比较酶联免疫吸附测定法与化学发光和比色检测法测定食品中的赭曲霉毒素 A。

Comparison of enzyme-linked immunosorbent assays with chemiluminescent and colorimetric detection for the determination of ochratoxin A in food.

机构信息

Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan.

出版信息

J Agric Food Chem. 2011 Feb 9;59(3):809-13. doi: 10.1021/jf103261u. Epub 2011 Jan 4.

PMID:21204536

Abstract

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for the determination of ochratoxin A (OTA) was developed using soybean peroxidase (SbP) in combination with 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) as a detection system. By varying the concentrations of the capture monoclonal anti-OTA antibody, a conjugate of OTA with SbP, and the composition of blocking buffers, the conditions of the immunoassay were optimized. Advantages of CL-ELISA were demonstrated by comparison with ELISA with colorimetric detection (COL-ELISA). The values of IC₁₀, IC₅₀, and working range (IC₂₀-IC₈₀) for CL-ELISA and COL-ELISA were 0.01, 0.08, and 0.02-0.3 ng/mL and 0.08, 0.58, and 0.17-2.2 ng/mL, respectively. The recovery values of CL-ELISA from three soybean spiked samples with OTA concentrations of 0.07, 0.1, and 0.15 ng/mL ranged from 72 to 125%. Determination of OTA in 21 various agricultural commodities showed that OTA in 8 examined samples was not detected by COL-ELISA. Furthermore, it was found that in 4 of these 8 samples the developed CL-ELISA determined OTA at levels from 0.96 to 4.64 ng/g.

摘要

建立了一种基于大豆过氧化物酶（SbP）结合 3-(10'-吩噻嗪基)丙烷-1-磺酸（SPTZ）和 4-吗啉吡啶（MORPH）作为检测系统的竞争化学发光酶联免疫吸附测定法（CL-ELISA），用于检测赭曲霉毒素 A（OTA）。通过改变捕获单克隆抗 OTA 抗体、OTA 与 SbP 的缀合物以及封闭缓冲液的组成，优化了免疫测定的条件。通过与比色检测酶联免疫吸附测定法（COL-ELISA）进行比较，展示了 CL-ELISA 的优势。CL-ELISA 和 COL-ELISA 的 IC₁₀、IC₅₀ 和工作范围（IC₂₀-IC₈₀）值分别为 0.01、0.08 和 0.02-0.3 ng/mL 和 0.08、0.58 和 0.17-2.2 ng/mL。从三个含有 OTA 浓度为 0.07、0.1 和 0.15 ng/mL 的大豆加标样品中，CL-ELISA 的回收率范围为 72-125%。对 21 种不同农产品中 OTA 的测定表明，COL-ELISA 未检测到 8 个检测样本中的 OTA。此外，发现开发的 CL-ELISA 在这 8 个样本中的 4 个样本中测定的 OTA 含量从 0.96 到 4.64 ng/g。