Development and characterisation of a monoclonal antibody to detect the mycotoxin roquefortine C

Roquefortine, also known as roquefortine C (ROQC) is a fungal secondary metabolite (mycotoxin) that is produced by some of the same Penicillia as the tremorgen penitrem-A (PEN-A). The two mycotoxins have been linked to sporadic cases of toxicosis in dogs, cattle, and humans, leading some to consider ROQC as a biomarker of PEN-A. Reported here are the development of a monoclonal antibody (mAb) and associated competitive enzyme-linked immunosorbent assay (ELISA) for the screening of ROQC in extracts of nuts (nut “milks”), and dog serum. The ELISA was sensitive for ROQC, with a level of 0.117 ng ml−1 inhibiting colour development by 50% (IC50), a limit of detection of 0.026 ng ml−1, and a dynamic range (IC20 to IC80) of 0.038 to 0.289 ng ml−1 in buffer. The assay was tolerant to significant levels of methanol. Recoveries from 4 types of nut milks spiked over the range of 0.25 to 2 ng ml−1 were in the range of 83.5% to 116%. A small survey of commercial nut “milks” and “creamers” indicated 4 of 35 samples contained ROQC at levels so low that they are unlikely to be significant to human health (<0.6 ng ml−1). The assay was also applied to canine serum. Recoveries from serum spiked over the range of 0.2 to 5 ng ml−1 ranged from 98.1% to 123%. The results suggest the ELISA can be applied to the screening of food products, such as nut extracts, as well as for the screening of serum from dogs suspected to be suffering from mycotoxin-induced tremors.

罗克福汀（roquefortine），又称罗克福汀C（roquefortine C，缩写ROQC），是一种真菌次生代谢产物（mycotoxin，真菌毒素），由与产震颤毒素penitrem-A（PEN-A）的部分青霉属菌株同源的青霉所合成。这两种真菌毒素均与犬、牛及人类的散发性中毒病例相关，因此部分研究者将ROQC视为PEN-A的生物标志物。
本文报道了一种用于筛查坚果提取物（坚果"奶"）及犬血清中ROQC的单克隆抗体（monoclonal antibody，mAb）及其配套的竞争性酶联免疫吸附试验（competitive enzyme-linked immunosorbent assay，ELISA）。
该ELISA对ROQC具有高灵敏度：在缓冲液体系中，其半数抑制浓度（IC50）为0.117 ng·ml⁻¹，检测限为0.026 ng·ml⁻¹，动态范围（IC20至IC80）为0.038~0.289 ng·ml⁻¹。该检测方法可耐受较高浓度的甲醇。
向4种坚果奶中添加浓度范围为0.25~2 ng·ml⁻¹的ROQC，其加标回收率为83.5%~116%。对市售坚果"奶"及"奶精"的小型调研显示，35份样本中有4份检出ROQC，浓度均低于0.6 ng·ml⁻¹，对人类健康暂无显著危害。
该检测方法同样应用于犬血清样本：向浓度范围为0.2~5 ng·ml⁻¹的加标血清中，回收率为98.1%~123%。
研究结果表明，该ELISA可用于坚果提取物等食品的筛查，同时也可用于疑似因真菌毒素引发震颤的犬只的血清筛查。