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Molecular Imaging of Reporter Genes
Published in Michel M. J. Modo, Jeff W. M. Bulte, Molecular and Cellular MR Imaging, 2007
Keren Ziv, Dorit Granot, Vicki Plaks, Batya Cohen, Michal Neeman
Enzymatic biotinylation of a target cell surface receptor can be used to yield a reporter gene that is detectable via an avidin-conjugated reporter probe. In mammalian cells, the endogenous enzyme biotin ligase can link biotin molecules to specific recognition sequences on biotin-accepting proteins (BAP/biotinylation sequence). Hence, the substance is a natural resident and neither is immunogenic nor requires an exogenously administered linker. The biotinylated sequence can then be imaged with contrast agents that are attached to avidin; however, one hindrance of extensive in vivo use of avidin is its immunogenicity.
Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Guesdon et al.119 developed two procedures for avidin-biotin-based immunocytochemistry (see Figure 24). The first technique (direct bridged avidin-biotin, or BRAB; Figure 24) made use of sequential applications of biotin-labeled antibody avidin (possessing four binding sites for biotin per molecule), and, finally, biotinylated enzyme and development. Guesdon et al.119 found that even extensive biotinylation of the antibody did not change its antigen-binding capacity. However, with biotinylation of antigens, up to 40% of antibody binding capacity could be lost. The catalytic activity of biotinylated enzymes was unchanged in the case of HRP and glucose oxidase, slightly decreased in the case of β-galactosidase, and greatly affected in the case of alkaline phosphatase. The second procedure (direct labeled avidin-biotin, or LAB technique; Figure 24) developed by Guesdon et al.119 made use of a first layer of biotinylated antibody and a second layer of enzyme-labeled avidin. The enzyme-labeling was carried out by a one-step glutaraldehyde procedure. Moreover, to reduce unspecific binding of their egg-white avidin, Guesdon et al.119 blocked the amino groups on the molecule partially by treatment with formaldehyde or glutaraldehyde. Such pretreated avidin preparations permitted use of low-ionic-strength buffers which otherwise had to be used to avoid excessive background. The triple-layer (BRAB) method was found to be slightly more sensitive than the two-layer (LAB) method.119 Different aspects of these early methods for biotinylation are discussed also by Bayer et al.15 A method involving periodate oxidation of oligosaccharide to an aldehyde that subsequently can react with biotin hydrazide was described for mouse monoclonal IgG and IgM antibodies by O’Shannessy et al.260
Generation of a novel fully human non-superagonistic anti-CD28 antibody with efficient and safe T-cell co-stimulation properties
Published in mAbs, 2023
Abdullah Elsayed, Christian Pellegrino, Louis Plüss, Frederik Peissert, Ramon Benz, Franziska Ulrich, Gudrun Thorhallsdottir, Sheila Dakhel Plaza, Alessandra Villa, Jacqueline Mock, Emanuele Puca, Roberto De Luca, Markus G. Manz, Cornelia Halin, Dario Neri
The gene encoding for the ECD of human CD28 with C-terminal AviTag™ (biotin ligase recognition sequence), 6× Histidine tag, and a human IgG1 Fc was cloned into the mammalian expression vector pcDNA3.1(+). An enterokinase cleavage site was incorporated before the Fc tag to potentially isolate the CD28 moiety from the Fc fusion. The protein was produced in CHO-S cells by transient gene expression (TGE) as previously described.59 Purification was performed by protein A affinity chromatography and SEC on a Superdex 200 increase 10/300 GL (GE Healthcare) to obtain a monomeric fraction. Site-specific enzymatic biotinylation was done using E. coli biotin ligase (BirA), as described before.60 The quality and functionality of the recombinant protein were assessed by SEC, SDS-PAGE, and ELISA using a commercial anti-CD28 mAb (clone: CD28.2, Biolegend; 302902).
Novel human monoclonal antibodies specific to the alternatively spliced domain D of Tenascin C efficiently target tumors in vivo
Published in mAbs, 2020
Lisa Nadal, Riccardo Corbellari, Alessandra Villa, Tobias Weiss, Michael Weller, Dario Neri, Roberto De Luca
The gene encoding for TNC-D was amplified and cloned into the bacterial expression vector pQE-12 with the BirA target sequence “BirA Substrate Peptide” (BSP), LHHILDAQKMVWNHR, to specifically biotinylate the antigen. Primers were designed to fuse the BSP tag sequence to the N-terminus of human TNC-D. PCR fragments were assembled and cloned into the expression vector by EcoRI and HindIII restriction site as described previously.51 The BSP-hTNC-D protein was expressed in E. coli TG-1 as described before.44,52 The protein biotinylation was carried with BirA enzyme, an E. coli enzyme able to achieve precise biotin modification. The biotinylation was carried in BirA buffer (10 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl2) following the protocol described by Fairhead et al.44
Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR
Published in mAbs, 2020
Jan Fredrik Simons, Yoong Wearn Lim, Kyle P. Carter, Ellen K. Wagner, Nicholas Wayham, Adam S. Adler, David S. Johnson
Yeast scFv FACS generally followed previously published protocols.6 Antigens represented the extracellular, soluble portions of each target protein and were procured in a lyophilized form from either R&D Systems (PD-1, 8986-PD-100; PD-L1, 9049-B7-100) (Minneapolis, MN) or Acro Biosystems (CTLA-4, CT4-H5229; OX40, OX0-H5224) (Newark, DE). Antigens were labeled with biotin using the EZ-LinkTM Sulfo-NHS-LC-Biotinylation Kit (A39257, Thermo Fisher Scientific, Waltham, MA). Briefly, 50–100 µg of lyophilized protein was resuspended over 8–12 hours in either phosphate-buffered saline (PBS) or H2O without agitation at 4°C. Biotin labeling was accomplished in aqueous solution by combining target proteins in a 1:50 molar ratio with NHS-LC-biotin reagent freshly prepared as a 9 mM stock. The reaction was incubated without agitation in the dark at 4°C for 2 hours after which a 7 kD MWCO Zeba desalting column (89890, Thermo Fisher Scientific, Waltham, MA) was used to remove excess biotin from the solution. Subsequently, the purified labeled proteins were quantified by Bradford assay (Thermo Fisher Scientific, Waltham, MA) using a standard curve of titrated bovine serum albumin (BSA) ampules (23209, Thermo-Fisher Scientific). Biotin labeling was verified by Western Blotting using Streptavidin-HRP (21130, Pierce, Waltham, MA).