Table Of ContentMethods in
Molecular Biology 1808
Alexander E. Kalyuzhny Editor
Handbook
of ELISPOT
Methods and Protocols
Third Edition
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
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Handbook of ELISPOT
Methods and Protocols
Third Edition
Edited by
Alexander E. Kalyuzhny
Bio-Techne, Inc., Minneapolis, MN, USA
Editor
Alexander E. Kalyuzhny
Bio-Techne, Inc.
Minneapolis, MN, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-8566-1 ISBN 978-1-4939-8567-8 (eBook)
https://doi.org/10.1007/978-1-4939-8567-8
Library of Congress Control Number: 2018946639
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Preface
Despite technological advancements and the availability of ultrasensitive analytical meth-
ods, ELISpot assay, which is technically quite simple, is firmly holding its ground for study-
ing the cytokine-secreting activity of immune system cells. Furthermore, this assay is gaining
a reputation as a robust and reliable research and diagnostic tool. To refresh readers’ mem-
ories, it is worth mentioning that the first edition of the Handbook of ELISpot was released
in 2005. Due to a strong interest from the scientific community, it was followed by a second
edition published in 2012. We were excited to learn that our second edition also received
strong positive feedback, which prompted us to continue with a third edition. It appears
that ELISpot remains to be a very dynamic technique that can be easily modified to meet
challenging experimental needs. However, the simplicity of ELISpot may be deceptive as it
requires a clear understanding of its bioassay and immunoassay components and how they
blend together. In addition to learning basic ELISpot technique, researchers should under-
stand the principles of analyzing ELISpot images and spot quantification, how to digest the
biological information from the images with arrays of spots, and how to perform statistical
analysis.
The third edition of the Handbook of ELISpot expands upon our first and second edi-
tions, and we are adhering to the same principles as before: the book should help research-
ers learn new protocols and become proficient in using this technique. Due to a strong
interest in multiplex ELISpot, our current volume includes numerous chapters on ELISpot’s
sibling, known as FluoroSpot, disclosing vast details on how to set, run, and analyze mul-
tiplex data. To address the challenges in studying and diagnosing infectious diseases, we
included chapters on using ELISpot for tuberculosis, influenza, Dengue virus, and feline
immunodeficiency virus analysis. Other chapters are focused on ELISpot for vaccine
research, essential controls, image analysis of spots, assay evaluation, ELISpot automation,
and challenges in analyzing antibody-secreting cells and designing the assay. As with the
first and second editions, the goal of compiling this volume was to make an additional tech-
nical reference and a troubleshooting guide both for researchers who are new to the field
and for experienced ELISpot users. All the chapters were written by experts who were
excited to have an opportunity to share their experience and skills with colleagues
worldwide.
I wish to express my sincere thanks to our contributing authors for committing their
time and effort to work on their chapters and submitting them in a timely manner. As the
book’s editor, I was privileged to connect with many exceptional and passionate scholars
from whom I learned a lot about advances in ELISpot technology.
We hope this book will serve as a stepping stone for novices as well as food for thought
for ELISpot experts.
Minneapolis, MN, USA Alexander E. Kalyuzhny
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Challenges in Developing Protein Secretion Assays at a Single-Cell Level . . . . . 1
Yoshitaka Shirasaki and Osamu Ohara
2 Mastering the Computational Challenges of Elispot Plate Evaluation . . . . . . . . 9
Sylvia Janetzki
3 Essential Controls for ELISpot Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Jodi Hagen and Alexander E. Kalyuzhny
4 Automatic Search of Spots and Color Classification in ELISPOT Assay . . . . . . 43
Sergey S. Zadorozhny and Nikolai N. Martynov
5 Four Color ImmunoSpot® Assays for Identification of Effector
T-Cell Lineages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Jodi Hanson, Diana R. Roen, and Paul V. Lehmann
6 Detection of Cross-Reactive B Cells Using the FluoroSpot Assay . . . . . . . . . . . 63
Peter Jahnmatz and Niklas Ahlborg
7 Multiplex ImmunoSpot® Assays for the Study of Functional
B Cell Subpopulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Diana R. Roen, Jodi Hanson, and Paul V. Lehmann
8 Detecting all Immunoglobulin Classes and Subclasses
in a Multiplex 7 Color ImmunoSpot® Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Richard Caspell and Paul V. Lehmann
9 Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental
Validation of the Multi-Color ImmunoSpot® Software Based
on Center of Mass Distance Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Alexey Y. Karulin, Zoltán Megyesi, Richard Caspell, Jodi Hanson,
and Paul V. Lehmann
10 Multi-Color FLUOROSPOT Counting Using ImmunoSpot®
Fluoro-X™ Suite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Zoltán Megyesi, Paul V. Lehmann, and Alexey Y. Karulin
11 B-Cell ELISpot Assay to Quantify Antigen-Specific Antibody-Secreting
Cells in Human Peripheral Blood Mononuclear Cells . . . . . . . . . . . . . . . . . . . . 133
Haw Hwai, Yi-Ying Chen, and Shiang-Jong Tzeng
12 Identification of Novel Mycobacterial Targets for Murine CD4+
T-Cells by IFNγ ELISPOT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Alison J. Johnson, Steven C. Kennedy, Tony W. Ng, and Steven A. Porcelli
vii
viii Contents
13 ELISPOT-Based “Multi-Color FluoroSpot” to Study Type- Specific
and Cross-Reactive Responses in Memory B Cells after Dengue
and Zika Virus Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Paulina Andrade, Josefina Coloma, and Eva Harris
14 Cultured ELISpot Assay to Investigate Dengue Virus Specific
T-Cell Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Chandima Jeewandara, Graham S. Ogg, and Gathsaurie Neelika Malavige
15 Ex Vivo ELISpot Assay to Investigate Dengue Virus Specific
T-Cell Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Gathsaurie Neelika Malavige
16 Ex Vivo ELISpot Assay to Investigate iNKT Cell Responses
in Acute Dengue Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Achala Indika Kamaladasa and Gathsaurie Neelika Malavige
17 Dendritic Cell-Based ELISpot Assay for Assessing T-Cell IFN-γ
Responses in Human Peripheral Blood Mononuclear Cells
to Dengue Envelope Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Peifang Sun and Monika Simmons
18 Utilization of Feline ELISpot to Evaluate the Immunogenicity
of a T Cell-Based FIV MAP Vaccine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Bikash Sahay, Alek M. Aranyos, Andrew McAvoy, and Janet K. Yamamoto
19 Detection and Quantification of Influenza A/H1N1 Virus-S pecific
Memory B Cells in Human PBMCs Using ELISpot Assay . . . . . . . . . . . . . . . . 221
Iana H. Haralambieva, Inna G. Ovsyannikova, Richard B. Kennedy,
and Gregory A. Poland
20 Towards a Full Automation of the ELISpot Assay for Safe and Parallelized
Immunomonitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
J. C. Neubauer, I. Sébastien, A. Germann, H. von Briesen,
and H. Zimmermann
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Contributors
Niklas ahlborg • Mabtech, Nacka Strand, Sweden; Department of Immunology,
Stockholm University, Stockholm, Sweden
PauliNa aNdrade • Division of Infectious Diseases and Vaccinology, School of Public
Health, University of California, Berkeley, CA, USA; University of San Francisco de
Quito, Quito, Ecuador
alek M. araNyos • Department of Infectious Diseases and Immunology,
College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
richard casPell • Cellular Technology Ltd., Shaker Heights, OH, USA
yi-yiNg cheN • Graduate Institute of Pharmacology, College of Medicine,
National Taiwan University, Taipei, Taiwan
JosefiNa coloMa • Division of Infectious Diseases and Vaccinology, School of Public
Health, University of California, Berkeley, Berkeley, CA, USA
a. gerMaNN • Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany
Jodi hageN • Bio-Techne, Minneapolis, MN, USA
Jodi haNsoN • Cellular Technology Ltd., Shaker Heights, OH, USA
iaNa h. haralaMbieva • Mayo Clinic Vaccine Research Group, Mayo Clinic, Rochester,
MN, USA
eva harris • Division of Infectious Diseases and Vaccinology, School of Public Health,
University of California, Berkeley, CA, USA
haw hwai • Department of Medicine, College of Medicine, National Taiwan University,
Taipei, Taiwan
Peter JahNMatz • Department of Medicine, Karolinska Institutet, Stockholm, Sweden;
Mabtech, Nacka Strand, Sweden
sylvia JaNetzki • ZellNet Consulting, Inc., Fort Lee, NJ, USA
chaNdiMa JeewaNdara • Centre for Dengue Research, Faculty of Medical Sciences,
University of Sri Jayewardenapura, Nugegoda, Sri Lanka
alisoN J. JohNsoN • Department of Microbiology and Immunology, Albert Einstein
College of Medicine, Bronx, NY, USA
alexaNder e. kalyuzhNy • Bio-Techne, Inc., Minneapolis, MN, USA
achala iNdika kaMaladasa • Centre for Dengue Research, Faculty of Medical Sciences,
University of Sri Jayewardenapura, Nugegoda, Sri Lanka
alexey y. karuliN • Cellular Technology Ltd., Shaker Heights, OH, USA
richard b. keNNedy • Mayo Clinic Vaccine Research Group, Mayo Clinic, Rochester,
MN, USA
steveN c. keNNedy • Department of Microbiology and Immunology, Albert Einstein
College of Medicine, Bronx, NY, USA
Paul v. lehMaNN • Cellular Technology Ltd., Shaker Heights, OH, USA
gathsaurie Neelika Malavige • Centre for Dengue Research, Faculty of Medical
Sciences, University of Sri Jayewardenapura, Nugegoda, Sri Lanka; MRC Human
Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,
Oxford, UK
ix
x Contributors
Nikolai N. MartyNov • MZ Computers Ltd, Moscow, Russian Federation
aNdrew Mcavoy • Department of Infectious Diseases and Immunology, College of
Veterinary Medicine, University of Florida, Gainesville, FL, USA
zoltáN Megyesi • Cellular Technology Ltd., Shaker Heights, OH, USA
J. c. Neubauer • Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany;
Fraunhofer Project Centre for Stem Cell Process Engineering, Wurzburg, Germany
toNy w. Ng • Department of Microbiology and Immunology, Albert Einstein College of
Medicine, Bronx, NY, USA
grahaM s. ogg • MRC Human Immunology Unit, Weatherall Institute of Molecular
Medicine, John Radcliffe Hospital, Oxford, UK
osaMu ohara • Laboratory for Integrative Genomics, RIKEN Center for Integrative
Medical Sciences, Tokyo, Japan; Department of Genome Research and Development,
Kazusa DNA Research Institute, Chiba, Japan; The Futuristic Medical Care Education
and Research Organization, Chiba University, Chiba, Japan
iNNa g. ovsyaNNikova • Mayo Clinic Vaccine Research Group, Mayo Clinic, Rochester,
MN, USA
gregory a. PolaNd • Mayo Clinic Vaccine Research Group, Mayo Clinic, Rochester, MN,
USA
steveN a. Porcelli • Department of Microbiology and Immunology, Albert Einstein
College of Medicine, Bronx, NY, USA; Department of Medicine, Albert Einstein College
of Medicine, Bronx, NY, USA
diaNa r. roeN • Cellular Technology Ltd., Shaker Heights, OH, USA
bikash sahay • Department of Infectious Diseases and Immunology, College of Veterinary
Medicine, University of Florida, Gainesville, FL, USA
i. sébastieN • Fraunhofer Project Centre for Stem Cell Process Engineering, Würzburg,
Germany
yoshitaka shirasaki • PRESTO, Japan Science and Technology Agency, Tokyo, Japan;
Department of Biological Science, Graduate School of Science, The University of Tokyo,
Tokyo, Japan; Laboratory for Integrative Genomics, RIKEN Center for Integrative
Medical Sciences, Tokyo, Japan
MoNika siMMoNs • Naval Medical Research Center, Silver Spring, MD, USA
PeifaNg suN • Henry Jackson Foundation for the Advancement of Military Medicine,
Bethesda, MD, USA
shiaNg-JoNg tzeNg • Graduate Institute of Pharmacology, College of Medicine, National
Taiwan University, Taipei, Taiwan
h. voN brieseN • Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany
JaNet k. yaMaMoto • Department of Infectious Diseases and Immunology, College of
Veterinary Medicine, University of Florida, Gainesville, FL, USA
sergey s. zadorozhNy • MZ Computers Ltd, Moscow, Russian Federation
h. ziMMerMaNN • Fraunhofer Institute for Biomedical Engineering, Sulzbach, Germany;
Fraunhofer Project Centre for Stem Cell Process Engineering, Würzburg, Germany;
Molecular and Cellular Biotechnology/Nanotechnology, Saarland University,
Saarbrucken, Germany; Facultad de Ciencias del Mar, Universidad Catolica del Norte,
Coquimbo, Chile
Chapter 1
Challenges in Developing Protein Secretion Assays
at a Single-Cell Level
Yoshitaka Shirasaki and Osamu Ohara
Abstract
In addition to direct physical interactions between/among cells, the secretion of humoral factors from
living cells is a critical process for cell-cell communications. A well-known extracellular signaling event is
mediated by immune cell cytokines/chemokines. Because cell-cell communication is crucial in immune
cell sociology, protein secretion assays first attracted a broad range of immunology interests. Now that we
have entered an era of systems biology, cell-cell interactions mediated by secreted molecules should be
revisited to understand the dynamics and homeostasis of the cell society as a whole. Of more importance,
recent advances in detection and microfluidics technologies enable us to monitor protein secretion in real
time rather than as a snapshot from the past, which gives us an opportunity to more deeply understand the
logic of mammalian cell sociology. This chapter reviews the recent progress in and future direction of pro-
tein secretion assays, particularly from a mammalian cell sociology viewpoint.
Key words Cell sociology, Protein secretion, Real-time monitoring, Single cell, Snapshot, Systems
biology
1 Introduction
It is well known that cell-cell communication plays a crucial role in
cellular dynamics and homeostasis. In particular, long-range inter-
actions among cells are key for homeostasis of the whole cell soci-
ety, and short-range interactions evoked by direct physical contact
between cells are indispensable routes for the local cell system.
Although long-range interactions can also be mediated physically
(e.g., through electric signals and/or cellular nanotubes), humoral
factors secreted from cells are the most prominent players for long-
range cell-cell interactions in this sense. This chapter focuses on
protein secretion as a representative humoral factor.
The concentration of secreted proteins is conventionally mea-
sured by enzyme-linked immunosorbent assay (ELISA) as a sum of
humoral factors secreted from many cells into culture media. Such a
measurement cannot clarify how many cells are contributing to
Alexander E. Kalyuzhny (ed.), Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, vol. 1808,
https://doi.org/10.1007/978-1-4939-8567-8_1, © Springer Science+Business Media, LLC, part of Springer Nature 2018
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