M ETHODS IN MOLECULAR BIOLOGY

Transcription

1 M ETHODS IN MOLECULAR BIOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes:

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3 ELISA Methods and Protocols Edited by Robert Hnasko Agricultural Research Service (ARS), U.S. Department of Agriculture, Albany, CA, USA

4 Editor Robert Hnasko Agricultural Research Service (ARS) U.S. Department of Agriculture Albany, CA, USA ISSN ISSN (electronic) Methods in Molecular Biology ISBN ISBN (ebook) DOI / Library of Congress Control Number: Springer New York Heidelberg Dordrecht London Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Cover illustration: Rendered three dimensional image of immunoglobulin (IgG1) protein as space filled and ribbon structure. Coordinates for IgG1 obtained from pdb1igy.pdb (Harris LJ, et al. J. Mol. Biol. 1998;275: ). Image by Robert Hnasko. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (

5 Pref ace 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 important and widely used diagnostic tool in medicine, animal health, botany, and quality assurance 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 defined conditions 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 difficult 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 refinement. No protocol is definitive 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 enzymatic reporter horseradish peroxidase (HRP). In subsequent chapters these authors demonstrate 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 Robert Hnasko v

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7 Contents Preface Contributors v ix 1 The Biochemical Properties of Antibodies and Their Fragments Robert M. Hnasko 2 Hybridoma Technology Robert M. Hnasko and Larry H. Stanker 3 Affinity Purification of Antibodies Robert M. Hnasko and Jeffery A. McGarvey 4 Bioconjugation of Antibodies to Horseradish Peroxidase (HRP) Robert M. Hnasko 5 Indirect ELISA Alice V. Lin 6 Direct ELISA Alice V. Lin 7 A Double-Sandwich ELISA for Identification of Monoclonal Antibodies Suitable for Sandwich Immunoassays Larry H. Stanker and Robert M. Hnasko 8 ELISpot and DC-ELISpot Assay to Measure Frequency of Antigen-Specific IFNγ-Secreting Cells Marcelo A. Navarrete 9 The Western Blot Thomas S. Hnasko and Robert M. Hnasko 10 Flow-Through Assay for Detection of Antibodies Using Protein-A Colloidal Gold Conjugate as a Probe Sreedevi Chennuru and Panduranga Rao Pavuluri 11 Multiplexed Microsphere Suspension Array-Based Immunoassays Andrew Lin, Alexandra Salvador, and J. Mark Carter 12 Multiplex Immunoassay: A Planar Array on a Chip Using the MagArray Technology Laurie M. Clotilde, Heng Yu, and M. Luis Carbonell 13 Lateral Flow Immunoassay Kathryn H. Ching 14 Immuno-PCR Assay for Sensitive Detection of Proteins in Real Time Xiaohua He and Stephanie A. Patfield 15 In Situ Proximity Ligation Assay (PLA) Sonchita Bagchi, Robert Fredriksson, and Åsa Wallén-Mackenzie vii

8 viii Contents 16 Tyramide Signal Amplification for Immunofluorescent Enhancement Lauren Faget and Thomas S. Hnasko 17 Correlative Microscopy for Localization of Proteins In Situ: Pre-embedding Immuno-Electron Microscopy Using FluoroNanogold, Gold Enhancement, and Low- Temperature Resin Daniela Boassa 18 Multiplex ELISA Using Oligonucleotide Tethered Antibodies Robert S. Matson 19 Gas Plasma Surface Chemistry for Biological Assays Khoren Sahagian and Mikki Larner Index

9 Contributors SONCHITA BAGCHI Department of Neuroscience, Uppsala biomedicinska centrum BMC, Uppsala University, Uppsala, Sweden DANIELA BOASSA National Center for Microscopy and Imaging Research and Center for Research on Biological Systems, University of California, San Diego, La Jolla, CA, USA M. LUIS CARBONELL MagArray Inc., Milpitas, CA, USA J. MARK CARTER Agricultural Research Service, U.S. Department of Agriculture, Albany, CA, USA SREEDEVI CHENNURU College of Veterinary Science, Sri Venkateswara Veterinary University, Proddatur, India KATHRYN H. CHING Crystal Bioscience, Emeryville, CA, USA LAURIE M. CLOTILDE MagArray Inc, Milpitas, CA, USA LAUREN FAGET 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, Uppsala, Sweden XIAOHUA HE Foodborne Toxin Detection and Prevention Research Unit (FTDP), Agricultural Research Service (ARS), Pacific West Area (PWA), Western Regional Research Center (WRRC), United States Department of Agriculture (USDA), Albany, CA, USA THOMAS S. HNASKO Department of Neurosciences, School of Medicine, University of California San Diego (UCSD), La Jolla, CA, USA ROBERT M. HNASKO Produce Safety and Microbiology Unit (PSM), Agricultural Research Service (ARS), Pacific West Area (PWA), Western Regional Research Center (WRRC), United States Department of Agriculture (USDA), Albany, CA, USA MIKKI LARNER Plasmatreat USA Inc., Belmont, CA, USA ANDREW LIN United States Food and Drug Administration (FDA), Alameda, CA, USA ALICE V. LIN Produce Safety and Microbiology Unit (PSM), Agricultural Research Service (ARS), Pacific West Area (PWA), Western Regional Research Center (WRRC), United States Department of Agriculture (USDA), Albany, CA, USA ROBERT S. MATSON QuantiScientifics LLC, Irvine, CA, USA JEFFERY A. MCGARVEY Foodborne Toxin Detection and Prevention Unit (FTDP), Agricultural Research Service (ARS), Pacific West Area (PWA), Western Regional Research Center (WRRC), United States Department of Agriculture (USDA), Albany, CA, USA MARCELO A. NAVARRETE Department of Hematology, Leiden University Medical Center, RC Leiden, The 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), Pacific West Area (PWA), Western Regional Research Center (WRRC), United States Department of Agriculture (USDA), Albany, CA, USA ix

10 x Contributors PANDURANGA RAO PAVULURI Ella foundation, Genome Valley, Turkapally, Hyderabad, Telangana, India KHOREN SAHAGIAN Plasmatreat USA Inc., Belmont, CA, USA ALEXANDRA SALVADOR Agricultural Research Service, U.S. Department of Agriculture, Albany, CA, USA LARRY H. STANKER Foodborne Toxin Detection and Prevention Unit (FTDP), Agricultural Research Service (ARS), Pacific West Area (PWA), Western Regional Research Center (WRRC), United States Department of Agriculture (USDA), Albany, CA, USA ÅSA WALLÉN-MACKENZIE Department of Neuroscience, Unit of Functional Neurobiology, Uppsala University, Uppsala, Sweden; Department of Comparative Physiology, Uppsala University, Uppsala, Sweden HENG YU MagArray Inc., Milpitas, CA, USA