The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Renai Road 199, Suzhou 215123, China.
Molecules. 2023 Jul 29;28(15):5743. doi: 10.3390/molecules28155743.
In this study, an extremely highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a newly produced monoclonal antibody (mAb) for the detection of ochratoxin A (OTA) in food samples was developed. OTA-Bovine serum albumin (BSA) conjugate was prepared and used as the immunogen for the production of the mAb. Among four hybridoma clones (8B10, 5C2, 9B7, and 5E11), the antibody from 8B10 displayed the highest affinity recognition for OTA. Based on the mAb (8B10), the IC and LOD of the ELISA for OTA were 34.8 pg mL and 1.5 pg mL, respectively, which was 1.53~147 times lower than those in published ELISAs, indicating the ultra-sensitivity of our assay. There was no cross-reactivity of the mAb with the other four mycotoxins (AFB, ZEN, DON, and T-2). Due to the high similarity in molecular structures among OTA, ochratoxin B (OTB), and ochratoxin C (OTC), the CR values of the mAb with OTB and OTC were 96.67% and 22.02%, respectively. Taking this advantage, the ELISA may be able to evaluate total ochratoxin levels in food samples. The recoveries of the ELISA for OTA in spiked samples (corn, wheat, and feed) were 96.5-110.8%, 89.5-94.4%, and 91.8-113.3%; and the RSDs were 5.2-13.6%, 8.2-13.0%, and 7.7-13.7% ( = 3), respectively. The spiked food samples (corn) were measured by ELISA and HPLC-FLD simultaneously. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation of y = 0.918x - 0.034 (R = 0.985, = 5) was obtained. These results demonstrated that the newly produced mAb-based ELISA was a feasible and ultra-sensitive analytical method for the detection of OTA in food samples.
在这项研究中，开发了一种基于新生产的单克隆抗体（mAb）用于检测食品样本中赭曲霉毒素 A（OTA）的超灵敏酶联免疫吸附测定法（ELISA）。制备了 OTA-牛血清白蛋白（BSA）缀合物，并将其用作产生 mAb 的免疫原。在四个杂交瘤克隆（8B10、5C2、9B7 和 5E11）中，抗体 8B10 对 OTA 表现出最高的亲和力识别。基于 mAb（8B10），ELISA 检测 OTA 的 IC 和 LOD 分别为 34.8 pg mL 和 1.5 pg mL，分别比已发表的 ELISA 低 1.53~147 倍，表明该测定法的超高灵敏度。该 mAb 与其他四种真菌毒素（AFB、ZEN、DON 和 T-2）没有交叉反应性。由于 OTA、赭曲霉毒素 B（OTB）和赭曲霉毒素 C（OTC）在分子结构上高度相似，mAb 与 OTB 和 OTC 的 CR 值分别为 96.67%和 22.02%。利用这一优势，ELISA 可能能够评估食品样本中的总赭曲霉毒素水平。ELISA 检测添加 OTA 的样品（玉米、小麦和饲料）的回收率分别为 96.5-110.8%、89.5-94.4%和 91.8-113.3%；RSD 分别为 5.2-13.6%、8.2-13.0%和 7.7-13.7%（n = 3）。同时通过 ELISA 和 HPLC-FLD 检测添加 OTA 的食品样品（玉米）。ELISA（x）和 HPLC-FLD（y）之间具有良好的相关性，线性回归方程为 y = 0.918x - 0.034（R = 0.985，n = 5）。这些结果表明，基于新生产的 mAb 的 ELISA 是一种可行的超灵敏分析方法，可用于检测食品样本中的 OTA。
