TY - GEN A1 - Hanack, Katja A1 - Schloer, Anja A1 - Holzloehner, Pamela A1 - Listek, Martin A1 - Bauer, Cindy A1 - Butze, Monique A1 - Micheel, Burkhard A1 - Hentschel, Christian A1 - Sowa, Mandy A1 - Roggenbuck, Dirk A1 - Schierack, Peter A1 - Fuener, Jonas A1 - Schliebs, Erik A1 - Goihl, Alexander A1 - Reinhold, Dirk T1 - Camelid nanobodies specific to human pancreatic glycoprotein 2 T2 - The journal of immunology N2 - 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. Y1 - 2016 SN - 0022-1767 SN - 1550-6606 VL - 196 SP - 313 EP - 328 PB - American Assoc. of Immunologists CY - Bethesda ER - TY - JOUR A1 - Roggenbuck, Dirk A1 - Borghi, Maria Orietta A1 - Somma, Valentina A1 - Buettner, Thomas A1 - Schierack, Peter A1 - Hanack, Katja A1 - Grossi, Claudia A1 - Bodio, Caterina A1 - Macor, Paolo A1 - von Landenberg, Philipp A1 - Boccellato, Francesco A1 - Mahler, Michael A1 - Meroni, Pier Luigi T1 - Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers JF - IEEE transactions on geoscience and remote sensing N2 - 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. KW - Antiphospholipid syndrome KW - Antiphospholipid antibody KW - Phospholipid binding proteins KW - Beta2-glycoprotein I KW - Line immunoassay Y1 - 2016 U6 - https://doi.org/10.1186/s13075-016-1018-x SN - 1478-6354 SN - 1478-6362 VL - 18 PB - BioMed Central CY - London ER - TY - GEN A1 - Roggenbuck, Dirk A1 - Borghi, Maria Orietta A1 - Somma, Valentina A1 - Büttner, Thomas A1 - Schierack, Peter A1 - Hanack, Katja A1 - Grossi, Claudia A1 - Bodio, Caterina A1 - Macor, Paolo A1 - von Landenberg, Philipp A1 - Boccellato, Francesco A1 - Mahler, Michael A1 - Meroni, Pier Luigi T1 - Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 436 KW - Antiphospholipid syndrome KW - Antiphospholipid antibody KW - Phospholipid binding proteins KW - Beta2 - glycoprotein I KW - Line immunoassay Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-407211 SN - 1866-8372 IS - 436 ER -