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Colorimetric ELISA with an acid–base indicator for sensitive detection of ochratoxin A in corn samples†
Analytical Methods ( IF 3.1 ) Pub Date : 2017-11-16 00:00:00 , DOI: 10.1039/c7ay01959a
Ke Pei 1, 2, 3, 4, 5 , Ying Xiong 1, 2, 3, 4, 5 , Xiangmin Li 2, 3, 4, 5 , Hu Jiang 2, 3, 4, 5 , Yonghua Xiong 1, 2, 3, 4, 5
Affiliation  

In this study, a direct competitive enzyme-linked immunosorbent assay (dcELISA) with an acid–base indicator was developed for the naked-eye detection of ochratoxin A (OTA). OTA-labeled urease conjugates were used as competing antigens, and the acid–base indicator of bromocresol purple (BCP) dye was employed as the signal reporter. When the competing antigen was captured by the anti-OTA antibody on the plate well, the urea substrate was catalyzed to generate NH3 molecules. The BCP dye produced a vivid color change from purple to light yellow because of a slight pH change in the solution, and hence, the indicator was suitable for the naked-eye detection of analytes. Under optimized conditions, the proposed method exhibited a high sensitivity for OTA naked-eye detection with a cutoff limit of 50 pg mL−1. The method also showed a good linear range of 12.5–100 pg mL−1 for OTA quantitative detection, with a half maximal inhibitory concentration of 31.4 pg mL−1, which is 11-fold lower than that of conventional HRP-based ELISA (IC50 = 354 pg mL−1). The reliability of the proposed colorimetric ELISA method for naked-eye detection indicated no false negative and only three false positive results from 70 tests. Intra- and inter-assays showed that the average recovery ranged from 83.7% to 111%, and the coefficient of variation (CV) ranged from 3.71% to 13.5%. The proposed method exhibited acceptable accuracy and precision for the quantitative detection of OTA in real corn samples. In summary, the developed acid–base indicator-based dcELISA offers a promising platform for resource-constrained countries for highly sensitive naked-eye detection of haptens.

中文翻译:

带有酸碱指示剂的比色ELISA用于玉米样品中och曲霉毒素A的灵敏检测

在这项研究中,开发了一种具有酸碱指示剂的直接竞争性酶联免疫吸附测定法(dcELISA),用于肉眼检测曲霉毒素A(OTA)。OTA标记的脲酶共轭物被用作竞争性抗原,溴甲酚紫(BCP)染料的酸碱指示剂被用作信号报告基因。当竞争抗原被抗OTA抗体捕获在平板孔上时,尿素底物被催化生成NH 3分子。由于溶液中的pH值发生轻微变化,BCP染料产生了从紫色到浅黄色的生动颜色变化,因此该指示剂适用于肉眼检测分析物。在优化的条件下,所提出的方法对OTA肉眼检测具有很高的灵敏度,截止限为50 pg / mL-1。该方法还显示了用于OTA定量检测的12.5–100 pg mL -1的良好线性范围,最大抑制浓度的一半为31.4 pg mL -1,比常规基于HRP的ELISA(IC)低11倍50 = 354 pg毫升-1)。所提出的用于肉眼检测的比色ELISA方法的可靠性表明,在70个测试中没有假阴性,只有3个假阳性结果。批内和批间分析表明,平均回收率在83.7%至111%之间,变异系数(CV)在3.71%至13.5%之间。所提出的方法对真实玉米样品中OTA的定量检测显示出可接受的准确性和精密度。总而言之,已开发的基于酸碱指示剂的dcELISA为资源有限的国家提供了一个有前途的平台,可对半抗原进行高灵敏度的裸眼检测。
更新日期:2017-11-16
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