| 31. |
- Hu, Francis Jingxin, et al.
(författare)
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Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody
- 2014
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 31:1, s. 35-43
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Tidskriftsartikel (refereegranskat)abstract
- One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.
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| 32. |
- Hu, Francis Jingxin, 1986-, et al.
(författare)
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Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
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Ingår i: New Biotechnology. - 1871-6784 .- 1876-4347.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Surface display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various display systems, phage display is by far the most popular, mainly thanks to its ability to harbor large library sizes. Here, we describe the first use of a grampositive host for display of a library of human antibody genes. The method allows for swift generation of binders by combining phage and gram-positive display, for its ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying specific low nanomolar scFv towards human HER2. The ranking and performance of the scFv isolated by flow sorting in surface immobilized form was retained when expressed as soluble scFv and analyzed by biolayer interferometry as well as after expression as full-length antibodies in mammalian cells. We also show the possibility to use gram-positive display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. We believe this combined approach has the potential for a more complete scan of the antibody repertoire and for swift affinity maturation of human antibody formats.
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| 33. |
- Häggmark, Anna, et al.
(författare)
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Classification of protein profiles from antibody microarrays using heat and detergent treatment.
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:5, s. 564-570
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Tidskriftsartikel (refereegranskat)abstract
- Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.
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| 34. |
- Jakobsen, L., et al.
(författare)
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Functional proteomics of the human centrosome
- 2010
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Ingår i: New Biotechnology. - : ELSEVIER SCIENCE BV. - 1871-6784 .- 1876-4347. ; 27, s. S82-S82
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 35. |
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| 36. |
- Johnsson, Ola, et al.
(författare)
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A novel feeding strategy for industrial fed-batch processes based on frequency content analysis
- 2012
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 29:Supplement, s. 11-11
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Konferensbidrag (refereegranskat)abstract
- Overflow metabolism, i.e. the production of metabolic by-products at a high glycolytic flux, is a recurring problem in fed-batch processes with many types of microorganisms. In the current study, a novel feeding strategy aimed at avoiding process failures due to overflow by-product formation was designed and implemented in a pilot-scale reactor (0.5 m3). The basic principle behind the strategy was to analyze the effects on the dissolved oxygen concentration by periodic variations in the inlet feed rate. The frequency spectrum of the dissolved oxygen signal was used to estimate the proximity of the system to the region where overflow metabolism occurs by examining the content in the relevant frequency range. A control variable based on the measured frequency content was subsequently used to control the feed rate. The only measurement required for this strategy is the dissolved oxygen level in the broth, for which robust, fast and precise probes are widely available in industrial fermentors today. The strategy was successfully implemented in pilot-scale processes for industrial enzyme production using Bacillus licheniformis. It was shown possible to run the process close to the optimal feed rate, indicated by very low amounts of acetate (the overflow metabolite) in the broth. In comparison to a reference strategy the new control strategy resulted in over 10% higher biomass yields.
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| 37. |
- Jönsson, Malin, et al.
(författare)
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CaRA – A multi-purpose phage display library for selection of calcium-regulated affinity proteins
- 2022
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Ingår i: New Biotechnology. - : Elsevier B.V.. - 1871-6784 .- 1876-4347. ; 72, s. 159-167
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Tidskriftsartikel (refereegranskat)abstract
- Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.
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| 38. |
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| 39. |
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| 40. |
- Klingström, Tomas, et al.
(författare)
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Workshop on laboratory protocol standards for the molecular methods database
- 2013
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 30:2, s. 109-113
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Tidskriftsartikel (refereegranskat)abstract
- Management of data to produce scientific knowledge is a key challenge for biological research in the 21st century. Emerging high-throughput technologies allow life science researchers to produce big data at speeds and in amounts that were unthinkable just a few years ago. This places high demands on all aspects of the workflow: from data capture (including the experimental constraints of the experiment), analysis and preservation, to peer-reviewed publication of results. Failure to recognise the issues at each level can lead to serious conflicts and mistakes; research may then be compromised as a result of the publication of non-coherent protocols, or the misinterpretation of published data. In this report, we present the results from a workshop that was organised to create an ontological data-modelling framework for Laboratory Protocol Standards for the Molecular Methods Database (MolMeth). The workshop provided a set of short- and long-term goals for the MolMeth database, the most important being the decision to use the established EXACT description of biomedical ontologies as a starting point.
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