title: Development of a recombinant antibody towards PAPP-A for immunohistochemical use in multiple animal species.
abstract: 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.
authors: Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors: Jakob H
authors: JH
authors: Mikkelsen
authors: Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors: Lasse B
authors: LB
authors: Steffensen
authors: Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. Electronic address: co@mb.au.dk.
authors: Claus
authors: C
authors: Oxvig
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D017124:Conserved Sequence; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D005557:Formaldehyde; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D011247:Pregnancy; D011266:Pregnancy-Associated Plasma Protein-A / Q000032:analysis* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000276:immunology; D013552:Swine; D016707:Tissue Fixation
chemical_list: D000939:Epitopes; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D005557:Formaldehyde; D011266:Pregnancy-Associated Plasma Protein-A
keywords: Affinity maturation; Immunohistochemistry; Pregnancy-associated plasma-protein-A; Recombinant antibody