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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.

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Related in: MedlinePlus

Using scFv-Fc antibody for immunofluorescence. Cells were immuno-labeled using either scFv (a, c, e, g) or scFv-hFc (b, d f, h) antibodies. scFv were detected using an anti-Myc tag antibody followed by Cy3-labeled anti-mouse antibodies and scFv-hFc using Cy3-labeled anti-human antibodies (red). Nuclei were stained using DAPI (blue). AA2 (a, b), TA10 (c, d), SF9 (e, f) and F2C (g, h) detect respectively Rab6-GTP, Giantin, alpha-Tubulin and nonmuscle MyosinIIA. scFv fused to a hFc portion keep their specificity and are, in general, more sensitive and easier to use than their monomeric scFv counterparts. Bar = 20 μm.
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Figure 2: Using scFv-Fc antibody for immunofluorescence. Cells were immuno-labeled using either scFv (a, c, e, g) or scFv-hFc (b, d f, h) antibodies. scFv were detected using an anti-Myc tag antibody followed by Cy3-labeled anti-mouse antibodies and scFv-hFc using Cy3-labeled anti-human antibodies (red). Nuclei were stained using DAPI (blue). AA2 (a, b), TA10 (c, d), SF9 (e, f) and F2C (g, h) detect respectively Rab6-GTP, Giantin, alpha-Tubulin and nonmuscle MyosinIIA. scFv fused to a hFc portion keep their specificity and are, in general, more sensitive and easier to use than their monomeric scFv counterparts. Bar = 20 μm.

Mentions: We then assessed the Fc-fused scFvs for detection of antigens by immunofluorescence. After fixation and permeabilization, HeLa cells were incubated with 4 different scFv or with their 4 scFv-hFc counterparts (Figure 2). For each antibody, the detection was at least as good and in general better after Fc fusion, and its usability improved. For example, while the anti-Rab6-GTP scFv scAA2 only gave good labeling when used in conjunction with very short washes [9], human hAA2 produced contrasted labeling using classical immunofluorescence protocols. Similarly, while the scFv TA10 can only detect Giantin when cells are fixed in methanol, the fusion antibody hTA10 can detect Giantin even when cells are fixed by paraformaldehyde (unpublished results). More generally, we systematically saw an increase in the sensitivity of scFv-Fc fusions compared to simple scFvs.


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)

Using scFv-Fc antibody for immunofluorescence. Cells were immuno-labeled using either scFv (a, c, e, g) or scFv-hFc (b, d f, h) antibodies. scFv were detected using an anti-Myc tag antibody followed by Cy3-labeled anti-mouse antibodies and scFv-hFc using Cy3-labeled anti-human antibodies (red). Nuclei were stained using DAPI (blue). AA2 (a, b), TA10 (c, d), SF9 (e, f) and F2C (g, h) detect respectively Rab6-GTP, Giantin, alpha-Tubulin and nonmuscle MyosinIIA. scFv fused to a hFc portion keep their specificity and are, in general, more sensitive and easier to use than their monomeric scFv counterparts. Bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Using scFv-Fc antibody for immunofluorescence. Cells were immuno-labeled using either scFv (a, c, e, g) or scFv-hFc (b, d f, h) antibodies. scFv were detected using an anti-Myc tag antibody followed by Cy3-labeled anti-mouse antibodies and scFv-hFc using Cy3-labeled anti-human antibodies (red). Nuclei were stained using DAPI (blue). AA2 (a, b), TA10 (c, d), SF9 (e, f) and F2C (g, h) detect respectively Rab6-GTP, Giantin, alpha-Tubulin and nonmuscle MyosinIIA. scFv fused to a hFc portion keep their specificity and are, in general, more sensitive and easier to use than their monomeric scFv counterparts. Bar = 20 μm.
Mentions: We then assessed the Fc-fused scFvs for detection of antigens by immunofluorescence. After fixation and permeabilization, HeLa cells were incubated with 4 different scFv or with their 4 scFv-hFc counterparts (Figure 2). For each antibody, the detection was at least as good and in general better after Fc fusion, and its usability improved. For example, while the anti-Rab6-GTP scFv scAA2 only gave good labeling when used in conjunction with very short washes [9], human hAA2 produced contrasted labeling using classical immunofluorescence protocols. Similarly, while the scFv TA10 can only detect Giantin when cells are fixed in methanol, the fusion antibody hTA10 can detect Giantin even when cells are fixed by paraformaldehyde (unpublished results). More generally, we systematically saw an increase in the sensitivity of scFv-Fc fusions compared to simple scFvs.

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