ELISA: Methods and Protocols

227 Pages·2015·9.948 MB·English

by  Robert Hnasko

Methods in
Molecular Biology 1318
Robert Hnasko Editor
ELISA
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
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ELISA
Methods and Protocols
Edited by
Robert Hnasko
Agricultural Research Service (ARS), U.S. Department of Agriculture, Albany, CA, USA
Editor
Robert H nasko
Agricultural Research Service (ARS)
U.S. Department of Agriculture
Albany, CA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-2741-8 ISBN 978-1-4939-2742-5 (eBook)
DOI 10.1007/978-1-4939-2742-5
Library of Congress Control Number: 2015943593
Springer New York Heidelberg Dordrecht London
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Cover illustration: Rendered three dimensional image of immunoglobulin (IgG1) protein as space fi lled and ribbon
structure. Coordinates for IgG1 obtained from pdb1igy.pdb (Harris LJ, et al. J. Mol. Biol. 1998;275:861–872). Image
by Robert Hnasko.
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Prefa ce
This book is intended as a practical biochemical guide to the Enzyme-Linked Immunosorbent
Assay (ELISA) used to detect a target substance in a liquid sample. The ELISA is an impor-
tant and widely used diagnostic tool in medicine, animal health, botany, and quality assur-
ance processes in food and beverage production. The adoption of the ELISA by these
industries is in part driven by the sensitivity and selectivity by which the assay can detect a
desired target analyte along with it potential for standardization, automation, and economy
of cost. The component parts of the ELISA are well suited for commercialization and
adaptable for target detection using a diverse range of platforms.
The antibody is central to the performance of an ELISA providing the basis of analyte
selection and detection. It is the interaction of antibody with analyte under defi ned condi-
tions that dictates the outcome of the ELISA, and deviations in those conditions will impact
assay performance. The aim of this manual is to provide the reader an overview of ELISA
techniques and their potential application along with the technical aptitude necessary to
design, run, and evaluate these immunoassays for the detection and quantitation of target
analytes in solution.
The origin of a given protocol is diffi cult to determine and appropriate references are
given when possible. Yet there will be many omissions as the pages of these protocols,
derived from use in our laboratories, pass through the many hands of revision and refi ne-
ment. No protocol is defi nitive and success often depends on the qualities of the antibodies
and samples.
This book begins with an introductory chapter that is intended to orient the reader on
the basic structure and function of immunoglobulins and their fragments. Arguably the
antibody is the most important component of any immunoassay, and the second chapter in
this book outlines the methodology to generate monoclonal antibodies using hybridoma
technology. It is intended to provide the reader with an appreciation of how one can use
hybridoma technology to generate and select for monoclonal antibodies that best meet the
criteria of a given immunoassay. It should impress upon the immunoassay developer the
value of an unlimited source of a stable characterized antibody and its commercial potential.
The third chapter provides the general methods used to purify antibodies, and the fourth
chapter provides one example of how to successfully conjugate an antibody to the enzy-
matic reporter horseradish peroxidase (HRP). In subsequent chapters these authors dem-
onstrate how to creatively use the properties of the antibody to identify, localize, and
quantify target analytes to answer questions and resolve problems. The reader will learn
how to use a variety of immunoassay strategies, reporters, and detection systems that will
undoubtedly facilitate their efforts to gain answers to their own questions. It is the goal of
this book to provide the technical information necessary for the reader to successfully use
the immunoassay as part of the discovery process.
Albany, CA, USA R obert Hnasko
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 The Biochemical Properties of Antibodies and Their Fragments . . . . . . . . . . . 1
Robert M. Hnasko
2 H ybridoma Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5
Robert M. H nasko and Larry H . S tanker
3 Affinity Purification of Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Robert M. H nasko and J effery A. M cGarvey
4 B ioconjugation of Antibodies to Horseradish Peroxidase (HRP). . . . . . . . . . . 4 3
Robert M . Hnasko
5 I ndirect ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Alice V. L in
6 D irect ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1
A lice V . Lin
7 A Double-Sandwich ELISA for Identification of Monoclonal
Antibodies Suitable for Sandwich Immunoassays. . . . . . . . . . . . . . . . . . . . . . . 69
Larry H. Stanker and Robert M. Hnasko
8 E LISpot and DC-ELISpot Assay to Measure Frequency
of Antigen-Specific IFNγ-Secreting Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Marcelo A . N avarrete
9 The Western Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 7
Thomas S. Hnasko and Robert M . Hnasko
10 Flow-Through Assay for Detection of Antibodies
Using Protein-A Colloidal Gold Conjugate as a Probe. . . . . . . . . . . . . . . . . . . 97
Sreedevi Chennuru and Panduranga R ao P avuluri
11 M ultiplexed Microsphere Suspension Array-Based Immunoassays . . . . . . . . . . 1 07
Andrew L in , A lexandra S alvador , and J. M ark Carter
12 Multiplex Immunoassay: A Planar Array on a Chip Using the MagArray™
Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Laurie M. C lotilde , Heng Yu , and M. L uis Carbonell
13 L ateral Flow Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 27
Kathryn H . Ching
14 Immuno-PCR Assay for Sensitive Detection of Proteins in Real Time . . . . . . . 1 39
Xiaohua H e and S tephanie A . P atfield
15 In Situ Proximity Ligation Assay (PLA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Sonchita Bagchi , Robert Fredriksson , and Åsa W allén-Mackenzie
vii
viii Contents
16 Tyramide Signal Amplification for Immunofluorescent Enhancement . . . . . . . 1 61
Lauren Faget and Thomas S . H nasko
17 C orrelative Microscopy for Localization of Proteins In Situ:
Pre-embedding Immuno-Electron Microscopy Using FluoroNanogold,
Gold Enhancement, and Low-T emperature Resin. . . . . . . . . . . . . . . . . . . . . . 173
Daniela B oassa
18 M ultiplex ELISA Using Oligonucleotide Tethered Antibodies. . . . . . . . . . . . . 1 81
Robert S . M atson
19 Gas Plasma Surface Chemistry for Biological Assays. . . . . . . . . . . . . . . . . . . . . 1 97
Khoren Sahagian and Mikki L arner
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 15
Contributors
SONCHITA BAGCHI • Department of Neuroscience, Uppsala biomedicinska centrum BMC ,
Uppsala University , U ppsala , Sweden
DANIELA BOASSA • National Center for Microscopy and Imaging Research and Center for
Research on Biological Systems, U niversity of California, San Diego , L a Jolla , C A , USA
M. LUIS CARBONELL • MagArray Inc., Milpitas, CA, USA
J. M ARK CARTER • Agricultural Research Service, U .S. Department of Agriculture ,
Albany, CA , U SA
SREEDEVI CHENNURU • College of Veterinary Science, S ri Venkateswara Veterinary
University , P roddatur , I ndia
KATHRYN H. CHING • Crystal Bioscience , Emeryville, C A , U SA
LAURIE M . C LOTILDE • MagArray Inc , M ilpitas , C A , U SA
LAUREN F AGET • Department of Neurosciences, School of Medicine, University of California
San Diego (UCSD), La Jolla, CA, USA
ROBERT FREDRIKSSON • Department of Neuroscience, Uppsala biomedicinska centrum BMC ,
Uppsala University , U ppsala , Sweden
XIAOHUA HE • Foodborne Toxin Detection and Prevention Research Unit (FTDP),
Agricultural Research Service (ARS), Pacifi c West Area (PWA), Western Regional
Research Center (WRRC), United States Department of Agriculture (USDA) , A lbany,
CA , USA
THOMAS S. HNASKO • Department of Neurosciences, School of Medicine , U niversity of
California San Diego (UCSD) , La Jolla , C A , U SA
ROBERT M . H NASKO • Produce Safety and Microbiology Unit (PSM), Agricultural Research
Service (ARS), Pacifi c West Area (PWA), Western Regional Research Center (WRRC),
United States Department of Agriculture (USDA) , A lbany, C A , USA
MIKKI LARNER • Plasmatreat USA Inc. , Belmont, C A , USA
ANDREW L IN • United States Food and Drug Administration (FDA) , A lameda, C A , USA
ALICE V. LIN • Produce Safety and Microbiology Unit (PSM), Agricultural Research
Service (ARS), Pacifi c West Area (PWA), Western Regional Research Center (WRRC),
United States Department of Agriculture (USDA) , A lbany, C A , U SA
ROBERT S . MATSON • QuantiScientifi cs LLC , I rvine , C A , U SA
JEFFERY A. MCGARVEY • Foodborne Toxin Detection and Prevention Unit (FTDP),
Agricultural Research Service (ARS), Pacifi c West Area (PWA), Western Regional
Research Center (WRRC), U nited States Department of Agriculture (USDA) , A lbany,
CA , U SA
MARCELO A. NAVARRETE • Department of Hematology, L eiden University Medical Center ,
RC Leiden, T he Netherlands; School of Medicine, University of Magallanes, Punta
Arenas, Chile
STEPHANIE A. PATFIELD • Foodborne Toxin Detection and Prevention Research Unit
(FTDP), Agricultural Research Service (ARS), Pacifi c West Area (PWA), Western
Regional Research Center (WRRC), United States Department of Agriculture (USDA) ,
Albany, C A , U SA
ix