Biomarker validation

Antibodies are widely used for the analysis of protein expression in cells in vivo and in vitro using a range of different methods. A variety of commercially available antibodies for all types of proteins and their respective post-translational modifications are available for this purpose. In general, these antibodies are produced using synthetic peptides, which exhibit a unique, specific sequence of the respective protein. The functionality, specificity and quality of the antibodies differ among the techniques that are used for the production and the individual batches. The quality of the antibodies directly affects the data that is collected and therefore the biological interpretation.

We offer a number of different techniques that can be used to characterize antibodies and determine kinetic constants.

• Peptide and protein microarrays
• Bead-based assay (Luminex Technology, BioPlex3D)
• Surface plasmon resonance (SPR, Biacore and bScreen)

In addition to antibody testing and characterization in general, there needs to be even better and more meticulous quality control mechanisms for antibodies used in clinical applications and for diagnostic testing.

The automated application of solutions and fluids both in contact and non-contact methods offers many advantages over manual dispensation. Automated handling is reproducible, cost-effective, and permits multi-parameter analyses on compact surfaces. The geometry, characteristics and physics behind dispensing and surface treatment play a decisive role whenever micro-volumes in the lower picoliter range are to be delivered. The experts at Fraunhofer IZI-BB therefore offer their comprehensive know-how, from the modification of surfaces, to the application of individual samples, to assay development.

Any individual application can be integrated optimally by using special nozzles or pins, in a volume field of 30 pL–90 nL. This way, even the smallest sample quantities can be employed effectively. Depending on requirements, DNA, RNA, proteins, peptides, micromolecules or also entire cells can be spotted.

These technologies have enabled Fraunhofer IZI-BB to spot human cells with a vitality rate <95%. Thus, miniaturized arrays of diverse cell lines can be used for small-compound analytics and drug/inhibitor screening. Additional possibilities include: protein/peptide/DNA/RNA microarrays, multiplex arrays in the 96Well scale, ivD and point-of-care applications.

The automated contactless application of almost every liquid and/or solution onto diverse surfaces is possible because of the diversity of dispensing technologies and the great expertise of our working group.

Increasing consumption of illegal drugs justifies the use of reliable and sensitive drug detection systems, e.g. for police and in emergency medicine. Currently, simple and reliable tests that can specifically determine various drugs in parallel (= multiplexing) are lacking. Existing systems usually can only detect substance classes in parallel.

The project’s goal is to develop an immunological technique capable of detecting several compounds within the required limits. This will be tested and validated in collaboration with the LKA in Berlin. For this purpose, nine of the most frequently consumed drugs were selected and antibodies were validated. Both colorimetric as well as fluorescence-based detection systems were established on various platforms. Qualitative and quantitative detection in serum is possible by means of blot methods on microtiter plates or on microarrays. Currently, ELISA can be used to reliably quantify three of the drugs in serum, without sample preparation.

The goal is to develop a user-friendly and sensitive platform that can establish itself as a reliable detection method for drug abuse.

The German Pain Society (Deutsche Schmerzgesellschaft) estimates that about 13 million people in Germany suffer chronic pain (as of August 2013). On a molecular level, emergence of pain or sensitization of the neurons is still poorly characterized. The origin of pain is based on the detection and processing of various signals in nociceptive neurons, whereby the interaction takes place along various signal paths. In the worst cases, signals are amplified, resulting in more intense pain. Pain transmission is subject to complex dynamics in the signalosome of these neurons. Some relevant pathways have already been described, but it is uncertain how crosstalk between the pathways actually takes place. In regard to a possible medical therapeutic approach, the understanding and analysis of this crosstalk is of great significance.

Decisive factors in the dynamics of the pathways and their crosstalk are post-translational modifications such as phosphorylation, which are of great significance as triggers to enzymatic activity or changed substrate specificity and thus lead to high dynamics within and between the signal cascades. The project involved developing the necessary high-throughput methods, such as peptide microarrays and bead-based assays – including simple rapid tests for quality control. The methods were used to test potential phosphorylation sites, allowing known phosphorylation sites to be confirmed and new ones to be identified. The data collected was combined with data from co-operation partners to create a new model of interaction between TRPV1 and ARMS.

Reduction of acute transplantation rejections and transplant survival rates could be improved significantly through the introduction of new immunosuppresives in recent decades. Immunosuppresives inhibit rejection by the cellular and humoral immune system. The human leukocyte antigen system (HLA) in particular plays a decisive role in the humoral immune response. The precise characterization of the anti-HLA antibodies can provide information on the individual risk of an anti-HLA antibody-mediated rejection, and hence make it possible to optimize the immunosuppressive therapy.

The objective is to apply all of the data to a system medicine-based diagnostic test, which detects individual immunologic risks in kidney transplant patients and allows for personalized treatments. Serum samples from kidney transplant patients before and after the transplantation can be examined for the presence of anti-HLA antibodies, and subsequently HLA epitope/binding specificity can be determined and kinetically characterized. The screening of patient samples for anti-HLA antibodies and the differentiation of the HLA antibody specificity is carried out with bead-based assays (Luminex FlexMap 3D). The HLA epitope/antigen binding specificities should be identified by means of single antigen beads.

Most approved drugs have a broad therapeutic spectrum and the desired therapeutic concentrations can be attained with a fixed dosage schedule. Others, however, have only a narrow therapeutic spectrum, meaning that there is very little leeway for adherence to the effective therapeutic concentration. An overdose results in toxicity and underdosing to ineffective treatment. In addition, the pharmacokinetic profile of certain drugs exhibits a high degree of inter-individual variation. This means that such drugs are absorbed, metabolized and excreted by each patient at a different rate.

The lack of methods and devices providing a quick and reliable way to measure drug levels in patients outside of medical facilities is, however, one of the biggest limitations today when it comes to the implementation of personalized treatment. The aim of the project is to develop a system that offers patients therapeutic drug monitoring for medications in their blood or urine from home and that submits the collected data directly and without errors to a clinical data management system.

The aim of the planned project is to develop an integrated system for DNA-based detection of biological contamination in fuels that requires minimal effort on the part of the user. Based on the preliminary work, the existing protocols and methods will be modified specifically for the system, combined with a new integrated procedure, coordinated and implemented in a technical solution for on-site sample collection (e.g. airports). Features of the new method:

• An integrated system solution
• Detection with minimal sample preparation
• Fast detection at the sampling site, e.g. at the airport, with a presentation of the results along with recommendations for action
• The simultaneous detection of multiple relevant organisms (multiplexing)
• Minimal user effort.

Compared to immunological methods already established on the market, this new technology is more specific, faster and more sensitive. At the same time, it is possible to detect a variety of relevant biological contaminants.

For many years, microarrays have been playing a major role in basic research and are becoming increasingly important for clinical applications and diagnostics. Various formats, detection modes and analytes are available. Microarrays can be used for DNA, proteins, peptides and small molecules, such as metabolites and drugs. DNA microarrays ware originally used in research and have now become a preferred instrument for gene expression, sequencing and the detection of mutations.

We have more than 10 years of experience in the design and production of microarrays – including surface and immobilization chemistry, handling of different dosage methods (contact and non-contact), assay design, detection and data analysis – all making us the ideal partner for any microarray application.

More information on microarrays can be found here.

• ELISA
• Lateral flow assay
• Label-free assays: Biacore, bScreen, Nanotemper
• Liquid handling and dispensing systems
• Various microarrays (production, assay development and evaluation)
• Various detection methods (e.g. optical, fluorescence, colorimetric)

• Bio-Plex® 3D Suspension Array System
• Non-contact liquid handling systems, such as I2-400
• bScreen
• Contact angle measurements, ellipsometry
• NanoTemper
• MTP Reader

Publications

• Hahn M.B., Meyer S., Schröter M.A., Seitz H., Kunte H.J., Solomun T. and Sturm H.,  Direct Electron Irradiation of DNA in Fully Aqueous Environment. Damage Determination in Combination with Monte Carlo Simulations, Phys.Chem.Chem.Phys., 2017, 19, 1798
• Schumacher S. and Seitz H., A Novel Immunoassay for Quantitative Drug Abuse Screening in Serum, J. Immunol. Methods 2016,  Vol 436: 34–40
• Schumacher S. and Seitz H., Quality control of Antibodies for diagnostic assays, New Biotechnology, N. Biotechnol. 2016. pii: S1871-6784(16)00014-5
• Morschheuser L., Mükusch S., Trusch M., Seitz H. and Rohn S. HPTLC as a fast screening tool for phosphorylation peptides, Journal of Chromatography B. 2016, 1008: 198–205
• Hahn M.B., Solomun T., Wellhausen R., Hermann S., Seitz H., Meyer S., Kunte H.J., Smiatek J. and Sturm H., Influence of the Compatible Solute Ectoine on the local water structure: Implications for the binding of the Protein G5P to DNA, J Phys Chem B. 2015, Vol 119(49):15212-20.
• Schumacher S. and Seitz H., Multiplex Approach for an Immunological Detection of Drug Abuse: A Validation Study, Mosbach Proceedings 2015, Toxichem Krimtech Special Issue Vol 82: 151-154
• Schumacher S., Muekusch S. and Seitz H. Up-to-Date Applications of Microarrays and their way to Commercialization, Microarrays 2015, Vol 4(2), 196-213
• Pratsch K., Wellhausen R. and Seitz H. Advances in the quantification of protein microarrays; Current Opinion in Chemical Biology. 2014, Vol 18: 16-20
• Solomun T., Seitz H. and Sturm H. Electron Irrdadiation of Immobilized DNA in Solution through a Silicon Nano-membrande; Radiation Physics and Chemistry 2013, Vol 88, 70-73
• Köhler K. and Seitz H. Validation Proceses of Serological Markers - a cross platform comparision; Sensors 2012, 12, 12710-12728; Special Issue Biochips
• Zorman, S., Seitz, H., Sclavi B. and Strick T. Topological characterization of the DnaA-oriC complex using single-molecule nanomanipulation, Nucleic Acids Res. 2012 May 11
• Solomun T., Sturm H., Wellhausen R., Seitz H. Complexion of a g5p-Protein with ssDNA immobilized on a Gold Surface: a Surface Plamson Resonance and Fluorescence Study, Chem. Phys. Lett. 2012; 533, 92-94
• Wellhausen R. and Seitz H. Facing Current Quantification Challenges in Protein Microarrays, J. Biomed. Biotech. 2012; Article ID 831347
• Köster DM., Haselbach D., Lehrach H and Seitz H. A DNAzyme based label-free detection system for miniaturized assays. Mol Biosyst. 2011; 7(10):2882-9.
• Schwibbert K., Marin-Sanguino A., Bagyan I., Heidrich G., Lentzen G., Seitz H., Rampp M., Schuster SC., Klenk HP., Pfeiffer F., Oesterheld D. and Kunte HJ A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581 (T). Environ. Microbiol. 2011 vol. 13 (8):1973-1994
• Solomun T., Seitz H. and Sturm H. DNA damage by low-energy electron impact: dependence on Guanine content. J Phys Chem B. 2009; 113:11557-9.
• Baek Y. S., Haas S., Hackstein H., Bein G., Hernandez-Santana M., Lehrach H., Sauer S. and Seitz H. Identification of novel transcriptional regulators involved in macrophage differentiation and activation in U937 cells. BMC Immunol. 2009; 10:18.E.
• Mayer-Enthart E., Sialelli J., Rurack K., Resch-Genger U., Köster D. and Seitz H. Towards improved biochips based on Rolling Circle Amplification - influences of the microenvironment on the fluorescence properties of labelled DNA oligonucleotides Ann N Y Acad Sci. 2008;1130:287-92
• Weigel C. and Seitz H. Bacteriophage replication modules, FEMS Microbiol. Rev. 2006; 30(3):321-81
• Seitz H., Politis A., Persson B.S., Horn S., Naslund A. And Werther M. Characterisation of regulatory DNA elements. Molecular & Cellular Proteomics 2006; 5(10): S94
• Seitz H., Hutschenreiter S., Hultschig C., Zeilinger C., Zimmermann B., Kleinjung F., Schuchhardt J., Eickhoff H. and Herberg F. Differential binding studies applying functional protein microarrays and surface plasmon resonance, Proteomics 2006; 6(19):5132-9
• Hultschig C., Kreutzberger J., Seitz H., Konthur Z., Buessow K., and Lehrach H. Recent advances of protein Microarrays, Curr Opin Chem Biol. 2006;10(1):4-10.
• Feilner T., Hultschig C., Lee J., Meyer S., Immink R.G.H., Koenig A., Possling A., Seitz H., Beveridge A., Scheel D., Cahill D.J., Lehrach H., Kreutzberger J. and Kersten B. High-throughput identification of potential Arabidopsis MAP kinases substrates, Mol Cell Proteomics 2005 Oct;4(10):1558-68
• Seitz H. and Hultschig C. Identification of protein-protein and protein-DNA interactions using functional microarrays, Molecular & Cellular Proteomics. 2005; 4(8): S55
• Sauer S., Lange B., Gobom J., Nyarsik L., Seitz H. and Lehrach H. Miniaturisation in functional genomics and proteomics, Nature Reviews Genetics 2005; 6, 465-476
• Feilner T., Kreutzberger J., Niemann B., Kramer A., Possling A., Seitz H. and Kersten B. Proteomic studies using microarrays. Current Proteomics, 2004; Vol. 1, No. 4, 283-295
• Buessow K., Quedenau C., Sievert V., Tischer J., Scheich C., Seitz H., Hieke B., Niesen F.H., Gotz F., Harttig U. and Lehrach H. A catalogue of human cDNA expression clones and its application to structural genomics, Genome Biology, 2004, 5(9):R71
• Seitz H., Hultschig C., Eickhoff H., Zeilinger C., Gotthold C., Borgmeier U. and Herberg F.W. Identification of protein-protein interactions using protein microarrays and surface plasmon resonance measurements, Molecullar & Cellular Proteomics, 2004, 3(10):S273
• Kersten B., Possling A., Blaesing F., Mirgorodskaya E., Gobom J. and Seitz H. Protein microarray technology and UV crosslinking combined with mass spectrometry for the analysis of protein-DNA interactions, Anal Biochem., 2004;331(2):303-13
• Seitz H., Krause M., Gotthold C., Borgmeier U., Kersten B., Luebbert C. and Heiser V. Application of protein array technology to study protein-protein interactions and protein-DNA interactions. Mollecular & Cellular Proteomics, 2003; 2(9):939
• Schmidt, F.,  Lueking, A.,  Nordhoff, E.,  Gobom, J., Klose, J., Seitz H., Egelhofer, V., Eickhoff, H., Lehrach, H. and Cahill, D. J. Generation of minimal protein identifiers of proteins from 2D gels and recombinant proteins. Electrophoresis, 2002; 23; 621 – 625
• Egelhofer V., Gobom J., Seitz H., Giavalisco P., Lehrach H. and Nordhoff E. Protein identification by MALDI-TOF-MS peptide mapping: a new strategy. Anal Chem., 2002; 74(8):1760-71.
• Weigel C. and Seitz H. Strand-specific loading of DnaB helicase by DnaA to a substrate mimicking unwound oriC. Mol Microbiol, 2002; 46(4):1149-56
• Seitz H., Welzeck M. and Messer W. A hybrid bacterial replication origin. EMBO Rep., 2001; 2(11):1003-6
• Messer W., Blaesing F., Jakimowicz D., Krause M., Majka J., Nardmann J., Schaper S., Seitz H., Weigel C., Wegrzyn G., Welzeck M and Zakrzewska-Czerwinska J. Bacterial replication initiator DnaA. Rules for DNA binding and roles of DnaA in origin unwinding and helicase loading. Biochimie, 2001; 83(1):5-12
• Glinkowska M., Konopa G., Wegrzyn A., Herman-Antosiewicz A., Weigel C., Seitz H., Messer W. and Wegrzyn G. The double mechanism of incompatibility between lambda plasmids and Escherichia coli dnaA(ts) host cells. Microbiology, 2001; 147(Pt 7): 1923-1928
• Seitz H., Weigel C. and Messer W. The interaction domains of the DnaA and DnaB replication proteins of Escherichia coli. Mol. Microbiol. 2000; 37(5):1270-1279
• Messer W., Blaesing F. Majka J., Nardmann J., Schaper S., Schmidt A., Seitz H., Speck C., Tungler D., Wegrzyn G., Weigel C., Welzeck M. and Zakrzewska-Czerwinska J. Functional domains of DnaA proteins. Biochimie, 1999; 81(8-9):819-825
• Duitman E.H., Hamoen L.W., Rembold M., Venema G., Seitz H., Saenger W., Bernhard F., Reinhardt R., Schmidt M., Ulrich C., Stein T., Leenders F. and Vater J. The mycosubtilin synthetase of Bacillus subtilis ATC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase, Proc. Nat. Acad. Science USA, 1999; 96(23):13294-13299
• Weigel C., Schmidt A., Seitz H., Tungler D., Welzeck M and Messer W. The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA. Mol. Microbiol., 1999; 34(1):53-66

(Co)-Editor of books

• »Enzymatic Nucleic Acids«, Advances in Biochemical Engineering/Biotechnology, XY, XY. and Seitz H., Springer Berlin Heidelberg New York, Springer Berlin Heidelberg, in preparation
• »Protein Microarrays – Methoden und Anwendungen«, Seitz H., Springer Spektrum, Springer Berlin Heidelberg New York, in press 2015, ISBN 978-3-642-34833-4
• »Biomarker Validation«, Schumacher S. and Seitz H., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2015, ISBN 978-3-527-33719-4
• »Molecular Diagnostics«, Advances in Biochemical Engineering/Biotechnology Vol. 133, Schumacher S. and Seitz H., Springer Berlin Heidelberg New York, Springer Berlin Heidelberg New York, May 2013, ISBN 978-3-642-37690-0
• »Protein-Protein-interactions«, Advances in Biochemical Engineering/Biotechnology Vol. 110, Werther M. and Seitz H., Springer Berlin Heidelberg New York, September 2008, ISBN 978-3-540-68820-4 
• »Analytics of Protein-DNA-interactions«, Advances in Biochemical Engineering/Biotechnology Vol 104., Seitz H., Springer Berlin Heidelberg New York, Jan 2007, ISBN 978-3-540-48147-8

Other publications

• Herrmann S. und Seitz H. (2015) Personalisierte Medizin/ Companion Diagnostika, GIT Verlag 08/2015
• Köhler K., Or-Guil M., Babel N. und Seitz H. (2011) Antikörper als Biomarker in der Diagnostik, Laborwelt
• Schumacher S., Köhler K., Dauber M., Jacob A. und Seitz H. (2011) Nun prüfe, wer sich bindet: Charakterisierung von Interaktionen mittels kinetischer Messungen GenomXpress 2/11
• Koester D., Mayer-Enthart E., Sialelli J., Rurack K., Resch-Genger U. und Seitz H. (2008) Hula Hoop für DNA – Eine hoch sensitive Detektionsmethode für DNA Microarrays  GenomXpress 1/08
• Eminli S. und Seitz H. (2005) »Methoden zur Charakterisierung von Protein-DNA Interaktionen« GenomXpress 3/05
• Eickhoff H. und Seitz H. (2004) »Protein microarray data bridging data sets from 2D gels and DNA microarrays« Proceedings of the European Science Foundation Workshop: Protein Arrays: Bridging the gap between Physics and Biomedicine, 2004, S. 11
• Marcus K., Hultschig C., Frank R., Herberg F., Schuchhardt J. and Seitz H. (2003) Innovative Forschungsansätze im NGFN Verbund »The Human Brain Proteome Project HBPP« GenomXpress 4/03

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