Greunke Kerstin, Spillner Edzard, Braren Ingke, Seismann Henning, Kainz Sabine, Hahn Ulrich, Grunwald Thomas, Bredehorst Reinhard
Institut für Biochemie und Lebensmittelchemie, Abteilung für Biochemie und Molekularbiologie, Universität Hamburg, Germany.
J Biotechnol. 2006 Jul 13;124(2):446-56. doi: 10.1016/j.jbiotec.2005.12.032. Epub 2006 Feb 21.
Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.
单克隆IgY有潜力成为诊断研究和治疗用途的独特工具，因为与哺乳动物抗体相比，禽类抗体由于其系统发育差异而具有多种优势。禽类免疫球蛋白基因多样化的机制使鸡成为产生重组单链抗体片段（scFv）以及高度多样化的Fab抗体文库的优良来源。然而，这些抗体片段的一个主要局限性是它们的单价形式，这损害了分子的功能亲和力，从而影响了它们在常用实验室方法中的适用性。在本研究中，我们构建了将禽类重组抗体片段转化为不同类型二价IgY抗体形式的载体。为了将已确立的哺乳动物单克隆抗体的特性与IgY恒定区的特性相结合，我们还构建了二价鼠/禽嵌合抗体构建体。当在HEK-293细胞中表达时，所有构建体均产生二价二硫键连接的抗体，通过凝集素印迹分析评估，其糖基化模式与天然IgY相似。通过一步法纯化后，对嵌合和完整的禽类二价抗体形式进行抗原结合分析以及与二抗试剂的相互作用分析。数据表明，所有抗体形式都具有可比的抗原结合特性以及禽类恒定区已确立的特性。
