School of Biotechnology and National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Anal Biochem. 2011 Mar 1;410(1):27-33. doi: 10.1016/j.ab.2010.11.009. Epub 2010 Nov 13.
Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody's assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.
哈尔伏革酮是一种抗原生动物药物，用于治疗家禽的球虫病，这是一种由艾美耳属寄生虫引起的传染性肠道疾病。为了确保食品不含任何哈尔伏革酮残留且对人类食用安全，有必要开发一种快速方法，以便在各种基质中检测低于最大残留限量（MRL）的残留量。为了满足这一需求，我们从哈尔伏革酮免疫鸡的 RNA 构建了免疫单链可变片段（scFv）文库，并通过噬菌体展示筛选出了哈尔伏革酮特异性 scFv。从文库中分离出的最佳克隆的酶联免疫吸附测定（ELISA）检测限为 30ng/ml。然而，某些食品中哈尔伏革酮的最低 MRL 可能低至 1ng/ml，远低于所选抗体的灵敏度。然后通过轻链改组对选定的抗体进行亲和力成熟，以进一步提高抗体的检测性能。从生物淘选库中分离出的哈尔伏革酮特异性重链池与从原始预淘选库中扩增的轻链库进行组装。这导致了一个重链偏向的文库，从中分离出一种 scFv，其检测哈尔伏革酮残留的灵敏度低至 80pg/ml，比原始 scFv提高了 185 倍。这种新的链改组 scFv 被纳入了经过验证的 ELISA（根据委员会法规 2002/657/EC）中，用于灵敏检测加标处理过的鸡蛋样品中的哈尔伏革酮。
