| 1. |
- Colwill, Karen, et al.
(författare)
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A roadmap to generate renewable protein binders to the human proteome
- 2011
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Ingår i: Nature Methods. - : Nature America Inc.. - 1548-7091 .- 1548-7105. ; 8:7, s. 551-8
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Tidskriftsartikel (refereegranskat)abstract
- Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.
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| 2. |
- Mersmann, Michael, et al.
(författare)
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Towards proteome scale antibody selections using phage display
- 2010
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Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 27:2, s. 118-128
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Tidskriftsartikel (refereegranskat)abstract
- In vitro antibody generation by panning a large universal gene library with phage display was employed to generate antibodies to more than 60 different antigens. Of particular interest was a comparison of pannings on 20 different SH2 domains provided by the Structural Genomics Consortium (SGC). Streamlined methods for high throughput antibody generation developed within the 'Antibody Factory' of the German National Genome Research Network (NGFN) were demonstrated to minimise effort and provide a reliable and robust source for antibodies. For the SH2 domains, in two successive series of selections, 2668 clones were analysed, resulting in 347 primary hits in ELISA. Half of these hits were further analysed, and more than 90 different scFv antibodies to all antigens were identified. The validation of selected antibodies by cross-reactivity ELISA, western blot and on protein microarrays demonstrated the versatility of the in vitro antibody selection pipeline to generate a renewable resource of highly specific monoclonal binders in proteome scale numbers with substantially reduced effort and time.
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