Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: Application to the four serotypes

Cabezas, Sheila; Rojas, Gertrudis; Pavón, Alequis; Álvarez, Mayling; Pupo, Maritza; Guillén, Gerardo; Guzmán, María G.

doi:10.1016/j.jviromet.2007.09.001

Cited by 25 publications

(15 citation statements)

References 41 publications

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“…The rapidity of this technique makes it ideal for generating novel antibodies for diagnostic and potentially therapeutic responses to outbreaks of emerging infectious disease such as SARS coronavirus and H5N1 Avian Influenza or genetically modified pathogens released in a bioterrorism incident. Since antibodies are highly specific and are capable of recognizing virtually every class of pathogen, including toxins, viruses, bacteria and fungi, they enable easy and rapid identification of pathogens (Nowakowski et al, 2002;Hayhurst et al, 2003;Paoli et al, 2004;Steiniger et al, 2007;Cabezas et al, 2008). Indeed, sandwich ELISAs utilizing high affinity mouse monoclonal antibodies have been developed against likely bioterrorist threats such as epsilon toxin of Clostridium botulinum and protective antigen, a toxin component of anthrax, with demonstrated toxin detection limits of 1-2 ng/ml (el Idrissi and Ward, 1992;Mabry et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays

Chan

Lim

et al. 2011

Journal of Immunological Methods

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Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development.

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Section: Introductionmentioning

confidence: 99%

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays

Chan

Lim

et al. 2011

Journal of Immunological Methods

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“…The use of phage display and a subtractive biopanning strategy directed the selection of binders towards serotype-specific epitopes, which is not feasible using immunisation strategies followed by conventional hybridoma technology. A previous report using phage display to isolate serotype-specific binders against NS1 did not incorporate a serotype-selective strategy, and isolation of serotype-specific binders was serendipitous and not successful for all four serotypes [28]. Serotype-specific antibodies have previously been isolated using immunisation and hybridoma technology, and these were used in a serotyping DENV NS1 ELISA [29–31].…”

Section: Discussionmentioning

confidence: 99%

Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay

et al. 2017

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The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability.

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“…As a basic functional unit of the antibody, the single-chain fragment variable (scFv) region maintains antigen specificity, and has a wide range of biomedical applications (Intorasoot et al, 2007;Bhatia et al, 2010). The phage library in which the scFv gene repertoires are expressed on the surface of phages becomes a powerful tool for isolation and identification of scFv molecules of interest (Cabezas et al, 2008;Tang et al, 2009). Here we describe a simple and rapid method for the generation and evaluation of a murine scFv library specific for porcine aminopeptidase N (pAPN), a common cellular receptor for TGEV and PEDV, using the T7Select Phage Display System.…”

Section: Discussionmentioning

confidence: 99%

Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system

Sun

Shi

Chen

et al. 2012

Journal of Virological Methods

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Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: Application to the four serotypes

Cited by 25 publications

References 41 publications

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays

Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay

Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system

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