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Über dieses Buch

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.

Inhaltsverzeichnis

Frontmatter

Chapter 1. Fundamentals and History of ELISA: The Evolution of the Immunoassays Until Invention of ELISA

Abstract
Current chapter reviews background and history of the immunoassays until invention of ELISA. In that perspective, important evolutions in the field such as side-chain theory, antigen-antibody theory, discovery of the antibody structure, invention of radioimmunoassay (RIA), and invention of enzyme linked immunosorbent assay (ELISA) are reviewed. The chapter, also describes the principles of the immune system such as antibody production in human body along with different classes of antibodies, as well as antigen-antibody coupling and specificity of such interaction. In that respect, the chapter also demonstrates sources for biomolecular interaction between the biomolecules. Dominant forces that are involved in physical interaction of the antigens and antibodies including hydrogen bonding (H-bonding), hydrophobic interaction, ionic attraction, and Van der Waals forces such as London dispersion force, dipole-dipole interaction, and ion-dipole interaction are introduced in great details.
Samira Hosseini, Patricia Vázquez-Villegas, Marco Rito-Palomares, Sergio O. Martinez-Chapa

Chapter 2. General Overviews on Applications of ELISA

Abstract
Current chapter reviews the applications of ELISA in various different fields including food industry, vaccine development, immunology (autoimmunity and humoral immunity), diagnosis (pregnancy, cancer and infectious diseases), toxicology, drug monitoring, pharmaceutical industry, and transplantation. Different examples related to each area are explained. ELISA was found to play major roles in all the mentioned disciplines.
Samira Hosseini, Patricia Vázquez-Villegas, Marco Rito-Palomares, Sergio O. Martinez-Chapa

Chapter 3. Step by Step with ELISA: Mechanism of Operation, Crucial Elements, Different Protocols, and Insights on Immobilization and Detection of Various Biomolecular Entities

Abstract
Current chapter describes the essential components of ELISA including the solid phase, the adsorbents (different types of target biomolecules), and the washing and blocking agents used in assay procedure. The chapter also reviews widely applied enzymes and substrates with their specific characteristics. To complete the assay, the chapter offers information regarding the stopping procedure and readout techniques such as colorimetric, fluorescence and luminescence, along with their reading instruments. To secure a high specificity, the chapter describes protocols for conducting different types of controls in the assay procedure. These controls are namely: positive, endogenous, negative, standard, and spike controls. The chapter subsequently describes available ELISA protocols including direct, indirect, sandwich, double sandwich, and competitive assays. Finally, this chapter is dedicated to reviewing immobilization techniques including physical, covalent, oriented strategies as well as immobilization via entrapment. In the case of covalent immobilization of the biomolecules, protein attachment via zero-length cross linkers and spacers (linear or branched) are described.
Samira Hosseini, Patricia Vázquez-Villegas, Marco Rito-Palomares, Sergio O. Martinez-Chapa

Chapter 4. Evaluation of the Detection Results Obtained from ELISA

Abstract
This chapter presents the most commons errors that typically occur when performing ELISA. Following every error, the chapter points out the possible reasons behind such errors and possible methods to overcome the problems. The chapter also describes key important parameters in assay evaluation including sensitivity, specificity, accuracy and limit of detection (LOD). Offering methods for calculation of the evaluation parameters, the chapter also provides insights and considerations for assessing the reliability of the assay. Such analysis is essential to upgrade a newly developed assay from the analytical relevance to the clinical application. Finally, the chapter provides information in regard to the measurable units in ELISA assay.
Samira Hosseini, Patricia Vázquez-Villegas, Marco Rito-Palomares, Sergio O. Martinez-Chapa

Chapter 5. Advantages, Disadvantages and Modifications of Conventional ELISA

Abstract
Nowadays ELISA is considered to be the troy horse for the routine clinical practice. This widely applied technique offers specific detection of a wide variety of target analytes in different kinds of samples. Since the invention of the technique four decades ago, ELISA has rapidly found various applications in food quality, environmental, biotechnological, and chemical disciplines among others. In spite of its many advantages, ELISA has certain limitations such as tedious/laborious assay procedure, and insufficient level of sensitivity in bio-recognition of challenging biomolecular entities such as microRNAs. A great number of research works has shown valuable attempts in addressing such shortages of ELISA through modification strategies. This chapter is dedicated to reviewing some of the main promising alternatives to the traditional ELISA. Paper- and fiber-based ELISAs, have shown great potentials for point-of-care (POC) applications due to their cost-effectiveness. Miniaturization of ELISA within micro-devices has increased the number and type of samples that can be analyzed, while much lower sample volume is required. Multiplexing was obtained as a result of micro and nano fabrication strategies and the integration of the assay within lab-on-chip (LOC) and lab-on-compact-disk (LOCD) devices. Taking advantage from a significantly vast surface area of the spheres, ultra-sensitive diagnosis was achieved by using micro-/nano-particles with different optical proprieties, sizes, synthetic variables and compositions. ELISA on the spot made possible to measure the biomolecules in vitro. Plasmonic ELISA offered detection strategies even with the aim of the naked eyes. Finally, the digital era has opened new windows of opportunity for ELISA, as the results of immunoassays can be recorded in remote/rural areas and subsequently analyzed by digital technologies or in centralized laboratories via mass data transfer.
Samira Hosseini, Patricia Vázquez-Villegas, Marco Rito-Palomares, Sergio O. Martinez-Chapa
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