Hybrotec GmbH

Schönau am Königssee, Germany

Hybrotec GmbH

Schönau am Königssee, Germany
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Eisold U.,University of Potsdam | Sellrie F.,UP Transfer GmbH | Schenk J.A.,UP Transfer GmbH | Schenk J.A.,Hybrotec GmbH | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.[Figure not available: see fulltext.] © 2015 Springer-Verlag Berlin Heidelberg

Inal S.,University of Potsdam | Kolsch J.D.,University of Potsdam | Sellrie F.,UP Transfer GmbH | Schenk J.A.,UP Transfer GmbH | And 4 more authors.
Journal of Materials Chemistry B | Year: 2013

We present two thermoresponsive water soluble copolymers prepared via free radical statistical copolymerization of N-isopropylacrylamide (NIPAm) and of oligo(ethylene glycol) methacrylates (OEGMAs), respectively, with a solvatochromic 7-(diethylamino)-3-carboxy-coumarin (DEAC)-functionalized monomer. In aqueous solutions, the NIPAm-based copolymer exhibits characteristic changes in its fluorescence profile in response to a change in solution temperature as well as to the presence of a specific protein, namely an anti-DEAC antibody. This polymer emits only weakly at low temperatures, but exhibits a marked fluorescence enhancement accompanied by a change in its emission colour when heated above its cloud point. Such drastic changes in the fluorescence and absorbance spectra are observed also upon injection of the anti-DEAC antibody, attributed to the specific binding of the antibody to DEAC moieties. Importantly, protein binding occurs exclusively when the polymer is in the well hydrated state below the cloud point, enabling a temperature control on the molecular recognition event. On the other hand, heating of the polymer-antibody complexes releases a fraction of the bound antibody. In the presence of the DEAC-functionalized monomer in this mixture, the released antibody competitively binds to the monomer and the antibody-free chains of the polymer undergo a more effective collapse and inter-aggregation. In contrast, the emission properties of the OEGMA-based analogous copolymer are rather insensitive to the thermally induced phase transition or to antibody binding. These opposite behaviours underline the need for a carefully tailored molecular design of responsive polymers aimed at specific applications, such as biosensing. © 2013 The Royal Society of Chemistry.

Stech M.,Fraunhofer Institute for Biomedical Engineering | Merk H.,RiNA GmbH | Schenk J.A.,UP Transfer GmbH | Schenk J.A.,Hybrotec GmbH | And 7 more authors.
Journal of Biotechnology | Year: 2013

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner. © 2012 Elsevier B.V.

Schenk J.A.,UP Transfer GmbH | Schenk J.A.,Hybrotec GmbH | Fettke J.,University of Potsdam | Lenz C.,UP Transfer GmbH | And 6 more authors.
Journal of Biotechnology | Year: 2012

The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14. kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem. © 2012 Elsevier B.V.

Kuhne M.,BAM Federal Institute of Materials Research and Testing | Dippong M.,BAM Federal Institute of Materials Research and Testing | Dippong M.,University of Potsdam | Flemig S.,BAM Federal Institute of Materials Research and Testing | And 6 more authors.
Journal of Immunological Methods | Year: 2014

A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. © 2014 Elsevier B.V.

Ettlinger J.,Fraunhofer Institute for Biomedical Engineering | Schenk J.A.,Hybrotec GmbH | Micheel B.,University of Potsdam | Ehrentreich-Forster E.,Fraunhofer Institute for Biomedical Engineering | Gajovic-Eichelmann N.,Fraunhofer Institute for Biomedical Engineering
Electroanalysis | Year: 2012

A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti-ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation-free progesterone immunoassay with a lower detection limit of 1ngmL -1 (3.18nmolL -1) in 1:2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium-PEG-progesterone tracer and a bioconjugate of one anti-progesterone and one anti-ferrocenium antibody. The immune complex is formed within 30s upon addition of progesterone, resulting in a total analysis time of 1.5min. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sellrie F.,UP Transfer GmbH | Graser E.,AJ Innuscreen GmbH | Lenz C.,UP Transfer GmbH | Hillebrand T.,AJ Innuscreen GmbH | And 2 more authors.
Biosensors and Bioelectronics | Year: 2013

First homogenous immunoassay for sequence-specific nucleic acid detection is developed. The assay bases on our finding that a fluorophore inserted into a DNA probe instead of one of the internal nucleotides may get protected from fluorescence quenching caused by an anti-fluorophore antibody, if the probe is hybridized with the target sequence. This ensures a positive signal in the antibody presence. The assay enables quantitative detection and may have potential for development of homogenous high-throughput platforms. © 2012 Elsevier B.V.

Tan C.,Fraunhofer Institute for Cell Therapy and Immunology | Schenk J.A.,HybroTec GmbH | Schenk J.A.,UP Transfer GmbH | Gajovic-Eichelmann N.,Fraunhofer Institute for Cell Therapy and Immunology | And 2 more authors.
Talanta | Year: 2015

A new homogeneous immunoassay for the detection of progesterone was developed to measure its concentration in human serum. We utilized the weak cross-reactivity of a monoclonal anti-progesterone antibody to an analog molecule (in this case β-estradiol) to create a mixture, in which the fluorescence-labeled antibody (AbF) and quencher-labeled BSA-estradiol (eBSAq) were at optimized equilibrium. At this stage, most antibodies were bound to eBSAq and the fluorescence of AbF was quenched. After adding samples containing free progesterone to the system, these would replace the eBSAq at the antigen-binding site. The fluorescence would be released. In contrast to conventional competitive immunoassays, the fluorescence signal increases with increasing progesterone concentration, greatly simplifying detection and calibration. The performance of the assay was very simple; there was only one mixing step; and other hormones like testosterone, estradiol or dehydroepiandrosterone (DHEA) do not interfere the assay. A wide linear range from 0.1 μg/L to 100 μg/L was achieved in buffer, with a LOD of 0.1 μg/L. In human serum the LOD was 5 μg/L, and the linear range was 5-500 μg/L. For this assay it is important to find the right combination of antibody and cross-reactive antigen. If such a combination could be defined, it is conceivable to apply this assay to a wide range of analytes. © 2014 Elsevier B.V. All rights reserved.

Sellrie F.,Fraunhofer Institute for Biomedical Engineering | Lenz C.,Fraunhofer Institute for Biomedical Engineering | Andersson A.,Fraunhofer Institute for Biomedical Engineering | Wilhelmsson L.M.,Chalmers University of Technology | And 2 more authors.
Talanta | Year: 2014

We report on the generation and analytical application of the monoclonal antibody G93-ED2 raised against the tricyclic fluorescent nucleoside analogue 1,3-diaza-2-oxophenoxazine (tC). G93-ED2 is specifically binding this deoxycytidine analogue and was found to raise its fluorescence intensity by a factor of 5. This unique feature makes it a valuable tool in fluorescence dependent immunoassays. G93-ED2 was successfully applied in a homogeneous fluorescence quenching immunoassay (DNA-Q) for the sequence specific determination of DNA. © 2014 Elsevier B.V.

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