黄曲霉毒素B1(AFB1),一种剧毒的霉菌毒素,总是被各种农产品污染。骆驼可变结构域的重链抗体(VHH)是免疫测定中值得注意的试剂,由于其优良的特点。骆驼动物的免疫是产生VHH的简单策略。在这项研究中,为了避免对大型动物的依赖,骆驼,基于鼠VH和骆驼VHH之间的身份,体外制备了针对AFB1的鼠抗体(cVHs),然后开发了AFB1的免疫测定法。通过替换框架区2(FR2)(cVH-FR2)中的保守疏水残基,选择鼠抗AFB1VH片段(VH-2E6)进行骆驼化,FR4区103位的点突变(cVH-103),和具有高AFB1亲和力VHH(cVH-Nb26)的CDR3接枝。cVH-Nb26作为从大肠杆菌表达的重折叠蛋白具有5mg/L的产量和从巴斯德毕赤酵母表达的10mg/L的产量。与抗AFB1单链片段可变(scFv)2E6相比,cVH-Nb26增强了AFB1结合相互作用的20倍以上。虽然cVH-Nb26的AFB1亲和力不能满足本形式的应用要求,本研究为体外制备骆驼化抗体提供了有效的策略,这可能是一种有前途的AFB1检测免疫试剂。
Aflatoxin B1 (AFB1), a highly toxic mycotoxin, always contaminated in a variety of agricultural products. Camelid variable domain of heavy chain antibody (VHH) is a noteworthy reagent in immunoassay, owing to its excellent characteristics. Immunization of camelid animals is a straightforward strategy to produce VHHs. In this study, to avoid the dependence on the large animals, the camelized, murine antibody (cVHs) against AFB1 was prepared in vitro based on the identities between murine VH and camelid VHH and then to develop an immunoassay for AFB1. A murine anti-AFB1 VH fragment (VH-2E6) was selected for camelization through replacement of conserved hydrophobic residues in framework region 2 (FR2) (cVH-FR2), point mutation at position 103 in the FR4 region (cVH-103), and CDR3-grafted with a high AFB1-affinity VHH (cVH-Nb26). The cVH-Nb26 had a yield of 5 mg/L as refolded protein expressed from Escherichia coli and 10 mg/L expressed from Pichia pastoris. Compared with anti-AFB1 single-chain fragment variable (scFv) 2E6, cVH-Nb26 performed more than 20-fold enhancement of AFB1-binding interactions. Although the AFB1-affinity of cVH-Nb26 cannot meet the application requirement in the present form, our study provides effective strategies for preparation of camelized antibody in vitro, which could be a promising immunoreagent for AFB1 detection.