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  - GEN
A1  - Maier, Natalia
A1  - Holzlöhner, Pamela
A1  - Hoenow, Anja
A1  - Scheunemann, Astrid
A1  - Weschke, Daniel
A1  - Hanack, Katja
T1  - Characterization of monoclonal antibodies generated by in vitro immunization
T2  - The journal of immunology
N2  - 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.
Y1  - 2016
SN  - 0022-1767
SN  - 1550-6606
VL  - 196
PB  - American Assoc. of Immunologists
CY  - Bethesda
ER  - 
TY  - GEN
A1  - Holzlöhner, Pamela
A1  - Butze, Monique
A1  - Hebel, Nicole
A1  - Weschke, Daniel
A1  - Schliebs, E.
A1  - Naumann, F.
A1  - Füner, J.
A1  - Micheel, Burkhard
A1  - Hanack, Katja
T1  - Monoclonal mouse antibodies against PBMC subpopulations of New World camelides
T2  - European journal of immunology
Y1  - 2016
SN  - 0014-2980
SN  - 1521-4141
VL  - 46
SP  - 1175
EP  - 1175
PB  - Wiley-Blackwell
CY  - Hoboken
ER  -