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Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

Schoonbroodt S, Frans N, DeSouza M, Eren R, Priel S, Brosh N, Ben-Porath J, Zauberman A, Ilan E, Dagan S, Cohen EH, Hoogenboom HR, Ladner RC, Hoet RM - Nucleic Acids Res. (2005)

Bottom Line: DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires.Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family.We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

View Article: PubMed Central - PubMed

Affiliation: Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium.

ABSTRACT
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

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Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing biotinylated GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.
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fig5: Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing biotinylated GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.

Mentions: The specificity of candidate clones selected from the library was further confirmed by ELISA as both phage-displayed Fab product and soluble Fab product, directly collected from cell supernatant or FPLC-purified (Figure 5). To allow soluble Fab production, the positive phage antibodies were recloned as described in Materials and Methods. Soluble Fabs were then produced at high yields from E.coli periplasm. The average yield of soluble recombinant protein after metal chelate chromatography purification was ∼100–400 μg purified Fab/L bacterial culture using shaker flasks. The specificity of the anti-GAPDH clones for binding to their target was then checked using ELISA (Figure 5). Two of the five specific phage antibodies (B1 and C1) were found reactive on GAPDH antigen as soluble Fab.


Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

Schoonbroodt S, Frans N, DeSouza M, Eren R, Priel S, Brosh N, Ben-Porath J, Zauberman A, Ilan E, Dagan S, Cohen EH, Hoogenboom HR, Ladner RC, Hoet RM - Nucleic Acids Res. (2005)

Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing biotinylated GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1131936&req=5

fig5: Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing biotinylated GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.
Mentions: The specificity of candidate clones selected from the library was further confirmed by ELISA as both phage-displayed Fab product and soluble Fab product, directly collected from cell supernatant or FPLC-purified (Figure 5). To allow soluble Fab production, the positive phage antibodies were recloned as described in Materials and Methods. Soluble Fabs were then produced at high yields from E.coli periplasm. The average yield of soluble recombinant protein after metal chelate chromatography purification was ∼100–400 μg purified Fab/L bacterial culture using shaker flasks. The specificity of the anti-GAPDH clones for binding to their target was then checked using ELISA (Figure 5). Two of the five specific phage antibodies (B1 and C1) were found reactive on GAPDH antigen as soluble Fab.

Bottom Line: DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires.Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family.We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

View Article: PubMed Central - PubMed

Affiliation: Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium.

ABSTRACT
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Show MeSH