کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2089271 1545783 2006 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Method for generation of in vivo biotinylated recombinant antibodies by yeast mating
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Method for generation of in vivo biotinylated recombinant antibodies by yeast mating
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 317, Issues 1–2, 20 December 2006, Pages 132–143
نویسندگان
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