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.

Generation of human monoclonal allergen-specific IgE and IgG antibodies from synthetic antibody libraries.

BACKGROUND: Allergen-specific IgE and IgG antibodies play pivotal roles in the induction and progression of allergic hypersensitivity reactions. Consequently, monoclonal human IgE and IgG4 antibodies with defined specificity for allergens should be useful in allergy research and diagnostic tests. We used combinatorial antibody libraries and subsequent recombinant production to make and assess IgE, IgG1, and IgG4 allergen-specific antibodies. METHODS: We used phage display to select a synthetic single-chain antibody fragment (scFv) library against 3 different allergens, from bee venom, bovine milk, and apple. The scFv obtained were converted into IgG1, IgG4, and IgE antibody formats and assessed for their biochemical properties by ELISA, immunoblotting, and fluorescence-activated cell sorting. RESULTS: Two different antibody formats for each IgG1, IgG4, and IgE antibody were produced in mammalian cells as disulfide-linked and glycosylated Ig, which were usable in allergen-specific ELISA assays and immunoblots. In addition, the recombinant IgE antibodies mediated the binding of allergens to HEK-293 cells transfected with the high-affinity IgE receptor, and this binding was blocked by corresponding IgG antibodies. CONCLUSIONS: The use of synthetic libraries for the generation of allergen-specific recombinant IgE and IgG antibodies should have broad applications in allergological research and diagnosis.

Comparative expression of different antibody formats in mammalian cells and Pichia pastoris.

Generation of recombinant antibody fragments has been advanced by phage display technology but their broad use in biochemical or analytical applications is often hindered by their univalence. For enhancement of functional affinity and overall applicability, the fusion of scFvs (single-chain variable fragments) to IgG constant domains has become an attractive approach. In order to evaluate characteristics and expression behaviour of different IgG-analogous antibody formats, we fused an scFv to different portions of the heavy chain constant region of human IgG1. Two types of antibodies, an scFv-C(H)2-3 antibody and an scFv-C(H)1-3 antibody, a new intermediate with retained C(H)1 domain, were generated with or without an affinity tag for purification. Additionally, the scFv was reconverted into the heterotetrameric IgG molecule. To allow a reliable comparison of expression behaviours of different antibodies, we established vector systems that allow isogenetic and efficient expression of the recombinant antibodies based on site-specific recombination. Upon recombinant expression in mammalian cells and the methylotrophic yeast Pichia pastoris, disulfide-linked and glycosylated oligomers were obtained. Establishment of isogenetic cell lines revealed that the presence of the C(H)1 domain is not critical for secretion efficiency. Reactivity of the different constructs with antigen and Fc receptors was verified by ELISA, surface plasmon resonance approaches, as well as FACS analysis of HEK-293 cells (human embryonic kidney cells) stably transfected with human FcgammaRI (high-affinity IgG receptor) (CD64). In summary, the results obtained provide evidence for comparable behaviour of the different antibody formats and the vectors for isogenetic expression will contribute to a broader application of phage display-derived antibodies.

Paratope-based protein identification by antibody and peptide phage display.

In this paper we report a novel application of single-chain antibody fragments (scFv) for protein identification utilizing the inherent information of the paratope for primary structure analysis. Combining the potential of antibody phage display and peptide phage display, selected scFvs are employed to select phage-displayed peptides mimicking an epitope of the protein of interest. Proof of principle is demonstrated by identification of the neuroblastoma protein NB-p260. This protein is recognized by apoptosis-inducing IgM antibodies present in the sera of healthy individuals. Identification of NB-p260 has been hindered by its high molecular weight in the range of 260-280kDa and its instability in purified protein preparations. Employing our approach, we subjected a human synthetic scFv library to selection using sodium dodecyl sulfate-denatured NB-p260. Specific scFvs were further used for selection of a heptapeptide phage display library. From analyzed clones, peptide sequences were identified, two of which could not be related to known proteins by conservative amino acid replacement and one of which, obtained from several clones, could be related to the actin-binding protein ABP278 after two conservative amino acid replacements. The identity of NB-p260 with ABP278 was verified by specific antibodies directed against the N and C termini of ABP278.