Mikkelsen, Jakob H3; Steffensen, Lasse B4; Oxvig, Claus4
1 Department of Molecular Biology and Genetics - Molecular Intervention, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University2 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University3 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University4 Department of Molecular Biology and Genetics - Molecular Intervention, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University
The metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), is increasingly recognized as a modulator of insulin-like growth factor (IGF) signaling; it cleaves IGF binding proteins causing the release of bioactive IGF. Accumulating evidence supports an important role of PAPP-A in both normal physiology and under different pathological conditions. However, antibodies for the detection of PAPP-A in non-human tissues have been lacking, although needed for use with several animal models which are currently being developed. To develop a monoclonal antibody suitable for the immunohistochemical detection of PAPP-A, we therefore selected a phage-derived scFv antibody, PAC1, specifically recognizing an epitope of PAPP-A, which is highly conserved between multiple animal species. We first converted this antibody into bivalent IgG, and verified its ability to recognize PAPP-A in sections of formalin-fixed and paraffin-embedded tissue. For increased sensitivity, affinity maturation to sub-nanomolar affinity was then carried out. The resulting recombinant antibody, PAC1-D8-mIgG2a, detects PAPP-A specifically and sensitively in human tissue. In addition, this antibody allows detection of PAPP-A in non-human species. We demonstrate its usefulness for the visualization of PAPP-A in murine and porcine tissues.
Journal of Immunological Methods, 2014, Vol 404, p. 33-40