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Steinau an der Straße, Germany

Kloke A.,Kohler | Fiebach A.R.,Kohler | Zhang S.,Kohler | Drechsel L.,Kohler | And 9 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2014

Assay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing. To close this gap, we present the LabTube platform, which is based on assay specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid phase extraction. Process control is achieved by a centrifugally-actuated ballpen mechanism. To demonstrate the workflow and functionality of the LabTube platform, we show two LabTube automated sample preparation assays from laboratory routines: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yields were observed compared to manual reference runs, while LabTube automation could significantly reduce the hands-on-time to one minute per extraction. This journal is © the Partner Organisations 2014. Source

Ladd Effio C.,Karlsruhe Institute of Technology | Wenger L.,Karlsruhe Institute of Technology | Otes O.,Karlsruhe Institute of Technology | Oelmeier S.A.,Karlsruhe Institute of Technology | And 3 more authors.
Journal of Chromatography A | Year: 2015

The demand for vaccines against untreated diseases has enforced the research and development of virus-like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, yeast and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction (ATPE). The benefits of ATPE have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of polyethylene (PEG) salt aqueous two-phase systems with a high capacity of up to 4.1. mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of PEG, the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage ATPE on a centrifugal partition chromatography (CPC) device in 500. mL scale. While single-stage ATPE enabled a DNA clearance of 99.6%, multi-stage ATPE improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage ATPE to 69.1% (40.1% VLP recovery) for the multi-stage ATPE. An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%. © 2015 Elsevier B.V. Source

Roggenbuck D.,GA Generic Assays GmbH | Reinhold D.,Otto Von Guericke University of Magdeburg | Wex T.,Otto Von Guericke University of Magdeburg | Goihl A.,Otto Von Guericke University of Magdeburg | And 9 more authors.
Clinica Chimica Acta | Year: 2011

Background: Crohn's disease (CD) is an inflammatory bowel disease (IBD) characterized by reactivity against microbial and self antigens. Zymogen granule glycoprotein 2 (GP2) was identified as the major autoantigen of CD-specific pancreatic autoantibodies (PAB). Methods: Human GP2 was expressed in the Spodoptera frugiperda 9 (Sf9) cell line using the baculovirus system, purified by Ni-chelate chromatography, and used as antigen for anti-GP2 IgA and IgG assessment by enzyme-linked immunosorbent assays (ELISA). Antibodies to mannan of Saccharomyces cerevisiae (ASCA), PAB, and anti-GP2 were investigated in sera of 178 CD patients, 100 ulcerative colitis (UC) patients, and 162 blood donors (BD). Results: Anti-GP2 IgG and IgA were found in 48/72 (66.7%) and 23/72 (31.9%) PAB positive and 5/106 (4.7%) and 1/106 (0.9%) PAB negative CD patients (p < 0.0001), respectively. CD patients displayed significantly higher reactivity to GP2 than UC patients and BD (p < 0.0001), respectively. Occurrence of anti-GP2 antibodies correlated with PAB reactivity (Spearmen's rho = 0.493, p < 0.00001). There was a significant relationship between the occurrence of ASCA IgG and anti-GP2 IgG (p = 0.0307). Conclusions: Anti-GP2 IgG and IgA constitute novel CD specific autoantibodies, the quantification of which could improve the serological diagnosis of IBD. © 2010 Elsevier B.V. Source

Berg A.,Karlsruhe Institute of Technology | Berg A.,Sanofi S.A. | Schuetz M.,Karlsruhe Institute of Technology | Schuetz M.,Diarect AG | And 3 more authors.
Food and Bioproducts Processing | Year: 2014

Characterization of protein solubility in downstream processing steps is important to either prevent protein aggregation, e.g. during inclusion body refolding, hydrophobic interaction chromatography and formulation or to decrease solubility, e.g. for selective precipitation or crystallization. In general we distinguish between thermodynamic solubility at equilibrium and kinetically driven apparent solubility. In our study we used a high throughput screening method established on a liquid handling robot to rapidly assess an apparent solubility of lysozyme and its dependence on parameters such as pH, ionic strength and additive concentration. Combinatorial effects were measured in a reasonable amount of time with high data density and low material consumption. Parameter interactions were observed between solvent pH and temperature. With increasing margin of pH from the isoelectric point, the effect of temperature was more pronounced. In addition, we found an influence of ionic strength on the additive induced changes in apparent solubility for all systems. PEG 300 and Tween 20 improved lysozyme apparent solubility at high salt concentrations. For sorbitol and sucrose, two distinct regions of maximum apparent solubility were found depending on the additive concentration. While an explanation for single parameter effects was possible, e.g. for pH by correlating net charge and solubility, this became difficult with increasing number of parameters. By reducing the experimental effort, it was possible to build a solid data basis to elucidate the mechanism of lysozyme aggregation and to find industrial relevant regions of increased solubility. Our approach is thus a powerful tool not only for process optimization but also for an increased understanding of precipitation. © 2013 The Institution of Chemical Engineers. Source

Richter C.,Diarect AG | Richter C.,Karlsruhe Institute of Technology | Konstantinidis K.,BadenBioTec GmbH | Asen I.,Diarect AG | And 3 more authors.
Engineering in Life Sciences | Year: 2014

Lyme borreliosis is the most common tick-borne disease in North America and Europe. A two-test approach (an ELISA followed by immunoblots) for testing current and past infection has been adopted in most countries. However, the heterogeneity of Borrelia antigens (ags) and the semiquantitative character of the immunoblot remain a limitation. By combining a microarray system with cell-free expression, we established a procedure for the expression and subsequent printing of different Borrelia ags onto several multiwell microarray plate surfaces. We successfully expressed and partially purified 11 immunodominant ags of Lyme borreliosis from different Borrelia species in a self-generated Escherichia coli cell-free system. Using sera from patients suffering from Lyme disease and different specific mAbs, proteins could be reproducibly detected on the microarray plates. To confirm the diagnostic outcome of the new assay, a comparison to the same cell-based expressed, purified, and printed Borrelia ags was performed. In summary, this approach serves as a proof of principle for the identification of potential biomarkers and offers the possibility of multiplex protein detection for specific diseases. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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