Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries.
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.
Deckers
Susanne
S
Institute of Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King Platz 6, Hamburg, Germany.
Braren
Ingke
I
Greunke
Kerstin
K
Meyer
Nadine
N
Rühl
Dana
D
Bredehorst
Reinhard
R
Spillner
Edzard
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D002645:Chickens; D004784:Environmental Monitoring / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006241:Haptens; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007136:Immunoglobulins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010851:Picrates / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014303:Trinitrotoluene / Q000276:immunology*
D000911:Antibodies, Monoclonal; D006241:Haptens; C016512:IgY; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D019151:Peptide Library; D010851:Picrates; D011993:Recombinant Fusion Proteins; D014303:Trinitrotoluene; C005858:picric acid