Biomarker Validation and Assay Development

The scope of tasks of this unit includes the development of specific assays for the validation of biomarkers as well as the development and adaptation of assays. A multitude of different spotting and dispensing technologies can be specifically employed to immobilise biomolecules on diverse surfaces such as microtiter plates and object slides or membranes. The application of samples can be determined and optimised with technologies such as contact angle measurement and ellipsometry. Kinetic analysis or thermodynamic measurements can be carried out for interactions of all types. All technologies are continuously being developed further for (customer-) specific applications. Among others, applications are system-biological projects, the kinetic analysis of antibodies, and the development of point-of-care applications, for example for drug and serum screening.

  • Manufacture (spotting) of customer-specific DNA, peptide and protein microarrays
  • Spotting on various materials, e.g. glass, plastic, membranes, microtiter plates, conductors, etc.
  • Benchmarking of diverse contact and non-contact spotters for the selection of an optimal system (reference laboratory for liquid dispensing systems)
  • Performance of customer-specific microarray experiments, evaluation and documentation
  • Optimisation of the experiments by thermodynamic and kinetic measurements
  • Transfer of other assay formats to microarrays
  • Development and establishment of assays for ELISA and microarrays
  • Colourmetric, fluorescence and electrochemical detection systems
  • Serum screening for the identification of disease-associated antibody profiles
  • Detection and validation of (potential) biomarkers in body fluids
  • Epitope mapping of antibodies
  • Bead based assays (Luminex platform)

Contact-free Dispensation of Liquids in the Picoliter Range

The automated application of solutions and liquids both in contact as well as non-contact processes 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 plays a decisive role whenever one needs to deliver micro-volumes in the lower picoliter range. The experts at Fraunhofer IZI-BB therefore offer their comprehensive know-how, from the modification of surfaces to the application of individual samples up to assay development.

Any individual application can be integrated optimally by using special nozzles or pins, in a volume field of 30pL–90nL. 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 targeted.

These technologies have enabled Fraunhofer IZI-BB to target human cells with a vitality rate <95%. Thus, miniaturised 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 work group.

Detection of Illegal Drugs in Multiplex Process

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 immunological detection systems capable of detecting several compounds within the required limits. This should 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 colourimetric 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. Micro-dispensing technologies reduce the consumption of sample materials and reagents.

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

NoPain

The German Pain Society (Deutsche Schmerzgesellschaft) estimates that about 13 million people in Germany suffer chronic pain (state: August 2013). On a molecular level, it is poorly understood how pain arises. 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. 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 take 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 goal of this project is to implement non-radioactive detection of potential phosphorylation sites on proteins or peptides by means of high-throughput platforms (microarrays and Lumiex platform).

Characterization of HLA Antigen Antibody Interactions

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 characterisation 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 optimise the immunosuppresive therapy.

The goal is the application of collected data to a systematic medicine-based diagnostic test that recognises the individual immunological risks of kidney transplant patients and that permits personalized therapies. 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 characterised. 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 specificity should be identified by means of antigens. Additionally, kinetic analyses can be used for anti-antibodies with the same antigen-binding specificity. The analysis is performed either by measuring the surface plasmon resonance (Flexchip) or through protein microarrays.

Bio-Plex® 3D Suspension Array System

Also better known by the name Luminex Platform. The Luminex Platform is a so-called multiplex bead-based system for the simultaneous analysis of up to 500 parameters. This platform employs up to 500 colour-coded beads (magnetic as well as non- magnetic). Any arbitrary molecule (e.g. DNA, RNA, peptides, proteins etc.) can be coupled to the beads and subsequently selected in a format similar to ELISA.

Biacore Flexchip

Up to 400 antibody-antigen interactions can be measured in parallel with the Biacore Flexchip system and classified with regard to their kinetic parameters. The association rate provides information about the length of the incubation time of the assays, while the dissociation rate and the equilibration constant characterise the stability of the complex. Both are criteria that are essential for the development of a diagnostic test. A kinetic ranking permits the identification of an antibody marker that shows high stability to its antigens and simultaneously requires a short incubation time for binding.

Non-contact liquid handling

The application of solutions and liquids on diverse surfaces falls under the category of contact-free handling of liquids. This can involve difficult solutions such as serum or lysates, but also standard analytes such as DNA, RNA, peptides and/or proteins. Two different devices are available, the sciFLEXARRAYER S5 and the instrument TWO 400. Application areas of these technologies are in point-of-care diagnostics, in-vitro diagnostics, the manufacturing of microarrays, peptide synthesis and many additional applications that profit from automatic liquid handling.

  • Pratsch K, Wellhausen R, Seitz H. Advances in the quantification of protein microarrays. Curr Opin Chem Biol Feb 2014; Vol 18; 16-20.
  • Solomun T, Seitz H, Sturm H. Electron Irrdadiation of Immobilized DNA in Solution through a Silicon Nano-membrande. Radiation Physics and Chemistry 2013, Vol 88, 70-73.
  • 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, Seitz H. Facing current quantification challenges in protein microarrays. J. Biomed. Biotechnol. 2012.
  • Zorman S, Seitz H, Sclavi B, Strick TR. Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation. Nucleic Acids Res. 2012 Aug;40(15):7375-83.
  • Köhler K, Seitz H. Validation processes of protein biomarkers in serum – a cross platform comparison. Sensors 2012; Vol. 9 12710-28.
  • Köster DM, Haselbach D, Lehrach H, Seitz H. A DNAzyme based label-free detection system for miniaturized assays. Mol Biosyst. Oct 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, Oesterhelt D, Kunte HJ. A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581 T. Environ Microbiol. 2011 Aug;13(8):1973-94.
  • Solomun T, Seitz H, Sturm H. DNA damage by low-energy electron impact: dependence on guanine content. J Phys Chem B. 2009 Aug 27;113(34):11557-9.
  • Baek YS, Haas S, Hackstein H, Bein G, Hernandez-Santana M, Lehrach H, Sauer S, Seitz H. Identification of novel transcriptional regulators involved in macrophage differentiation and activation in U937 cells. BMC Immunol. 2009 Apr 2;10:18.
  • Mayer-Enthart E, Sialelli J, Rurack K, Resch-Genger U, Köster D, Seitz H. Toward improved biochips based on rolling circle amplification – influences of the microenvironment on the fluorescence properties of labeled DNA oligonucleotides. Ann N Y Acad Sci. 2008;1130:287-92.
  • Hultschig C, Kreutzberger J, Seitz H, Konthur Z, Buessow K, Lehrach H. Recent advances of protein Microarrays. Curr Opin Chem Biol. 2006;10(1):4-10.
  • Seitz H, Politis A, Persson BS, Horn S, Naslund A, 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, Herberg FW. Differential binding studies applying functional protein microarrays and surface plasmon resonance. Proteomics. 2006 Oct;6(19):5132-9.
  • Weigel C, Seitz H. Bacteriophage replication modules. FEMS Microbiol Rev. 2006 May;30(3).
  • Hultschig C, Kreutzberger J, Seitz H, Konthur Z, Büssow K, Lehrach H. Recent advances of protein microarrays. Curr Opin Chem Biol. 2006 Feb;10(1):4-10.
  • Seitz H, Hultschig C. Identification of protein-protein and protein-DNA interactions using functional microarrays. Molecular & Cellular Proteomics. 2005; 4(8): S55.
  • Feilner T, Hultschig C, Lee J, Meyer S, Immink RG, Koenig A, Possling A, Seitz H, Beveridge A, Scheel D, Cahill DJ, Lehrach H, Kreutzberger J, Kersten B, Feilner T, Hultschig C, Lee J, Meyer S, Immink RG, Koenig A, Possling A, Seitz H, Beveridge A, Scheel D, Cahill DJ, Lehrach H, Kreutzberger J, Kersten B. High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates. Mol Cell Proteomics. 2005 Oct;4(10):1558-68.
  • Sauer S, Lange BM, Gobom J, Nyarsik L, Seitz H, Lehrach H. Miniaturization in functional genomics and proteomics. Nat Rev Genet. 2005 Jun;6(6):465-76.
  • Seitz H, Krause M, Gotthold C, Borgmeier U, Kersten B, Luebbert C, Heiser V. Application of protein array technology to study protein-protein interactions and protein-DNA interactions. Molecullar & Cellular Proteomics, 2004, 3(10):S273.
  • Seitz H, Hultschig C, Eickhoff H, Zeilinger C, Gotthold C, Borgmeier U, Herberg FW. Identification of protein-protein interactions using protein microarrays and surface plasmon resonance measurements. Molecullar & Cellular Proteomics, 2004, 3(10):S273.
  • Feilner T, Kreutzberger J, Niemann B, Kramer A, Possling A, Seitz H, Kersten B. Proteomic studies using microarrays. Current Proteomics, 2004; Vol. 1, No. 4, 283-295.
  • Büssow K, Quedenau C, Sievert V, Tischer J, Scheich C, Seitz H, Hieke B, Niesen FH, Götz F, Harttig U, Lehrach H. A catalog of human cDNA expression clones and its application to structural genomics. Genome Biol. 2004;5(9):R71.
  • Kersten B, Possling A, Blaesing F, Mirgorodskaya E, Gobom J, Seitz H. Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein-DNA interactions. Anal Biochem. 2004 Aug 15;331(2):303-13.
  • Weigel C, Seitz H. Strand-specific loading of DnaB helicase by DnaA to a substrate mimicking unwound oriC. Mol Microbiol. 2002 Nov;46(4):1149-56.
  • Egelhofer V, Gobom J, Seitz H, Giavalisco P, Lehrach H, Nordhoff E. Protein identification by MALDI-TOF-MS peptide mapping: a new strategy. Anal Chem. 2002 Apr 15;74(8):1760-71.
  • Schmidt F, Lueking A, Nordhoff E, Gobom J, Klose J, Seitz H, Egelhofer V, Eickhoff H, Lehrach H, Cahill DJ. Generation of minimal protein identifiers of proteins from two-dimensional gels and recombinant proteins. Electrophoresis. 2002 Feb;23(4):621-5.
  • Messer W, Blaesing F, Jakimowicz D, Krause M, Majka J, Nardmann J, Schaper S, Seitz H, Speck C, Weigel C, Wegrzyn G, Welzeck M, Zakrzewska-Czerwinska J. Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading. Biochimie. 2001 Jan;83(1):5-12.
  • Glinkowska M, Konopa G, Wegrzyn A, Herman-Antosiewicz A, Weigel C, Seitz H, Messer W, Wegrzyn G. The double mechanism of incompatibility between lambda plasmids and Escherichia coli dnaA(ts) host cells. Microbiology. 2001 Jul;147(Pt 7):1923-8.
  • Seitz H, Welzeck M, Messer W. A hybrid bacterial replication origin. EMBO Rep. 2001 Nov;2(11):1003-6.
  • Seitz H, Weigel C, Messer W. The interaction domains of the DnaA and DnaB replication proteins of Escherichia coli. Mol Microbiol. 2000 Sep;37(5):1270-9.
  • Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, Saenger W, Bernhard F, Reinhardt R, Schmidt M, Ulrich C, Stein T, Leenders F, 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, Tüngler D, Welzeck M, Messer W. The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA. Mol Microbiol. 1999 Oct;34(1):53-66.
  • Messer W, Blaesing F, Majka J, Nardmann J, Schaper S, Schmidt A, Seitz H, Speck C, Tüngler D, Wegrzyn G, Weigel C, Welzeck M, Zakrzewska-Czerwinska J. Functional domains of DnaA proteins. Biochimie. 1999 Aug-Sep;81(8-9):819-25.