College of Chemistry, Chemical Engineering & Materials Science, Soochow University, Renai Road 199, Suzhou 215123, China.
School of Biology & Basic Medical Science, Soochow University, Renai Road 199, Suzhou, 215123, China.
Analyst. 2024 Jan 15;149(2):442-450. doi: 10.1039/d3an01779f.
Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN-BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC and LOD of the ELISA for ZEN were 0.37 ng mL and 0.04 ng mL, respectively, which were 1.6-308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol), the CR values of the mAb with the homologs were within 3.59%-105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46-105.48% with RSDs of 4.87-10.11%. A good correlation between ELISA () and HPLC-FLD () with the linear regression equation = 1.0589 + 1.43815 ( = 0.998, = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.
玉米赤霉烯酮（ZEN）是一种广泛存在于农产品中的最具毒性的霉菌毒素之一。本研究建立了一种灵敏的酶联免疫吸附测定法（ELISA），结合免疫亲和柱提取，用于检测食品和饲料样品中的 ZEN。首先合成了一种含有羧基的 ZEN 衍生物，然后将其与牛血清白蛋白（BSA）偶联。用形成的 ZEN-BSA 缀合物作为免疫原，生产针对 ZEN 的单克隆抗体（mAb）。成功筛选出能够分泌针对 ZEN 的抗体的杂交瘤克隆（1G5）。基于该 mAb，ELISA 法检测 ZEN 的 IC 和 LOD 分别为 0.37ng/mL 和 0.04ng/mL，分别比已发表的 ELISA 低 1.6-308.1 倍，表明该方法具有很高的灵敏度。该 mAb 与其他四种霉菌毒素（展青霉素、黄曲霉毒素 B1、脱氧雪腐镰刀菌烯醇和呕吐毒素）无交叉反应性。由于 ZEN 及其同系物（α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉烯酮、α-玉米赤霉醇、β-玉米赤霉醇）在分子结构上高度相似，该 mAb 与同系物的 CR 值在 3.59%-105.71%之间。利用大量的 mAb，通过将 mAb 固定在 Sepharose-4B 凝胶上并将其填充到 SPE 柱中，制备了免疫亲和柱。使用免疫亲和柱提取 ZEN 加标样品（玉米、小麦、饲料），并用 ELISA 和 HPLC-FLD 同时进行测定。ELISA 法测定 ZEN 加标样品的回收率为 92.46-105.48%，相对标准偏差（RSD）为 4.87-10.11%。ELISA（）和 HPLC-FLD（）之间具有良好的相关性，线性回归方程为 = 1.0589 + 1.43815（ = 0.998， = 6）。为了验证适用性，还将新开发的基于 mAb 的 ELISA 法应用于从当地市场随机采集的一些实际样品。结果证明，新生产的 mAb 基 ELISA 法是一种用于检测食品和饲料样品中 ZEN 的可行且灵敏的方法。
