Alpaca Immunoglobulins OCTOBER 2012 Alpaca Immunoglobulins by Andrew Padula Oct 2012 RIRDC Publication No 14-065 RIRDC Project No PRJ-007770 © 2012 Rural Industries Research and Development Corporation. All rights reserved. ISBN 978-1-74254-681-0 ISSN 1440-6845 Alpaca immunoglobulins Publication no. 14-065 Project No. PRJ-007770 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165. Researcher Contact Details Name: Andrew Padula Address: 26 Howitt Ave, Bairnsdale, Victoria, 3875 Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au ii Foreword This research explores an alternative use for alpaca, other than for fibre production. Fibre production has been the traditional saleable product from alpaca, along with trade in live animals and, to a lesser extent, alpaca meat. Blood contains molecules called immunoglobulins, which have a number of medical uses, including antivenom production. Australia has over 130,000 alpaca which represent a vast, untapped resource for using the animals to produce unique therapeutic immunoglobulins. This research has examined the potential of the alpaca immunoglobulins for a specific application: snake antivenom production. There are two main benefactors of this research. Firstly, the Australian alpaca industry stands to benefit from having an alternative use for the alpaca, as a producer of high value niche market products. Secondly, the benefits are to the global community in terms of safer and more effective therapeutic immunoglobulin products. Alpaca belong to the camelid family. Camels have recently been shown to produce a unique form of immunoglobulins in their blood. These immunoglobulins have been shown in the camel to be more heat stable, less allergenic than horse or sheep immunoglobulins, and better at inhibiting some enzymes. These properties, especially the lower allergenicity, identify camelid immunoglobulins as potentially having significant advantages over horse or sheep immunoglobulins. This project was undertaken as a proof of concept study. No similar work has been conducted anywhere in the world. Much of what was undertaken was developed on basic science principles. The key findings of this research are that alpaca can be used successfully to produce therapeutic immunoglobulins. This project explored one specific type of immunoglobulin product; antibodies produced against snake venoms, for antivenom production. In this project, we have demonstrated that alpaca can be immunised, without harm to the animal, against highly toxic and low molecular weight compounds. We have shown that alpaca produce strong immune responses to low doses of a range of snake venoms. We have developed the technology to monitor these specific immune responses. We have developed a system for harvesting of the alpaca blood and separating the serum. We have successfully adapted a process used in horses to concentrate these immunoglobulins during processing. We have obtained the necessary animal ethics approvals to test the protective nature of these immunoglobulins in mice that are challenged with doses of venom. Finally, we have shown that these immunoglobulins are truly protective against the lethal effects of snake venoms. This study has proved the concept that alpaca can make potent therapeutic antibodies against snake venoms. However, further work is required before this could be commercialised. This report is an addition to RIRDC’s diverse range of over 2000 research publications and it forms part of our Rare Natural Animal Fibres R&D program, which aims to conduct RD&E for new and developing animal industries that contribute to the profitability, sustainability and productivity of regional Australia. Most of RIRDC’s publications are available for viewing, free downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313. Craig Burns Managing Director Rural Industries Research and Development Corporation iii About the author Andrew Padula is a veterinarian with a PhD in veterinary physiology from the University of Melbourne. He owns, and runs, a veterinary practice in Bairnsdale, Victoria. Acknowledgments This project would not have been possible without the enthusiasm and financial support of the Australian Alpaca Association (AAA). The AAA loaned various equipment for use in the project. In particular, Fiona and Ian Vanderbeek, of Alcazar Suri Stud, have been outstanding in helping to make this project successful, and in supplying alpaca. Thanks also to Jenny McDavitt, Merungle Alpaca Stud, Paul and Fran Haslin of Elysion Alpaca, Paul Cramley and Linda Davies of Pacofino, and Ros and Michael Davis of Elimbari Alpaca for supplying animals for use at various stages in the project. Technical aspects of the project were supported by Frank Madaras and Peter Mirtschin of Venom Science Pty Ltd, Tanunda, South Australia. The uniqueness of the alpaca necessitated starting from scratch with some aspects of this work, and Frank in particular helped with numerous issues. Thank you Dr Tim Kuchel of the Institute of Veterinary and Medical Sciences, Adelaide, South Australia, for advice on various technical aspects of antibody production. Thank you to Prof. Jose Maria Gutierrez, University of Costa Rica, Instituto Clodomiro Picado, Costa Rica for enthusiastic technical advice. Thanks to Nathan Dunstan of Venom Supplies Pty Ltd, South Australia for supply of the venoms used in the project. The venoms are the essential component required to make antibodies, and without the help of those who place themselves at risk with venomous snakes, this work could not happen. Thank you to Dr Ken Winkel, Director of the Australian Venom Research Unit, University of Melbourne. Ken has been part of many fruitful and informed discussions on the topic of snake venoms and treatments. We are fortunate to have such expertise in Australia. Thank you to Max Campbell and Ian Temby, of the Department of Primary Industries Wildlife and Small Institutions Animal Ethics Committee, for working with me the get the necessary approvals to undertake this work. I am most appreciative of the ‘working together’ approach with the delicate animal ethics issues associated with snake venoms and their application in animals. Thank you to Catheryn Obrien, veterinarian at Walter Eliza Hall Institute, for generous donation of technical knowledge and experience in working with mice. Thank you to the Animal Welfare Science Centre, University of Melbourne, for assistance with obtaining animal ethics approvals. Thank you to staff at Bairnsdale Animal Hospital: Kylee, Lisa, Debbie and Stephanie, for putting your time into this project. In particular, I would like to thank Allan Quirke, aka “the human clamp”, for the hours of helping with the field work with the alpaca. His abilities in the field and many useful tips are most appreciated. Thank you to Julie Bird of RIRDC for commissioning this project. iv Abbreviations µg: Microgram µL: Microlitre µm: Micrometre AAA: Australian Alpaca Association CSL: Commonwealth Serum Laboratory ELISA: Enzyme Linked Immunosorbent Assay g: Gram h: hour HCIgG: Heavy chain only immunoglobulin IgA: Immunoglobulin A IgG: Immunoglobulin G Kg: Kilogram M: Molar mg/mL: milligram per millilitre mg: Milligram mL: millilitre MW: molecular weight NaCl: Sodium Chloride nm: Nanometre PCV: packed cell volume PBS: Phosphate Buffered Saline PLA2: Phospholipase A2 U/mL: Units per millilitre U: Units °C: degrees Celsius v Contents Foreword ............................................................................................................................................... iii About the Author.................................................................................................................................. iv Acknowledgments................................................................................................................................