时间分辨荧光免疫层析法定量检测谷物中黄曲霉毒素B1和玉米赤霉烯酮残留

建立了一种快速定量检测谷物产品中黄曲霉毒素(Aflatoxin B1,AFB1)和玉米赤霉烯酮(Zearalenone,ZEN)的时间分辨荧光免疫层析方法。采用时间分辨荧光微球标记黄曲霉毒素B1抗体和玉米赤霉烯酮抗体,研究了如p H值、标记抗体浓度、荧光探针使用量、检测T线包被原浓度、质控C线羊抗鼠Ig G浓度、样品前处理方法等因素对时间分辨荧光免疫层析方法灵敏度的影响。结果表明:AFB1的检出限为0.80 ng/mL,线性范围(IC20~IC80)为0.81~5.67 ng/mL,半抑制浓度(IC50)为2.15 ng/mL。在ZEN检出限为4.58 ng/mL,线性范围(IC20~IC80)为4.76~85.60 ng/mL,半抑制浓度(IC50)为20.19 ng/mL。方法特异性良好,与T-2毒素、脱氧雪腐镰刀菌烯醇、伏马毒素、赭曲霉毒素A多种真菌毒素交叉率小于10%。通过选择玉米、麦麸、大豆、小麦进行添加回收试验,AFB1的添加回收率在97.1%~108.7%之间,ZEN的添加回收率在92.8%~109.1%之间,相对标准偏差小于15%。选取经HPLC-MS/MS检测过的FAPAS标准质控样本进行测试,检测结果与其结果一致。在实际产品检测对比中,与市售胶体金免疫层析卡,ELISA试剂盒的检测结果基本一致。本方法操作简单快速、可定量,检测过程约25 min,适用于谷物样品中黄曲霉毒素B1和玉米赤霉烯酮的现场快速筛。

Development of a Time-Resolved Fluorescence Immunochromatographic Assay for the Quantitative Determination of AFB1 and ZEN in Grain

A time-resolved fluorescence immunochromatographic assay for the quantitative determination of AFB1 and ZEN in grain was developed.Anti-AFB1 and anti-ZEN were labelled with time-resolved fluorescent microsphere as tracers.The assay conditions,including pH value of labelling,concentrations of antibody for labeling,concentration of the coating antigen(T line)and the goat-anti-mouse IgG(C line),as well as the sample pretreatment were optimized.Under the optimal condition,the assay showed the AFB1 limit of detection and detection range were 0.80 ng/mL and 0.81~5.67 ng/mL,respectively;the AFB1 IC50 was 2.15 ng/mL;the assay showed the ZEN limit of detection and detection range were 4.58 ng/mL and 4.76~85.60 ng/mL,respectively,the ZEN IC50 was 20.19 ng/mL.The cross-reaction rates with T-2、DON、FB1、OTA were less than 10%,indicating a good specificity of the proposed assay.Recovery test was done by using maize,soybean,wheat bran and wheat sample.The AFB1 recoveries ranged from 97.10%to 108.7%with RSDs below 15%and the ZEN recoveries ranged from 92.8%to 109.1%with RSDs below 15%.The results of the FAPAS had a good agreement with those of UPLC-MS/MS.The results had a good agreement with colloidal gold immunochromatography and ELISA.In conclusion,theproposed assay was convenient、rapid、accurate and sensitive.The detection process is about 25 min.It is suitable for rapid screening of AFB1 and ZEN in grain samples.