Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University , Wuhan 430070, China.
Anal Chem. 2013 Nov 19;85(22):10992-9. doi: 10.1021/ac402608e. Epub 2013 Oct 31.
A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) μg/mL, 1000-fold more sensitive than that reported previously (1 μg/mL). The fusion protein was able to detect fungal concentrations below 1 μg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 μg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.
一种灵敏且特异的分析方法，用于检测无处不在的产毒黄曲霉属真菌，对于监测和控制黄曲霉毒素至关重要。通过噬菌体展示技术，从黄曲霉中分离出了四种针对可溶性细胞壁蛋白（SCWPs）的高反应性鸡单链可变片段（scFv）。scFv 抗体 AfSA4 对黄曲霉和寄生曲霉均显示出最高的活性，并通过免疫荧光定位特异性识别其细胞壁的表面靶标。分子建模揭示了抗体表面上独特的紧凑基序，主要涉及 L-CDR2 和 H-CDR3。表面等离子体共振测量结果表明，与单独的 AfSA4 相比，与碱性磷酸酶融合的 AfSA4 具有更高的结合能力和 6 倍的亲和力。免疫印迹分析表明，融合蛋白与来自两种真菌的 SCWP 成分具有良好的结合能力。与平行生成的作为捕获抗体的抗曲霉单克隆抗体 mAb2A8 进行的直接夹心酶联免疫吸附试验表明，两种真菌的检测限低至 10(-3) μg/mL，比之前报道的（1 μg/mL）灵敏 1000 倍。融合蛋白能够检测到玉米和花生颗粒中真菌浓度低于 1 μg/g 的污染样品，其灵敏度比目前报道的（10 μg/g）至少高 10 倍。因此，该融合蛋白可用于快速、简单、特异性地诊断田间和储存的食品/饲料中曲霉属的污染。
