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Antibodies, Volume 3, Issue 2 (June 2014), Pages 182-252

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Research

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Open AccessArticle The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag)
Antibodies 2014, 3(2), 182-191; doi:10.3390/antib3020182
Received: 11 February 2014 / Revised: 18 March 2014 / Accepted: 20 March 2014 / Published: 2 April 2014
Cited by 3 | PDF Full-text (645 KB) | HTML Full-text | XML Full-text
Abstract
We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag) is tightly bound by a commercial antibody when expressed at the C-term end of
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We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag) is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique. Full article
(This article belongs to the Special Issue Antibody Constructs)
Open AccessArticle Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications
Antibodies 2014, 3(2), 215-231; doi:10.3390/antib3020215
Received: 19 February 2014 / Revised: 10 April 2014 / Accepted: 28 April 2014 / Published: 9 May 2014
Cited by 1 | PDF Full-text (1099 KB) | HTML Full-text | XML Full-text
Abstract
Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA) and a ~32 kDa subunit B (RTB). There are fears of ricin being used as a weapon for warfare
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Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA) and a ~32 kDa subunit B (RTB). There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs), previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR) array biosensor (ProteOn XPR36) indicated three distinct, non-competitive binding epitopes (“bins”). The association (ka) and dissociation (kd) rate constants and binding affinities (KD) of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (KD) ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab) fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications. Full article
(This article belongs to the Special Issue Antibody Engineering)
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Review

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Open AccessReview Role of B Cells in Breaking and Maintaining Tolerance to Clotting Factor VIII in Congenital and Acquired Hemophilia A
Antibodies 2014, 3(2), 192-204; doi:10.3390/antib3020192
Received: 29 October 2013 / Revised: 1 March 2014 / Accepted: 24 March 2014 / Published: 8 April 2014
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Abstract
Immune responses directed against clotting factor FVIII (FVIII) seriously complicate treatments for patients with hemophilia A. This response can manifest in congenital hemophilia A patients who generate inhibitor antibodies that bind and inactivate “transplanted” replacement FVIII, as well as in acquired hemophiliacs, whose
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Immune responses directed against clotting factor FVIII (FVIII) seriously complicate treatments for patients with hemophilia A. This response can manifest in congenital hemophilia A patients who generate inhibitor antibodies that bind and inactivate “transplanted” replacement FVIII, as well as in acquired hemophiliacs, whose immune systems have lost tolerance to self-FVIII. Regardless of the mechanism by which production of anti-FVIII inhibitor antibody is triggered, the maintenance of this deleterious response in both congenital and acquired hemophiliacs likely relies upon FVIII specific memory B cells. In this review, the similarities and differences in the kinetics, specificities, and subclasses of antibodies produced in response to allo- and auto-FVIII is outlined. A brief description of the immune cell interactions that contribute to maintenance of antibody response, focusing on development of memory B cells and/or long lived plasma cells is also presented. As current treatments for inhibitor antibodies are not successful in all patients, a better understanding of the functions and persistence of memory B cells specific for FVIII is required. Herein, both clinical and experimental data regarding the effects of immune tolerance induction on memory B cell subpopulations is discussed. Finally, the outcomes of B cell-specific depletion via rituximab in hemophilia and other autoimmune diseases are discussed to highlight insights into the subpopulations of memory B cells that contribute to the development and maintenance of successful tolerance to FVIII. Full article
(This article belongs to the Special Issue B Cells and Immunological Tolerance)
Open AccessReview Engineered Bovine Antibodies in the Development of Novel Therapeutics, Immunomodulators and Vaccines
Antibodies 2014, 3(2), 205-214; doi:10.3390/antib3020205
Received: 24 February 2014 / Revised: 5 April 2014 / Accepted: 25 April 2014 / Published: 9 May 2014
Cited by 3 | PDF Full-text (428 KB) | HTML Full-text | XML Full-text
Abstract
Some bovine antibodies across all classes are unique, such as the CDR3 of the variable heavy-domain (VH CDR3), which is exceptionally long (up to 66 amino acids), unlike most conventional antibodies where the VH CDR3 loops range from 10 to 25 amino acids.
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Some bovine antibodies across all classes are unique, such as the CDR3 of the variable heavy-domain (VH CDR3), which is exceptionally long (up to 66 amino acids), unlike most conventional antibodies where the VH CDR3 loops range from 10 to 25 amino acids. The exceptionally long VH CDR3 is encoded by unusually long germline IGHD genes together with insertion of novel “a” nucleotide rich conserved short nucleotide sequence (CSNS) specifically at the IGH V-D junction. Such an exceptionally long VH CDR3 confers unique “knob and stalk” structural architecture where the knob, formed by intra-VH CDR3 disulfide bridges, is separated by 20 Å solvent exposed stalk composed of anti-parallel beta strands. The substitution of the knob with cytokines, such as, erythropoietin and granulocyte colony stimulating factor 3 (granulocyte colony stimulating factor), results in expression of functional fusion proteins with enhanced pharmacokinetics. The beta stranded stalk can be substituted with other rigid structures, for example, repeat alpha helices to form coiled-coil that mimics the beta-stranded stalk and, thus, opens opportunities for insertion of this structure in the CDRs of antibodies across species. Given the versatility of such a structural platform in bovine antibody VH CDR3, it provides the opportunity for the development of new generation of diagnostics, therapeutics, vaccines and immunomodulating drugs. Full article
(This article belongs to the Special Issue Antibody Engineering)
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Open AccessReview Refolding Technologies for Antibody Fragments
Antibodies 2014, 3(2), 232-241; doi:10.3390/antib3020232
Received: 20 January 2014 / Revised: 26 April 2014 / Accepted: 13 May 2014 / Published: 23 May 2014
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Abstract
Refolding is one of the production technologies for pharmaceutical grade antibody fragments. Detergents and denaturants are primarily used to solubilize the insoluble proteins. The solubilized and denatured proteins are refolded by reducing the concentration of the denaturants or detergents. Several refolding technologies have
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Refolding is one of the production technologies for pharmaceutical grade antibody fragments. Detergents and denaturants are primarily used to solubilize the insoluble proteins. The solubilized and denatured proteins are refolded by reducing the concentration of the denaturants or detergents. Several refolding technologies have been used for antibody fragments, comprising dilution, dialysis, solid phase solvent exchange and size exclusion chromatography, as reviewed here. Aggregation suppressor or folding-assisting agents, including arginine hydrochloride, ionic liquids and detergents or denaturants at low concentrations, are included in the refolding solvent to enhance refolding yield. Full article
(This article belongs to the Special Issue Antibody Constructs)
Open AccessReview Efficient Expression of Antibody Fragments with the Brevibacillus Expression System
Antibodies 2014, 3(2), 242-252; doi:10.3390/antib3020242
Received: 13 March 2014 / Revised: 2 May 2014 / Accepted: 12 May 2014 / Published: 26 May 2014
Cited by 1 | PDF Full-text (550 KB) | HTML Full-text | XML Full-text
Abstract
Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for
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Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for production in bacteria. They also are useful in applications requiring intracellular delivery and for further engineering toward molecules possessing multiple custom functions. An expression system based on Brevibacillus is characterized by high efficiency and simple genetic recombination for secretory production. The Brevibacillus expression system has been successfully utilized for the efficient production of antibody fragments, e.g., scFvs (single-chain antibody fragments) comprising heavy-chain and light-chain variable domains, linked by a spacer sequence. Expression in fusion with a Halobacterium-derived secretory protein was shown to confer enhanced productivity. In the case of Fabs, productivity as high as 100 mg/L was accomplished in a simple system, i.e., shake flask cultures. The Brevibacillus expression system offers several advantages, shared by other bacterial systems, such as E. coli, in particular, for the ease in genetic engineering and culture production. Full article
(This article belongs to the Special Issue Antibody Constructs)

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