Establishment of hapten‐specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries

Abstract

Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy

“…Moreover, the enormous diversity of the synthetic antibody libraries available today means that immune animals are not necessarily needed to derive suitable binders. Indeed, frameworks of synthetic human [64] and avian [65] variable regions have been successfully converted to their IgY derivatives and produced in eukaryotic hosts.…”

Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library

“…Phage display is an efficient method for the preparation of monoclonal antibodies from both immune and nonimmune sources, without the restraints of conventional hybridoma approach (Deckers et al, 2009). The application of phage display technology for the generation of monoclonal antibodies against even the most difficult targets, such as small molecules, represents a promising alternative to traditional hybridoma technology (Garet et al, 2010).…”

Developing Cell-Specific Antibodies to Endothelial Progenitor Cells Using Avian Immune Phage Display Technology

“…chicken scFvs can be readily produced in large quantities and are tagged for rapid isolation and detection using highly specific secondary antibodies. in addition, these scFvs can be converted to igG 29,30 and humanized. 27 codon-optimized gUG1 had improved yields of recombinant protein that facilitated an additional FPlc gel permeation chromatography-based desalting step after nickel-agarose purification.…”