z-logo
Premium
Establishment of hapten‐specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries
Author(s) -
Deckers Susanne,
Braren Ingke,
Greunke Kerstin,
Meyer Nadine,
Rühl Dana,
Bredehorst Reinhard,
Spillner Edzard
Publication year - 2009
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20080032
Subject(s) - antibody , recombinant dna , monoclonal antibody , phage display , peptide library , escherichia coli , immunoglobulin light chain , microbiology and biotechnology , hapten , hybridoma technology , immunoassay , biology , protein engineering , chemistry , affinity maturation , computational biology , biochemistry , enzyme , gene , peptide sequence , immunology
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.

This content is not available in your region!

Continue researching here.

Having issues? You can contact us here