免疫亲和层析技术协同酶联免疫吸附法检测赭曲霉毒素A

Detection of Ochratoxin A Using Immunoaffinity Chromatography Combined with Enzyme-Linked Immunosorbent Assay

利用免疫亲和层析技术(immunoaffinity chromatography,IAC)对样品中赭曲霉毒素A(ochratoxin A,OTA)进行捕获与浓缩,利用酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法对IAC捕获的目标物OTA进行测定。IAC与ELISA中的抗OTA单克隆抗体来自不同的克隆株,分属不同独特型,与OTA结合靶点的选择性存在差异,IAC与ELISA协同检测,可以有效过滤OTA结构类似物造成的干扰,提高免疫分析的特异性与灵敏度。该方法用于加标样品测定时,OTA的检出限为0.2 ng/g,定量限为0.4 ng/g,OTA的平均回收率为75.9%,与单一的ELISA法相比,该方法虽然回收率略低,但灵敏度可以显著提高,达到ELISA法的60倍。通过对49份样品的实际检测,该方法检出阳性样品与国标法的符合率达到100%,漏检率为0%,由此可见,该方法在准确性上表现出明显的优势。作为一种综合免疫分析技术,该方法不需要大型仪器设备,对操作环境没有严格要求,便于基层实验室对样品中OTA的分析。

Ochratoxin A(OTA)in samples was captured and concentrated by immunoaffinity chromatography(IAC)and detected by enzyme-linked immunosorbent assay(ELISA).The anti-OTA monoclonal antibodies used in IAC and ELISA came from different clone strains,belonging to different idiotypes,and there were differences between their selectivity for binding to OTA targets.The combined use of IAC and ELISA could effectively filter out the interference from OTA analogues,thus improving the specificity and sensitivity of immunological analysis.The limit of detection(LOD)for OTA in spiked samples was 0.2 ng/g,the quantification limit was 0.4 ng/g,and the average recovery was 75.9%.Although the recovery of the IAC-ELISA method was slightly lower than that of ELISA,the sensitivity was increased by 60 times.For 49 real samples,the proportion of samples that tested positive by this combined method was 100%consistent with the result of the national standard method,with a missed detection rate of 0%.Therefore,the developed method exhibited significant advantages in accuracy.As a comprehensive immunoassay,this method does not require any large-scale instruments or equipment,and has no strict requirement on the operating environment,thereby making it easy for grassroot laboratories to analyze OTA in samples.