Enzyme-linked Immunosorbent Assay (ELISA) -  Samira Hosseini,  Sergio O. Martinez-Chapa,  Marco Rito-Palomares,  Patricia Vazquez-Villegas

Enzyme-linked Immunosorbent Assay (ELISA) (eBook)

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2017 | 1. Auflage
124 Seiten
Springer Singapore (Verlag)
978-981-10-6766-2 (ISBN)
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This book offers comprehensive information on all aspects of ELISA, starting with the fundamentals of the immune system. It also reviews the history of analytical assays prior to the advent of ELISA (enzyme-linked immunosorbent assay) and addresses the materials of choice for the fabrication of the platforms, possible biomolecular interactions, different protocols, and evaluation parameters. The book guides readers through the respective steps of the analytical assay, while also familiarizing them with the possible sources of error in the assay. It offers detailed insights into the immobilization techniques used for protein attachment, as well as methods for evaluating the assay and calculating the key parameters, such as sensitivity, specificity, accuracy and limit of detection. In addition, the book explores the advantages and shortcomings of the conventional ELISA, as well as various approaches to improving its performance. In this regard, merging and integrating other technologies with widely known ELISAs have opened new avenues for the advancement of this immunoassay. Accordingly, the book provides cutting-edge information on integrated platforms such as ELISpot, plasmonic ELISAs, sphere-/bead-based ELISAs, paper-/fiber-based ELISAs and ELISA in micro-devices. 



Dr. Samira Hosseini obtained her B.Sc. degree in Applied Physics, her M.Sc. degree in Polymer Chemistry and her PhD degree in Biomedical Engineering. Currently, she is working as a Postdoctoral Researcher at the School of Engineering and Sciences, Tecnológico de Monterrey, Mexico. She is the author/co-author of more than 15 ISI indexed scientific publications, 12 book chapters and the inventor/co-inventor of 4 Intellectual properties. She is a member of the Mexican National System of Researchers. Her main areas of research are surface engineering, interface science, bioanalytical assays and polymeric materials. 

Dr. Patricia Vázquez-Villegas obtained her Bachelor's degree in Food Sciences Engineering program in 2008 and her PhD in Engineering Sciences from Tecnológico de Monterrey, Monterrey, México in 2013 majoring in Biotechnology. She made a one-year internship in 2012 at the University of British Columbia, Vancouver, Canada under the supervision of Dr. Charles A. Haynes. Since 2014 she holds a Postdoc position in Tecnológico de Monterrey where she is working in the development and application of platforms for purification and detection of different kinds of biomarkers. Dr. Vázquez is the author/co-author of 8 ISI indexed scientific publications, 1 book chapter and the inventor/co-inventor of 4 intellectual properties. She is a member of the Mexican National System of Researchers. Her main contribution had been in the field of continuous recovery of biomolecules using liquid-liquid extraction.

Professor Rito-Palomares obtained his PhD in Chemical Engineering from The University of Birmingham, United Kingdom in 1995. Following a sabbatical in the Department of Chemical Engineering at the University of Cambridge, United Kingdom (2001)., Prof. Rito-Palomares is currently the Director of the Biotechnology Centre and a Full Professor at Tecnologico de Monterrey, Mexico. He is a member of the Mexican Academy of Sciences, scientific committee of the International Foundation for Science (IFS, Sweden) as well as Mexican Research Council. He is the author/co-author of more than 100 ISI indexed scientific publications, 4 book chapters and inventor/co-inventor of 5 Intellectual properties that present his research work in the Bio-separation field. His main research contributions have been in the areas of biochemical recovery of biological products, and production, characterization and recovery of chemically modified proteins and the potential purification of stem cells. 

Sergio O. Martinez is Professor of the Electrical and Computer Engineering Department at Tecnológico de Monterrey, Monterrey Campus; and researcher and chair of the Sensors and Devices area of the National School of Engineering and Sciences at the same Institute. Sergio O. Martínez received the BS degree in Electronics and Communications Engineering and the MS degree in Control Engineering both from Tecnológico de Monterrey, Campus Monterrey, Mexico in 1983 and 1985, respectively. He obtained a MS degree in Electronics from Philips International Institute, Eindhoven, Netherlands in 1990; and the PhD degree in Microelectronics from National Polytechnic Institute of Grenoble, France in 2002. Prof. Martínez-Chapa is a member of the Mexican National System of Researchers since 2004 as well as member of the Mexican Academy of Sciences since 2010. His recent research activities, focused on Micro/Nanotechnologies, include Microfabrication Processes for Particle Manipulation Devices, Electrokinetically driven Microdevices for Manipulation of Suspended Particles in Fluidic Media, and Modeling and Design of surface plasmon resonance (SPR)-based Biosensing Platforms. He has authored/co-authored 75 peer-reviewed publications, 7 book chapters and the inventor/co-inventor of 2 granted intellectual properties.


This book offers comprehensive information on all aspects of ELISA, starting with the fundamentals of the immune system. It also reviews the history of analytical assays prior to the advent of ELISA (enzyme-linked immunosorbent assay) and addresses the materials of choice for the fabrication of the platforms, possible biomolecular interactions, different protocols, and evaluation parameters. The book guides readers through the respective steps of the analytical assay, while also familiarizing them with the possible sources of error in the assay. It offers detailed insights into the immobilization techniques used for protein attachment, as well as methods for evaluating the assay and calculating the key parameters, such as sensitivity, specificity, accuracy and limit of detection. In addition, the book explores the advantages and shortcomings of the conventional ELISA, as well as various approaches to improving its performance. In this regard, merging and integrating other technologieswith widely known ELISAs have opened new avenues for the advancement of this immunoassay. Accordingly, the book provides cutting-edge information on integrated platforms such as ELISpot, plasmonic ELISAs, sphere-/bead-based ELISAs, paper-/fiber-based ELISAs and ELISA in micro-devices.

Preface 6
Acknowledgements 7
Contents 8
1 Fundamentals and History of ELISA: The Evolution of the Immunoassays Until Invention of ELISA 11
Abstract 11
1.1 Evolution of the Immunoassays Until Invention of ELISA 11
1.1.1 Side Chain Theory 11
1.1.2 Antigen-Antibody Binding Theory 12
1.1.3 Discovery of Antibody Structure 13
1.1.4 Invention of Radioimmunoassay (RIA) 13
1.1.5 Invention of Enzyme Linked Immunosorbent Assay (ELISA) 14
1.2 Principles of the Immune System 15
1.2.1 Antibody Production in Human Body 15
1.2.2 Different Types of Antibodies 15
1.2.2.1 Immunoglobulin G (IgG) 16
1.2.2.2 Immunoglobulin A (IgA) 17
1.2.2.3 Immunoglobulin M (IgM) 18
1.2.2.4 Immunoglobulin D (IgD) 19
1.2.2.5 Immunoglobulin E (IgE) 19
1.2.3 Antigen-Antibody Coupling 20
1.2.3.1 Specificity of the Antigen-Antibody Coupling 21
1.3 Biomolecular Interactions Between Antibody and Antigen 21
1.3.1 Hydrogen Bonding 21
1.3.2 Hydrophobic Interaction 22
1.3.3 Ionic Attraction 22
1.3.4 Van der Waals Forces 24
1.3.4.1 London Dispersion Force 24
1.3.4.2 Dipole-Dipole Interaction 24
1.3.4.3 Ion-Dipole Interaction 25
References 25
2 General Overviews on Applications of ELISA 29
Abstract 29
2.1 Applications of ELISA 29
2.1.1 Food Industry 29
2.1.2 Vaccine Development 30
2.1.3 Immunology 30
2.1.3.1 Autoimmunity 31
2.1.3.2 Humoral Immunity 31
2.1.4 Diagnosis 32
2.1.4.1 Pregnancy Test 32
2.1.4.2 Cancer Detection 33
2.1.4.3 Detection of the Infectious Diseases 33
2.1.5 Toxicology 34
2.1.6 Drug Monitoring and Pharmaceutical Industry 35
2.1.7 Transplantation 35
References 36
3 Step by Step with ELISA: Mechanism of Operation, Crucial Elements, Different Protocols, and Insights on Immobilization and Detection of Various Biomolecular Entities 40
Abstract 40
3.1 Mechanism of Operation 40
3.2 Different Elements of the Assay 42
3.2.1 Solid Phase 42
3.2.2 Adsorbents 42
3.2.2.1 Target Biomolecules 43
3.2.3 Washing Agents 43
3.2.4 Blocking Agents 43
3.2.5 Enzymes and Substrates 43
3.2.5.1 Different Types of Enzyme 46
3.2.5.2 Different Types of Substrate 48
3.2.6 Stopping Process 49
3.2.7 Reading Techniques 51
3.2.7.1 Colorimetric Assay 51
3.2.7.2 Fluorescent Assay 51
3.2.7.3 Luminescent Assay 52
3.2.8 Reading Apparatus 52
3.2.9 Controls 53
3.2.9.1 Positive Controls 53
3.2.9.2 Endogenous Positive Control 53
3.2.9.3 Negative Controls 53
3.2.9.4 Standard Controls 53
3.2.9.5 Spike Controls 53
3.3 Different Protocols 54
3.3.1 Direct ELISA 54
3.3.2 Indirect ELISA 54
3.3.3 Sandwich ELISA 54
3.3.4 Double Sandwich ELISA 56
3.3.5 Competitive ELISA 56
3.4 Initial Interaction of the Biomolecules with the Surface 56
3.5 Immobilization Techniques for Protein Attachment 58
3.5.1 Physical Immobilization 58
3.5.2 Immobilization via Entrapment 59
3.5.3 Covalent Immobilization 59
3.5.3.1 Immobilization via Zero-Length Cross Linker 60
3.5.3.2 Immobilization via Spacers 61
3.5.4 Oriented Immobilization 61
References 62
4 Evaluation of the Detection Results Obtained from ELISA 66
Abstract 66
4.1 Conducting a Reliable Assay 66
4.1.1 Sources of Errors 67
4.1.2 Troubleshooting 67
4.2 Key Parameters in ELISA Evaluation 71
4.2.1 Sensitivity 72
4.2.2 Specificity 72
4.2.3 Accuracy 72
4.2.4 Limit of Detection (LOD) 73
4.3 Measurable Units in ELISA 73
References 74
5 Advantages, Disadvantages and Modifications of Conventional ELISA 76
Abstract 76
5.1 Significance of Conventional ELISA 77
5.2 Shortages of Conventional ELISA 77
5.3 Materials of Choice for Fabrication of ELISA Well Plates 78
5.4 Different Types of ELISA Well Plates 79
5.5 Modified ELISA Platforms 80
5.5.1 ELISA on Coated Platforms 81
5.5.2 ELISpot 83
5.5.3 Plasmonic ELISA 85
5.5.4 Sphere-/Bead-Based ELISA 89
5.5.5 Paper-Based ELISA 93
5.5.6 Fiber-Based ELISA 99
5.5.7 ELISA in Micro-Devices 107
5.5.8 Other Strategies 114
5.5.8.1 Smart Devices in ELISA 114
5.5.8.2 Digital ELISA 116
5.5.8.3 Aptamers as Antibody Substitutes in ELISA 117
5.5.8.4 Multiple and Portable ELISA 117
References 117

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