Development of Enzyme Linked Immunosorbent Assay for Determination of Zearalenone in Animal Feeds

가축사료 중 Zearalenone 분석을 위한 Enzyme Linked Immunosorbent Assay법의 개발

  • Published : 1991.12.01

Abstract

We examined to develop the enzyme linked immunosorbent assay (ELISA) for determination of zearalenone in animal feeds. Zearalenone was first converted to 6'-(carboxymethyl) zearalenone oxime(zearalenone oxime) to get a coupling site and then conjugated to bovine serum albumin(BSA) for use as immunogen and to horseradish peroxidase(HRP) for use as enzyme marker. Antibody against zearalenone was obtained after 11 weeks of immunization of rabbit with zearalenone oxime-BSA. Cross reactivity of the antibody with ${\alpha}-zearalanol,\;{\beta}-zearalenol,\;{\alpha}-zearalanol\;and\;{\beta}-zearalanol$ were 168, 46, 26 and 20% respectiviely. A simple procedure was devised for the screening of zearalenone in feeds using ELISA. Feeds samples(5g) were extracted by blending with 25 ml of methanol-phospate butTered saline-dimethylformate(70 : 29 : 1) and the extract was filtered and aqueous filterate analyzed. It took only 1 hours to do whole procedure for the analysis of zearalenone in feeds by the direct competitive ELISA, and detectable limit was 1-100 ppb. Using this procedure, only 4 of 24 feed samples showed positive results with 3.93-7.43 ppb levels.

가축사료중 zearalenone 분석을 위한 enzyme linked immunosorbent assay(ELISA) 법의 개발을 위해 우선 zearalenone의 항원성을 증폭시키기 위해 zearalenone oxime 유도체를 합성한 다음 bovine serum albumin(BSA)와 conjugate를 만들고, 이를 항원으로 토끼에 면역시켜 11주에 zearalenone에 특이한 항체를 얻어냈다. 생성된 항체를 zearalenone외에 ${\alpha}-zearalenol$과는 강한 cross reactivity를 나타내었고 ${\beta}-zearalenol,\;{\alpha}-zearalenol\;및\;{\beta}-zearalenol$과는 약간의 반응을 보였으며 확립된 ELISA 조건은 당므과 같다. 먼저 시료를 methanol-phosphate buffered saline-dimethyl formate(70 : 29: 1)을 4배 첨가하여 blending 한 다음 Whatman No. 4를 통한 여액을 ELISA시료로 사용하였다. 효소 반응시간과 발색시간은 각각 $37^{\circ}C$에서 30분과 15분이었고, 흡광도는 410nm에서 ELISA reader로서 측정하였으며, 측정한계는 1~100 ppb로 매우 낮았다. 확립된 ELISA 조건으로 실제시료의 zearalenone오염도는 측정결과 24개 시료 중 4개의 시료가 양성반응을 보였고 그 함량범위는 $3.93~7.43\;\mu\textrm{g}/kg$이었다.

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