Fusarium속이 생성하는 zearalenone 측정을 위한 Indirect Competitive ELISA의 확립

Establishment of Indirect Competitive ELISA for the Detection of Zearalenone Produced by Fusarium sp.

Zearalenone 검출을 위하여 Z-M-26 hybridoma cell을 mouse의 복강에 투여한 후 생산, 정제한 항체와 합성한 Zearalenone-oxime-OVA conjugate를 이용하여 ELISA법을 확립하였다. Carbonyl buffer로 희석한 Zearalenone-oxime-OVA conjugate를 4$^{\circ}C$에서 하룻밤 coating 하고 1% BSA용액으로 하룻밤 blocking한 다음, 1,000배 희석한 항체를 Zearalenone 또는 시료와 혼합하여 하룻밤 반응시키는 것이 효과적이었다. 또한 2차 항체와 기질용액의 반응시간은 각각 1시간, 30분이 적당하였고, 발색된 반응액 450nm에서 측정하였다. 이 분석법의 결과 0.1~100 ppb의 Zearalenone이 측정 가능하였으며, 본 실험에서 확립한 indirect competitive ELISA법은 농산물 중 Zearalenone 분석에 효과적으로 활용할 수 있으리라 생각된다.

An enzyme-linked immunosorbent assay (ELISA) was established for the detection of zearalenone by using monoclonal antibodies produced by Z-M-26 hybridoma cells when injected into a mouse and zearalenone-oxime-OV A conjugate. Zearalenone-oxime-OV A conjugates were diluted with carbonyl buffer, coated to 96 well microtiter plates at $4^{\circ}C$ overnight and blocked with 1% BSA overnight. One thousand times diluted antibody solution together with standard zearalenone or sample was added to 96-well microtiter plates and stood overnight. A secondary antibody conjugated with HRP was added and an hour later, enzyme substrate (TMBZ) solution was added for color develpment. Mter 30 minutes, coloring reaction was terminated by adding 2 N $H_2S0_4$ and the O.D. was measured at 450 nm. Detection range of this method was about 0.1~100 ppb. The established indirect competitive ELISA method was suitable for a rapid and effective analysis of zearalenone in agricultural products.