Alvarez-Rueda Nidia, Behar Ghislaine, Ferré Virginie, Pugnière Martine, Roquet Françoise, Gastinel Louis, Jacquot Catherine, Aubry Jacques, Baty Daniel, Barbet Jacques, Birklé Stéphane
Inserm, Université de Nantes, Nantes Atlantique Universités, U601, Département de Recherche en Cancérologie, 9 quai Moncousu, F-44000 Nantes, France.
Mol Immunol. 2007 Mar;44(7):1680-90. doi: 10.1016/j.molimm.2006.08.007. Epub 2006 Sep 29.
Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.
通过对一只羊驼（小羊驼）进行免疫，获得了对甲氨蝶呤（MTX）具有特异性的单域抗体。使用噬菌体展示技术从免疫羊驼文库中淘选特异性VHH结构域（V-D-J区域）。通过固定化金属亲和色谱法从大肠杆菌（C41菌株）周质中纯化出对MTX具有特异性的抗体片段，表达水平约为10mg/L。经SDS-PAGE和蛋白质印迹分析后，发现一条约16,000Da的单条带，对应于VHH片段，而竞争ELISA证明其与可溶性MTX具有选择性结合。表面等离子体共振（SPR）分析表明，抗MTX VHH结构域对MTX-血清白蛋白偶联物的亲和力在纳摩尔范围内（29 - 515nM）。通过IMGT/V-QUEST发现，编码抗MTX VHH的基因与先前报道的羊驼和人类IGHV种系基因相似。七个抗MTX CDR3序列中的V-D和D-J连接重排表明它们起源于三个不同的祖B细胞。我们的结果表明，骆驼科动物单域抗体能够与低分子量水溶性半抗原进行高亲和力结合。此外，这些抗MTX VHH为羊驼单域抗体的抗原结合库如何在没有VL的情况下为半抗原提供结合位点提供了新的见解。与鼠重组抗体相比，这种类型的单域抗体在生产率以及与人类IGHV3亚组基因的序列相似性方面具有优势。
