کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2089271 | 1545783 | 2006 | 12 صفحه PDF | دانلود رایگان |

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of Kd = 4.8 × 10− 9 M and Kd = 5.1 × 10− 9 M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.
Journal: Journal of Immunological Methods - Volume 317, Issues 1–2, 20 December 2006, Pages 132–143