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1.  Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display 
BMC Biotechnology  2012;12:29.
Background
Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS) or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity.
Results
A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv) against these five antigens were selected using antibody phage display and characterised.
Conclusion
In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.
doi:10.1186/1472-6750-12-29
PMCID: PMC3423037  PMID: 22703709
2.  Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity 
BMC Biotechnology  2009;9:60.
Background
Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.
Results
In this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 × 108 clones), that included the λ light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (IC50 = 23 ± 3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity).
Conclusion
Results of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication.
doi:10.1186/1472-6750-9-60
PMCID: PMC2716335  PMID: 19563687
3.  A multi-Fc-species system for recombinant antibody production 
BMC Biotechnology  2009;9:14.
Background
Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies.
Results
We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody.
Conclusion
Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.
doi:10.1186/1472-6750-9-14
PMCID: PMC2654441  PMID: 19245715
4.  Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV) 
BMC Biotechnology  2008;8:66.
Background
Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.
Results
In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent.
Conclusion
For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.
doi:10.1186/1472-6750-8-66
PMCID: PMC2543005  PMID: 18764933
5.  Single chain Fab (scFab) fragment 
BMC Biotechnology  2007;7:14.
Background
The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.
Results
Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised.
Conclusion
A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.
doi:10.1186/1472-6750-7-14
PMCID: PMC1829395  PMID: 17346344

Results 1-5 (5)