The Complement System: Methods and Protocols

384 Pages·2014·7.135 MB·English

by  Mihaela Gadjeva (auth.), Mihaela Gadjeva (eds.)

Methods in
Molecular Biology 1100
Mihaela Gadjeva Editor
The
Complement
System
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

The Complement System
Methods and Protocols
Edited by
Mihaela Gadjeva
Division of Infectious Diseases, Brigham and Women’s Hospital,
Harvard Medical School, Harvard University, Boston, MA, USA
Editor
Mihaela Gadjeva
Division of Infectious Diseases
Brigham and Women’s Hospital
Harvard Medical School
Harvard University
Boston , MA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-723-5 ISBN 978-1-62703-724-2 (eBook)
DOI 10.1007/978-1-62703-724-2
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2013950908
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Dedication
To Prof. Kenneth Reid in recognition of his outstanding and fundamental work in
Complement Biology.
To Prof. Gerald Pier in gratitude for his continuous advice and generous support.
To my family who are my inspiration.

Pref ace
The complement system is a key system for immune surveillance and homeostasis.
Discoveries during the past 20 years of complement research yielded detailed knowledge of
the molecular structure of complement components, the mechanisms of activation, and
regulation of the system in health and disease. The objective of the current book is to
describe methods to study the soluble components and receptors involved in the comple-
ment system, including new methodological aspects to assay complement function and
activation in humans and in model animals. This collection of more than 30 individual
chapters describes variety of protocols to purify and analyze the activity of the individual
complement components. Of particular interest to clinical workers are the sections focused
on the detection of complement SNPs, the clinical methods to evaluate complement system
activation, and data interpretation. The introduction portions of each of the chapters high-
lights the current concepts and scientifi c principles based on which the individual assays are
build, whereas the references include key papers that are selected to broaden reader’s
knowledge of the specifi c topics.
I hope that this collection of well-established “classical” assays and recently developed
“new” assays to analyze the complement system activation will be useful to a wide audience
of scientists, interested in complement research including complementologists, new entrants
to the fi eld as well as clinician–scientists, biochemists, molecular biologists, and
immunologists.
I would like to thank John Walker, Series Editor, and Humana Press, for the opportu-
nity to assemble this outstanding collection of articles dedicated to analysis of the comple-
ment system. In addition, I would like to thank the contributing authors for taking time
from their busy schedules to create easy-to-follow and well-presented chapters.
Boston, MA , USA M ihaela Gadjeva
vii

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Mihaela Gadjeva
2 Evaluation of Complement Function by ELISA. . . . . . . . . . . . . . . . . . . . . . . . 11
Anja Roos and Jörgen Wieslander
3 Classical Complement Pathway Component C1q: Purification
of Human C1q, Isolation of C1q Collagen-Like and Globular Head
Fragments and Production of Recombinant C1q—Derivatives.
Functional Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Mihaela Kojouharova
4 Classical Complement Pathway Components C1r and C1s:
Purification from Human Serum and in Recombinant Form
and Functional Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Véronique Rossi, Isabelle Bally, Monique Lacroix, Gérard J. Arlaud,
and Nicole M. Thielens
5 Expression and Purification Methods for the Production
of Recombinant Human Complement Component C2. . . . . . . . . . . . . . . . . . 61
Angela W. Norton, Paolo G.V. Martini, Lynette C. Cook,
Scott Alderucci, Dianna M. Lundberg, Susan M. Fish, Charles Bedard,
John Gill, Arthur O. Tzianabos, and Michael F. Concino
6 Purification and Characterization of Human and Mouse Complement C3. . . . 75
Marieta M. Ruseva and Meike Heurich
7 Purification of Human Complement Protein C5. . . . . . . . . . . . . . . . . . . . . . . 93
Lars Sottrup-Jensen and Gregers Rom Andersen
8 Quantification of Complement C5b-9 Binding
to Cells by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Oren Moskovich and Zvi Fishelson
9 Challenges and Opportunities in Fractionation of Recombinant
Human Mannan-Binding Lectin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Thomas Vorup-Jensen
10 Genotyping of FCN and MBL2 Polymorphisms
Using Pyrosequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Lea Munthe-Fog, Hans Ole Madsen, and Peter Garred
ix