@article{SchloerHolzloehnerListeketal.2018, author = {Schl{\"o}r, Anja and Holzl{\"o}hner, Pamela and Listek, Martin and Grieß, Cindy and Butze, Monique and Micheel, Burkhard and Hentschel, Christian and Sowa, Mandy and Roggenbuck, Dirk and Schierack, Peter and F{\"u}ner, Jonas and Schliebs, Erik and Goihl, Alexander and Reinhold, Dirk and Hanack, Katja}, title = {Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2}, series = {New biotechnology}, volume = {45}, journal = {New biotechnology}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1871-6784}, doi = {10.1016/j.nbt.2018.03.006}, pages = {60 -- 68}, year = {2018}, abstract = {Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified as a major autoantigenic target in Crohn's disease patients. It was reported recently that a long (GP2a) and a short (GP2b) isoform of GP2 exist and that in the outcome of inflammatory bowel diseases (IBD) GP2-specific autoantibodies probably appear as new serological markers for diagnosis and therapeutic monitoring. To investigate this further and in order to establish diagnostic tools for the discrimination of both GP2 isoforms, a set of different murine monoclonal and camelid recombinant single domain antibodies (camelid VHH) was generated and validated in various enzyme-linked immunosorbent assay (ELISA) formats, immunofluorescence on transgenic cell lines and immunohistochemistry on monkey pancreas tissue sections. Out of six binders identified, one was validated as highly specific for GP2a. This murine monoclonal antibody (mAb) was used as capture antibody in construction of a sandwich ELISA for the detection of GP2a. Camelid VHHs or a second murine mAb served as detection antibodies in this system. All antibodies were also able to stain GP2a or GP2b on transgenic cell lines as well as on pancreatic tissue in immunohistochemistry. The KD values measured for the camelid VHHs were between 7 nM and 23pM. This set of specific binders will enable the development of suitable diagnostic tools for GP2-related studies in IBD.}, language = {en} } @misc{ListekHoenowGossenetal.2020, author = {Listek, Martin and H{\"o}now, Anja and Gossen, Manfred and Hanack, Katja}, title = {A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch Naturwissenschaftliche Reihe}, number = {865}, issn = {1866-8372}, doi = {10.25932/publishup-45989}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-459893}, pages = {14}, year = {2020}, abstract = {The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.}, language = {en} } @article{RoggenbuckBorghiSommaetal.2016, author = {Roggenbuck, Dirk and Borghi, Maria Orietta and Somma, Valentina and Buettner, Thomas and Schierack, Peter and Hanack, Katja and Grossi, Claudia and Bodio, Caterina and Macor, Paolo and von Landenberg, Philipp and Boccellato, Francesco and Mahler, Michael and Meroni, Pier Luigi}, title = {Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers}, series = {IEEE transactions on geoscience and remote sensing}, volume = {18}, journal = {IEEE transactions on geoscience and remote sensing}, publisher = {BioMed Central}, address = {London}, issn = {1478-6354}, doi = {10.1186/s13075-016-1018-x}, pages = {14}, year = {2016}, abstract = {Background: Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). Methods: Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (beta 2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-beta 2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human beta 2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human beta 2GPI or after CL-micelle absorption. Results: Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM a beta 2GPI and aCL. Anti-CL and anti-beta 2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and beta 2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized beta 2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-beta 2GPI humoAbs. Conclusions: The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.}, language = {en} } @misc{HanackSchloerHolzloehneretal.2016, author = {Hanack, Katja and Schloer, Anja and Holzloehner, Pamela and Listek, Martin and Bauer, Cindy and Butze, Monique and Micheel, Burkhard and Hentschel, Christian and Sowa, Mandy and Roggenbuck, Dirk and Schierack, Peter and Fuener, Jonas and Schliebs, Erik and Goihl, Alexander and Reinhold, Dirk}, title = {Camelid nanobodies specific to human pancreatic glycoprotein 2}, series = {The journal of immunology}, volume = {196}, journal = {The journal of immunology}, publisher = {American Assoc. of Immunologists}, address = {Bethesda}, issn = {0022-1767}, pages = {313 -- 328}, year = {2016}, abstract = {Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified to be a major autoantigenic target in Crohn's disease patients. It was discussed recently that a long and a short isoform of GP2 exists whereas the short isoform is often detected by GP2-specific autoantibodies. In the outcome of inflammatory bowel diseases, these GP2-specific autoantibodies are discussed as new serological markers for diagnosis and therapeutic monitoring. To investigate this further, camelid nanobodies were generated by phage display and selected against the short isoform of GP2 in order to isolate specific tools for the discrimination of both isoforms. Nanobodies are single domain antibodies derived from camelid heavy chain only antibodies and characterized by a high stability and solubility. The selected candidates were expressed, purified and validated regarding their binding properties in different enzyme-linked immunosorbent assays formats, immunofluorescence, immunohistochemistry and surface plasmon resonance spectroscopy. Four different nanobodies could be selected whereof three recognize the short isoform of GP2 very specifically and one nanobody showed a high binding capacity for both isoforms. The KD values measured for all nanobodies were between 1.3 nM and 2.3 pM indicating highly specific binders suitable for the application as diagnostic tool in inflammatory bowel disease.}, language = {en} } @article{RoggenbuckGoihlHanacketal.2017, author = {Roggenbuck, Dirk and Goihl, Alexander and Hanack, Katja and Holzloehner, Pamela and Hentschel, Christian and Veiczi, Miklos and Schierack, Peter and Reinhold, Dirk and Schulz, Hans-Ulrich}, title = {Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2}, series = {Clinical chemistry and laboratory medicine : journal of the Forum of the European Societies of Clinical Chemistry - the European Branch of the International Federation of Clinical Chemistry and Laboratory Medicine}, volume = {55}, journal = {Clinical chemistry and laboratory medicine : journal of the Forum of the European Societies of Clinical Chemistry - the European Branch of the International Federation of Clinical Chemistry and Laboratory Medicine}, publisher = {De Gruyter}, address = {Berlin}, issn = {1434-6621}, doi = {10.1515/cclm-2016-0797}, pages = {854 -- 864}, year = {2017}, abstract = {To better understand emerging adults' perceptions of family interactions and value transmission to the next generation, we examined Hmong American emerging adults' reflections on their parents' parenting. Participants discussed what parenting practices they would do differently and others they hoped to emulate with their future adolescent children. Thirty Hmong American emerging adults (18-25 years; M = 21.2 years; 50\% female) participated in interviews that focused retrospectively on the parent-adolescent relationship. Results revealed that emerging adults wanted to parent differently in three ways: less pressure about education, fewer restrictions, and more open communication. Emerging adults imagined being a similar parent in four ways: promoting education, promoting life values, giving guidance, and offering love and support. The findings highlight parenting practices that Hmong American emerging adults plan on transmitting (and not transmitting) to their own children, offering a glimpse into the type of parents the emerging adults may become.}, language = {en} } @misc{MaierHolzloehnerHoenowetal.2016, author = {Maier, Natalia and Holzl{\"o}hner, Pamela and Hoenow, Anja and Scheunemann, Astrid and Weschke, Daniel and Hanack, Katja}, title = {Characterization of monoclonal antibodies generated by in vitro immunization}, series = {The journal of immunology}, volume = {196}, journal = {The journal of immunology}, publisher = {American Assoc. of Immunologists}, address = {Bethesda}, issn = {0022-1767}, pages = {2}, year = {2016}, abstract = {Monoclonal antibodies are highly valuable tools in biomedicine but the generation by hybridoma technology is very time-consuming and elaborate. In order to circumvent the consisting drawbacks an in vitro immunization approach was established by which murine as well as human monoclonal antibodies against a viral coat protein could be developed. The in vitro immunization process was performed by isolation of murine hematopoietic stem cells or human monocytes and an in vitro differentiation into immature dendritic cells. After antigen loading the cells were co-cultivated with naive T and B lymphocytes for three days in order to obtain antigen-specific B lymphocytes in culture, followed by fusion with murine myeloma cells or human/murine heteromyeloma cells. Antigen-specific hybridomas were selected and the generated antibodies were purified and characterized in this study by ELISA, western blot, gene sequencing, affinity measurements. Further the characteristics were compared to a monoclonal antibody against the same target generated by conventional hybridoma technology. Isotype detection revealed a murine IgM and a human IgG4 antibody in comparison to an IgG1 for the conventionally generated antibody. The antibodies derived from in vitro immunization showed indeed a lower affinity for the antigen as compared to the conventionally generated one, which is probably based on the significantly shorter B cell maturation (3 days) during the immunization process. Nevertheless, they were suitable for building up a sandwich based detection system. Therefore, the in vitro immunization approach seems to be a good and particularly fast alternative to conventional hybridoma technology.}, language = {en} } @article{ListekHoenowGossenetal.2020, author = {Listek, Martin and H{\"o}now, Anja and Gossen, Manfred and Hanack, Katja}, title = {A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line}, series = {Scientific Reports}, volume = {10}, journal = {Scientific Reports}, publisher = {Macmillan Publishers Limited, part of Springer Nature}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-020-58571-w}, pages = {12}, year = {2020}, abstract = {The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.}, language = {en} } @article{KabaMaierSchliebeOhleretal.2015, author = {Kaba, Hani E. J. and Maier, Natalia and Schliebe-Ohler, Nicole and Mayer, Yvonne and Mueller, Peter P. and van den Heuvel, Joop and Schuchhardt, Johannes and Hanack, Katja and Bilitewski, Ursula}, title = {Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein}, series = {Journal of microbiological methods}, volume = {108}, journal = {Journal of microbiological methods}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0167-7012}, doi = {10.1016/j.mimet.2014.11.003}, pages = {61 -- 69}, year = {2015}, abstract = {We selected the immunogenic cell wall beta-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells.}, language = {en} } @misc{RoggenbuckBorghiSommaetal.2016, author = {Roggenbuck, Dirk and Borghi, Maria Orietta and Somma, Valentina and B{\"u}ttner, Thomas and Schierack, Peter and Hanack, Katja and Grossi, Claudia and Bodio, Caterina and Macor, Paolo and von Landenberg, Philipp and Boccellato, Francesco and Mahler, Michael and Meroni, Pier Luigi}, title = {Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers}, series = {Postprints der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe}, number = {436}, issn = {1866-8372}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-407211}, pages = {14}, year = {2016}, abstract = {Background Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). Methods Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (β2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-β2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human β2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human β2GPI or after CL-micelle absorption. Results Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized β2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-β2GPI humoAbs. Conclusions The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.}, language = {en} } @article{LuetkecosmannFaupelPorstmannetal.2019, author = {Luetkecosmann, Steffi and Faupel, Thomas and Porstmann, Silvia and Porstmann, Tomas and Micheel, Burkhard and Hanack, Katja}, title = {A cross-reactive monoclonal antibody as universal detection antibody in autoantibody diagnostic assays}, series = {Clinica chimica acta}, volume = {499}, journal = {Clinica chimica acta}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0009-8981}, doi = {10.1016/j.cca.2019.09.003}, pages = {87 -- 92}, year = {2019}, abstract = {Diagnostics of Autoimmune Diseases involve screening of patient samples for containing autoantibodies against various antigens. To ensure quality of diagnostic assays a calibrator is needed in each assay system. Different calibrators as recombinant human monoclonal antibodies as well as chimeric antibodies against the autoantigens of interest are described. A less cost-intensive and also more representative possibility covering different targets on the antigens is the utilization of polyclonal sera from other species. Nevertheless, the detection of human autoantibodies as well as the calibration reagent containing antibodies from other species in one assay constitutes a challenge in terms of assay calibration. We therefore developed a cross-reactive monoclonal antibody which binds human as well as rabbit sera with similar affinities in the nanomolar range. We tested our monoclonal antibody S38CD11B12 successfully in the commercial Serazym (R) Anti-Cardiolipin-beta 2-GPI IgG/IgM assay and could thereby prove the eligibility of S38CD11B12 as detection antibody in autoimmune diagnostic assays using rabbit derived sera as reference material.}, language = {en} }