Thomas Boehm1, Sophie Pils2, Elisabeth Gludovacz3, Helen Szoelloesi4, Karin Petroczi4, Otto Majdic5, Andrea Quaroni6, Nicole Borth7, Peter Valent8, Bernd Jilma4. 1. Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. Electronic address: thomas.boehm@meduniwien.ac.at. 2. Department of Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. 3. Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. 4. Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. 5. Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. 6. Department of Biomedical Sciences, Veterinary Research Tower, Cornell University, Ithaca, NY 14853-6401, USA. 7. Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. 8. Department of Internal Medicine I, Division of Hematology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
Abstract
OBJECTIVES: Diamine oxidase (DAO) is essential for extracellular degradation of histamine. For decades activity assays with inherent limitations were used to quantify the relative amounts of DAO. No reference DAO standard is available. Absolute DAO amounts cannot be determined. Controversy exists, whether DAO is circulating or not in non-pregnant individuals. The role of DAO as biomarker in various diseases is ambiguous. It is not clear, whether precise quantification of human DAO antigen using commercially available enzyme-linked immunosorbent assays (ELISAs) is possible. The objective was to develop a precise and robust ELISA to quantify DAO in various biological fluids. DESIGN AND METHODS: A research prototype ELISA was established using a mouse monoclonal antibody for capturing and a polyclonal rabbit serum IgG fraction for the detection of human DAO. The limit of blank (LoB), limit of detection (LoD) and estimated limit of quantification (eLoQ) and normal DAO concentrations in serum and plasma were determined. RESULTS: The LoB, LoD and eLoQ derived from 42 standard curves are 0.27, 0.48 and 0.7ng/mL respectively. The detection range using the LoD as the lower and the highest DAO standard as the upper boundary is 0.5 to 450ng/mL. Serum and plasma mean/median concentrations are between 0.5 and 1.5ng/mL in healthy volunteers (n=58) and mastocytosis patients (n=19) and plateau at approximately 145ng/mL (n=16) during pregnancy. Accurate quantification was not influenced by heparin (DAO is a heparin-binding protein), lipaemic or hemolytic serum. The measured DAO antigen concentrations are in close agreement with published enzymatic activity data using radioactive putrescine as substrate. CONCLUSIONS: This research prototype ELISA is able to reliably and accurately quantify human DAO in different biological fluids. The potential of DAO as biomarker in various diseases can be evaluated.
OBJECTIVES:Diamine oxidase (DAO) is essential for extracellular degradation of histamine. For decades activity assays with inherent limitations were used to quantify the relative amounts of DAO. No reference DAO standard is available. Absolute DAO amounts cannot be determined. Controversy exists, whether DAO is circulating or not in non-pregnant individuals. The role of DAO as biomarker in various diseases is ambiguous. It is not clear, whether precise quantification of humanDAO antigen using commercially available enzyme-linked immunosorbent assays (ELISAs) is possible. The objective was to develop a precise and robust ELISA to quantify DAO in various biological fluids. DESIGN AND METHODS: A research prototype ELISA was established using a mouse monoclonal antibody for capturing and a polyclonal rabbit serum IgG fraction for the detection of humanDAO. The limit of blank (LoB), limit of detection (LoD) and estimated limit of quantification (eLoQ) and normal DAO concentrations in serum and plasma were determined. RESULTS: The LoB, LoD and eLoQ derived from 42 standard curves are 0.27, 0.48 and 0.7ng/mL respectively. The detection range using the LoD as the lower and the highest DAO standard as the upper boundary is 0.5 to 450ng/mL. Serum and plasma mean/median concentrations are between 0.5 and 1.5ng/mL in healthy volunteers (n=58) and mastocytosispatients (n=19) and plateau at approximately 145ng/mL (n=16) during pregnancy. Accurate quantification was not influenced by heparin (DAO is a heparin-binding protein), lipaemic or hemolytic serum. The measured DAO antigen concentrations are in close agreement with published enzymatic activity data using radioactive putrescine as substrate. CONCLUSIONS: This research prototype ELISA is able to reliably and accurately quantify humanDAO in different biological fluids. The potential of DAO as biomarker in various diseases can be evaluated.
Authors: Elisabeth Gludovacz; Daniel Maresch; Leonor Lopes de Carvalho; Verena Puxbaum; Laurenz J Baier; Leander Sützl; Gabriela Guédez; Clemens Grünwald-Gruber; Barbara Ulm; Sophie Pils; Robin Ristl; Friedrich Altmann; Bernd Jilma; Tiina A Salminen; Nicole Borth; Thomas Boehm Journal: J Biol Chem Date: 2017-11-29 Impact factor: 5.486
Authors: Philipp Velicky; Karin Windsperger; Karin Petroczi; Sophie Pils; Birgit Reiter; Tamara Weiss; Sigrid Vondra; Robin Ristl; Sabine Dekan; Christian Fiala; David E Cantonwine; Thomas F McElrath; Bernd Jilma; Martin Knöfler; Thomas Boehm; Jürgen Pollheimer Journal: Sci Rep Date: 2018-04-20 Impact factor: 4.379
Authors: Oriol Comas-Basté; Sònia Sánchez-Pérez; Maria Teresa Veciana-Nogués; Mariluz Latorre-Moratalla; María Del Carmen Vidal-Carou Journal: Biomolecules Date: 2020-08-14
Authors: Thomas Boehm; Birgit Reiter; Robin Ristl; Karin Petroczi; Wolfgang Sperr; Thomas Stimpfl; Peter Valent; Bernd Jilma Journal: Allergy Date: 2019-01-01 Impact factor: 13.146
Authors: Thomas Boehm; Matthias Karer; Peter Matzneller; Nina Buchtele; Franz Ratzinger; Karin Petroczi; Christian Schoergenhofer; Michael Schwameis; Heinz Burgmann; Markus Zeitlinger; Bernd Jilma Journal: Int J Immunopathol Pharmacol Date: 2020 Jan-Dec Impact factor: 3.219