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- Carlsson, Fredrika, et al.
(författare)
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Novel antibody specificities targeting glycoprotein B of cytomegalovirus identified by molecular library technology
- 2009
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 25:6, s. 429-436
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies provide some protection against cytomegalovirus-mediated disease. All aspects of antibody recognition of important viral antigens are however not fully appreciated. Glycoprotein B (gB), a key protein in the viral membrane, participates in viral infection and it is a component of prototype vaccines. By using combinatorial antibody library and selection technology, novel antibody specificities targeting gB have now been isolated. We define a monoclonal antibody fragment able to recognize site I of antigenic domain (AD) 2, a poorly immunogenic epitope targeted by potent virus-neutralizing antibodies, in a way that is different from the binding of antibodies induced by infection but similar to those induced by vaccination. We also describe the existence of a novel epitope overlapping site I of AD-2 and AD-1, the immunodominant epitope of gB. These specificities, derived by molecular engineering, will be useful for the future assessment of humoral immune responses against this opportunistic viral infection.
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- Löfdahl, Per-Åke, 1959-, et al.
(författare)
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Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
- 2009
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Ingår i: New Biotechnology. - Amsterdam : Elsevier. - 1871-6784 .- 1876-4347. ; 26:5, s. 251-259
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Tidskriftsartikel (refereegranskat)abstract
- Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naive 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the P-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira (R)), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PICA is a promising methodology for stringent selection of binders from complex naive libraries to yield high affinity reagents with selective target binding characteristics.
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- Mandenius, Carl-Fredrik, et al.
(författare)
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Mechatronic design methodology for biotechnology products
- 2009
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Ingår i: New Biotechnology. - Amsterdam : Elsevier. - 1871-6784 .- 1876-4347. ; 25:Suppl. 1, s. S190-S190
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Biotechnology products can be divided into (1) biologics, which comprise those metabolites, biopolymers, and cell structures that are produced through biological processes, and (2) biotechnical machines, which are apparatuses and devices that transform, change, or analyze ‘biological specimens’ for specific purposes, often by using the biological systems per se. The first category has been thoroughly treated in bioengineering theory and practice while the second has been very scarcely investigated.In this presentation is described how the general design science theory can be applied when designing a technical system where biological species or components have the key role in the engineering design solutions. We have named these systems bio-mechatronic systems, since they are combined design achievements between traditional electronic and mechanical sub-systems and the biological systems, and where biological molecules and/or active microbial or cellular components influence the design solutions in a complex way.The purpose is to demonstrate that biotechnology and bioengineering related design can utilize and benefit from other commonly used design tools in, for example, mechanical and electric engineering. These tools should result in shorter development times and a reduction of the need for prototype testing and verification.Four examples will be presented, all well-known biotechnology products in the pharmaceutical and clinical areas: (1) a protein purification system, (2) a bioreactor system, (3) a biosensor instrument, and (4) an embryonic stem cell manufacturing systems.
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