Limits...
A multi-Fc-species system for recombinant antibody production.

Moutel S, El Marjou A, Vielemeyer O, Nizak C, Benaroch P, Dübel S, Perez F - BMC Biotechnol. (2009)

Bottom Line: 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 opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS UMR144, 26 rue d'Ulm, F75248 Paris Cedex 05, France. Sandrine.Moutel@curie.fr

ABSTRACT

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.

Show MeSH

Related in: MedlinePlus

Immunofluorescence using natural or multi-species anti-Myc 9E10 antibodies. Hela cells expressing Myc- and GFP-tagged CLIP-170 were immuno-labeled using either the natural mouse monoclonal antibody Myc1-9E10 (a-c) or the recombinant human (d-f), rabbit (g-i) or mouse (j-l) versions of 9E10 scFv fused to Fc domains. 9E10 antibodies were detected using Cy3-labeled secondary antibodies. GFP fluorescence was directly imaged in the green channel (green, a, d, g, h). The overlays (c, f, u, j) show the GFP fluorescence in green and 9E10 detection of Myc-tagged proteins in red. Nuclei, stained using DAPI, are shown in blue. This experiment shows that recombinant 9E10 antibodies efficiently detect Myc tags by immunofluorescence and that the multi-species strategy can be used to change the species of natural antibodies. Bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654441&req=5

Figure 4: Immunofluorescence using natural or multi-species anti-Myc 9E10 antibodies. Hela cells expressing Myc- and GFP-tagged CLIP-170 were immuno-labeled using either the natural mouse monoclonal antibody Myc1-9E10 (a-c) or the recombinant human (d-f), rabbit (g-i) or mouse (j-l) versions of 9E10 scFv fused to Fc domains. 9E10 antibodies were detected using Cy3-labeled secondary antibodies. GFP fluorescence was directly imaged in the green channel (green, a, d, g, h). The overlays (c, f, u, j) show the GFP fluorescence in green and 9E10 detection of Myc-tagged proteins in red. Nuclei, stained using DAPI, are shown in blue. This experiment shows that recombinant 9E10 antibodies efficiently detect Myc tags by immunofluorescence and that the multi-species strategy can be used to change the species of natural antibodies. Bar = 20 μm.

Mentions: Finally, we showed that multi-species antibody technology can also be adapted to natural antibodies, widening their use. The 9E10 scFv (directed against the widely used Myc tag) has been isolated from Myc1-9E10 hybridoma cells before [10]. We sub-cloned it into our 3 versions of pFUSE-Fc and produced h9E10, r9E10 and m9E10 in CHO cells. The resulting multispecies 9E10 were then tested by immunofluorescence using HeLa cells transiently expressing Myc-tagged GFP-CLIP-170 [11] (Figure 4). This experiment showed that human and rabbit 9E10 were as efficient as the original mouse 9E10 in their ability to detect the overexpressed protein.


A multi-Fc-species system for recombinant antibody production.

Moutel S, El Marjou A, Vielemeyer O, Nizak C, Benaroch P, Dübel S, Perez F - BMC Biotechnol. (2009)

Immunofluorescence using natural or multi-species anti-Myc 9E10 antibodies. Hela cells expressing Myc- and GFP-tagged CLIP-170 were immuno-labeled using either the natural mouse monoclonal antibody Myc1-9E10 (a-c) or the recombinant human (d-f), rabbit (g-i) or mouse (j-l) versions of 9E10 scFv fused to Fc domains. 9E10 antibodies were detected using Cy3-labeled secondary antibodies. GFP fluorescence was directly imaged in the green channel (green, a, d, g, h). The overlays (c, f, u, j) show the GFP fluorescence in green and 9E10 detection of Myc-tagged proteins in red. Nuclei, stained using DAPI, are shown in blue. This experiment shows that recombinant 9E10 antibodies efficiently detect Myc tags by immunofluorescence and that the multi-species strategy can be used to change the species of natural antibodies. Bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654441&req=5

Figure 4: Immunofluorescence using natural or multi-species anti-Myc 9E10 antibodies. Hela cells expressing Myc- and GFP-tagged CLIP-170 were immuno-labeled using either the natural mouse monoclonal antibody Myc1-9E10 (a-c) or the recombinant human (d-f), rabbit (g-i) or mouse (j-l) versions of 9E10 scFv fused to Fc domains. 9E10 antibodies were detected using Cy3-labeled secondary antibodies. GFP fluorescence was directly imaged in the green channel (green, a, d, g, h). The overlays (c, f, u, j) show the GFP fluorescence in green and 9E10 detection of Myc-tagged proteins in red. Nuclei, stained using DAPI, are shown in blue. This experiment shows that recombinant 9E10 antibodies efficiently detect Myc tags by immunofluorescence and that the multi-species strategy can be used to change the species of natural antibodies. Bar = 20 μm.
Mentions: Finally, we showed that multi-species antibody technology can also be adapted to natural antibodies, widening their use. The 9E10 scFv (directed against the widely used Myc tag) has been isolated from Myc1-9E10 hybridoma cells before [10]. We sub-cloned it into our 3 versions of pFUSE-Fc and produced h9E10, r9E10 and m9E10 in CHO cells. The resulting multispecies 9E10 were then tested by immunofluorescence using HeLa cells transiently expressing Myc-tagged GFP-CLIP-170 [11] (Figure 4). This experiment showed that human and rabbit 9E10 were as efficient as the original mouse 9E10 in their ability to detect the overexpressed protein.

Bottom Line: 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 opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS UMR144, 26 rue d'Ulm, F75248 Paris Cedex 05, France. Sandrine.Moutel@curie.fr

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus