ng/ml. Table 1. Intra-(reproducibility) and inter-assay variability (repeatability) of the BoNT/A ELISA's. Results are expressed as OD 405 (± standard deviation) Intra variability a (±0.0513) Inter -variability b (±0.1431) ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml mean r 2 c (±0.0312) (±0.0245) (±0.0531) (±0.0151) (±0.0486) (±0.0020) (±0.0070) (±0.0038) (±0.0227) (±0.0043) (±0.0029) (±0.0050) a To measure intra-assay variability, three independent assays were performed by the same operator in a single day. b To measure inter-assay variability, three independent assays were performed on separate days by the same operator. c Correlation coefficients (r 2 ) describe the linear regression lines to the standard curves (±0.0049) (±0.0040) (±0.0004) (±0.0026)

5 Development of Capture ELISA for BoNT/A Detection 123 Table 2. Comparison of the sensitivity between capture ELISA and mouse LD 50 for BoNT/As prepared from various origins BoNT/A originated from Mouse i.p. LD 50 /mg Detection limit by capture ELISA (ng/ml) Conversion of sensitivity of capture ELISA to LD 50 Wako Co. (commercial) 2.00 * LD 50 ATCC3502 (in this study) 2.70 * LD 50 ATCC19397 (in this study) 5.00 * LD 50 Figure 3. Detection of BoNT/A in 10% human serum by capture ELISA. Three different 10% human serum in assay buffer was spiked with BoNT/A at final concentrations of 32.25, and ng/ml. Results are expressed as OD 405 (± standard deviation). Correlation coefficients (r 2 ) describe the linear regression lines to the standard curves. The r 2 value was determined as (±0.0026) and (±0.0002) for assay buffer and 10% human serum, respectively. 를각각첨가하여 capture ELISA를수행하였으며, 그결과 assay buffer만으로실험하여설정한 standard curve와매우유사한양상을나타내었다 (p<0.05) (Fig. 3). 5. Capture ELISA 와 mouse bioassay 의비교 Capture ELISA와 mouse bioassay 간민감도를비교하기위하여 Reed and Muench법 (12) 에의해 LD 50 를결정한 2 가지의서로다른 origin의 A형독소및상업적으로판매하는보툴리눔독소 (Wako사) 를사용하여 detection limit를결정하였다. 이들독소에대한 detection limit는 0.061~0.488 ng/ml로서, 마우스에서의생물활성도와유사한민감도를나타내었다 (0.165~2.441 LD 50 ) (Table 2). 고 보툴리눔독소는자연계에서생성되는가장강력한독소로서역사적으로생물학적무기로적용하기위한 찰 시도가여러차례이루어진바있다 (7). 현재보툴리눔중독증에대한국제적인표준진단법은 mouse bioassay 이며, 이방법은보툴리눔독소탐지에대한민감도 (sensitivity) 및특이도 (specificity) 가매우우수하나하나의샘플에대하여많은수의마우스가요구되고결과를얻기까지오랜시간 (3~4일이상 ) 이소요된다는점에서생물테러발생시적용하기에한계가있다. 따라서본연구에서는다량의사람혈청으로부터보툴리눔 A형독소를신속정확하게탐지할수있는 in vitro 독소스크리닝법을개발하고자하였으며, 이를위하여면역학적기법에바탕을둔 capture ELISA법을적용하고자하였다. 보툴리눔 A형독소탐지용 capture ELISA법은토끼혈중 IgG를 capture antibody로, biotin-conjugated monoclonal antibody를 detection antibody로사용함으로써개발하였다. 새로개발한 capture ELISA법을실제보툴리눔중독증환자의혈청에적용하기위해서는 standard curve를기준으로하여 BoNT/A에대한특이적탐지능을보유하고있어야하며, 실험데이터가안정적으로재현 (reproducibility) 되어야하고, 인체유래가검물로부터독소검출 (detectability) 이가능하여야한다. 이와더불어현재이용되고있는 mouse bioassay와유사한민감도 (sensitivity) 를보유하고있어야만 capture ELISA를독소스크리닝을위한수단으로서사용할수있다. 본연구에서새로개발한 capture ELISA법이위와같은조건을충족시키는지확인하기위하여먼저 capture ELISA의민감도를조사한결과, BoNT/A에대한 detection limit는 ng/ml로서 Chiao 등 (4) 이보고한 capture ELISA의 detection limit (5 ng/ml) 보다약 10배이상우수한민감도를나타내었으며, Szilagyi 등 (16) 이보고한 capture ELISA와는유사한 detection limit (0.5 ng/ml) 를나타내었다. 또한 detection antibody인 monoclonal antibody에 biotin을결합시킴으로써 capture ELISA의민감도가약 4 배증가하였으며, 이는 biotin을결합시킨항체를사용할경우민감도가증가된다는이전보고 (9) 와동일한결과

6 124 Y J Kim, et al. 이다. 또한본 capture ELISA법의독소탐지에대한반복실험결과, BoNT/A의농도 0부터 ng/ml에대한 intra-variation 및 inter-variation은흡광도 (OD 405 ) 절대값의 1~11% 로 Szilagyi 등 (16) 이보고한 capture ELISA의 variation 범위와유사한것으로나타났다. 본연구에서개발한 capture ELISA는 BoNT/A의농도 0~31.25 ng/ml 범위에서직선회귀곡선 (r 2 =0.9951~0.9999) 을나타냄으로써보툴리눔독소량에대하여비례적으로반응함을확인하였다 (Fig. 1B). 즉, 이는 capture ELISA의실험결과값 (OD 405 ) 으로부터미지의샘플에포함되어있는 BoNT/A의양을예측할수있음을의미한다. 또한 capture ELISA는실험결과를얻기까지약 8시간정도소요되며, 따라서검사당일샘플내보툴리눔 A형독소존재여부스크리닝이가능하므로 mouse bioassay의결과를얻기까지소요되는기간을단축할수있다는장점이있다. 사람에서는 7가지보툴리눔독소혈청형중 A, B 및 E형이주로보툴리눔중독증을유발하며 (15), 한국의경우 A형및 B형독소에의한보툴리눔중독증확진환자가발생한바있다 (5,17). 이들혈청형간에는 30~60% 의 sequence homology가존재하나 (10) 각혈청형에따라서로다른항원성을나타내기때문에환자에서의독소혈청형을알아야적절한항독소처치가가능하므로혈청형을확인하는것이중요하며, 따라서개발된보툴리눔 A형독소탐지법은 A형독소에대한 specificity를보유하고있어야한다. 본연구에서확립한 capture ELISA법은 0에서 ng/ml의농도범위에대하여보툴리눔독소 B 및 E형에대한교차반응없이보툴리눔 A형독소만을탐지함으로써 (Fig. 2) 보툴리눔독소 A형만을특이적으로검출할수있음을확인하였다. 보툴리눔중독증에대한 outbreak 혹은생물테러가발생하였을경우가장일반적으로의심환자의분변, 혈청및발생역학적관련성을나타내는음식물이주요실험실진단가검물로사용된다 (16). 따라서 capture ELISA 법이환자가검물로부터의독소탐지능이있는지확인하기위하여인체유래혈청이첨가된 assay buffer에인위적으로 BoNT/A를첨가하여실험을수행하였으며, 그결과 assay buffer만으로실험하여설정한 standard curve와유사한양상을나타내었다 (p<0.05) (Fig. 3). 이는본 capture ELISA법이생물테러발생시영향받은사람의혈청으로부터극미량의보툴리눔 A형독소를용이하게탐지할 수있음을보여주는결과라할수있다. 마지막으로 capture ELISA와 mouse bioassay 간민감도를 3가지의서로다른 origin의독소에대하여비교한결과, 이들독소에대한 detection limit 는 0.061~0.488 ng/ml로서마우스에서의생물활성도와유사한민감도를나타내었다 (0.165~ LD 50 ) (Table 2). 결론적으로, 본연구에서는토끼 polyclonal antibody 및 biotinylated monoclonal antibody를이용한보툴리눔 A형독소탐지용고감도스크리닝법을개발하여반복성, 재현성, 특이성및사람혈청에서의독소탐지능을확인함으로써검증하였다. 또한새로개발한 capture ELISA법이 mouse bioassay와유사한민감도를나타냄으로써향후생물테러발생시 mouse bioassay에대한대체수단으로서다량의사람혈청에대한보툴리눔 A형독소동시스크리닝에매우유용하게활용할수있을것으로판단된다. 나아가보툴리눔독소증의심환자발생시초기단계에신속정확한결과를도출해냄으로써실험실진단에대한중요한 supportive data로서도활용될수있을것으로사료된다. 참고문헌 1) Boroff D, Fleck U: Statistical analysis of a rapid in vivo method for the titration of the toxin of Clostridium botulinum. J Bacteriol 92: , ) Cai S, Singh BR, Sharma S: Botulism diagnostics: from clinical symptoms to in vitro assays. Crit Rev Microbiol 33: , ) Center for Disease Control and prevention: Botulism in the United States, pp Handbook for epidemiologists, clinicians, and laboratory workers. Center for Disease Control and Prevention, Atlanta GA ) Chiao DJ, Wey JJ, Tang SS: Monoclonal antibody-based enzyme immunoassay for detection of botulinum neurotoxin type A. Hybridoma 27: 43-47, ) Chung GT, Kang DH, Yoo CK, Choi JH, Seong WK: The first outbreak of botulism in Korea. Korean J Clin Microbiol 6: , ) Doellgast GJ, Triscott MX, Beard GA, Bottoms JD, Cheng T, Roh BH, Roman MG, Hall PA, Brown JE: Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal ampli-

7 Development of Capture ELISA for BoNT/A Detection 125 fication via enzyme-linked coagulation assay. J Clin Microbiol 31: , ) Geissler E, Moon JE van Courtland: Biological and toxin weapons: Research, development and use from the middle ages to Sipri Chemical & Biological Warfare Studies No. 18. Oxford University Press Inc, New York, ) Greenfield RA, Brown BR, Hutchins JB, Iandolo JJ, Jackson R, Slater LN, Bronze MS: Microbiological, biological, and chemical weapons of warfare and terrorism. Am J Med Sci 323: , ) Heckels JE, Virji M: Separation and purification of surface components. pp In Bacterial cell surface techniques, Hancock IC, Poxton IR (Ed), John Wiley & Sons, Chichester, England, ) Lacy DB, Stevens RC: Sequence homology and structural analysis of the clostridial neurotoxins. J Mol Biol 291: , ) Malizio CJ, Goodnough MC, Johnson EA: Purification of Clostridium botulinum type A neurotoxin. pp 27-39, Bacterial toxins: methods and protocols, Vol. 145, Holst O (Ed), Humana Press, Totowa, NJ, ) Reed LJ, Muench H: A simple method of estimating fifty percent endpoints. Am J Hyg 27: , ) Sharma SK, Ferreira JL, Eblen BS, Whiting RC: Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies. Appl Environ Microbiol 72: , ) Shone C, Wilton-Smith P, Appleton N, Hambleton P, Modi N, Gatley S, Melling J: Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay. Appl Environ Microbiol 50: 63-67, ) Simpson LL: Botulinum neurotoxin and tetanus toxin. Academic Press, San Diego, ) Szilagyi M, Rivera VR, Neal D, Merrill GA, Poli MA: Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B. Toxicon 38: , ) Yi HA, Lim JG, Lee JB, Her JH, Kim HA, Shin YE, Cho YW, Lee H, Yi SD: A familial outbreak of food-borne botulism. J Korean Neurol Assoc 22: , 2004.

Lumbar spine

Lumbar spine CT 32 111 DOI : 10.3831/KPI.2010.13.2.111 Lumbar Spine CT 32 Received : 10. 05. 23 Revised : 10. 06. 04 Accepted : 10. 06. 11 Key Words: Disc herniation, CT scan, Clinical analysis The Clinical