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Development of Direct Competitive Enzyme-Linked Immunosorbent Assay using Monoclonal Antibody (MAb) against Sulfamthazine (SMZ) and Establishment of Application Condition for Milk Sample

Shim, Won-Bo (Division of Applied Life Science(Brain Korea 21 program), Graduate School of Gyeongsang National University)
Mun, Chun-Sun (Food & Risk Standarization Team, The Bureau of Risk Management, KFDA)
Kim, Jung-Sook (Division of Applied Life Science(Brain Korea 21 program), Graduate School of Gyeongsang National University)
Choe, Ju-Mi (Division of Applied Life Science(Brain Korea 21 program), Graduate School of Gyeongsang National University)
Kim, Ji-Hun (National Institute of Agricultural Science and Technology, Rural Development Administration)
Park, Seon-Ja (Division of Applied Life Science(Brain Korea 21 program), Graduate School of Gyeongsang National University)
Kang, Sung-Jo (Division of Applied Life Science(Brain Korea 21 program), Graduate School of Gyeongsang National University)
Chung, Duck-Hwa (Division of Applied Life Science(Brain Korea 21 program), Graduate School of Gyeongsang National University)

Publication Information

Korean Journal of Food Science and Technology / v.38, no.2, 2006 , pp. 176-182 More about this Journal

Abstract

Sensitive and specific monoclonal antibody (MAb) was produced from hybridoma (1H11-5) obtained by fusion of myeloma cell (V653) and spleen cell isolated from mouse immunized sulfamthazine (SMZ)-HG-KLH. Direct competitive ELISA was developed for rapid detection of SMZ in milk samples using MAb against SMZ with optimized conditions between MAb and SMZ-HG-HRP conjugate, and applicable conditions for analysis of milk samples were established. Detection range of immunoassay was 0.1 to 100 ppb. Recoveries from spiked raw milk and processed milk samples averaged 82.1-120.7 and 82.1-97.1%, respectively.

본 연구는 축산농가에서 가축의 세균성 질병의 예방과 치료 및 성장촉진을 목적으로 주로 사용되고 있으며, 축산물과 유가공품에 잔류 가능성이 높은 sulfamethazine(SMZ)을 검출할 수 있는 직접경쟁 효소면역분석법(direct competitive ELISA)의 개발과 이를원유 및 시판우유 분석에 이용하는 것을 목적으로 하였다. 면역항원의 합성은 hemiglutarate와 hemisuccinate를 이용하여 SMZ-hemiglutarate(SMZ-HG)와 SMZ-hemisuccinate(SMZ-HS) hapten을 유도한 후 단백질 KLH와 STI를 결합시켜 마우스 면역에 사용하였고, SMZ-HG-KLH를 이용하여 면역한 마우스에서 가장 높은항체 생성정도를 나타내었다. 세포융합과 cloning을 실시하여 총15종의 hybridoma cell line을 확보하였고 그 중 가장 경쟁성이 뛰어나고 민감도가 높은 1H11-5 hybridoma를 선택하고 대량 생산하였다. 생산된 항체는 sulfamethazine에만 54%의 교차 반응성을 나타내었고, 다른 설파계 항생제와는 반응을 하지 않는 특이성이 높은 항체로 확인되었고 이를 이용하여 확립된 direct competitive ELISA법은 검출범위가 0.1-100 ppb 수준으로 기존에 보고된 ELISA보다 민감도가 높았다. 민감도와 특이성이 높은 direct competitive ELISA를 이용하여 원유와 시판우유를 분석하기 위한 전처리법을 확립하여 회수율을 확인한 결과 원유의 경우 82-121%까지 회수가 되는 것으로 나타났으며, 시판유의 경우는 82-97%까지 회수가 되는 것으로 나타나 우유 샘플 중에 미량 잔류하는 SMZ를 신속하고 정확하게 분석할 수 있을 것으로 사료되었다. 이러한 연구결과를 종합해 볼 때 확립된 direct competitive ELISA법은 우유 시료뿐만 아니라 모든 축산물에 잔류 가능성이 높은 SMZ의 분석을 신속하고 정확하게 분석이 가능할 것으로 사료되었고 확립된 direct competitive ELISA법의 안정화 조건을 확립하고 응용한다면 외국으로부터 생산 및 수입되는 ELISA kit을 대체할 수 있는 저가형 국산화 ELISA kit의 상용화가 가능할것으로 사료되었다.

Keywords

sulfamethazine; antibiotic; monoclonal antibody; ELISA; milk;

Citations & Related Records

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