Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2

Publication year: 2018
Authors: Schlör A. a, Holzlöhner P. a, Listek M. a, Griess C. a, Butze M. a, Micheel B. a, Hentschel Ch. b, Sowa M. b, Roggenbuck D. b,c, Schierack P. c, Füner J. d, Schliebs E. d, Goihl A. e, Reinhold D. e, Hanack K. a
Affiliations:

a - Department of Biochemistry and Biology, Immunotechnology group, University of Potsdam, Potsdam, Golm, Brandenburg, Germany
b - GA Generic Assays GmbH, Dahlewitz, Brandenburg, Germany
c - Institute of Biotechnology, Faculty II, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Brandenburg, Germany
d - preclinics GmbH, Potsdam, Brandenburg, Germany
e - Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University, Magdeburg, Saxony-Anhalt, Germany

Published in: New Biotechnology, 2018
doi: 10.1016/j.nbt.2018.03.006

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 KDvalues 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.


MP-SPR keywords: antibody interactions, binding affinity, ELISA, immobilized membrane glycoprotein, kinetics, recombinant single domain antibodies (VHH) binding