利用SD大鼠抗血清建立瘦肉精ciELISA检测方法的研究

以克伦特罗(CL)为竞争半抗原,人工合成的克伦特罗-卵清白蛋白(CL-OVA)为包被抗原,两者与一定量的SD大鼠抗CL血清反应,建立快速定量测定CL含量的间接竞争酶联免疫吸附检测(ciELISA)方法。结果表明,理想的包被抗原质量浓度为3μg/mL,抗CL血清稀释倍数为1∶1.6×105。绘制出标准曲线,线性检测范围为4.88~5 000ng/mL,线性回归方程为y=-0.295 2x+1.160 6,r2=0.990 2,加标平均回收率为95.45%。该方法敏感性高,特异性强,操作简便,对于研制瘦肉精残留检测试剂盒有重要意义。     

Development of a polyclonal antibody-based indirect competitive ELISA for determination of sterigmatocystin in wheat and corn flours

Sterigmatocystin (STC) is a toxic secondary metabolite produced by more than 50 fungal species, including Aspergillus flavus, A. parasiticus, A. nidulans, and A. versicolor. The Joint FAO/WHO Expert Committee on Food Additives concluded that sterigmatocystin is genotoxic and carcinogenic with the critical effect determined to be carcinogenicity. The present study describes a simple method to prepare hapten and immunogens in order to generate polyclonal antibodies against this metabolite. We developed a sensitive and specific polyclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ciELISA) for monitoring STC in wheat and corn flours without the need for derivatisation of STC or clean-up of samples by immunoaffinity chromatography for quantification. The half inhibitory concentration (IC₅₀) of the established method was 4.52 ± 0.81 ng mL^?1, with the limit of detection (IC₁₀) being 0.19 ± 0.04 ng mL^?1 in wheat and corn flour matrices with the coefficient of variation of less than 22%.The assay was very specific to STC and showed no cross-reactivity with its analogue structures. The ELISA allowed for up to 5% methanol without significant influence on the IC₅₀ value. Validation of the assay was performed by spiking STC into a blank flour matrix and the recoveries were in the range of 75.3 % to 104.5 % with a coefficient of variation less than 15%. A small retail survey was conducted by purchasing wheat (n = 8) and corn flours (n = 2) from local grocery stores. All of these retail samples were negative for STC using the developed ELISA method and were confirmed by LC-MS/MS. We demonstrated a rapid, simple, and reliable method for screening STC in wheat and corn flours.     

Development of a sensitive polyclonal antibody-based competitive indirect ELISA for determination of citrinin in grain-based foods

ABSTRACT

A sensitive competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for the detection and quantification of citrinin (CIT) in grain-based food samples. The limit of quantification (IC₂₀) of the established method was 0.10 ± 0.02 ng mL^?1, with the limit of detection (IC₁₀) being 0.04 ± 0.007 ng mL^?1 in wheat and corn flour matrices with a coefficient of variation (CV) less than 20%. The assay was very specific to CIT and showed no cross-reactivity with other mycotoxins (OTA, T-2 toxin, HT-2 toxin, DON, patulin and zearalenone). In spiked wheat and corn flours, the recoveries ranged from 86.6% to 115.6% with CVs of less than 20%. The effectiveness of this method was verified by participating in a proficiency test (PT) from the Food Analysis Performance Assessment Scheme (FAPAS) 17181 corn flour. A successful z-score (?0.6) for this PT sample showed that the present method is comparable to the instrumental methods used by other laboratories in the PT testing scheme. A small survey of grain-based foods was conducted using this method and CIT was detected in 43% of the samples up to a concentration of 17.7 ng g^?1. This method is suitable for sensitive and rapid quantitation of citrinin in wheat and corn matrices.     

己烯雌酚残留检测ELISA试剂盒的研制及应用

在实验室前期制备的己烯雌酚多克隆抗体基础上建立间接竞争ELISA检测方法,研制出己烯雌酚残留ELISA快速检测试剂盒,并对其性能参数进行测定。结果表明,所选用的抗体对己烯雌酚亲和力高,除了与双烯雌酚、己烷雌酚的交叉反应率分别为6.83%和2.56%外,与其他结构类似物或功能类似物交叉反应率均小于0.01%;己烯雌酚残留检测的标准曲线为y=-28.71x+59.37,R2为0.9895,线性范围为0.08~8.17ng/mL,检测限IC10为0.05ng/mL,灵敏度IC50为1.09ng/mL,样品的加标回收率为80.04%~107.91%,平均批内和批间变异系数分别为6.41%和8.83%;与HPLC测定方法比对具有较高的相符性,相关系数R2为0.9882;该试剂盒在4℃下至少可保存6个月,可用于批量猪肉、鸡肉、鱼肉、饲料等样品中己烯雌酚残留的检测。