van Wyngaardt Wouter, Malatji Teresiah, Mashau Cordelia, Fehrsen Jeanni, Jordaan Frances, Miltiadou Dubravka, du Plessis Dion H
Immunology Division, Onderstepoort Veterinary Institute, Onderstepoort, 0110, Republic of South Africa.
BMC Biotechnol. 2004 Apr 1;4:6. doi: 10.1186/1472-6750-4-6.
Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers.
With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA.
The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.
从大型组合文库中筛选出的抗体片段在诊断和治疗方面有众多应用。现有的大多数抗体库源自人类免疫球蛋白基因。然而，其他物种的基因也可使用。由于基因转换引入多样性的方式，仅用两组引物就能轻松获取鸡的天然抗体库。
考虑到体外诊断应用，我们构建了一个大型重组丝状噬菌体文库，该文库展示了源自鸡重链和轻链可变区组合配对的单链抗体片段。部分重链中包含合成随机化的互补决定区。从该文库中筛选出了识别半抗原、蛋白质和病毒颗粒的单链抗体片段。使用表面等离子体共振测定了三种不同抗体片段的亲和力。其中两种处于低纳摩尔范围，一种处于亚纳摩尔范围。为说明文库中抗体的实用价值，将噬菌体展示的单链片段整合到旨在检测非洲马瘟和蓝舌病病毒颗粒的酶联免疫吸附测定（ELISA）中。在竞争性ELISA中检测到了病毒抗体。
本文所述的源自鸡的噬菌体文库有望成为针对多种抗原的重组抗体片段的通用来源。它有潜力提供可应用于研究和诊断的单克隆试剂。对于体外应用，基于禽类供体的天然噬菌体文库可能被证明是现有可选抗体库的有用补充。
