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基于单克隆抗体的 ELISA 和侧向流动免疫分析法定量检测赭曲霉毒素（OTA 和 OTB）的研究

Development of ELISA and Lateral Flow Immunoassays for Ochratoxins (OTA and OTB) Detection Based on Monoclonal Antibody.

机构信息

Fujian Key Laboratory of Pathogenic Fungi and Mycotoxins, Fujian Agriculture and Forestry University, Fuzhou, China.

School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.

出版信息

Front Cell Infect Microbiol. 2020 Mar 6;10:80. doi: 10.3389/fcimb.2020.00080. eCollection 2020.

DOI:10.3389/fcimb.2020.00080
PMID:32211342
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7067699/
Abstract

Ochratoxins were important secondary metabolites secreted by fungi, and OTA and OTB are mainly significant mycotoxin, having toxic effects on humans and animals. Therefore, it is important to establish a rapid, sensitive, and precise method for ochratoxins detection and quantification in real samples. In this study, a stable monoclonal antibody (mAb) that recognizing both OTA and OTB toxins was employed for the establishment of indirect competitive ELISA (ic-ELISA), colloidal gold nanoparticles (CGNs), and nanoflowers gold strips (AuNFs) for detection of ochratoxins in real samples. A 6E5 hybridoma cell line stable secreting mAb against both OTA and OTB toxins was obtained by fusion of splenocytes with myeloma SP2/0 cells. The 6E5 mAb had a high affinity (3.7 × 10 L/mol) to OTA, and also showed similar binding activity to OTB. The optimized ic-ELISA resulted in a linear range of 0.06-0.6 ng/mL for ochratoxins (OTA and OTB) detection. The IC50 was 0.2 ng/mL and the limit of detection (LOD) was 0.03 ng/mL. The mean recovery rate from the spiked samples was 89.315 ± 2.257%, with a coefficient variation of 2.182%. The result from lateral flow immunoassays indicated that the LOD of CGNs and AuNFs were 5 and 1 μg/mL, respectively. All these results indicated that the developed ic-ELISA, CGNs, and AuNFs in this study could be used for the analysis of the residual of ochratoxins (OTA and OTB) in food and agricultural products.

摘要

赭曲霉毒素是真菌分泌的重要次级代谢产物，OTA 和 OTB 是主要的真菌毒素，对人类和动物具有毒性作用。因此，建立一种快速、灵敏、准确的方法来检测和定量实际样品中的赭曲霉毒素非常重要。在本研究中，采用了一种能够同时识别 OTA 和 OTB 毒素的稳定单克隆抗体（mAb），用于建立间接竞争 ELISA（ic-ELISA）、胶体金纳米粒子（CGNs）和纳米金花金条（AuNFs），以检测实际样品中的赭曲霉毒素。通过脾细胞与骨髓瘤 SP2/0 细胞融合，获得了一株稳定分泌针对 OTA 和 OTB 毒素的 mAb 的 6E5 杂交瘤细胞系。6E5 mAb 对 OTA 具有高亲和力（3.7×10 L/mol），对 OTB 也表现出相似的结合活性。优化后的 ic-ELISA 对 OTA 和 OTB 的检测线性范围为 0.06-0.6 ng/mL。IC50 为 0.2 ng/mL，检测限（LOD）为 0.03 ng/mL。从加标样品中回收的平均回收率为 89.315±2.257%，变异系数为 2.182%。侧向流动免疫测定的结果表明，CGNs 和 AuNFs 的 LOD 分别为 5 和 1 μg/mL。所有这些结果表明，本研究中开发的 ic-ELISA、CGNs 和 AuNFs 可用于分析食品和农产品中残留的赭曲霉毒素（OTA 和 OTB）。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/e4acff0d380b/fcimb-10-00080-g0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/6e3f47f4a822/fcimb-10-00080-g0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/6c0d3538451b/fcimb-10-00080-g0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/a3c1bcf9fc31/fcimb-10-00080-g0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/30d9f2c7985a/fcimb-10-00080-g0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/db3a9ec26d64/fcimb-10-00080-g0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/e4acff0d380b/fcimb-10-00080-g0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/6e3f47f4a822/fcimb-10-00080-g0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/6c0d3538451b/fcimb-10-00080-g0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/a3c1bcf9fc31/fcimb-10-00080-g0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/30d9f2c7985a/fcimb-10-00080-g0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/db3a9ec26d64/fcimb-10-00080-g0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8a94/7067699/e4acff0d380b/fcimb-10-00080-g0006.jpg

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